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Article
Protein templated biomimetic silica nanoparticles. Erienne Jackson, Mariana Ferrari, Carlos Cuestas-Ayllon, Rodrigo Fernandez-Pacheco, Javier Perez-Carvajal, Jesus Martinez de la Fuente, Valeria Grazu, and Lorena Betancor Langmuir, Just Accepted Manuscript • DOI: 10.1021/la504978r • Publication Date (Web): 05 Mar 2015 Downloaded from http://pubs.acs.org on March 10, 2015
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Protein templated biomimetic silica nanoparticles.
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Erienne Jackson1, Mariana Ferrari1, Carlos Cuestas-Ayllon2, Rodrigo Fernández-Pacheco2,
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Javier Perez-Carvajal3, Jesús M. de la Fuente4, Valeria Grazú2*, Lorena Betancor1*
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1
Laboratorio de Biotecnología, Facultad de Ingeniería-Universidad ORT, Montevideo, Uruguay
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Instituto de Nanociencia de Aragon (INA), Universidad de Zaragoza, 50018 Zaragoza, Spain
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Instituto de Ciencia de Materiales de Madrid, CSIC, Sor Juana Inés de la Cruz, 3, Cantoblanco,
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28049 Madrid, Spain
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4
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Cerbuna 12, 50009 Zaragoza, Spain.
Instituto de Ciencia de Materiales de Aragón (ICMA), CSIC-Universidad de Zaragoza, c/Pedro
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Institute of Nano Biomedicine and Engineering, Key Laboratory for Thin Film and
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Microfabrication Technology of the Ministry of Education, Research Institute of Translation
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Medicine, Shanghai Jiao Tong University, Dongchuan Road 800, 200240 Shanghai, People’s
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Republic of China.
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*co-corresponding authors:
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[email protected] 17
Laboratorio de Biotecnología
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Facultad de Ingeniería
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Universidad ORT Uruguay
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Cuareim 1447, 1100 Montevideo
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Phone: +59829021505 int 1322
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[email protected] Nanoimmunotech S.L., Edif CIEM, Avda de la Autonomia 50003 Zaragoza
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Instituto de Nanociencia de Aragon
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University of Zaragoza
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C/ Mariano Esquillor s/n
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Zaragoza 50018
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Spain
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Phone: +34 976762982
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ABSTRACT
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Biomimetic silica particles can be synthesized as a nanosized material within minutes in a
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process mimicked from living organisms such as diatoms and sponges. In this work, we
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have studied the effect of bovine serum albumin (BSA) as a template to direct the synthesis
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of silica nanoparticles (NPs) with the potential to associate proteins on its surface. Our
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approach enables the formation of spheres with different physicochemical properties.
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Particles using BSA as a protein template were smaller ( ̴250–380 nm) and were more
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monodispersed than those lacking the proteic core ( ̴700–1000 nm) as seen by dynamic
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light scattering (DLS), scanning electron microscopy (SEM), and environmental scanning
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electron microscope (ESEM) analysis.The absence of BSA during synthesis produced silica
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nanoparticles without any porosity that was detectable by nitrogen adsorption, whereas
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particles containing BSA developed porosity in the range of 4–5 nm which collapsed on
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removable of BSA thus producing smaller pores. These results were in concordance with
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pore size calculated by high resolution transmission electron microscopy (HTEM).
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Reproducibility of the BSA-templated nanoparticles properties was determined by
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analyzing four batches of independent synthesizing experiments that maintained their
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properties. The high positive superficial charge of the nanoparticles facilitated the
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adsorption under mild conditions of a range of proteins from an E. coli extract and a
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commercial preparation of laccase from Trametes versicolor. All the proteins were
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quantitatively desorbed. Experiments conducted showed the reusibility of the particles as
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supports for ionic adsorption of the biomolecules. The protein loading capacity of the BSA
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based biomimetic particles was determined using laccase as 98.7 ± 6.6 mg.g-1 of particles.
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Keywords: silica nanoparticles, biomimetic silica, nanosupports, protein ionic adsorption.
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1. INTRODUCTION
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Silica nanoparticles have gained considerable interest among researchers due to their vast
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potential in biomedicine. Properties such as biocompatibilty, low toxicity and scalable
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availability have fuelled studies that span from applications in cell differentiation1to drug or
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siRNA delivery systems2 and imagenology3,4. Moreover, the precise control of silica
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particle size, porosity, crystallinity and shape strengthen their possibilities in
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nanobiotechnology5. However, traditional methods for the synthesis of silica nanoparticles
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often require time consuming processes, use of organic solvents and/or surfactants, high
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temperatures and other harsh sol-gel processing conditions5. Efforts should be focused on
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the search for new synthetic routes for silica nanospheres involving lower costs and lower
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environmental
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biocompatibility.
