synthesis of sub-10 nm solid lipid nanoparticles for topical and biomarker detection applications

RESEARCH PAPER

Synthesis of sub-10 nm solid lipid nanoparticles for topicaland biomarker detection applications

Xiomara Calderon-Colon • Marcia W. Patchan •

Mellisa L. Theodore • Huong T. Le • Jennifer L. Sample •

Jason J. Benkoski • Julia B. Patrone

Received: 4 September 2013 / Accepted: 6 January 2014 / Published online: 22 January 2014

� Springer Science+Business Media Dordrecht 2014

Abstract Solid lipid nanoparticles (SLNs) are a

promising platform for sensing in vivo biomarkers due

to their biocompatibility, stability, and their ability to

carry a wide range of active ingredients. The skin is a

prominent target organ for numerous inflammatory

and stress-related biomarkers, making it an excellent

site for early detection of physiological imbalance and

application of sensory nanoparticles. Though smaller

particle size has generally been correlated with

increased penetration of skin models, there has been

little attention paid to the significance of other

nanoparticle synthesis parameters with respect to their

physical properties. In this study, we demonstrate the

synthesis of sub-10 nm SLNs by the phase inversion

temperature (PIT) method. These particles were

specifically designed for topical delivery of hydrogen

peroxide-detecting chemiluminescent dyes. A system-

atic design of experiments approach was used to

investigate the role of the processing variables on SLN

form and properties. The processing variables were

correlated with the SLN properties (e.g., dye solubil-

ity, phase inversion temperature, particle size, poly-

dispersity, melting point, and latent heat of melting).

Statistical analysis revealed that the PIT method,

while allowing total control over the thermal proper-

ties, resulted in well-controlled synthesis of ultra-

small particles, while allowing great flexibility in the

processing conditions and incorporated compounds.

Keywords Solid lipid nanoparticle (SLN) �Design of experiments (DOE) � Topical delivery �Biomarker detection � Nanobiotechnology

Introduction

The skin is a prominent target organ for numerous

inflammatory and stress-related signals that are altered

during disease, and it offers a rich environment for

biomarker exploration, making it an excellent site for

early detection of physiological imbalance. Unfortu-

nately, the stratum corneum is a formidable barrier to the

penetration of sensory compounds (Schafer-Korting

X. Calderon-Colon (&) � M. W. Patchan �M. L. Theodore � H. T. Le � J. L. Sample �J. J. Benkoski (&) � J. B. Patrone (&)

Research and Exploratory Development Department, The

Johns Hopkins University Applied Physics Laboratory,

11100 Johns Hopkins Road, Laurel, MD 20723, USA

e-mail: [email protected]

J. J. Benkoski


J. B. Patrone


M. W. Patchan


M. L. Theodore


H. T. Le


J. L. Sample


123

J Nanopart Res (2014) 16:2252

DOI 10.1007/s11051-014-2252-2

et al. 2007). Its complex structure and composition are

finely tuned to prevent breaching by a wide variety of

toxins and microbes. The same factors also impair the

absorption of sensory compounds. The corneocytes of

the outer skin layers (stratum corneum) are arranged in a

‘‘brick and mortar’’ structure, characterized by parallel,

overlapping layers (Wertz and Downing 1989). They

are embedded in a highly hydrophobic lipid matrix

primarily consisting of ceramides, cholesterol, choles-

terol esters, and fatty acids (Moser et al. 2001). This

layered structure consequently has the rare ability to

block both the hydrophilic and hydrophobic penetrants.

Further limiting permeation is the constant sloughing of

corneocytes.

Despite these challenges, the skin remains a highly

desirable site not only for biomarker detection, but

also for the topical delivery of therapeutics. There are

three possible routes of penetration beyond the stratum

corneum: transcellular, intercellular, and via hair

follicles. Ideally, topically applied formulations are

designed to penetrate the skin to a certain degree,

achieve a significant residence time, and deliver an

active compound, all the while remaining non-irritat-

ing. Thus, particles must be rationally designed to

penetrate beyond the stratum corneum and persist

within the skin despite routine washing and natural

skin sloughing. Small particle size has been indicated

by several studies to increase penetration within skin

models (Ryman-Rasmussen et al. 2006, 2007; Vogt

et al. 2006; Rancan et al. 2012).

