Solid Lipid Nanoparticles: A New and Effective Delivery .../media/Knowledge Center/Science Reports/Conference... · and Effective Delivery System for Bioactives in Foodsin Foods Jochen

Solid Lipid Nanoparticles: A New and Effective Delivery System for

Bioactives in FoodsBioactives in Foods

Jochen Weiss*Julian McClements, Thrandur Helgason, Tarek Awad, Eric Decker

*Food Structure and Functionality LaboratoriesDepartment of Food Science and BiotechnologyUniversity of HohenheimGarbenstrasse 25, 70599 Stuttgart, Germany

IFT International Food Nanoscience ConferenceJune 6th, 2009, Anaheim, CA

1Food Structure and Functionality Laboratories

Presentation OverviewPresentation Overview• Foreword• Solid Lipid Nanoparticles (SLN)

– What are they?– How are they manufactured?

P ti d P bl f SLN• Properties and Problems of SLN– Polymorphic Transitions– Gelation

• Stabilization of SLN• Stabilization of SLN– Pre-Crystallization– Post-Cryzstallization

• Application ExamplesApplication Examples– Omega-3 Encapsulation– b-Carotene Encapsulation

• Conclusions

Foreword: The ‘Structural’ Food Science Revolution

SimpleMultiple

The Lipid Family The Biopolymer Family

Solid Lipid

Simple Droplets

Emulsions

Solid LipidParticles

The Surfactant Family

CoatedDroplets Pickering

Emulsions

Improved understanding of nanoscalarassembly processes have led to this

explosion !!!

Many more …

Food Structure and Functionality Laboratories

3

pAdapted from Julian McClements

I. What Are “Solid Lipid Nanoparticles” (SLN)??

• Liquid lipid in emulsion is replaced

Emulsionliquidlipid (oil)

emulsion is replaced by high melting point lipid

• Glycerides or waxes exchangedegradation y

suitable• Typical medium size

ranges from 50 - 500 SurfactantLayer

lipophiliccompound g

nm • At small sizes, crystal

structures become

Layer p

No exchangeLess degradation

dependent on surfactant and size

• PolymorphismSolid Lipid

Nanoparticlesolidlipid


Why Solid Lipid Nanoparticles?• Better control over release kinetics of encapsulated

compound– Engineering via size and lipid composition

M lti t i

Conventional Carrier

– Melting can serve as trigger• Enhanced bioavailability of entrapped bioactives• Chemical protection of labile incorporated

compounds• Much easier to manufacture than biopolymeric

20-50 mdc/dtcs• Much easier to manufacture than biopolymeric

nanoparticles– No special solvents required– Wider range of base materials (lipids)– Conventional emulsion manufacturing methods

Microcarrier

2 5Conventional emulsion manufacturing methods applicable

• Raw materials essential the same as in emulsions• Very high long-term stability• Application versatility: Nanocarrier

2-5 mdc/dtcs

pp y– Can be subjected to commercial sterilization

procedures– Can be freeze-dried to produce powdered

formulation

200 nmdc/dtcsDissolution velocitySaturation solubility


Manufacturing of SLNg• Three different approaches:

Hot homogenization– Hot homogenization homogenization at elevated temperatures

– Hot microemulsification F ti f i l i t l t d t t 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 encapsulatedyp p p• Scale-up procedures vary greatly between the different

processes


Production of SLN by Melting of Carrier Lipid and

Dispersing of Bioactive

Hot Homogenization• Hot homogenization can be carried out by high

Dispersion of Bioactive‐Lipid in Hot Surfactant Solution

g y gpressure homogenizers or high intensity ultrasound

• Metal contamination a possibilty wit high-intensity ultrasound coated probe

Coarse Pre‐emulsion Formation (Ultraturax)

intensity ultrasound coated probe• Production of nanoemulsions at elevated

temperatures requires ability to thermostat the homogenization chambersT i l li id t t b t 5 10%

Microfluidization at T > Tm

• Typical lipid contents between 5-10%, successful production of up to 40% reported

• 3-5 passes at 500-1500 barsHot Oil‐in‐Water Nanoemulsions

Solidification by ControlledSolidification by Controlled Cooling

Solid Lipid Nanoparticles

Note: Small particle size and presence of emulsifiers retards lipid crystallization – sample may remain as

shelf-stable supercooled melt for months/yearsNanoparticles shelf-stable supercooled melt for months/years


II. Properties and Issues Surrounding SLN …

Issues with SLN!Localization of bioactives?

