ORIGINAL PAPER

Solid Lipid Nanoparticles: Effect of Carrier Oil and EmulsifierType on Phase Behavior and Physical Stability

Cheng Qian • Eric Andrew Decker •

Hang Xiao • David Julian McClements

Received: 14 January 2011 / Revised: 26 May 2011 / Accepted: 8 June 2011 / Published online: 24 June 2011

� AOCS 2011

Abstract The impact of surfactant type and carrier oil

type on the phase behavior and physical stability of

emulsified tripalmitin was investigated. Solid lipid nano-

particles (SLNs) were prepared by homogenizing lipid and

aqueous phases at a temperature (&80 �C) above the

melting point of tripalmitin, and then cooling the resulting

oil-in-water emulsion to induce lipid droplet crystalliza-

tion. When stored at 37 �C, tripalmitin particles had good

long-term stability (d \ 150 nm) when coated with Tween

20, but were prone to aggregation and gelation when

coated with modified starch (MS). Conversely, when stored

at B20 �C tripalmitin particles coated by MS were more

stable to aggregation/gelation than those coated by Tween

20. Blending tripalmitin with low melting point lipids

(either medium chain triglycerides or orange oil) prior to

homogenization led to a considerable alteration in the SLN

phase behavior and stability. DSC measurements indicated

that the presence of the carrier oils reduced the crystalli-

zation temperature, melting temperature, and melting

enthalpy of tripalmitin. In addition, the carrier oils

improved the stability of SLNs to particle aggregation and

gelation, although some particle coalescence still occurred.

These results have important implications for formulating

colloidal delivery systems for utilization within the food

and other industries.

Keywords Solid lipid nanoparticles � Polymorphic

transitions � Crystallization � Melting � Emulsions �Orange oil � Tripalmitin � MCT

Introduction

The potential of solid lipid nanoparticles (SLNs) to

encapsulate, protect, and deliver bioactive lipophilic food

components has been demonstrated in a number of studies

[1–4]. SLN suspensions consist of fully or partially crys-

talline lipid nanoparticles suspended within an aqueous

continuous phase. The SLNs are usually coated with a layer

of emulsifier molecules to facilitate their formation and

enhance their long-term stability.

One of the main proposed advantages of SLNs for

encapsulating lipophilic components is their ability to

retard the diffusion of molecules through the solidified

lipid phase, which may increase the retention and chemical

stability of encapsulated components [5]. SLNs may

therefore be particularly useful for encapsulating and pro-

tecting chemically labile food lipids, such as b-carotene,

lycopene, citral, and x-3 fatty acids. On the other hand, the

utilization of SLNs in the food industry is currently limited

because they are often prone to particle aggregation and

gelation [6]. The instability of SLN suspensions to aggre-

gation and gelation has been attributed to a-to-b poly-

morphic transitions of emulsified triacylglycerols [7, 8].

These polymorphic transitions lead to a change in particle

shape (from roughly spherical to disc-like), which causes a

large increase in the oil–water surface area [9, 10]. Con-

sequently, particle aggregation can occur between exposed

hydrophobic patches on different particles. For this reason,

it is important to include sufficient levels of an emulsifier

that rapidly forms a protective coating around the lipid

C. Qian � E. A. Decker � H. Xiao � D. J. McClements (&)

Department of Food Science, University of Massachusetts,

Amherst, MA 01003, USA

e-mail: mcclements@foodsci.umass.edu

123

J Am Oil Chem Soc (2012) 89:17–28

DOI 10.1007/s11746-011-1882-0

droplets after they crystallize [11, 12]. In particular, it has

been suggested that forming a thick steric layer around the

lipid particles may be an effective means of avoiding

particle aggregation and gelation [13].

Other potential limitations of SLN have also been

attributed to their highly ordered lipid crystalline struc-

ture: they have limited loading capacity and they are

prone to expel encapsulated lipophilic materials during

storage [14]. Recently, it has been shown that some of

these limitations can be overcome by utilizing a mixture

of lipids with different melting characteristics [1, 15, 16].

Traditionally, SLNs were created using a pure lipid

phase with a sharp melting point (e.g., a pure triacyl-

glycerol), which led to the formation of a highly ordered

crystalline structure in the solidified particles. This

highly ordered structure tended to expel any encapsulated

components into the surrounding media. In contrast, if a

mixed lipid phase with a range of melting points is used,

then a more disordered crystalline structure is formed

inside the particles [1, 15, 16]. These more disordered

particles tend to have a higher loading capacity for

lipophilic components, exhibit less expulsion of lipo-

philic components during storage, and show less particle

aggregation and gelation [16, 17]. This type of delivery

system is sometimes referred to as a nanostructured lipid

carrier (NLC).

The nature of the ingredients used to formulate SLN or

NLC systems significantly affects their physicochemical

properties, stability, and release characteristics [14]. Some

of the most important parameters that have to be consid-

ered when selecting a lipid phase are: the melting point

range; crystal morphology; viscosity; and, polarity. A

number of the most important characteristics to consider

when selecting an appropriate emulsifier system are:

reduction in interfacial tension; adsorption kinetics; ability

to interfere with nucleation and crystal growth; and, ability

to prevent particle aggregation [1, 18, 19].

