Solid Lipid Nanoparticles as Novel Nutraceutical Carriers in Foods
Jochen Weiss*Julian McClements, Thrandur Helgason, Tarek Awad, Eric Decker
*Food Structure and Functionality LaboratoriesDepartment of Food Science & BiotechnologyUniversity of HohenheimGarbenstrasse 21, 70599 Stuttgart, Germany
Emulsion Workshop
November 13-14th, 2008, Amherst, MA
1Food Structure and Functionality
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Drivers for the Development of Bioactive
Crarrie Systems
• FUNCTIONAL FOODS
• Combination of Food Science and Technology, Nutrition and Consumer Sciences to Develop Functional Foods
• Goal: Improved health and life quality of population trough consumption of health-promoting foods
• Reduced costs and burden for health care systems
• Inclusion of bioactive compounds
• BUT: low bioavailability and high chemical instability of many bioactives
1998 2000 2002 2004 2006 2008
Ma
rket
Siz
e (
US
$ B
illio
n)
0
10
20
30
40
50
60
70
Functional Foods
� Global Market Size $50-60 Billions(Mintel & Euromonitor, 2005)
� USA > $20 Billions
� 8% worldwide growth annually
� 14% US growth annually USA
� Global Marketsize: $100 Billion in 2012
2Food Structure and Functionality
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Bioactive Food
Components
Cell Division And Growth
Apoptosis
DNA Repair
Inflammation
Hormon-Regulation
Cancer Metabolism
Energy Balances
• Isoprenoides– Carotenoides, Saponines, Tocotrienoles,
Tocopheroles
• Fatty Acids– MUFA, PUFA, ω-3, CLA
• Phenolic Compounds– Flavonoles, Flavonones, Anthrocyanines, Lignins,
Tannins
• Proteines / Amino Acids– Isothiocynate, Allyl-S Components, Capsaicinoides
• Polysaccharides– Ascorbic Acid, Oligosaccharides
• Minerals
• ........
Compounds chemically diverse, may be unstable upon introduction in food, often lipophilic �partitioning, low bioavailability and stability
3Food Structure and Functionality
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“Solid Lipid Nanoparticles” (SLN)
• Liquid lipid in emulsion is replaced by high melting point lipid
• Glycerides or waxes suitable
• Typical medium size ranges from 50 - 500 nm
• At small sizes, crystal structures become dependent on surfactant and size
• Polymorphism
Emulsion
Solid LipidNanoparticle
SurfactantLayer
liquidlipid (oil)
solidlipid
lipophiliccompound
exchangedegradation
No exchangeLess degradation
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Why Solid Lipid Nanoparticles?• Better control over release kinetics of encapsulated
compound– Engineering via size and lipid composition– Melting can serve as trigger
• Enhanced bioavailability of entrapped bioactives• Chemical protection of labile incorporated
compounds• Much easier to manufacture than biopolymeric
nanoparticles– No special solvents required– Wider range of base materials (lipids)– Conventional emulsion manufacturing methods
applicable• Raw materials essential the same as in emulsions• Very high long-term stability• Application versatility:
– Can be subjected to commercial sterilization procedures
– Can be freeze-dried to produce powdered formulation
Conventional Carrier
Microcarrier
Nanocarrier
20-50 µµµµm
2-5 µµµµm
200 nm
dc/dt
dc/dt
dc/dt
cs
cs
csDissolution velocitySaturation solubility
5Food Structure and Functionality
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Lipids Used in Manufacturing of SLN
Triglycerides Hard fat types (Co-)Emulsifiers
� Tricaprin
� Trilaurin
� Trimyristin
� Tripalmitin
� Tristearin
� Hydrogenated coco-glycerides
� Witepsol™ W/H 35, H42, E85
� Glycerol monostearate (Imwitor™), behenate (Compritol™)
� Palmitostearate (Precirol™)
� Stearic acid
� Palmitic acid
� Decanoic acid
� Behenic acid
� Soybean Lecithin
� Egg lecithin
� Phosphatidylcholine
� Polaxomer 188, 182, 407, 908
� Polysorbate 20, 60, 80
� Sodium cholate, glycocholate
� Butanol
� Butyric acid
� Taurocholic acid sodium salt
Our work focuses on production of stable SLN with food bioactives
6Food Structure and Functionality
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Manufacturing of SLN
• Three different approaches:– Hot homogenization
� homogenization at elevated temperatures
