Biomimetic silica nanospheres: a versatile nanotool for protein immobilization.

Biomimetic silica nanospheres: a versatile nanotool for protein immobilization. Erienne Jackson 1, Mariana Ferrari 1, Valeria Grazú 2, Jesús Martinez de la Fuente 3, Lorena Betancor 1. 1 Laboratorio de Biotecnología, Universidad ORT Uruguay, Montevideo, Uruguay; 2 Universidad de Zaragoza, Zaragoza, Spain; 3 Institute of Materials Science, Spanish Research Council, Aragón, Spain, Institute of Nano Biomedicine and Engineering. Biotechnology Department Lorena Betancor, PhD betancor@ort.edu.uy

Diatoms inspirate nanotechnology + silicic acid Kroger et al., Science 1999

Biomimetic silica: support for protein immobilization Silaffin peptide or poly amine molecule + Solution of Silicic acid + enzyme

Biomimetic silica: support for protein immobilization Enzyme Application Polyamine molecule Immobilization efficiency (%) Butyrylcholinesterase Organophosphate hydrolase Organophosphate acetic anhydrolase Hydroxylaminobenzene mutase Biosensor for nerve agent detection Biosensor for nerve agent detection or decontamination system Biocatalysis of amino-substituted aromatics R5 90 R5 25 R5 75 R5 80 Soybean peroxidase Biocatalysis R5 60 Nitrobenzene nitroreductase Activation of prodrugs PEI / Lys 48 / 21 Glucose oxidase Biosensor, Biocatalysis R5 / PEI / Lys 51/ 71/ 24 b-galactosidase Biocatalysis R5 / PEI / Lys 45 / 42 / 16 Catalase Biosensors and biosynthesis R5 75 Betancor and Luckarift, (2008) Trends Biotechnol. 26, (10), 566

Protein entrapment in biosilica nanoparticles. Rapid. Immobilization occurs within seconds. Mild. Formation of the particles occurs at room temperature and ph 8. Green. No requirement of organic solvents or high temperatures. Nanosized. 300 to 500 nm, Lower diffusion limitations and higher volumetric activities. Robust. Physical properties suitable for flow-through applications. Stabilizing. Numerous enzymes have been stabilized by entrapment in this support. Polymorphous. Shapes can be tailored by varying the conditions of silica deposition. Betancor et al. (2006) Chem. Comm. (34), 3640. Betancor and Luckarift, (2008) Trends Biotechnol. 26, (10), 566 Betancor et al. (2006) Biomacromolecules., 7(9), 2631.

New perspectives in biomimetic silica nanoparticles (a) (b) Foto 15 Tetramethyl orthosilicate Polyethylenimine (PEI) Buffer phosphate 0.1M, ph 8 + + Na 2 HPO 4 = Betancor and Luckarift, (2008) Trends Biotechnol. 26, (10), 566

New perspectives in biomimetic silica nanoparticles (b) Protein templated nanoparticles Foto 15 Sample Size (nm) DLS PDI* Z potential (mv) Silica-PEI 798.7 ± 100.8 0.639 ± 0.2 38 ± 0.8 Foto 15 Silica-PEI-BSA 374.4 Foto 12 ± 36.4 0.409 ± 0.1 32 ± 0.7 Tetramethyl orthosilicate Polyethylenimine (PEI) Buffer phosphate 0.1M, ph 8 + + Na 2 HPO 4 + = Foto 12 protein Jackson et. Protein templated biomimetic silica nanoparticles. (2015) Langmuir. Submitted Protein entrapment

Protein concentration in the supernatant (mg/ml) Proteinconcentrationin the supernatant (mg/ml) Use of external particle surface for protein immobilization: Adsorption of E coli protein extract. KDa 1 2 3 4 5 6 7 8 9 10 200 116 97 66 45 29 2,5 2,0 1,5 1,0 0,5 0,0 0 50 100 150 200 Time (min) Desorption of E coli protein extract. KDa 1 2 3 4 5 6 7 1,8 1,6 116 97 66 45 29 1,4 1,2 1,0 0,8 0,6 0,4 0,2 0,0 0 200 400 600 800 1000 1200 NaCl (mm)

Immobilization of Laccase from Trametes versicolor Betancor, L., et al. (2013). ChemCatChem, 5(1), 46.

Activity (UI) Activiy (UI) Adsorption of Laccase from Trametes versicolor 16 14 12 10 8 6 4 2 0 Adsorption 0 10 20 30 40 50 60 70 t (min) Adsorption 0,1 0,09 0,08 0,07 0,06 0,05 0,04 0,03 0,02 0,01 0 Desorption 0 200 400 600 800 1000 1200 [NaCl] (mm) Desorption 13.4 ± 1.5 mg 15.3 ± 0.5 mg Loading capacity using Laccase Tv. mg protein/g particles Silica PEI 42.8 ± 7.4 Silica PEI-BSA 57.3 ± 4.5

New perspectives in biomimetic silica nanoparticles Use of external particle surface for protein immobilization through covalent interaction Tetramethyl orthosilicate Polyethylenimine (PEI) BSA Silica PEI+BSA Glutaraldehyde Enzyme + + + + NH 2 Covalent linkage