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The use of biomimetic approaches in the production of inorganic nanostructures could be of
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great interest to the scientific and industrial community due to the relatively mild physical
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conditions needed for their synthesis6, 7.In particular, biomimetic silica can be synthesized
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as a nanostructured material with divergent morphologies within minutes under mild and
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green conditions8, 9. Inspired from silaffin proteins used by unicellular diatoms10, several
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studies have identified alternate aminated molecules, as candidates for inducing silica
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precipitation from precursor compounds in vitro8,
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structure-directing templates via self-assembling into larger aggregates13.Influence of
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templates in silica biomineralization has been studied in vitrousing biological and non-
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biological molecules. For example bacterial flagella proved to be a promising biotemplate
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for developing morphology-controlled synthesis of silica nanotubes14, and chiral cationic
impact
while
simultanelously
maintaining
11, 12
reproducibility
and
.Silafins also serve in vivo as
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gelators induced the formation ofhelical mesoporous silica nanotubes14,
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demonstrate that the study of templates on biomimetic silica formation not only augments
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its possible applications but also help unveil details of its mechanism of biomimetic
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nucleation of silica.
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The benign reaction conditions associated with biomimetic silica formation in vitroare
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compatible with protein immobilization; any molecule present in the synthetic reaction
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mixture is entrapped due to the rapid formation of a network of fused silica nanospheres.
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The encapsulation of biomolecules within biomimetic silica particles has been investigated
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for a wide variety of enzymes
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coentrapment of different enzymatic functionalities20.The is no literature till date on the
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studies of interaction of proteins on the surface of biomimetic silica nanoparticles.
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Physicochemical studies of the nanoparticle surface are fundamental for the preparation of
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protein-nanoparticle composites. The nature of the interaction between the protein and the
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nanoparticle may have implications on their applications21,22. Strategies that allow changes
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in particle properties may be of substantial interest as this may impact the manner in which
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they “crosstalk” with proteins.
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In this work, we have studied the influence of a biological template on the physicochemical
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properties of the biomimetic silica nanospheres. We have developed a novel approach for
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the synthesis of biomimetic silica that may produce smaller and more homogeneous
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nanoparticles (Scheme 1). Moreover, we have explored the characteristics of the final
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material with a view of the on possible nanobiotechnological applications, wherein,
8, 16, 17, 18, 19
15
. These works
and has even proved successful for the
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properties such as size, dispersion, surface charge, and surface interactions with protein are essential.
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Scheme 1: Standard and novel approach for the synthesis of silica nanoparticles. TMOS,
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Tetramethyl orthosilicate. PEI, Polyethylenimine. BSA Bovine Serum albumin
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2. EXPERIMENTAL
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Polyethylenimine(PEI) (Mw 1300), Bradford reagent, molecular weight marker (M.W.
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30,000–200,000), protamine, Laccase from Trametes versicolor (EC 1.10.3.2) (activity of
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≥10 units per mg protein)and 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
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diammonium salt (ABTS) were from Sigma-Aldrich (St. Louis, MO). Tetramethyl
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orthosilicate was from MERCK (Whitehouse Station, NJ). Sodium phosphate dibasic and
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sodium acetate were from Biopack (Buenos Aires, Argentina). Sodium dodecyl sulfate
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(SDS) and Triton X-100 were from AppliChem (Darmstadt, Germany). EDTA disodium
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was from J.T Baker (Mexico). BSA solution was from New England Biolabs (Ipswich,
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MA). Gel filtration PD10-Columns were from GE Healthcare (Buckimghamshire, UK).
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All other chemicals were analytical grade reagents.
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2.1 Synthesis of biomimetic silica particles
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A stock solution of polyethylenimine (PEI, 10%) was prepared in deionized water. Silicic
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acid was prepared by hydrolyzing tetramethyl orthosilicate (TMOS, 1 M) in 1 mM
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hydrochloric acid. The precipitation mixture consisted of 10 mL 0.1M sodium phosphate
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dibasicbuffer pH 8.0, 2.5 mL hydrolyzed TMOS and 2.5 mL PEI 10%. The mixture was
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agitated for 5 min at room temperature. The resultant silica particles (Si-PEI) were pelleted
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by centrifugation for 10 min (4600g), washed twice with sodium phosphate dibasic buffer
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0.025M, pH 7.0 and sonicated for 5 min to improve monodispersity. For Silica-PEI-BSA
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particles, the precipitation mixture consisted of 10 mL of sodium phosphate dibasic buffer
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0.1M pH 8.0 with 1 mg.mL-1 BSA, 2.5 mL hydrolyzed TMOS (1M) and 2.5 mL of PEI
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10%.
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2.2 General procedures for nanoparticle characterization
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The morphology and particle-size distribution of the resulting nanoparticles (NPs) were
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characterized by SEM in a field emission FEI Inspect F operated at 10 and 5 kV. Aliquots
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of the different lyophilized nanoparticles were immobilized on the SEM sample holder
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using a double sided carbon tape. The samples were then immediately sputter-coated with
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gold before observation. Particle-size distribution was evaluated from several micrographs
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using an automatic image analyzer. Approximately 100 particles were selected for further
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consideration, which resulted in a stable size-distribution statistic. The nanoparticles were
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also analyzed under low atmospheric pressure and in the presence of water vapor using
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ESEM. Nanoparticles were added on a sample holder and mounted onto Peltier cooling
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stages set at 3 ºC. The relative humidity of the chamber was modified from 50 % to 100 %.