– Hot microemulsification
� Formation of microemulsion at elevated temperatures
– Cold homogenization
� Homogenization at low temperatures using milling processes
• Each process has advantages and disadvantages
• Selection of suitable process predominantly governed by type of compound to be encapsulated
• Scale-up procedures vary greatly between the different processes
7Food Structure and Functionality
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Production of SLN by Hot Homogenization
• Hot homogenization can be carried out by high pressure homogenizers or high intensity ultrasound
• Metal contamination a possibilty wit high-intensity ultrasound � coated probe
• Production of nanoemulsions at elevated temperatures � requires ability to thermostat the homogenization chambers
• Typical lipid contents between 5-10%, successful production of up to 40% reported
• 3-5 passes at 500-1500 bars
Dispersion of Bioactive-Lipid in
Hot Surfactant Solution
Coarse Pre-emulsion Formation
(Ultraturax)
Microfluidization
at T > Tm
Hot Oil-in-Water Nanoemulsions
Solidification by Controlled
Cooling
Melting of Carrier Lipid and Dispersing of Bioactive
Solid Lipid Nanoparticles
Note: Small particle size and presence of emulsifiers retards lipid crystallization – sample may remain as
shelf-stable supercooled melt for months/years
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Sound Great, BUT ……..
Issues with SLN!
� Kinetic instabilities
� Crystal structure: polymorphic transitions
� SLN dispersion stability: creaming
� Microphase separations during crystallization
� Loading & formulation
� A lot of Expertise is needed
Localization of bioactives?
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Crystal Structures of Triglyceride SLNs
• SLN structure depends on underlying crystal structure of matrix
• Different possible association configurations of individual chains
• Gives rise to longitudinal stacking of TAG molecules in lamellae
• α, β’ and β crystals � hexagonal, cubic and orthogonal crystals with different latices spacing
• Temperature profiles during production and storage essential
αααα ββββ’ ββββββββ
2.54Å
4.1-4.2Å
3.8 Å
4.6 Å
2L 3L
4.15Å
Fatty Acid Chain
End view
hexagonal cubic orthogonal
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The Issue of Polymorphic Transformations
� When polymorphic transitions occur, the lipid crystals rearrange to assume a more ordered state
� Ostwald’s step rule states:
� Thermodynamically less stable phase are initially formed and a stepwise phase changes to more stable phases follows
� Thus, the α-form form transitions to β’ and finally to β
� These crystals have different morphologies!
Himawan, C., V.M. Starov, and A.G.F. Stapley, Advances in Colloid and Interface Science, 2006. 122(1-3): p. 3-33.
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Why are Polymorphic Transitions a Problem?
5oC
30 min. 75oC
Fluid SLN at 5°C Gel at 5°C
Oiling off !!
Melting
Coalesced Droplets
After the initial formation of SLN, the suspensions increasingly lose fluidity due to particle aggregation. This gelation process is highly time and
temperature sensitive
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Polymorphic Transitions Depend on Storage Temperature
Helgason, T., et al., Journal of Food Hydrocolloids, 2007.
Stored at 1°C Stored at 5°C
Sto
rage
Sto
rage
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Time (min)
0 20 40 60 80 100 120 140
∆H
SL
N/ ∆
HC
(%
)
0
20
40
60
80
1°C
5°C
10°C
Time (min)
0 20 40 60 80G
* [P
a*s
]1e-3
1e-2
1e-1
1e+0
1e+1
1e+2
1e+3
1e+4
1e+5
1oC
5oC
10oC
Polymorphic Transitions Correlate Directly with Increases in Gel Strength
αααα
ββββ
The ratio of melt enthalphy of stable SLN (DHSLN) to melt enthalpy of coalesced/separated droplets increases with increasing holding temperature indicating a more rapid polymorphic
transformation in SLN (αααα to ββββ). This corresponds to a simultaneous increase in G’
TTcocoTTSLNSLN
TTcc
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Proposed Mechanism of SLN Destabilization
Awad, T., et al., Food Biophysics, 2007; Helgason, T., et al., Journal of Food Hydrocolloids, 2007.