Lipase sources Immobilization of lipases on biomimetic silica nanoparticles. Rhizomucor miehei lipase(rml) Thermomyces lanuginosus lipase (TLL) Bacillus thermocatenolatus lipase(btl2 Advantages of nanosized supports in biocatlysis: Large functional surface area Increased enzyme loading Reduction in diffusion limitations Enhanced particle mobility

Lipase immobilization in/on biomimetic silica nanoparticles Enzyme Strategy Immobilization (%) Yield (%) RML Physical entrapment 90,0 0, 8 24,0 0,6 Covalent linkage 87,0 0, 4 84,0 0,7 TLL BTL 2 Physical entrapment 40,0 0,9 30,0 0,7 Covalent linkage 74,0 0,9 100 1 Physical entrapment 91,0 0,1 47,00 0,09 Covalent linkage 95,0 0,1 47,00 0,08 Fig. 1 TEM image of silica nanoparticles

Residual activity (%) Thermal stability (60 C) of lipase preparations Residual activity (%) Enzyme Strategy Half life time (min) RML soluble enzyme 2,5 ± 0,2 Physical entrapment 70 ± 1 Covalent linkage 60 ± 1 TLL BTL2 soluble enzyme 140 ± 1 Physical entrapment 160 ± 1 Covalent linkage 360 ± 5 soluble enzyme 25 ± 1 Physical entrapment 125 ± 5 Covalent linkage 63 ± 1 RML Physical entrapment 100 RML Covalent linkage 100 90 80 70 60 50 40 30 20 10 0 RML Si RML Sol 0 20 40 60 80 100 120 T(min) 80 60 40 20 0 RML SiGlu RML Sol 0 50 100 150 200 250 300 T (min)

Residual activity (%) Residual activity (%) Residual activity (%) Stability of RML immobilized against solvents. Stability in 50% ethanol Stability in 50% ethanol 120 100 120 100 80 80 60 60 40 40 20 20 0 0 h 1 h 72 h 0 0 h 1 h 72 h Stability in 100% tert-butanol 120 100 80 60 40 20 0 0 h 1 h 96 h Stabilities were performed at 25 C incubating 13 IU/mL

AU AU AU Ascorbic palmitate synthesis Sin título 0,024 0,022 0,020 0,018 0,016 Ascorbic acid/palmitic Acid 0,014 0,012 0,010 0,008 0,006 0,004 0,002 0,000 Sin título 2,00 4,00 6,00 8,00 10,00 12,00 14,00 Minutes 0,190 Sample Name: sustrato de RML; Vial: 1; Injection: 4; Channel: 486 ; Date Acquired: 9/24/2014 12:32:18 PM UYT 0,180 Lipase Ascorbic acid Palmitic acid Ascorbic palmitate HO H 2 O 0,170 Reported by User: System Project Name: Defaults Report 0,160Method: Sin título Date Printed: Report Method ID: 101 10/14/2014 Ascorbic 0,150 4:32:58 PM America/Montevideo palmitate 0,140 0,130 0,120 0,110 0,100 0,090 0,080 0,070 0,060 0,050 0,040 0,030 0,020 0,010 0,000-0,010 Sin título 2,00 4,00 6,00 8,00 10,00 12,00 14,00 Minutes Sample Name: PA 5 mm reaccion 6-10-2014; Vial: 1; Injection: 4; Channel: 486 ; Date Acquired: 10/7/2014 2:54:41 PM UYT 0,013 Reaction after 1 hour at 37 C 0,012 Reported by User: System Project Name: Defaults Report Method: Sin título Date Printed: Report 0,011Method ID: 101 10/14/2014 4:44:43 PM America/Montevideo 0,010 6 % conversion 0,009 0,008 0,007 0,006 0,005 0,004 0,003 0,002 0,001 0,000-0,001 2,00 4,00 6,00 8,00 10,00 12,00 14,00 Minutes Sample Name: RML 1h ; Vial: 1; Injection: 1; Channel: 486 ; Date Acquired: 9/24/2014 2:34:51 PM UYT After 24 hours of reaction course, the immobilized preserved 80% of activity measured with pnpb RP-HPLC analysis: Luna C18 column, 250 x 4.56, 1 ml/min, Methanol: H2O: Acetic acid, 95:5:0,5 (v:v:v), UV detection at 266 nm Reported by User: System Project Name: Defaults Report Method: Sin título Date Printed: Report Method ID: 101 10/14/2014 4:38:12 PM America/Montevideo

GFP immobilization in biomimetic GFP immobilization silica nanoparticles 100% immobilization Minimal decrease in fluorescence after immobilization ph stability of entrapped GFP

Conclusions:.An inert protein template (BSA) enabled the rapid synthesis of positively charged disperse 200-300 nm particles as opposed to the synthesis without template which produced larger and less size homogenous nanoparticles. The particles synthesized in the presence of BSA reversibly and ionically adsorbs a range of proteins.. Glutaraldehyde functionalization of silica nanoparticles provided the possibility of covalently attached proteins on the particle surface. Silica particles provided stability against temperature, ph, organic solvents. In the case of lipasas it also provided operational stability..

ACKNOWLEDGMENTS Protein technology group Mariana Ferrari, PhD candidate Erienne Jackson, MSc candidate Josefina Louge, Research assistant Collaborators Dr. Jesús Martinez de la Fuente Dr. Valeria Grazú