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Specimens were examined with a working distance of 5.5 mm and a low accelerating
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voltage of 10 kV at low pressure to reduce beam damage.
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For HRTEM examinations, a single drop (10 µL) of the aqueous solution (0.1 mg.mL-1) of
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BSA and non BSA templated particles were placed onto a copper grid coated with a carbon
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film. The grid was air dried for several hours at room temperature. TEM analysis was
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carried out in a FEI Tecnai F30 working at 300 kV.
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Thermogravimetric analysis (TGA) was performed using a TA STD 2960 simultaneous
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DTA-DTGA instrument in air or in nitrogen atmosphere and at a heating rate of 10ºC min-1.
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DLS and Z-potential measurements were performed on a Brookhaven Zeta PALS
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instrument at 25 ºC. Each sample was measured thrice, combining 10 runs per
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measurement.
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Texturalproperties of Silica-PEI and Silica-PEI-BSA were characterized by nitrogen
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adsorption isotherms at -196ºC of the samples using automatic adsorption equipment
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(ASAP2010 micromeritics®).Before the measurements, samples were lyophilized and then
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calcined under air fluxin a HOBERSAL® oven at 600ºC for 6 hours, using a heating rate of
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7ºC min-1.Lyophilized and calcined samples were then outgassed under vacuum at 150ºC
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until the pressure was stable and lower than 5 mercury microns. The apparent BET surface
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area (S0BET) was calculated by fitting nitrogen adsorption data to the BET equation.
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Themicropore volume (Vm) was calculated by the t-plot method. Barrett-Joyner-Halenda
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(BJH) method was also applied in order to determine the pore size distribution in the
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mesoporerange (2–50 nm). The total pore volume was directly recorded from the isotherm.
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The free space measurements were performed using helium gas. Nitrogen and helium gases
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used in the experiments were 99.9995% pure.
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2.3 Determination of protein concentration
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For E. coli experiments, protein concentration was determined by Bradford reagent 23 using
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the protocol recommended by the manufacturer, using BSA as a standard. For laccase
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experiments, protein concentration was determined spectrophotometrically measuring
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absorbance at 595nm specific of the T1 Cu located at the active site and using an ԐM =
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4400M-1cm-1.
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2.4 Determination of laccase enzyme activity
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Activity of Trametes versicolor (Tv)laccase was measured spectrophotometrically
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following the increment in the absorbance at 405nm generated by the hydrolysis of ABTS
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(0.38mM) in 25mM sodium acetate buffer pH 4.5 at 25°C. One enzyme unit (IU) was
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defined as the amount of enzyme able to produce 1 µmol of product per minute in the
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above defined conditions.
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2.5 Adsorption of Escherichia coli total protein extract to Si-PEI-BSA nanoparticles
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E. coli BL21(DE3) cells harboring pUC18 were inoculated into Luria-Bertani (LB) medium
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supplemented with 100 µg/ml ampicillin and incubated overnight at 37°C with continuous
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shaking (200 rpm), and then harvested by centrifugation at 5000 rpm for 15 min. Pellets
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were then re-suspended in a lysis buffer (PBS, lysozyme 1 mg.mL-1, EDTA disodium salt
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0.1mM). The lysate was kepton ice for 30 min thereafter 0.2% Triton X-100 was added.
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This step was followed by sonication (130 Watt ultrasonic processor, VCX130, SONICS &
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MATERIALS, INC.,Newtown, USA). The lysate was then centrifuged at 17000g for 60
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min at 4°C and the soluble fraction was collected.
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0.5 g of silica was then added to a 2.5 mL solution of 10 mM sodium phosphate buffer pH
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7.0 and 2.5 mL of the lysate soluble fraction. The mixture was incubated for 60 min at
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room temperature. Aliquots of supernatant collected by centrifugation (5 min, 5000g) were
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taken at intervals of 10, 20, 30 and 60 min, and thereafter the mixture was incubated
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overnight at 4°C with mild agitation and the final aliquot of supernatant was obtained.
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Protein concentration was measured for each aliquot and the particles were washed thrice
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with 10 mM sodium phosphate buffer pH 7.0.
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2.6 Adsoption of Tv laccase to silica nanoparticles
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Enzyme stock solutions were prepared by dissolving the lyophilized enzyme in buffer (25
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mM sodium phosphate buffer, pH 7.0).The stock solutions were purified by gel filtration
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using PD-10 columns in 10 mM sodium phosphate buffer, pH 7.0.
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1 g of silica was added to a solution of 2.5 mL 10 mM sodium phosphate buffer pH 7.0 and
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2.5mL of gel filtrated laccase (10 mg.mL-1). The mixture was incubated for 60 min at room
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temperature. Aliquots of the supernatant collected by centrifugation (5min 5.000g) were
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taken at intervals of 10, 20, 30, and 60 min. The mixture was incubated overnight at 4°C
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with mild agitation and the final aliquot of supernatant was obtained. The adsorbed laccase
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was then washed thrice with 10 mM sodium phosphate buffer, pH 7.0. Enzyme activity and
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protein concentration were measured for each aliquot.