Most of the studies on SLN and NLC in the past have

been carried out on systems suitable for application within

the pharmaceutical industry. Many of the ingredients used

to formulate pharmaceutical delivery systems are unsuit-

able for widespread utilization within the food and bever-

age industry. For this reason, one of our main objectives

was to determine whether stable SLN and NLC suspen-

sions could be formed using food grade lipids and emul-

sifiers. Tripalmitin was used as a high melting point lipid,

while either medium chain triglycerides (MCT) or orange

oil was used as a low melting point lipid (‘‘carrier oil’’).

The nature of the emulsifier was investigated by comparing

a small molecule surfactant (Tween 20) that adsorbs rap-

idly and forms thin interfacial layers, with a modified

starch (MS) that adsorbs slowly but forms thick interfacial

layers. This study should lead to a better understanding of

the factors influencing the formation and performance of

food-grade SLN and NLC systems.

Materials and Methods

Materials

Tripalmitin was purchased from Fluka (Buchs, Switzer-

land). Sodium phosphate monobasic and sodium phosphate

dibasic were purchased from Fisher Scientific (St. Clair

Shores, MI, USA). Sodium azide and polyethylene glycol

sorbitan monolaurate (Tween 20) were purchased from

Sigma-Aldrich Chemical Co. (St Louis, MO, USA).

Orange oil was provided by Givaudan (East Hanover, NJ,

USA). Modified starch (MS, PURITY GUMTM

Ultra) was

provided by the National Starch Company (Bridgewater,

NJ, USA). Medium chain triglycerides (Miglyol 812 N)

were provided by Sasol Germany GmbH (Eatontown, NJ,

USA). This MCT oil consists of triglycerides with mainly

C8 and C10 fatty acids obtained by fractionation of vege-

table oils. All materials were used without further

purification.

Methods

Solution Preparation

Phosphate buffer (pH 7.0) was prepared by dissolving

4 mM sodium phosphate (monobasic) and 6 mM sodium

diphosphate (dibasic) in distilled water. 0.02% w/w sodium

azide was used as a preservative to prevent microbial

growth. Aqueous surfactant solutions were prepared by

dispersing either 1.5% (w/w) Tween 20 or 5% (w/w)

modified starch in phosphate buffer and stirring overnight

at ambient temperature.

SLN and NLC Preparation

SLNs and NLCs consisting of 10% (w/w) lipid phase and

90% (w/w) aqueous emulsifier solution were prepared

using a hot high-pressure homogenization method [20].

The lipid phase consisted of either pure tripalmitin or tri-

palmitin mixed with carrier oil (30 wt% MCT or orange

oil) at a temperature of 80–85 �C, which is above the

melting point of tripalmitin. The lipid phase was then

mixed with aqueous emulsifier solution held at 80–85 �C

using a hand-held high speed blender at 30% power for

1.5 min (model SDT-1810, EN shaft, Termar Co., Cin-

cinnati, OH, USA) to produce a coarse emulsion. The hot

coarse emulsion was then homogenized further by passing

it five cycles at 9,000 psi through a microfluidizer

(Microfluidics, Newton, MA, USA) maintained at 80–85 �C

18 J Am Oil Chem Soc (2012) 89:17–28

123

to prevent fat solidification during the homogenization

procedure. The whole process took less than 5 min per

sample. The emulsion (200 ml) was split into three samples

that were placed into closed opaque plastic bottles and then

stored in temperature controlled incubators at 5, 20 and

37 �C, respectively. The cooling rate used was approxi-

mately 1–2 �C per min.

Particle Size Determination

Particle size measurements were performed using a static

light scattering instrument (Mastersizer 2000, Malvern

Instrument Ltd., Westborough, MA, USA). Measurements

are reported as the volume-length mean diameter,

d43 ¼P

nid4i =P

nid3i , where ni is the number of particles

of diameter di. The particle size distribution of some

samples was also assessed by dynamic light scattering

using a dynamic light scattering instrument (Malvern

Zetasizer NanoZS, Malvern Instrument Ltd., Westborough,

MA, USA). Samples were diluted 100 times in buffer

solution prior to analysis to avoid multiple scattering

effects. All measurements were performed at ambient

temperature (&25 �C).

Differential Scanning Calorimetry

A differential scanning calorimeter (DSC; Q1000, TA

Instruments, New Castle, DE, USA) was used to study lipid

crystallization and polymorphic transitions of different

formulations during several cool-heat cycles at constant

cooling and heating rates (5 �C/min for heating and cool-

ing). An aliquot of emulsion (8–10 mg) was placed in a

hermetic aluminum pan and sealed. A sealed empty pan

was used as a reference. All samples were heated from 5 to

80 �C, and then cooled back to 5 �C.

