(11) solid lipid nanoparticles (jw) - umass...

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

Laboratories

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


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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


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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


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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


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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


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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


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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


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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)


Laboratories

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


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



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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


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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!


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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


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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


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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)


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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!!!


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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


Laboratories

(11) solid lipid nanoparticles (jw) - umass...

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