Kinetic instabilities Crystal structure:

polymorphicpolymorphictransitions

SLN dispersion stability: creamingy g

Microphaseseparations during crystallization

Loading & formulation

A lot of Expertise is needed


Crystal Structures of T i l id SLN

Fatty Acid Chain

Triglyceride SLNs

SLN d d

End viewhexagonal cubic orthogonal

• SLN structure depends on underlying crystal structure of matrixDiff t ibl i ti• Different possible association configurations of individual chains

• Gives rise to longitudinal stacking f TAG l l i l ll

4.1-4.2Å

3.8 Å

4.6 Å4.15Å

of TAG molecules in lamellae • , ’ and crystals hexagonal,

cubic and orthogonal crystals with diff t l ti i

’

different latices spacing• Temperature profiles during

production and storage essential2L 3L


The Issue of Polymorphic T f tiTransformations

When polymorphic transitions When polymorphic transitions occur, the lipid crystals rearrange to assume a more ordered state

Ostwald’s step rule states: Ostwald s step rule states: Thermodynamically less stable phase are initially formed and a stepwise phase changes toa stepwise phase changes to more stable phases follows

Thus, the α‐form formtransitions to β’ and finally to ββ y 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.


Why are Polymorphic Transitions a Problem?

5oC

Oiling off !!

Melting5oC

30 min. 75oC

Melting

Fluid SLN at 5°C Gel at 5°C Coalesced DropletsFluid SLN at 5°C Gel at 5°C Coalesced Droplets

After the initial formation of SLN, the suspensions increasingly lose fluidity due to particle aggregation. This gelation process is highly time and

i itemperature sensitive


Polymorphic Transitions Depend on Storage TemperatureTemperature

Stored at 1°C Stored at 5°C

Storage

Storage

Helgason, T., et al., Journal of Food Hydrocolloids, 2007.


Polymorphic Transitions Correlate Directly with Increases in Gel Strength

801°C 5°C 10°C

1e+4

1e+5

1oC 5oC

Increases in Gel Strength

TTcocoTTSLNSLN

HC

(%)

40

60

Pa*

s]

1e+1

1e+2

1e+3 10oC

TTcc

HS

LN/

20

40

G*

[P

1e-1

1e+0

1e+1

0 20 40 60 80 100 120 1400

0 20 40 60 801e-3

1e-2

Time (min) Time (min)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 ( to ). ( )

This corresponds to a simultaneous increase in G’


Proposed Mechanism of SLN D t bili tiDestabilization

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 heatingthat eventually lead to coalescence upon heating


Morphological Changes Due to Polymorphic Transitions Have Been Observed by Othersy

• Dramatic morphological changes during storage have been observed even in initially stable SLN

TEM of SLN Preparation after 1 year storage

e e a y s ab e Spreparations after long-term storage

• The influence of crystal form on shape of crystallized lipid droplets has been observed by Bunjes andhas been observed by Bunjes and coauthors

Dubes et al, European Journal of Pharmaceutics and Biopharmaceutics, 2003, Vol. 55, 279-282 polymorph (platelets)

Needle-shape crystals


e

A Last Issue: Loading Capacity…

tal V

olum

e

1.0

1.2

332 2

32

4 43 3

43

core

total

r r rVRV r

Idealized core-shell particle (e.g. -3 loaded TAG SLN

with TAG shell)

~ SLN Regime

ume

to T

ot

0.6

0.8 32

32

3

1r r

r

r1

r2

Mi i l L di

Shel

l Vol

u

0.4~Transparency Boundary

~ Minimal Loading Boundary

100 200 300 400 500

Rat

io o

f S

0.0

0.2e.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

Particle Size (nm)100 200 300 400 500

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

a 1 wt% emulsion made of fishoil!

y g(recommended daily allowances) must be delivered


III. Approaches to Stabilization of SLN Modulation via Surfactant Choice- Modulation via Surfactant Choice -

• Choice of surfactants in formation of stable SLN critical: Initial crystal structure (pre solidification):– 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, such emulsifiers may have substantial impact on the resulting crystal structurestructure

– 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


Influence of Surfactant on Crystallization of SLN (Pre-Solidification Influence)

• Use of long-chain fatty acid containing phospholipids lowers p p psupercooling tendency

• Solidification of PL prior to TAG solidification alters crystallization b h ibehavior

• Modification of Tc thus possible through appropriate choice of emulsifier

DSC heating curves of SLNs after controlled cooling

emulsifier• General retardation of

polymorphic transitions in the presence of saturated and eggsaturated and egg lecithin

Bunjes and Koch, 2005, J. Cont. Release, Vol. 107, 229-243


Influence of Surfactant Type on SLN Formation (Tween 20 40 60 & 80) Pre Crystallization(Tween 20, 40, 60 & 80) – Pre-Crystallization

First Cooling Cycle Second Cooling Cycle

Tween 80Tween 80

Tween 60

Tween 40

Tween 60

Tween 40

Tween 20 Tween 20

Surfactant type influences the crystal structures generated!Surfactant type influences the crystal structures generated!