Rheology Test

Storage modulus (G0) and loss modulus (G00) of emulsions

undergoing sol-to-gel transitions were measured using a

dynamic shear rheometer (Malvern Kinexus rheometer,

Westborough, MA, USA). A constant strain of 0.1% and

frequency of 1 Hz were used as described previously

[2, 21]. All the samples were loaded at 5 �C, and heated to

80 �C, then cooled down to 5 �C at a constant heating and

cooling rate of 5� C/min.

Visual Observations

Sample fluidity/gelation was also determined by simple

visual observation when they were stored at 37, 20 and

5 �C from 1 to 30 days. All samples were sealed in plastic

tubes, which were inverted to determine whether they

remained fluid (flowed to the bottom) or became solid like

(stayed at the top).

Statistical Analysis

All measurements were repeated three times using tripli-

cate samples. Means and standard deviations were calcu-

lated from this data.

Results and Discussion

Crystallization of Bulk Oil Phases

Knowledge of the thermal behavior of tripalmitin in bulk

oil phases is useful for interpreting its behavior in emul-

sified oil phases, and therefore we characterized the ther-

mal transitions of tripalmitin in the absence and presence of

carrier oils using DSC. The lipid phases were initially

heated to 80 �C to melt the tripalmitin, and then subjected

to cool-heat cycles (Fig. 1a–c).

Pure Tripalmitin

Upon cooling a sample containing only tripalmitin from

80 �C, a sharp exothermic peak was observed around

39 �C (Fig. 1a), which is due to crystallization into the

a-form [22]. Upon heating from 5 �C, several endothermic

and exothermic peaks were observed (Fig. 1a), which can

be assigned to various phase transitions [2]. The endo-

thermic peak at 44 �C corresponds to melting of the

a-form; the exothermic peak at 47 �C corresponds to

recrystallization of the a-melt into the more stable b0-form;

the small endothermic peak at 50 �C is related to melting

of the b0-form; the exothermic peak at 53 �C corresponds

to recrystallization of the a-melt into the most stable

b-form; the largest endothermic peak at 65 �C corresponds

to the melting of the b-form. This suggested that the bulk

tripalmitin persisted in the a-form under the experimental

cooling/heating conditions used in this study, i.e., the rel-

atively short holding time between fat crystallization and

melting.

Tripalmitin & Carrier Oils

The thermal behavior of tripalmitin was changed appre-

ciably in the presence of the two carrier oils, i.e., 30%

MCT (Fig. 1b) or 30% orange oil (Fig. 1c). Upon cooling

from 80 �C, a single exothermic peak (192 J/g of tripal-

mitin) was observed around 34 �C for the system con-

taining 30% MCT, which was about 5 �C lower than in the

absence of MCT (Fig. 1a). The position of this peak sug-

gests that the tripalmitin initially crystallized into the

J Am Oil Chem Soc (2012) 89:17–28 19

123

a-form. Upon heating from 5 �C, a single endothermic peak

(202 J/g of tripalmitin) was observed around 61 �C, which

was about 4 �C lower than the equivalent peak measured for

pure tripalmitin. Presumably this peak corresponded to the

melting of the b-form of tripalmitin. These results indicate

that both the melting and crystallization temperature of tri-

palmitin were reduced appreciably in the presence of 30%

MCT. This effect can be described by the Henderson–

Hasselbach equation, which predicts that the melting point

of a high melting point substance decreases in the presence

of a low melting point substance [23]. This phenomenon

occurs because some of the high melting fraction (in this

case tripalmitin) dissolves in the low melting fraction

(in this case MCT). A fairly similar general behavior was

observed for the system containing 30% orange oil, but the

effects were even more pronounced.

For the system containing 30% orange oil, a small

exothermic peak (4.8 J/g of tripalmitin) was observed upon

cooling at around 38 �C and a much larger exothermic

peak (204 J/g of tripalmitin) was observed at 29 �C

(Fig. 1c). These peaks probably correspond to crystalliza-

tion of tripalmitin into the a-form. The larger exothermic

peak occurred at a temperature that was about 10 �C below

that observed for pure tripalmitin. Upon heating from 5 �C,

a single endothermic peak (211 J/g of tripalmitin) was

observed around 57 �C, which was about 8 �C lower than

the equivalent peak measured for pure tripalmitin. Pre-

sumably this peak corresponded to the melting of the

-16

-12

-8

-4

0

4

8

12

16

20

24

28

32

0 10 20 30 40 50 60 70 80

Hea

t Flo

w (

W/g

)

Temperature (ºC)

Heating

Cooling

Crystallization

Melting

Heating

Cooling

Crystallization

a b

c

Melting

Fig. 1 a DSC thermograms of pure bulk tripalmitin samples heated

from 5 to 80 �C, and then cooled from 80 to 5 �C. b DSC

thermograms of bulk samples containing 70% tripalmitin and 30%

MCT heated from 5 to 80 �C, and then cooled from 80 to 5 �C. c DSC

thermograms of bulk samples containing 70% tripalmitin and 30%

orange oil heated from 5 to 80 �C, and then cooled from 80 to 5 �C

20 J Am Oil Chem Soc (2012) 89:17–28

123

b-form of tripalmitin. These results indicate that orange oil

was more effective at reducing the melting and crystalli-

zation temperature of tripalmitin than MCT oil.