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2.7 Desorption of proteins adsorbed to silica nanoparticles
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For desorption, silica particles containing adsorbed proteins were incubated with 10 mM
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sodium phosphate buffer, pH 7.0 with 25, 50, 75, 100, and 300 mM NaCl ( w/v 1:10 for
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laccase experiments and 1:25 for E. coli extract experiments). For NaCl concentrations of
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1M, the particles desorbed with 300 mM were washed thrice and resuspended in 10 mM
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sodium phosphate buffer, pH 7.0, 1M NaCl. In each case, aliquots of the supernatant
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collected by centrifugation (10 min, 4600g) were taken and enzyme activity and protein
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concentration were determined.
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Visualization of adsorbed and desorbed protein patterns were done by polyacrylamide gel
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electrophoresis (SDS PAGE, 10%) followed by staining with Coomassie Brilliant Blue R-
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250. Besides,in the case of the adsorbed proteins, 0.1 g of silica after adsorption was
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washed thrice with 10 mM sodium phosphate buffer pH 7.0 and then incubated with sample
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eletrophoretic buffer (R 1:3) for 5 min at 100°C.
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2.8 Determination of the loading capacity of the nanoparticles
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0.025 g of Si-PEI and Si-PEI-BSA nanoparticles were added to a solution of 0.5 mL 10
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mM sodium phosphate buffer pH 7.0 with Tv laccase 11mg.mL-1. Also, 0.025 g of
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MANAE agarose was added to a solution of 0.5 mL 10 mM sodium phosphate buffer pH
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7.0 with Tv laccase 11mg.mL-1. These mixtures were then incubated overnight at 4°C with
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mild agitation. Aliquots of the supernatant collected by centrifugation (5min 5000g) were
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taken. Enzyme activity was measured for each aliquot.
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The results shown in this paper correspond to the mean value of the four independent
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experiments.
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3. RESULTS AND DISCUSSION
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In nature, as the presence of a template during synthesis alters the properties of the biosilica
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materials, we explored the use of a proteic inert BSA template during silica formation in
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vitro. Our hypothesis included that silica precipitation was achieved using PEI as the
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required aminated catalyst for the synthesis.PEI is a branched polymer bearing primary and
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secondary amines that has previously demonstrated its potential for the formation of
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nanosized silica particles19. Interestingly, the use of BSA as a template generated particles
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with half the hydrodynamic radius than the particles without BSA (Table 1).
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Table 1. Dynamic light scattering and net charge analysis of nanoparticles synthesized with
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different templates.
Sample Silica-PEI Silica-PEI-BSA
DLS Size (nm)
Z potential PDI*
(mV)
798.7 ± 100.8 0.639 ± 0.2 38 ± 0.8 374.4 ± 36.4
0.409 ± 0.1 32 ± 0.7
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*Values given in number
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BSA has net negative charge (pI 4.7) at pH 8.0 and the synthesis was conducted in this
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conditions. Thus, PEI might cover the surface of a BSA core due to ionic interaction and
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therefore become organized as a compact sphere that directs silica precipitation. The
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difference in size could be advantageous for alternative applications of the biomimmetic
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particles24, 25.In particular, large superficial areas increase the loading capacity of surface
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immobilized biomolecules (eg. proteins, nucleic acids, drugs) which would be highly
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beneficial for biotechnological and biomedical applied purposes as it allows obtaining more
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efficient biocatalysts and enhancing the therapeutic efficacy of drugs.
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Both BSA and non BSA templated particles showed good colloidal stability with Z
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potentials of 32 ± 0.7 mV and 38 ± 0.8 mV, respectively at a neutral pH. Recommended
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values of Z potential for stable colloid suspension must be superior to ± 30 mV26. Values of
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Z potential were above 30 mV from pH3–pH 6 (Supplementary Information Figure S1).
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Analysis by SEM showed that in the presence of BSA, biomimetic silica formed as
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preferentially loose particles (Figure 1a) with a nanosized diameter concentrated within the
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range of ̴280-300 nm (Figure 1b). Whereas, the absence of BSA during silica precipitation
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prompted the formation of interconnected randomly agglutinated particles (Figure 1c ) of
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approximately 700–1400 nm (Figure 1d) thus the range was not as concentrated as in the
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presence of BSA.
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Figure 1
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This result is in accordance with those obtained for DLS (Table 1) that showed much larger
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hydrodynamic diameters for Si-PEI than for Si-PEI- BSA particles from the mean value
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obtained from four different batches of synthesis. Polydispersity index (PI) analysis also
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show a wider size distribution in the case of non BSA templated particles. All these results
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demonstrated that nanoparticles containing BSA were more homogeneous in size
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distribution (Table1).This data confirms our hypothesis of an ordered arrangement of PEI
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on the surface of BSA molecules thus directing the formation of more compact particles.
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High-resolution TEM analysis of silica particles provided evidence for spherical
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morphology nanoparticles with uniform nanopores (Figure 2). Nanopore size was estimated
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at 2.6–3.4 nm for Si-PEI-BSA and 2.8-4.4 nm for Si-PEI. Morphology observed was
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spherical for Si-PEI-BSA nanoparticles with a confirmed distribution in sizes as shown by
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SEM and DLS (Figure 2b). In the case of Si-PEI nanoparticles were found to be coalesced
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at times.
