Engineering Conferences International ECI Digital Archives Design and Manufacture of Functional Microcapsules and Engineered Products Proceedings 4-5-2016 Encapsulation of biological materials Alexander Routh University of Cambridge, afr10@cam.ac.uk Follow this and additional works at: http://dc.engconfintl.org/microcapsules Recommended Citation Alexander Routh, "Encapsulation of biological materials" in "Design and Manufacture of Functional Microcapsules and Engineered Products", Chair: Simon Biggs, University of Queensland (Aus) Co-Chairs: Olivier Cayre, University of Leeds, UK Orlin D. Velev, North Carolina State University, USA Eds, ECI Symposium Series, (2016). http://dc.engconfintl.org/microcapsules/14 This Abstract and Presentation is brought to you for free and open access by the Proceedings at ECI Digital Archives. It has been accepted for inclusion in Design and Manufacture of Functional Microcapsules and Engineered Products by an authorized administrator of ECI Digital Archives. For more information, please contact franco@bepress.com.
Encapsulation for Biological Material Alex Routh Dept of Chemical Engineering and Biotechnology BP Institute University of Cambridge 1
About the BP Institute Formed in 1999 with an endowment from BP 6 academics in 5 Cambridge departments Prof Andy Woods Earth Sciences Dr Stuart Clarke Chemistry Dr Jie Li Engineering Dr Colm Caulfield DAMTP Dr Jerome Neufeld Earth Sciences/DAMTP Dr Alex Routh Chemical Engineering 2
Outline Colloidosomes How to make them Release profiles Biological encapsulation Yeast Lactic acid bacteria Enzymes Release by Shear Dilution Acid Metal shells Targeted delivery 3
Encapsulation: Colloidosome Formation Continuous phase (organic) Emulsification 1 Shell stabilisation by sintering 2 Transfer & redispersion Dispersed phase (aqueous) Solvent miscible with interior fluid Bacteria Latex particles Surfactant 4
Optical Microscopy Water-in-oil microcapsules Dried colloidosome microcapsules - scale ~ 10µm - - scale ~ 10µm - 5
SEM Colloidosome microcapsules - scale ~ 10µm - Coagulated latex particles forming single particle layer shell - scale ~ 200nm - Langmuir 25(1): 159-166 2009 6
Effect of Sintering Time 5 min 30 min 60 min - scale for all images ~ 500nm - These colloidosomes have been sintered as shown while maintaining a constant sintering temperature of 7 C above the glass transition temperature (Tg) of the polymer. Langmuir 25(1): 159-166 2009 7
Colloidosomes dye release experiment Relese rate seems to be invariant to sintering time 100 Release Fraction (%) 80 60 40 20 5 min 30 min 60 min 0 0 5 10 15 20 25 Time (mins) Langmuir 25(1): 159-166 2009 8
Colloidosomes for biological encapsulation Colloidosomes are poor for encapsulation of small molecules e.g perfumes in washing powders want an inorganic shell for this Colloidosomes are inherently porous with holes around the size of the base colloidal particles Like a string shopping bag Ideal for biological encapsulation Nutrients can freely diffuse in and out Simple biologically friendly production self assembly production 9
Yeast in colloidosomes Fluorescence & transmission confocal microscopy images of a colloidosome encapsulating viable Baker s Yeast cells stained with FUN 1 10
Yeast in colloidosomes Time series of optical microscope images showing yeast cells moving and dividing within a colloidosome Langmuir, 28(2):1169-1174 2012 11
Glucose trends Glucoseconcentration / mmol 8 7 6 5 4 3 2 1 0 0 100 200 300 400 500 Time / hrs Colloidosomes encapsulating yeast (single latex stage) Water-core colloidosomes Unencapsulated yeast Unencapsulated yeast exposed to 0.25% Virkon for 10 min Colloidosomes encapsulating yeast (double latex stage) Langmuir, 28(2):1169-1174 2012 12