The addition of the carrier oils clearly had a pronounced

influence on the thermal behavior of bulk tripalmitin. In

particular, incorporation of the carrier oils caused a

decrease in the melting and crystallization temperatures of

tripalmitin, as well as accelerating the transition from the

a- to b-polymorphic form. One would expect that these

effects would have an impact on the formation, stability

and properties of SLNs prepared using tripalmitin and

carrier oils.

Effect of Emulsifiers on Tripalmitin SLN Properties

Phase Behavior and Aggregation Stability

Previous studies have shown that emulsions containing

solidified tripalmitin particles are highly unstable to

aggregation when the tripalmitin undergoes a a-to-b poly-

morphic transition [2, 21]. The origin of this effect has been

attributed to the change in shape of the tripalmitin particles

from spherical (a-form) to disk-like (b-form), which leads

to an increase in oil–water contact area and increased par-

ticle–particle interactions through hydrophobic attraction.

For this reason, we prepared SLN suspensions in which the

lipid particles were present in the b-form, so as to determine

the potential of improving SLN stability using emulsifiers

or carrier oils. This was achieved by preparing tripalmitin

oil-in-water emulsions at 80 �C using the hot homogeni-

zation method, holding them at 37 �C overnight (to promote

fat crystallization), heating them to 60 �C (to promote the

a-to-b polymorphic transition), and then cooling them to

5 �C. The samples were then heated to 80 �C to establish

that the tripalmitin was actually in the b-form, and then

cooled to 5 �C to determine if the particles had aggregated

during the heat–cool cycle.

DSC thermograms of tripalmitin suspensions containing

two kinds of emulsifiers (modified starch or Tween 20)

were recorded during a heat–cool cycle (5 to 80 to 5 �C) to

determine the influence of emulsifier type on phase

behavior and particle stability (Fig. 2a, b). Upon heating

from 5 �C, the tripalmitin particles coated by Tween 20

(Fig. 2a) and modified starch (Fig. 2b) both exhibited a

single endothermic peak around 62 �C, which corresponds

to melting of the b-form of the crystals (Fig. 1a). This

demonstrated that the tripalmitin was in the most stable

polymorphic form at the beginning of the heating scans,

which indicated that there had been a rapid transition from

the a to b-polymorphic forms in the emulsified tripalmitin

(Fig. 2a), which was not observed in the bulk tripalmitin

(Fig. 1a). Upon subsequent cooling from 80 �C, two exo-

thermic peaks were observed at 20.5 and 39.8 �C in the

tripalmitin emulsions containing Tween 20. As described

in previous studies, the lower peak (TC1 & 20.5 �C) cor-

responds to crystallization of emulsified tripalmitin,

whereas the higher peak (TC2 & 39.8 �C) corresponds to

crystallization of destabilized tripalmitin present as either

large droplets or bulk oil [6, 24–26]. Droplet coalescence

or oiling-off increases the volume of each lipid particle,

which means that nucleation occurs predominantly through

a heterogeneous mechanism, rather than through a homo-

geneous mechanism [27, 28]. These results show that the

Tween 20-coated particles were highly unstable to coa-

lescence and/or oiling off during cool-heat cycles as has

been reported by others [25].

Fig. 2 a DSC thermograms of pure emulsified tripalmitin samples

stabilized by Tween 20 heated from 5 to 80 �C, and then cooled from

80 to 5 �C. b DSC thermograms of pure emulsified tripalmitin

samples stabilized by modified starch heated from 5 to 80 �C, and

then cooled from 80 to 5 �C

J Am Oil Chem Soc (2012) 89:17–28 21

123

The system containing tripalmitin particles coated by

modified starch showed somewhat different behavior dur-

ing cooling: a small exothermic peak was observed around

40.0 �C and a much larger exothermic peak around 30.0 �C

(Fig. 2b). Presumably, the peak at 40.0 �C corresponds to

crystallization of destabilized tripalmitin droplets, whereas

the peak at 30.0 �C corresponds to crystallization of stable

tripalmitin droplets. These results suggest that the tripal-

mitin droplets coated with modified starch were more

resistant to droplet coalescence than those coated by Tween

20. This effect could be attributed to the fact that the

modified starch formed a thicker interfacial coating around

the tripalmitin particles than the Tween 20, thereby

reducing the tendency for particle aggregation to occur

[25, 29].

Gelation

Rheological measurements were also performed to monitor

the effect of the two different emulsifiers on the gelation of

tripalmitin SLN suspensions. We measured the temperature

dependence of the complex shear modulus (G*) of tripal-

mitin suspensions stabilized by either Tween 20 or modi-

fied starch (Fig. 3). In general, the complex shear modulus

provides a measure of the rigidity of a material. In our

experiments, an increase in G* indicates the formation of a

particle network in the SLN suspensions. Tripalmitin

oil-in-water emulsions were cooled to 5 �C to promote fat

crystallization and then stored overnight. Samples were

then placed in the rheometer measurement chamber at

5 �C, and then subjected to a heat–cool cycle at a con-

trolled rate using an oscillation test.