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Figure 2
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Both the types of NPs were also analyzed by ESEM. The original characteristics of material
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samples analyzed by ESEM might be preserved, as they do not need to be desiccated and
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coated with gold. Besides, it is possible to examine the samples in the presence of water
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vapor, thus obviating the need for a hard vacuum. ESEM micrographs taken at different
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percentages of relative humidity (from 50–100%) showed that the hydration process did not
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significantly alter the 3D morphology of both types of NPs despite their high content of
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organic matter (nearly 40% determined by TGA analysis) (Figure 3 and 4). The size range
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of the nanoparticles was also maintained (Supplementary Information Figure S2 and S3)
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Figure 3
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281 282
Figure 4
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In order to characterize the textural properties of Si-PEI and Si-PEI-BSA nanoparticles,
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samples were lyophilized (lyo) and then calcined (cal).Nitrogen adsorption isotherm of Si-
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PEI-lyo present a type II/III isotherm according to the deBoer classification which is
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characteristic of a non-specific adsorption27(Figure 5).
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Figure 5
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The absence of microporosity deduced from the t-plot method indicates thatthe organic
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compound (PEI) is located on the surface of the nanoparticles or inside the nanoparticles
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but blocks the access to the sensing gas. Once the sample is calcined and the organic
294
compounds removed (Si-PEI-cal), the isotherm can be classified as type II.S0BET increase
295
from 10 m2.g-1 to 40 m2.g-1 and the absence of microporosity is maintained (Table 2).
296
Table 2. Textural properties of lyophilized (lyo) and calcinated (cal) samples. Vm Total Volume External Surface Area (m2/g) (cm3/g) (cm³/g STP)
Sample
SBET (m2/g)
Silica-PEI lyo
10
10
-*
22
Silica-PEI cal
46
46
-*
37
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Silica-PEI-BSA lyo
21
21
-*
36
Silica-PEI-BSA cal
35
14
0.0062
41
297
* Non detectable
298
The difference between the kneeof the isotherms suggests a higher affinity between the
299
adsorbent and adsorbate on the calcined material.The organic compound was mainly
300
located on the surface of the particles and once PEI molecules were removed the external
301
surface area increases thus a larger surface area of silica is now available for nitrogen
302
adsorption. The apparent difference between these results and HTEM measurements could
303
be explained assuming that PEI molecules were also located inside the silica nanoparticles,
304
blocking the access of the sensing gas in the lyophilized sample and thus enabling the
305
measurement of mesoporosity by BJH method. These pores could collapse during
306
calcinations leading to non-porous silica nanoparticles or even porous nanoparticles with
307
minuscule pores that are not detected by the limitation of the nitrogen adsorption technique.
308
LyophilizedSi-PEI-BSA shows a type II isotherm with a mesopore distribution centered at
309
4.5 nm, which is in congruity with this material with the pore size distribution calculated by
310
HTEM. Nitrogen can flow inside these pores even though they still contain the protein.
311
Once the material is calcined, the external surface area decreases (Table 2) and the BJH
312
method cannot be applied. Interestingly, a micropore volume (less than 2 nm) (Table 2) can
313
be determined. This can be explained by the collapse of mesopores when the proteins are
314
denatured during the heating process, hence promoting smaller pores in the range of
315
micropores.
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316
After nanoparticles characterization Si-PEI-BSA particles were assayed in the interaction
317
with several proteins. Testing the nature of the interaction nanoparticles with biological
318
samples is significant for their putative biotechnological application.
319
As an initial approach, nanoparticles were made to interact with a complex protein mixture.
320
If ionic adsorption was the sole interaction between proteins with nanoparticles, a complete
321
desorption of the biological molecules would be expected after incubation with salt. The
322
strength of ionic molecule adsorption on surfaces depends on the number of moieties
323
involved in the interaction. Thus, poorly negatively charged molecules or small proteins
324
that offer less surface area for interaction would desorb at low salt concentrations.
325
Experiments were conducted with different batches of silica synthesized particles offering a
326
protein extract from E. coli to the particles. As the experiment was performed in batches, it
327
was possible to analyze the differential adsorption of the proteins from the supernatant by
328
SDS-PAGE (Figure 6a). As expected, the electrophoretic pattern demonstrated that those
329
with strong negative superficial charge were absorbed rapidly. There were no significant
330
differences in the supernatants from 90–120 min (Figure 6a) and judging by the slope of the
331
curve showing protein content in the supernatant vs time, proteins were absorbed mainly in
332
the first 30 min of the experiment (Figure 6b).
333
Figure 6
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334 335
The adsorbed proteins were also analyzed by electrophoresis of the Si-PEI-BSA particles
336
after boiling them directly in the eletrophoretic buffer containing high concentration of SDS
337
and mercaptoethanol. This strong treatment ensures desorption of any adsorbed proteins.
338
The analysis showed that there was a preferential adsorption of certain proteins on the
339
particles as the pattern is significantly different from that of the initial protein extract
340
(Figure 6a).