SLN destabilization occurs via a complex combination of polymorphic transitions, morphological changes and aggregation
that eventually lead to coalescence upon heating
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Morphological Changes Due to Polymorphic Transitions Have Been Observed by Others
• Dramatic morphological changes during storage have been observed even in initially stable SLN preparations after long-term storage
• The influence of crystal form on shape of crystallized lipid droplets has been observed by Bunjes and coauthors
Dubes et al, European Journal of Pharmaceutics and Biopharmaceutics, 2003, Vol. 55, 279-282
TEM of SLN Preparation after 1 year storage
Needle-shape crystals
ββββ polymorph (platelets)
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Approaches to Stabilization of SLN - Modulation via Surfactant Choice -
• Choice of surfactants in formation of stable SLN critical: – Initial crystal structure (pre-solidification):
• Surfactants with liquid lipid tails will form a fluid membrane around the solidifying lipids upon crystallization. In this case crystallization is not initiated/aided by the surfactants.
• Surfactants with solid lipid tails may interact with the solidifying lipid matrix and act as nuclei. At small droplet diameters, suchemulsifiers may have substantial impact on the resulting crystalstructure
– Polymorphic transitions (post-solidification)• Surfactant concentration and type may have an influence on the
kinetics of polymorphic transitions after crystallization.
– Dispersion stability (post-solidification)• Insufficient surfactant may result in aggregation of the dispersion
due to hydrophobic interactions
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Influence of Surfactant on Crystallization of SLN (Pre-Solidification Influence)
• Use of long-chain fatty acid containing phospholipids lowers supercooling tendency
• Solidification of PL prior to TAG solidification alters crystallization behavior
• Modification of Tc thus possible through appropriate choice of emulsifier
• General retardation of polymorphic transitions in the presence of saturated and egg lecithin
DSC heating curves of SLNs after controlled cooling
Bunjes and Koch, 2005, J. Cont. Release, Vol. 107, 229-243
18Food Structure and Functionality
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Bunjes and Koch, 2005, J. Cont. Release, Vol. 107, 229-243
Influence of Surfactant on the Crystallization Temperature (Pre-
Solidification Influence)
• S100: Soybean Lecithin
• DMPC:Dimyristoyl-PC
• DPPC:Dipalmitoyl-PC
• E100:Egg yolk lecithin
• S100-3:hydrogenated Lecithin
19Food Structure and Functionality
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Influence of Surfactant Type on SLN Formation (Tween 20, 40, 60 & 80) – Pre-Crystallization
Tween 80
Tween 60
Tween 40
Tween 20
Tween 60
Tween 40
Tween 20
Tween 80
First Cooling Cycle Second Cooling Cycle
Surfactant type influences the crystal structures generated!
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Modulation of Polymorphic Transitions by Post-Addition of Surfactant
• SLN were initially manufactured with 10% tripalmitin and 2% Tween 20
• Immediately after homogenization SDS was added
• Addition of SDS at high concentration increasingly stabilized the α- and β´- form
0% SDS
0,01% SDS
0,05% SDS
0,1% SDS
0,5% SDS
1% SDS
2,5% SDS
5% SDS
30°C 40°C 50°C 60°C 70°C
Helgason, T., et al., Journal of Food Hydrocolloids, 2007.
SD
S C
once
ntra
tion
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Can Addition of Surfactants Post-Solidification Help Stabilize the Dispersion?