The tripalmitin suspensions stabilized by Tween 20 had

a high G* value at 5 �C indicating that they formed a

strong gel (Fig. 3). Visual observation of these systems

confirmed that they were gelled at this temperature—when

test tubes containing them were inverted the tripalmitin

suspensions did not flow. These measurements indicated

that the Tween 20-coated tripalmitin particles aggregated

with each other and formed a strong gel network.

G* remained fairly constant around 30 kPa when this

system was heated from 5 to 20 �C, which indicated that

the particle network remained intact over this temperature

range. However, when the temperature was raised further,

there was a steep decline in G* until it reached &0 kPa at

40 �C. The DSC data indicated that the tripalmitin should

have remained solid across this temperature range

(Fig. 2a), which suggests that there may have been a

change in particle morphology or particle–particle inter-

actions with increasing temperature. At the end of the

heating process (80 �C), we observed a layer of oil on top

of the emulsions, which is indicative of extensive droplet

coalescence.

In contrast, the tripalmitin suspension containing parti-

cles stabilized by modified starch did not show any sign of

particle aggregation and gelation, with the shear modulus

remaining close to zero across the entire temperature range

studied (Fig. 3). These results indicate that a thick coating

of modified starch around the tripalmitin particles was able

to inhibit extensive particle aggregation under the condi-

tions used in the rheology experiments.

Effect of Carrier Oil on Tripalmitin SLN Properties

In this section, we examined the influence of incorporating

carrier oil (either orange oil or MCT) on the phase behavior

and stability of the emulsified tripalmitin. We hypothesized

that the incorporation of liquid carrier oil would lead to a

less perfect crystal structure being formed within the lipid

phase, leading to the formation of NLC suspensions. The

two carrier oils used were selected because they are both

low melting point food-grade oils with different physico-

chemical characteristics: MCT is highly non-polar,

whereas orange oil is relatively polar.

Oil-in-water emulsions were formed by homogenizing

a hot lipid phase (70% tripalmitin and 30% carrier oil) with

a hot aqueous phase (emulsifier and water) together as

described earlier. A sample of emulsified tripalmitin was

placed in the DSC instrument at 5 �C, and then its phase

behavior was recorded when it was subjected to a heat–

cool cycle (5 to 80 to 5 �C) to determine the influence of

carrier lipid on phase transitions and particle stability.

During the heating stage, only one endothermic peak

was observed at about 58 �C for the Tween 20-stabilized

systems containing either MCT (Fig. 4a) or orange oil

Fig. 3 Temperature dependence of the complex shear modulus of

emulsified tripalmitin samples stabilized by either Tween 20 or

modified starch heated from 5 to 80 �C

22 J Am Oil Chem Soc (2012) 89:17–28

123

(Fig. 4b), which corresponded to the b-form melting tem-

perature. This melting temperature (Tm) was appreciably

lower than the melting temperature of the b-form in

particles containing pure tripalmitin, i.e., 62 �C (Fig. 2a).

This result suggests that the presence of MCT or orange oil

lowered the melting temperature of the b-form of emulsi-

fied tripalmitin, as was observed for bulk tripalmitin

(Fig. 1). The crystallization behavior of the tripalmitin

was also influenced by the presence of the carrier oils.

For the sample containing orange oil, only a single exo-

thermic peak was observed at a relatively low temperature

(&10 �C), indicating that the particles were relatively

stable to coalescence and oiling-off after heat–cool cycling.

For the sample containing MCT (Fig. 4a), a large exo-

thermic peak was observed at a relatively low temperature

(TC1 & 15 �C) and a small exothermic peak was observed

at a higher temperature (TC2 & 35 �C) during the cooling

cycle, which indicated that the majority of the particles

were stable to aggregation, but that some coalescence and/

or oiling-off had occurred.

The crystallization temperature (TC1) of the lipid drop-

lets was appreciably less in the presence of carrier oils than

in the absence (Table 1). In the system containing MCT

(Fig. 4a), TC1 decreased from 20 to 15 �C; while in the

system containing orange oil (Fig. 4b), TC1 decreased from

20 to 10 �C, compared to the particles containing pure

tripalmitin (Fig. 3). These results suggest that the liquid

carrier oil inhibited the formation of tripalmitin crystals,

possibly by acting as a liquid solvent for the solid tripal-

mitin crystals, as discussed earlier for the bulk lipid phase.

The samples stabilized by modified starch (Fig. 5a, b)

exhibited fairly similar trends to those stabilized by Tween

20 (Fig. 4a, b). The melting temperature of the b-form was

appreciably lower for particles containing either carrier oil

than for particles containing pure tripalmitin (Fig. 2a). The

crystallization temperature was lower in the presence of

carrier oil than in its absence: TC1 decreased from 29 to

25 �C for MCT (Fig. 5a) and from 29 to 15 �C for orange

oil (Fig. 5b). The enthalpy change per unit mass of tri-

palmitin associated with the melting of the b-form

appeared to decrease somewhat in the presence of the

carrier oils (Table 1), which may be because the carrier oil

dissolved some of the solid tripalmitin involved in the

transition or it changed the crystal structure [2].