341
The particles containing adsorbed proteins were incubated with increasing amounts of NaCl
342
and the desorbed proteins were analyzed by SDS-PAGE (Figure 7a). Increasing the
343
concentrations of NaCl enabled desorption of different proteins corresponding to diverse
344
interaction intensities with the particles. Protein elution started at 100 mM NaCl with
345
preferential desorption of small proteins in accordance with a weaker interaction with the
346
nanoparticles.
347
Desorption was quantitative reaching100.9 ± 5.4 % protein recovery after incubation with
348
1M NaCl (Figure 7b).
349
Figure 7
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350 351
This experiment also proved that ionic interaction was the sole “communication” between
352
proteins and the particles. Particles were completely devoid of adsorbed proteins after being
353
incubated with NaCl (Supplementary File Figure S2). The stripped support was assayed
354
again in the adsorption of proteins from E. coli extract and similar results, in terms of
355
protein adsortion and desorbtion were obtained (Supplementary File Figure S4).
356
Reusability constitutes a fundamental premise for ionic exchangers. Our results
357
demonstrate the feasibility of reusing Si-PEI-BSA particles as ionic exchange material.
358
The same experiment as for the E. coli protein extract was carried out with a solution of a
359
commercial well characterized protein: a laccase from Trametes versicolor28. Studies of
360
interaction of nanoparticles with a purified model protein may provide a better insight into
361
charge specific binding and allow for rigorous studies of the maximum loading capacity.
362
The laccase offered to the silica particles (0.4 mg.g-1) was adsorbed within the first 10 min
363
of the experiment as expected by its low pI of 4.5 and high negative net superficial charge.
364
As laccases are often inhibited by Cl- desorption was carried out by a shift to pH 3.0and
365
followed by measuring the increase of protein concentration in the supernatant. Again,
366
quantitative amounts of enzyme were obtained demonstrating that all the adsorbed protein
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367
had been successfully desorbed (Supplementary File Figure S2). Colloidal stability of the
368
particles at pH 3.0 was improved as Z potential values were above 40 mV (Supplementary
369
File Figure S2).Finally, we studied the loading capacity of the particles synthesized with a
370
protein template using laccase as model protein (96 KDa). Load of up to 98.7 ± 6.6 mg.g-1
371
of silica particles was obtained for Si-PEI-BSA spheres.
372
4. CONCLUSION
373
The data summarized herein demonstrates that the use of an inert protein template (BSA)
374
enabled the rapid synthesis of positively charged dispersed (250-380 nm) particles as
375
opposed to the synthesis without a template which produced larger and smaller sized
376
homogenous nanoparticles. Characterization of different batches of nanoparticles
377
demonstrated the reproducibility of the synthetic strategy. The particles synthesized in the
378
presence of BSA reversibly and ionically adsorb a range of proteins. The nanoparticles
379
proved successful in their reutilization which added to their ease of preparation and cost-
380
effective green synthesis which is a remarkable advantage in its use as a nanosupport for
381
surface integration of biomolecules. Although, improved precise control of the nanoparticle
382
properties have yet to be developed they present a promising perspective for their
383
functionalization with proteins and their use thereof for biomedical, biosensing or
384
biocatalytical applications.
385
5. SUPPLEMENTARY MATERIALS
386
Supplementary information includes a figure of the potential of Si-PEI-BSA and Si-PEI at
387
varied pH and SDS-PAGE of the desorbed supports after incubation with NaCl, SDS, and
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388
silica reutilization. Additionally, it includes graphical representations of size distribution of
389
Si-PEI-BSA and Si-PEI nanoparticles at different percentages of relative humidity.
390
This material is available free of charge via the internet at http://pubs.acs.org.
391
6. ACNOWLEDGEMENTS
392
J.M. de la Fuente thanks ARAID for financial support. We also thank R. Barriosand
393
T.García-Somolinos;(Textural characterization facility at the Materials Science Institute of
394
Madrid, Spanish National Research Council) for having carried out the N2
395
adsorption/desorption experiments. E. Jackson and L. Betancor are grateful to the National
396
Research and Innovation Agency (ANII) and Universidad ORT Uruguay. JPerez-Carvajal
397
thanks the MINECO (Spain) support BES-2010-038410. The authors would also like to
398
thank Sara Rivera for technical support, Dr. C.R.I. Crick for his contributions to the paper.
399
7. REFERENCES
400
(1) Bao, X.; Wei, X.; Wang, Y.; Jiang, H.; Yu, D.; Hu, M. Effect of silica-based
401
nanomaterials and their derivate with PEGylation on cementoblasts. Annals of Biomedical
402
Engineering 2014,42 (8), 1781-1789.
403
(2) Kell, A. J.; Barnes, M. L.; Slater, K.; Lavigne, C. Functionalised silica nanoparticles
404
stable in serum-containing medium efficiently deliver siRNA targeting HIV-1 co-receptor
405
CXCR4 in mammalian cells. International Journal of Nano and Biomaterials 2012,4 (3-4),
406
223-242.
407
(3) Liu, X.; Qian, H.; Ji, Y.; Li, Z.; Shao, Y.; Hu, Y.; Tong, G.; Li, L.; Guo, W.; Guo, H.