Added Tween 20
(%)
Liquid Solid
d43 Stdev d43 Stdev d32 Stdev d32 Stdev
0 0.770 0.085 0.163 0.006 Gel X Gel X
0.01 0.677 0.051 0.160 0.000 Gel X Gel X
0.025 0.837 0.412 0.163 0.006 Gel X Gel X
0.05 0.680 0.046 0.163 0.006 Gel X Gel X
0.075 0.683 0.012 0.163 0.006 Gel X Gel X
0.1 0.950 0.471 0.163 0.006 Gel X Gel X
0.5 0.783 0.159 0.167 0.006 Gel X Gel X
1 0.643 0.136 0.163 0.006 9.187 6.430 0.197 0.015
2.5 0.990 0.546 0.163 0.006 7.413 4.924 0.193 0.015
5 0.997 0.197 0.167 0.006 4.077 1.269 0.193 0.006
Addition of surfactant appears to help stabilize the dispersion
Helgason et al., Langmuir, 2008 (in Print)
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TwTotal Concentration (%)
0 1 2 3 4 5 6 7T
wa
q/T
wT
ota
l (%
)10
20
30
40
50
60 Liquid
Solid
Tween 20 Added (%)
0 1 2 3 4 5 6 7
Tw
een
20
De
tecte
d (
%)
0
1
2
3
4
Liquid
Solid
Evidence of Additional Surfactant Adsorption Upon Solid-Liquid Transitions
Solidification of droplets results in decreases in Tween 20 in the aqueous phase, suggesting additional absorption of the surfactant to the newly formed interfaces
Helgason et al., Langmuir, 2008 (In Print)
23Food Structure and Functionality
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Temperature (°C)
5 10 15 20 25 30 35 40
Hyd
rod
yn
am
ic R
ad
ius (
nm
)
200
300
400
500
600
700
800
900
100
1000
1% Tween 20 added
2% Tween 20 added
6% Tween 20 added
Crystallization in the Presence of Excess Surfactant
• In the presence of excess surfactant (2/6 wt%), particles grew upon solification, but did not aggregate
• In this case, dispersion remained stable
• If insufficient surfactant was present, particles aggregated rapidly upon cooling
Cooling
Crystallization
Helgason et al., Langmuir, 2008 (In Print)
Aggregation
Stable Dispersion
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What About Crystal Structures?(Post-Solidification)
0%
0.01%
0.05%
0.1%
1%
2.5%
5%
20 30 40 50 60 70
Heating enthalpy of tripalmitin SLN after addition of Tween 20 after
storage for 24 hours at 20°C
Cooling enthalpy of tripalmitin SLN after addition of Tween 20 after melting
at 75°C
0%
0.01%
0.05%
0.1%
1%
2.5%
5%
20 30 40 50 60 70
At increased added Tween 20 concentrations, more complex melting behavior suggesting alternative crystal structures
Helgason et al., Langmuir, 2008 (In Print)
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Proposed Mechanisms of Surfactant Modulation
• Pre-solidification:– Surfactants may act as seeds for the crystallization depending on their
molecular structure (liquid/solid tails) and the droplet size (no clear boundary, gradual modifications of crystal structures apparent)
– Sufficient surfactants must be available to form the liquid dispersion –which is less than the conc. required for solid dispersions
Liquid Tail Surfactants Solid Tail Surfactants
d < ~150 nm d >> ~150 nm d < ~150 nm d >> ~150 nm
26Food Structure and Functionality
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Proposed Mechanisms of Surfactant Modulation
• Post-solidification:
– Surfactants can aid stabilization of SLN dispersions by (a) modulating polymorphic transitions and (b) stabilizing generatedcrystals
Addition of
Surfactant
At low surfactant concentration
Cool to 5°C
At increased surfactant concentration
Low/no
excess
surfactant
Increased surface,
excess surfactant
adsorbs to interface
Polymorphic transitions, uncovered surfaces, aggregation
Solid lipid
Liquid
lipid
Crystallization
Cool to 5°C
CrystallizationExcess surfactant
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Issues Surrounding Loading with Bioactives
• Incorporated bioactive molecule can increase the complexity of the lipid matrix
• Linear bioactive molecules are more likely to fit inside the crystal matrix
• Nonlinear bioactive molecules likely disrupt the crystal order
• Bioactive molecules can be incorporated between fatty acid chains, between lipid layers and in imperfections in the crystals page
• Little problems in case of a low loading ration, but at high loading ratios, bioactives may be expelled from the matrix during crystallization
• Localization of bioactive??? Loading capacity???