Modulation of Polymorphic Transitions by Post-Addition of SurfactantAddition of Surfactant

• SLN were initially f t d ith

5% SDS

manufactured with 10% tripalmitin and 2% Tween 20 Immediately after

1% SDS

2,5% SDS

SD

S• Immediately after homogenization SDS was added

• Addition of SDS at 0,1% SDS

0,5% SDS

S C

oncent• Addition of SDS at high concentration increasingly stabilized the α- and 0 01% SDS

0,05% SDStration

stabilized the α and β´- form 0% SDS

0,01% SDS

30°C 40°C 50°C 60°C 70°C30 C 40 C 50 C 60 C 70 CHelgason, T., et al., Journal of Food Hydrocolloids, 2007.


Can Addition of Surfactants Post-Solidification Help Stabilize the Dispersion?Help Stabilize the Dispersion?

Added Tween 20

Liquid Solid

(%) d43 Stdev d43 Stdev d32 Stdev d32 Stdev0 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 X0.025 0.837 0.412 0.163 0.006 Gel X Gel X0.05 0.680 0.046 0.163 0.006 Gel X Gel X0.075 0.683 0.012 0.163 0.006 Gel X Gel X0 1 0 950 0 471 0 163 0 006 G l X G l X0.1 0.950 0.471 0.163 0.006 Gel X Gel X0.5 0.783 0.159 0.167 0.006 Gel X Gel X1 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 0152.5 0.990 0.546 0.163 0.006 7.413 4.924 0.193 0.0155 0.997 0.197 0.167 0.006 4.077 1.269 0.193 0.006

Addition of surfactant appears to help stabilize the dispersionpp p pHelgason et al., Langmuir, 2008 (in Print)


Evidence of Additional Surfactant Adsorption Upon Solid-Liquid Transitions

60 Liquid Solid %

)

4

LiquidSolid

q

wTo

tal (

%)

40

50

So d

Det

ecte

d (

2

3Solid

Twaq

/Tw

30

een

20 D

1

2

0 1 2 3 4 5 6 710

20

0 1 2 3 4 5 6 7

Tw

0

TwTotal Concentration (%)Tween 20 Added (%)

Solidification of droplets results in decreases in Tween 20 in the aqueous phase, suggesting additional absorption of the surfactant to the newly formed interfacesgg g p y

Helgason et al., Langmuir, 2008 (In Print)


Crystallization in the Presence of Excess Surfactant

nm)

800900

10001% Tween 20 added

Surfactant

• In the presence of f t t

Aggregation

adiu

s (n

400

500

600

700 2% Tween 20 added6% Tween 20 added

excess surfactant (2/6 wt%), particles grew upon solification but did Cooling

nam

ic R

300

400solification, but did not aggregate

• In this case, dispersion

Cooling

C t lli ti

ydro

dyn

200dispersion remained stable

• If insufficient surfactant was

CrystallizationStable Dispersion

Temperature (°C)5 10 15 20 25 30 35 40

Hy

100surfactant was present, particles aggregated rapidly upon cooling Temperature ( C)upon cooling



What About Crystal Structures?(Post-Solidification)

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

2 5%

5% 5%

0.1%

1%

2.5%

0 1%

1%

2.5%

0%

0.01%

0.05%

0%

0.01%

0.05%

0.1%

20 30 40 50 60 70

0%

20 30 40 50 60 70

At increased added Tween 20 concentrations, more complex melting behavior suggesting alternative crystal structurescrystal structures



Proposed Mechanisms of Surfactant Modulation• Pre solidification:• 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 Surfactantsq

d < ~150 nm d >> ~150 nm d < ~150 nm d >> ~150 nm


Proposed Mechanisms of Surfactant Modulation• Post-solidification:

– Surfactants can aid stabilization of SLN dispersions by (a) modulating polymorphic transitions and (b) stabilizing generated g p y p ( ) g gcrystals