Lactic acid bacteria in colloidosomes Encapsulation of lactic acid bacteria Maintain viability through simulated gastric and intestinal conditions Probiotic yoghurts Health effects dependent on survival of probiotic bacteria through stomach 13
Avoiding killing or damaging the bacteria Ethanol causes cell membrane damage Need to alter method of aggregating colloidal latex particles Salt causes colloidal aggregation Lactic acid bacteria have a relatively high tolerance to salt Success with a combination of NaCl and Ethanol 14
Encapsulated viable Lactic Acid Bacteria Green fluorescence = viable undamaged bacteria Transmission and confocal microscopy images of colloidosomes encapsulating Lactic acid bacteria stained with fluorescent molecular probes 15
Encapsulated Lactic Acid Bacteria: Glucose and ph trends Langmuir 28(46):16007-16014 2012 16
Encapsulated Lactic Acid Bacteria: Protection at low ph Langmuir 28(46):16007-16014 2012 17
Enzymes in Colloidosomes We want Survival for months in washing detergent Ease of release on dilution and shear Added to washing liquids to break down fats Enzymes are denatured by the surfactants Does encapsulation provide protection? Release upon Acid Shear Dilution Coating with CaCO 3 to provide absolute barrier 18
Unsealed colloidosomes will leak Enzymes are smaller than bacteria they release from standard colloidosomes Unsealed colloidosomes will release amylase enzyme over 30 hours Langmuir 30:1939-1948 2014 United Kingdom Patent Application No. 1319923.7 19
Production of colloidosomes with inorganic shell Simple to make with inorganic shells add Na 2 CO 3 and CaCl 2 to the two aqueous phases Langmuir 30:1939-1948 2014 United Kingdom Patent Application No. 1319923.7 20
SEM images No CaCO3 25% CaCO3 40% CaCO3 40% CaCO3 21
Release with acid With a dye: release on application of acid 25% encapsulation yield Langmuir 30:1939-1948 2014 United Kingdom Patent Application No. 1319923.7 22
Final release upon application of shear With a dye: increase in CaCO 3 in shell needs larger shear to cause release Increasing shear Langmuir 30:1939-1948 2014 United Kingdom Patent Application No. 1319923.7 23
Triggered release on shear or dilution Enzyme can be released by shear Enzyme can be released by dilution Langmuir 30:1939-1948 2014 United Kingdom Patent Application No. 1319923.7 24
Sealed colloidosomes protect enzyme Release activity upon shear Unencapsulated enzymes and unsealed colloidosomes display reduced activity Langmuir 30:1939-1948 2014 United Kingdom Patent Application No. 1319923.7 25
Aqueous core colloidosomes with a metal shell Dye release % Silver Coated Colloidosomes: AgNO3 in wash solution, reducing agent in core a b 100 Blank sample 90 80 70 60 Polymer shell 50 c d 40 30 Release silver shell 20 10 0 Silver shell 0 50 100 150 200 250 300 350 400 450 500 550 Time/h Optical microscope image, SEM images and TEM image of silver shell colloidosomes: (a) optical microscope image; (b and c) SEM images; and (d) TEM image (1)dye release % (dye) (2)dye release % (dye+latex) (3)dye release % (dye+latex+silver) (4)dye release % (polymer shell colloidosomes) (5)dye release % (silver shell colloidosomes) (6)dye release % (triggered silver shell colloidosomes) Release of Allura Red dye from colloidosomes sealed with a silver shell, polymer shell and unsealed 26
Aqueous core colloidosomes with a metal shell SEM images of spherical silver shell colloidosomes SEM images of gold coated colloidosomes Thiol chemistry (including SH structure) Tumor Colloidosome One potential application of metal coated colloidosomes - targeted drug delivery 27
Conclusions Colloidosomes are a biologically benign method of encapsulation Range of biological material can be encapsulated shell locking step requires species specific method Continued biological activity is observed Metal shell can be placed around colloidosome Colloidosomes can be targeted to specific proteins 28
Acknowledgements Grace Yow Polly Keen Qian Sun 29