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Particle Size (nm)
100 200 300 400 500
Ra
tio o
f S
hell
Volu
me
to T
ota
l V
olu
me
0.0
0.2
0.4
0.6
0.8
1.0
1.2
The Issue of Loading Capacity
With decreasing size, the amount of material that can be loaded in the particle decreases. In Foods, this can be a severely limiting issue since RDAs
(recommended daily allowances) must be delivered
( )
( )
33
2 2
3
2
3
2
3
2
4 4
3 34
3
1
core
total
r r rV
RV
r
r r
r
π π
π
− − ∆= =
− ∆= −
r1
r2
Idealized core-shell particle (e.g. ω-3 loaded TAG SLN
with TAG shell)
~ SLN Regime
~Transparency Boundary
~ Minimal Loading Boundary
e.g. at R=0.5, rSLN~60 nm �maximally allowed size to maintain an RDA of 300 mg in a 1 wt% emulsion made of fishoil!
29Food Structure and Functionality
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Addition of Large Quantities of Bioactives Influences Crystallization
(e.g. Tripalmitin SLN With ω-3 Fatty Acids)
10 20 30 40 50 60 70
Temperature (oC)
Hea
t fl
ow
(J/
g)
1.00
0.00
0.05
0.10
0.25
0.05
0.75
y = -15.936x + 40.037
R2 = 0.9765
y = -10.855x2 - 1.569x + 64.069
R2 = 0.9954
10
20
30
40
50
60
70
80
0 0.2 0.4 0.6 0.8 1
Φ ω−3
Tc,T
m (
oC
)
In bulk tripalmitin in the presence of ωωωω-3 fatty acids – significant decreases in melting and crystallization temp (50% loading desired)
Melt temperature
Crystallization temp.
ωω ωω-3
fat
ty a
cid
co
nte
nt
Melting
30Food Structure and Functionality
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How Does This Affect Production of SLN???
-15
-12.5
-10
-7.5
-5
-2.5
0
2.5
5
0 20 40 60 80Temperature (
oC)
Hea
t fl
ow
(J/
g)
Cool1
Heat
Cool2
-15
-10
-5
0
5
10
0 20 40 60 80Temperature (
oC)
Hea
t fl
ow
(J/
g)
Cool1
Heat
Cool2
Formation of αααα-crystals suppressed, formation of thermodynamically stable ββββ promoted.
Without Fish Oil With 0.25% Fish Oil
Tween 20 Stabilized
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Dispersion Stability of SLN in the Presence of ω-3 Fatty Acids
• Crystallized nanoemulsion with >25% w-3 fatty acids DO NOT aggregate
• Indicates that morphological changes associated with polymorphic transitions are suppressed.
Time (min)
0 10 20 30 40 50 60
Z-a
vare
ge
siz
e (
nm
)
130
140
150
160
170
180
190
200
0% ω-3
10% ω-3
25% ω-3
32Food Structure and Functionality
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Rheology of SLN Containing ω-3 Fatty Acids
• ω-3 fatty acid containing SLN did not show a noticeable increase in complex modulus
• The sample remained fluid during the first cooling process and also during a subsequent additional heating and cooling cycle.
1.E-02
1.E+00
1.E+02
1.E+04
0 20 40 60 80 100
Temperature (oC)
G*
(Pa)
+0.00
+0.25
+0.25 (melting)
33Food Structure and Functionality
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Potential Mechanism of Stabilization of SLN By ω-3 Fatty Acids
Liquid oil inside the crystal matrix retards the shape change
0% ω-3
>25% ω-3
Solid lipid
Liquid
lipid
Crystallization
Crystallization
Tripalmitin crystal covered by surface ω-3 fatty acids
Tripalmitin crystal containing micro-dispersed ω-3 fatty acidsActual structure as yet unkown!!!
34Food Structure and Functionality
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Conclusions• SLN are a promising new delivery system for the food industry due
to the fact that:– Large scale production possible, no organic solvents needed– High concentrations of functional compounds can be achieved– Lyophilization possible– Spray drying for lipids with Tm > 70ºC to yield powders
• Solid lipid nanoparticles are non-trivial systems with potentially complex structures that include variations in– Particle morphology, – Internal particle microstructure – Internal crystal structure
• Substantial issues that need to be addressed– Dispersion stability– Polymorphic transitions– What is the exact microstructure? � chemical stability
• Manufactures need to consider:– Lipid matrix compositional changes upon inclusion of bioactive– Choice of surfactant!!!! – Manufacturing conditions
35Food Structure and Functionality
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