408
Mesoporous silica-coated NaYF4 nanocrystals: Facile synthesis, in vitro bioimaging and
409
photodynamic therapy of cancer cells. RSC Advances 2012,2 (32), 12263-12268.
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Page 25 of 30 29
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410
(4) Natarajan, S. K.; Selvaraj, S. Mesoporous silica nanoparticles: Importance of surface
411
modifications and its role in drug delivery. RSC Advances 2014,4 (28), 14328-14334.
412
(5) Liberman, A.; Mendez, N.; Trogler, W. C.; Kummel, A. C. Synthesis and surface
413
functionalization of silica nanoparticles for nanomedicine. Surface Science Reports 2014,69
414
(2–3), 132-158.
415
(6) Tong, Z.; Yang, D.; Xiao, T.; Tian, Y.; Jiang, Z. Biomimetic fabrication of g-
416
C3N4/TiO2 nanosheets with enhanced photocatalytic activity toward organic pollutant
417
degradation. Chemical Engineering Journal 2015,260, 117-125.
418
(7) Briggs, B. D.; Pekarek, R. T.; Knecht, M. R. Examination of transmetalation pathways
419
and effects in aqueous Suzuki coupling using biomimetic Pd nanocatalysts. Journal of
420
Physical Chemistry C 2014,118 (32), 18543-18553.
421
(8) Betancor, L.; Luckarift, H. R. Bioinspired enzyme encapsulation for biocatalysis.
422
Trends in Biotechnology 2008,26 (10), 566-572.
423
(9) Jin, R.-H.; Yao, D.-D.; Levi, R. Biomimetic Synthesis of Shaped and Chiral Silica
424
Entities Templated by Organic Objective Materials. Chemistry – A European Journal
425
2014,20 (24), 7196-7214.
426
(10) Kröger, N.; Poulsen, N. Diatoms - From cell wall biogenesis to nanotechnology. 2008;
427
Vol. 42, pp 83-107.
428
(11) Zane, A. C.; Michelet, C.; Roehrich, A.; Emani, P. S.; Drobny, G. P. Silica
429
morphogenesis by lysine-leucine peptides with hydrophobic periodicity. Langmuir 2014,30
430
(24), 7152-7161.
431
(12) Belton, D. J.; Patwardhan, S. V.; Annenkov, V. V.; Danilovtseva, E. N.; Perry, C. C.
432
From biosilicification to tailored materials: Optimizing hydrophobic domains and
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433
resistance to protonation of polyamines. Proceedings of the National Academy of Sciences
434
2008,105 (16), 5963-5968.
435
(13) Gröger, C.; Lutz, K.; Brunner, E. Biomolecular self-assembly and its relevance in
436
silica biomineralization. Cell Biochemistry and Biophysics 2008,50 (1), 23-39.
437
(14) Li, D.; Qu, X.; Newton, S. M. C.; Klebba, P. E.; Mao, C. Morphology-controlled
438
synthesis of silica nanotubes through pH- and sequence-responsive morphological change
439
of bacterial flagellar biotemplates. Journal of Materials Chemistry 2012,22 (31), 15702-
440
15709.
441
(15) Xiaobing, W.; Xianfeng, P.; Huanyu, Z.; Yuanli, C.; Yongmin, G.; Baozong, L.; Kenji,
442
H.; Yonggang, Y. The formation of helical mesoporous silica nanotubes. Nanotechnology
443
2008,19 (31), 315602.
444
(16) Song, X.; Jiang, Z.; Li, L.; Wu, H. Immobilization of β-glucuronidase in lysozyme-
445
induced biosilica particles to improve its stability. Frontiers of Chemical Science and
446
Engineering 2014.
447
(17) Sheppard, V. C.; Scheffel, A.; Poulsen, N.; Kröger, N. Live diatom silica
448
immobilization of multimeric and redox-active enzymes. Applied and Environmental
449
Microbiology 2012,78 (1), 211-218.
450
(18) Kuan, I. C.; Chuang, C. A.; Lee, S. L.; Yu, C. Y. Alkyl-substituted methoxysilanes
451
enhance the activity and stability of d-amino acid oxidase encapsulated in biomimetic
452
silica. Biotechnology Letters 2012,34 (8), 1493-1498.
453
(19) Berne, C.; Betancor, L.; Luckarift, H. R.; Spain, J. C. Application of a microfluidic
454
reactor for screening cancer prodrug activation using silica-immobilized nitrobenzene
455
nitroreductase. Biomacromolecules 2006,7 (9), 2631-2636.
ACS Paragon Plus Environment
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Langmuir
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(20) Betancor, L.; Luckarift, H. R. Co-immobilized coupled enzyme systems in
457
biotechnology. Biotechnology and Genetic Engineering Reviews 2010,27, 95-114.
458
(21) Monopoli, M. P.; Åberg, C.; Salvati, A.; Dawson, K. A. Biomolecular coronas provide
459
the biological identity of nanosized materials. Nature Nanotechnology 2012,7 (12), 779-
460
786.
461
(22) Majouga, A.; Pichugina, D.; Ananieva, I.; Kurilova, S.; Shpigun, O.; Kuz'Menko, N.;
462
Zyk, N. New separation materials based on gold nanoparticles. Journal of Manufacturing
463
Technology Management 2010,21 (8), 950-955.