Measurements of the shear modulus versus temperature

indicated that there was no gelation of the samples con-

taining either carrier oil during heating and cooling cycles

(i.e., the shear modulus remained close to zero at all tem-

peratures), and only a small amount of oil was observed on

Fig. 4 a DSC thermograms of emulsified samples containing 70%

tripalmitin and 30% MCT stabilized by Tween 20 heated from 5 to

80 �C, and then cooled from 80 to 5 �C. b DSC thermograms of

emulsified samples containing 70% tripalmitin and 30% stabilized by

Tween 20 orange oil heated from 5 to 80 �C, and then cooled from 80

to 5 �C

Table 1 Summary of thermal properties (phase transition tempera-

tures and enthalpies) of tripalmitin oil-in-water suspensions contain-

ing different emulsifiers and carrier oils

Carrier oil Tm

(�C)

DHm

(J/g)

TC1

(�C)

TC2

(�C)

DHC1

(J/g)

DHC2

(J/g)

Tween 20

None 62.9 182 20.2 39.5 53 77

30% MCT 57.9 163 14.7 35.2 103 52

30% Orange oil 56.5 175 10.3 – 171 –

Modified Starch

None 62.4 202 29.3 39.6 96 5

30% MCT 59.6 180 24.8 – 153 –

30% Orange oil 55.6 121 15.1 – 56 –

The enthalpies are reported per unit mass of tripalmitin

J Am Oil Chem Soc (2012) 89:17–28 23

123

top of the samples after they had been heated to 80 �C after

a cool–heat cycle (data not shown). This suggests that the

addition of relatively small amounts of carrier oil (30%)

prevented extensive particle aggregation and gelation,

thereby increasing the physical stability of SLNs.

Impact of Isothermal Holding Temperature on Long

Term Stability

The DSC and rheology measurements described above

were carried out under dynamic conditions, i.e., as the

temperature was scanned upward or downward at a con-

trolled rate. In practice, samples are often stored at a fixed

temperature for extended periods, and therefore we

examined the influence of isothermal storage on the

behavior of tripalmitin suspensions containing different

carrier oils and emulsifiers.

Tripalmitin suspensions were prepared using the hot

homogenization method, cooled to three different storage

temperatures (5, 20, and 37 �C), and then held for periods

of up to 1 month to determine their long-term stability. The

visual appearance and texture of the samples was recorded

during storage at the three different temperatures (Table 2,

Fig. 6). The mean particle diameters of the samples stored

at 37 �C were also measured before and after storage.

Immediately after preparation all of the samples contained

relatively small particles, with mean particle diameters

\200 nm for the suspensions containing modified starch

and \150 nm for the suspensions containing Tween 20.

The influence of emulsifier and carrier oil type on the

physical stability of the tripalmitin suspensions depended

strongly on storage temperature.

37 �C: At a storage temperature of 37 �C, all suspen-

sions containing Tween 20 maintained their small particle

diameters (d \ 150 nm) (Fig. 7a) and remained fluid

(Fig. 6c), suggesting that they were stable to particle

aggregation and gelation. On the other hand, suspensions

containing modified starch showed appreciable changes in

their properties during storage. There was evidence of

some crystalline material on top of the samples containing

pure tripalmitin particles after 1 day storage and a distinct

increase in viscosity (Table 2). The sample viscosity con-

tinued to increase over the next few days until a firm gel

was formed after 6 days storage (as indicated by no sample

flow when the test tubes were inverted). The presence of

carrier oils improved the stability of the suspensions con-

taining modified starch but there were still some observed

changes in their physical properties. There was evidence of

crystal formation on top of the system containing MCT

after 1 day storage, and there was a noticeable increase in

the viscosity of the system containing orange oil after

2 days storage. In addition, there was an appreciable

increase in the mean particle diameter of both suspensions

after 1 month storage: d & 660 nm for samples containing

MCT and d & 220 nm for samples containing orange oil

(Fig. 7b). These results suggest that the non-ionic surfac-

tant (Tween 20) provided better physical stability than the

biopolymer emulsifier (modified starch) when the suspen-

sions were stored at 37 �C. In addition, they indicate that

addition of carrier oils (either MCT or orange oil) greatly

improved the stability of systems prone to particle

aggregation.

20 �C: At a storage temperature of 20 �C, the suspen-

sions containing pure tripalmitin particles coated by Tween

Fig. 5 a DSC thermograms of emulsified samples containing 70%

tripalmitin and 30% MCT stabilized by modified starch heated from 5

to 80 �C, and then cooled from 80 to 5 �C. b DSC thermograms of

emulsified samples containing 70% tripalmitin and 30% orange oil

stabilized by modified starch heated from 5 to 80 �C, and then cooled

from 80 to 5 �C

24 J Am Oil Chem Soc (2012) 89:17–28

123

20 formed a highly viscous fluid after 1 day storage,

formed a soft gel after 6 days storage, and formed a hard

gel after 1 month storage (Fig. 6b). This suggested that

they were highly unstable to particle aggregation and

gelation. However, when carrier oils (either MCT or

orange oil) were incorporated into the tripalmitin particles

the suspensions became stable to particle aggregation and

gelation, based on the fact that there was no increase in

mean particle diameter (d \ 150 nm) and no evidence of

an increase in viscosity. Presumably, the incorporation of

the carrier oils prevented the large change in particle shape

that is believed to be responsible for particle aggregation

and gelation in pure triacylglycerol systems [7, 8]. Again,

there were appreciable differences in the physical stability

of the samples depending on emulsifier type. The suspen-

sions containing pure tripalmitin particles coated by mod-

ified starch formed a highly viscous fluid after 1 day

storage with evidence of some crystalline material on top.