At low surfactant concentration At increased surfactant concentration

Low/no excess

f

Liquid lipid

Addition of SurfactantCool to 5°C

surfactant

CrystallizationCool to 5°C

Crystallizationf

Increased surface, excess surfactant

Polymorphic transitions, uncovered

f i

Solid lipidCrystallization

Excess surfactant

adsorbs to interfacesurfaces, aggregation


V. Application ExamplesCase 1: Omega 3 Fatty AcidsCase 1: Omega-3 Fatty Acids

1.00y = 15 936x + 40 037

80Melting

w (J

/g)

0.25

0.050.75

y = -15.936x + 40.037R2 = 0.9765

50

60

70

(o C)

Melt temperature

d co

nten

tMelting

Hea

t flo

w

0 00

0.05

0.10

30

40

50

T c,T

m (

Crystallization temp.

-3

fatty

aci

d

10 20 30 40 50 60 70

0.00y = -10.855x2 - 1.569x + 64.069

R2 = 0.995410

20

0 0 2 0 4 0 6 0 8 1

10 20 30 40 50 60 70Temperature (oC)

0 0.2 0.4 0.6 0.8 1

In bulk tripalmitin in the presence of -3 fatty acids – significant

d i lti d t lli ti t (50% l di d i d)decreases in melting and crystallization temp (50% loading desired)


How Does This Affect Production of SLN???

5 Cool110 Cool1

Without Fish Oil With 0.25% Fish OilTween 20 Stabilized

2 50

2.55

(J/g

)

Cool1HeatCool25

10

J/g)

Cool1HeatCool2

Tween 20 Stabilized

10-7.5

-5

-2.5

Hea

t flo

w (

-5

0

Hea

t flo

w (J

-15-12.5

-10

0 20 40 60 80-15

-10

0 20 40 60 80

H

0 20 40 60 80Temperature (oC)0 20 40 60 80

Temperature (oC)

Formation of -crystals suppressed, formation of thermodynamically t bl t dstable promoted.


Dispersion Stability of SLN in the Presence of -3 Fatty Acids3 Fatty Acids

Cr stalli ed 190

200

• Crystallized nanoemulsion with >25% w-3 fatty acids (n

m)

180

190

DO NOT aggregate• Indicates that

morphological areg

e si

ze

160

170

0% -3 10% -3 morphological

changes associated with polymorphic

Z-av

a

140

150

25% -3

transitions are suppressed.

0 10 20 30 40 50 60130

140

Time (min)


Rheology of SLN Containing -3 Fatty Acidsgy g y

• -3 fatty acid 1 E+04+0.00+0 25 3 fatty acid

containing SLN did not show a noticeable increase

1.E+04

)

+0.25+0.25 (melting)

noticeable increase in complex modulus

• The sample i d fl id d i

1.E+02

G*

(Pa)

remained fluid during the first cooling process and also d i b t

1.E+00

during a subsequent additional heating and cooling cycle.

1.E-020 20 40 60 80 100

Temperature (oC)Temperature ( C)


Potential Structure of SLN Containing -3 Fatty AcidsFatty Acids

0% ω-3Solid lipid

Liquid lipid

Crystallization

>25% ω-3

lipid

Tripalmitin crystal covered by surface -3

Liquid oil inside the crystal matrix Crystallization

yfatty acids

Tripalmitin t lretards the

shape change

y crystal containing micro-dispersed -3 pfatty acidsActual structure as yet unkown!!!


Case 2: -Carotene in SLNLiq. Surf.

Liq. Surf.

Solid Surf.

Solid Surf.

Miglyol& -carotene

Tripalmitin & -carotene

(0 1%)

Code Surfactant system Main surfactant (w/w)

Co-surfactant (w/w) Lipid (w/w)

Hydrogenated 2 4% Phospholipon 0 6% 10%

Solid

(0.1%) (0.1%)

HLPPP Hydrogenatedlecithin

2.4% Phospholipon80H

0.6% Taurodeoxycholate

10% Tripalmitin

ULPPP Unsaturated lecithin 2.4% Alcolec PC 75 0.6% Taurodeoxycholate

10% Tripalmitin

Tw60PPP Tween 60 1.4% Tween 60 0.6% Taurodeoxycholate

10% Tripalmitin

d Matrix

RDA:3-6 mg-day -carotene

y p

Tw80PPP Tween 80 1.4% Tween 80 0.6% Taurodeoxycholate

10% Tripalmitin

HLM Hydrogenated lecithin

2.4% Phospholipon80H

0.6% Taurodeoxycholate

10% Miglyol

ULM Unsat rated lecithin 2 4% Alcolec PC 75 0.6% 10%

Liqui

Translates to:

3-6 g SLN/day ULM Unsaturated lecithin 2.4% Alcolec PC 75 Taurodeoxycholate Miglyol

Tw60M Tween 60 1.4% Tween 60 0.6% Taurodeoxycholate

10% Miglyol

Tw80M Tween 80 1.4% Tween 80 0.6% Taurodeoxycholate

10% Miglyol

d Matrix

3 6 g SLN/day

Food Structure and Functionality Laboratories 32

Crystallization of SLN with -carotene

TW60PPPUsing surfactants that solidify prior Onset 24.4°C

TW80PPP

Flow

to the matrix increases the crystallization Onset 21.0°C

ULPPPExoth

Hea

t Ftemperature of the SLNThe system with

Onset 21.1°C

HLPPP Cooling

hermal

The system with hydrogenated lecithin crystallized at the

Onset 30.3°C

0 10 20 30 40 50 60 70

Temperature (°C)

Figure 1. DSC thermographs of the initial cooling of

crystallized at the highest temperature

Technologie Funktioneller Lebensmittel33

g g p gdifferent surfactant system

Melting Analysis of SLN

TW80PPPMelting peak at 40ºC indicates presence of α-

TW60PPPExo

Flow

sub-cell crystals Present in high-melting surfactant-stabilized

Onset 40 9°CULPPP

HLPPP

othermalH

eat Fparticles

More complex melting indicated more complex

Onset 40.9 C

HLPPP

Heating

crystal structureSurface initiated crystallization?

Onset 42.1°C

20 30 40 50 60 70 80

Temperature (°C)Figure 2. Melting thermographs after 1 day of

t t 20°C f SLN ith t

Increased rigidity of the interface?


storage at 20°C for SLN with carotene

What About Gelation and Shape Changes?

nm) 200

HLPPPULPPP

No aggregation or gelation was observed

Rad

ius

(n

180

ULPPPTw80PPPTw60PPPHydrodynamic radius

increased but much less so in SLN that

33.4% incr.

21.9% incr.

odyn

amic

140

160less so in SLN that had been manufactured with hi h lti

Cooling18.5% incr.

0 10 20 30 40 50

Hyd

ro

120

high melting surfactants

2.8% incr.

Temperature (°C)

Figure 4. Size increase of all surfactant systems, during cooling from 45-5°C.

Apparently, SLN remain spherical with solid surfactants


during cooling from 45 5 C.

β-Carotene Stability in SLN

ent (

%)

100

120Measured as relative decrease in

ne C

onte

60

80HLPPPULPPP

concentrationDramatic improvement in

-Car

oten

40

60 Tw80PPPTw60PPP

improvement in stability of β-carotene in HLPPP

t

0 5 10 15 20 25

Rel

.-

0

20systemsTween 60 performed better than Tween

Storage Time at 20oC (Days)0 5 10 15 20 25

Figure 5. β-carotene breakdown over time at 20°C, using tripalmitin as an lipid matrix

better than Tween80, but less well than phospholipids


using tripalmitin as an lipid matrix

Mechanism of Bioactive Stabilization in SLN

β-carotene is ll d h th

β -carotene

expelled when the particle transitions to achieve a thermodynamically

Crystallization and storage

y ymore favorable form

Particle

Liquid Surfactant

Particle crystallizes in a crystal form that is well suited to

Crystallization and storage

maintain the β -carotene dispersed

Solid Surfactant

β -carotene


Solid Surfactant

V. ConclusionsCo c us o s• SLN are a promising nanoscaler delivery system for the

food industry due to the fact that:ood dus y due o e ac a– Large scale production possible, no organic solvents needed– High concentrations of functional compounds can be achieved– Lyophilization possible

Spray drying for lipids with T > 70ºC to yield powders– Spray drying for lipids with Tm > 70ºC to yield powders• Solid lipid nanoparticles are non-trivial systems with

potentially complex structures that include variations inParticle morphology– Particle morphology,

– Internal particle microstructure – Internal crystal structure

• Manufactures need to consider:Manufactures need to consider:– Lipid matrix compositional changes upon inclusion of bioactive– Choice of surfactant!!!! – Manufacturing conditions


Documents

Solid Lipid Nanoparticles: A New and Effective Delivery .../media/Knowledge Center/Science Reports/Conference... · and Effective Delivery System for Bioactives in Foodsin Foods Jochen