464
(23) Bradford, M. M. A rapid and sensitive method for the quantification of microgram
465
quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976,72,
466
248-254.
467
(24) Fuentes, M.; Mateo, C.; Pessela, B. C. C.; Batalla, P.; Fernandez-Lafuente, R.; Guisán,
468
J. M. Solid phase proteomics: Dramatic reinforcement of very weak protein-protein
469
interactions. Journal of Chromatography B: Analytical Technologies in the Biomedical and
470
Life Sciences 2007,849 (1-2), 243-250.
471
(25) Pessela, B. C. C.; Fuentes, M.; Mateo, C.; Munilla, R.; Carrascosa, A. V.; Fernandez-
472
Lafuente, R.; Guisan, J. M. Purification and very strong reversible immobilization of large
473
proteins on anionic exchangers by controlling the support and the immobilization
474
conditions. Enzyme and Microbial Technology 2006,39 (4), 909-915.
475
(26) Romero-Pérez, A.; García-García, E.; Zavaleta-Mancera, A.; Ramírez-Bribiesca, J. E.;
476
Revilla-Vázquez, A.; Hernández-Calva, L. M.; López-Arellano, R.; Cruz-Monterrosa, R. G.
477
Designing and evaluation of sodium selenite nanoparticles in vitro to improve selenium
478
absorption in ruminants. Veterinary Research Communications 2010,34 (1), 71-79.
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(27) Broekhoff, J. C. P.; De Boer, J. H. Studies on pore systems in catalysts: X.
480
Calculations of pore distributions from the adsorption branch of nitrogen sorption isotherms
481
in the case of open cylindrical pores B. Applications. Journal of Catalysis 1967,9 (1), 15-
482
27.
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(28) Betancor, L., Johnson, G. and Luckarift, H. Stabilized Laccases as Heterogeneous
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Bioelectrocatalysts. Chem Cat Chem 2012.
485
486
Figure Legends
487
Figure 1. Scanning electron microscopy (SEM) of (a) silica particles with PEI as template;
488
(b) graphical representation of size distribution; (c) SEM of silica particles with PEI and
489
BSA as template and (d) graphical representation of size distribution.
490 491
Figure 2. High Resolution Transmission electron microscopy (HTEM) of (a) and (c) silica
492
particles with only PEI (Si-PEI) and (b) and (d) silica particles with PEI and BSA as
493
template (Si-PEI-BSA).
494
Figure 3. ESEM micrographs of Si-PEI-BSA taken at different percentages of relative
495
humidity: (a) 50%, (b) 70%, (c) 80% and (d) 100%. For clarification purposes, 50% of
496
humidity was equivalent to the atmospheric moisture.
497
Figure 4. ESEM micrographs of Si-PEI taken at different percentages of relative humidity:
498
(a) 50%, (b) 70%, (c) 80% and (d) 100%. For clarification purposes, 50% of humidity was
499
equivalent to the atmospheric moisture.
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500
Figure 5. Nitrogen adsorption isotherms at -196ºCofSi-PEI (a) and Si-PEI-BSA (b). Open
501
triangles correspond to lyophilized (lyo) samples where open circles were also calcined
502
(cal).
503
Figure 6. (a) SDS-PAGE (12%) of samples from the adsorption of E. coli total protein
504
extract to Si-PEI-BSA nanoparticles. Lane 1: molecular weight marker; Lane 2: E. coli total
505
protein extract; Lane 3: E. coli total protein extract diluted 10 times; Lane 4: supernatant
506
after 10 min; Lane5: after 20 min; Lane6: after 30 min; Lane7: after 60min; Lane 8: empty
507
Lane 9: supernatant after 90min; Lane 10: proteins adsorbed to silica at the end of
508
experiment. (b) Curve corresponding to the variation of the protein concentration in the
509
supernatant with time in the adsorption of E. coli total protein extract to silica
510
nanoparticles.
511
Figure 7. (a) SDS-PAGE (12%) of samples from the desorption of E. coli total protein
512
extract adsorbed to SI-PEI-BSA nanoparticles with different concentrations of NaCl. Lane
513
1: molecular weight marker; Lane 2: proteins adsorbed to silica before desorption; Lane 3:
514
desorption of proteins with 25 mM NaCl; Lane 4: with 50 mM NaCl; Lane5: with 100 mM
515
NaCl; Lane 6: with 300 mM NaCl; Lane7: with 1 M NaCl. (b) Curve representing the
516
desorption of E. coli total protein extract adsorbed to silica nanoparticles with different
517
concentrations of NaCl.
518
519
520
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Synthetic strategy 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42
Silica PEI
Langmuir
a)
• 700-1000nm •b)Agglutinated particles • Good colloidal stability
Page 30 of 30
Tetramethyl orthosilicate
+
Polyethylenimine (PEI)
Synthetic strategy
a)
Silica PEI+BSA
b)
Tetramethyl orthosilicate
+ Polyethylenimine (PEI)
+ BSA
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• 250-380 nm •More homogeneous in size distribution • More structured • Larger superficial areas • Good colloidal stability