The viscosity of these samples was observed to increase

over time until they formed a gel after 6 days storage.

These results indicate that samples containing pure tripal-

mitin particles were again highly susceptible to particle

aggregation and gelation. The incorporation of carrier oils

(either MCT or orange oil) into the tripalmitin particles

improved the stability of the suspensions somewhat, but

there was still some evidence of physical instability. Small

crystals were observed on top of the samples containing

MCT after 1 day storage, while there was evidence of a

slight increase in viscosity in the samples containing

orange oil after 2 days storage.

5 �C: At a storage temperature of 5 �C, suspensions

containing pure tripalmitin particles coated by Tween 20

formed hard gels after 1 day storage indicating that

extensive particle aggregation and network formation had

occurred (Fig. 6a). On the other hand, the suspensions

containing pure tripalmitin particles coated by modified

starch were viscous fluids after 1 day storage, and only

gelled after 5 days storage (as evidenced by no flow when

the test tubes were inverted). The addition of carrier oils to

the tripalmitin particles coated by Tween 20 completely

inhibited particle aggregation and gelation: there was no

observed increase in mean particle diameter (d \ 150 nm)

or sample viscosity. However, there was still some evi-

dence of instability in the tripalmitin suspensions contain-

ing carrier oils and modified starch: small crystals were

observed on top of the samples containing MCT after 1 day

storage, and a slight increase in sample viscosity was

observed in the samples containing orange oil after 2 days

storage.

Our results clearly indicate that the physical stability of

SLN suspensions containing tripalmitin particles is highly

dependent on emulsifier type, carrier oil type, and storage

temperature. At relatively low storage temperatures (5 and

20 �C), suspensions containing pure tripalmitin were

highly unstable to particle aggregation and gelation irre-

spective of emulsifier type. This effect can be attributed to

Table 2 Description of observed changes in the morphology and rheology of SLN during isothermal storage at different holding temperatures

Modified starch Tween 20

(a) Oil phase: pure tripalmitin

37 �C Small crystals on surface after 1st day; viscous fluid

after 4th day; partial gelation after 7th day

No observed change in appearance or rheology

20 �C Thick viscous fluid after 1st day; partial gelation

after 4th day; Completely gelled at 6th day

Thick viscous fluid after 1st day;

Soft gel after 6th day

5 �C Thick viscous fluid after 1st day;

completely gelled at 5th day

Hard gel after 1st day

(b) Oil phase: 70% tripalmitin; 30% MCT

37 �C Small crystals on surface after 1st day;

no subsequent change in appearance or rheology

No observed change in appearance or rheology

20 �C Small crystals on surface after 1st day;

no subsequent change in appearance or rheology

No observed change in appearance or rheology

5 �C Small crystals on surface after 1st day;

no subsequent change in appearance or rheology

No observed change in appearance or rheology

(c) Oil phase: 70% tripalmitin; 30% orange oil

37 �C Slight increase in viscosity after 2–3 days;

no subsequent change in appearance or rheology

No observed change in appearance or rheology

20 �C Appreciable increase in viscosity after 2–3 days;

no subsequent change in appearance or rheology

No observed change in appearance or rheology

5 �C Large increase in viscosity after 2–3 days;

no subsequent change in appearance or rheology

No observed change in appearance or rheology

J Am Oil Chem Soc (2012) 89:17–28 25

123

the change in particle shape that occurs during the a-to-bpolymorphic transition of the tripalmitin, which increases

the exposure of non-polar surfaces to the surrounding

aqueous phase, leading to increased hydrophobic attraction

between particles [7, 8]. The utilization of modified starch,

rather than Tween 20, retarded the rate of gelation in the

pure tripalmitin suspensions at these low storage temper-

atures, but gelation still occurred eventually. Presumably

the modified starch formed a thick polymeric layer around

the lipid particles that partially inhibited their ability to

come into close contact, but over time the particles were

still able to come sufficiently close to aggregate.

At relatively high storage temperatures (37 �C), sus-

pensions containing pure tripalmitin particles coated by

Tween 20 were stable to particle aggregation and gelation,

whereas those coated by modified starch were not. The

improved stability of the Tween 20 suspensions at the

higher storage temperature is consistent with the rheology

measurements, which indicated that the strength of gels

formed at low temperatures decreased appreciably when

they were heated above about 37 �C (Fig. 3). The physi-

cochemical origin of this effect is currently unknown, and

may be due to changes in the behavior of the tripalmitin or

5 ºC

23 ºC

37 ºC

a

b

c

Fig. 6 Photographs of the visual appearance of samples stored at

different temperatures (5, 20 and 37 �C) after inversion of the test-

tube. From left-to-right the samples were: a Tween 20 stabilized pure

tripalmitin SLN; b MS stabilized pure tripalmitin SLN; c Tween 20

stabilized tripalmitin with 30% w/w MCT; d MS stabilized tripalmitin

with 30% w/w MCT; e Tween 20 stabilized tripalmitin with 30% w/w

orange oil; f MS stabilized tripalmitin with 30% w/w orange oil

0

100

200

300

400

500

600

700

Mea

n P

arti

cle

Dia

met

er (

nm)

Storage Time

70%PPP+30%MCT

70%PPP+30%OO

Modified Starch

0

100

200

300

400

500

600

700

Fresh made Two weeks Four weeks

Fresh made Two weeks Four weeks

Mea

n P

arti

cle

Dia

met

er (

nm)

Storage Time

100%PPP

70%PPP+30%MCT

70%PPP+30%OO

Tween 20

a

b

Fig. 7 a Impact of storage time on the mean particle diameters of

emulsified tripalmitin stabilized by Tween 20 in the absence and

presence of carrier oils (37 �C). b Impact of storage time on the mean

particle diameters of emulsified tripalmitin stabilized by modified

starch in the absence and presence of carrier oils (37 �C)

26 J Am Oil Chem Soc (2012) 89:17–28

123

the surfactant with temperature. Observation of the crys-

tallization temperature of emulsified tripalmitin in the DSC

thermograms suggests that the tripalmitin may not

have fully crystallized when it was stored at 37 �C in the

Tween-stabilized emulsions (Fig. 2a), but that it was in the

modified starch-stabilized emulsions (Fig. 2b). This

observation was supported by DSC measurements of the

enthalpy changes of the samples during cooling. The

Tween-stabilized system showed a small exothermic peak

corresponding to crystallization of some liquid tripalmitin,

whereas the modified starch-stabilized systems showed no

evidence of an exothermic peak, which indicated that all

the tripalmitin was already fully crystallized at this storage

temperature.

The presence of carrier oils in the lipid phase greatly

improved the physical stability of the tripalmitin suspen-

sions. For the Tween 20 coated particles, the addition of

either 30% MCT or orange oil completely suppressed

particle aggregation and gelation. For the modified starch

coated particles, the carrier oils prevented gel formation,

but there was still some evidence of instability, such as

crystal formation on top of the MCT samples and an

increase in viscosity in the orange oil samples. Initially,

we postulated that modified starch would be more effec-

tive at stabilizing SLN suspensions than Tween due to its

ability to form a thick polymeric coating around the lipid

particles. However, our experimental data suggests that

modified starch was not effective at providing long term

stability to SLN suspensions, particularly at elevated

storage temperatures. Modified starch consists of large

surface-active polymers that would be expected to adsorb

to oil–water interfaces relatively slowly. It is therefore

possible that when the tripalmitin particles changed shape

from spherical to disk-like the modified starch molecules

were not able to adsorb rapidly enough to prevent particle

aggregation. Alternatively, modified starch is known to be

less surface-active than small molecule surfactants such as

Tween 20, and therefore it is possible that it partly des-

orbed from the tripalmitin particle surfaces when they

changed from liquid to solid thereby making them less

stable to aggregation. Clearly, more detailed experimental

work is required to establish the physicochemical origin

of the observed differences between the two types of

emulsifiers.

Conclusions

Our results show that the physical stability of tripalmitin

SLNs can be improved by altering the emulsifier type or by

incorporating low melting point carrier oils. Pure tripal-

mitin particles are highly unstable to particle aggregation

and gelation at relatively low storage temperatures where

the tripalmitin is known to crystalize, which is attributed to

the change in particle shape associated with a a-to-b-

polymorphic transformation. The addition of carrier oils to

the SLN suspensions reduced the melting and crystalliza-

tion behavior of the tripalmitin particles, as well as their

stability to aggregation and gelation. Incorporation of 30%

MCT or orange oil to the lipid phase greatly improved their

stability to particle aggregation and gelation, especially for

the particles stabilized by Tween 20. The use of modified

starch as an emulsifier could retard the rate of particle

gelation at low storage temperatures, but gelation still

occurred eventually. Overall, our results indicated that

physically stable tripalmitin SLNs can be formulated by

using Tween 20 as an emulsifier and by adding carrier oils

(MCT or orange oil) to the lipid phase prior to homoge-

nization. These SLN suspensions may prove to be useful

delivery systems for lipophilic bioactive food components,

such as oil soluble vitamins, carotenoids, and other nutra-

ceuticals. In future studies, it would be useful to examine

the ability of these SLN to incorporate, protect, and release

encapsulated lipophilic components.

Acknowledgments This material is based upon work partly sup-

ported by a United States Department of Agriculture, AFRI-NIFA

Grant and Hatch Grant.

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