Recent advances in nanotechnology have enabled

the development of nanometer-sized particles for

improved skin penetration (Figen et al. 2010; Mei

et al. 2003; Liu et al. 2007). Nanoparticles have

accordingly generated great interest within the phar-

maceutical and cosmetic industries as vehicles for the

delivery of therapeutic and sensory compounds. Solid

lipid nanoparticles (SLNs) are one of the main classes

of lipid-based nanoparticles that have emerged from

these efforts. Unlike quantum dots and most polymeric

nanoparticles, SLNs are comprised entirely of resorb-

able, nontoxic compounds. The resulting biocompat-

ibility makes them particularly well-suited for

dermatological applications (Puglia and Bonina

2012; Papakostas et al. 2011; Korting and Schafer-

Korting 2010). SLNs are composed of solid lipids

(0.1–30 % w/w) dispersed in an aqueous medium, and

stabilized with a surfactant (0.5–5 % w/w). SLNs

offer many advantages for topical delivery including

biocompatibility, stability, and potential for controlled

release of active ingredients. SLNs are particularly

attractive for delivering hydrophobic compounds. In

one example, Mei et al., observed a 3.459 increase in

the steady-state flux and permeability coefficient of

triptolide in rat skin relative to pure triptolide alone

(Mei et al. 2003). Despite intense research, questions

remain as to the penetration and permanence of these

particles in skin (Baroli 2010). We have recently

demonstrated the controlled diffusion of dyed SLNs

from bacterial cellulose into skin in vivo (Patchan

2013). However, more investigation is necessary to

optimize skin penetration by varying specific SLN

properties. Further investigation on how drug (or dye)

solubility influences skin penetration remains impor-

tant for a more complete understanding of this delivery

vehicle.

SLNs are commonly synthesized by high energy

methods such as ultrasonication, microfluidization,

and high-pressure homogenization (Figen et al. 2010).

The complexity of such methods has delayed the broad

adoption of SLN technology by creating the percep-

tion that nanoemulsions are expensive and difficult to

produce (Tadros et al. 2004). Furthermore, the harsh

processing conditions are of concern for active

compounds that are sensitive to extreme temperature,

pressure, or shear stress.

To this end, the Solans group has developed the

more gentle phase inversion temperature (PIT)

method (Tadros et al. 2004; Sevcikova et al. 2011;

Forgiarini et al. 2000). In the PIT method, the

composition remains constant while the temperature

is changed. One simply stirs the heated solution as it

cools to room temperature. This method is made

possible by the temperature sensitivity of nonionic

surfactants. The polyethylene glycol (PEG) head

group of the surfactant molecule becomes increasingly

swollen with water at decreasing temperature. With

the alkane tail remaining roughly constant in size, the

spontaneous curvature of the micelles reverses sign at

the phase inversion temperature. At this temperature,

the mixture inverts from a water-in-oil emulsion to an

oil-in-water emulsion. The interfacial tension also

reaches a minimum at this temperature, favoring a fine

dispersion. The droplets then solidify with continued

cooling of the oil phase below its melting temperature.

In this paper, we demonstrate the generation of sub-

10 nm SLNs using this technique. We further dem-

onstrate loading of the SLNs with a wide range of

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lipophilic compounds, and the effects of processing

variables on nanoparticle characteristics.

SLNs have the potential to serve as a universal

platform for the topical delivery of lipophilic com-

pounds. To test this hypothesis, we have chosen a set

of peroxide-detecting chemiluminescent compounds

with differing solubility and chemical structure as a

model system. This chemical system is designed to

efficiently react with hydrogen peroxide to give a

fluorescent signal. It consists of 1,10-oxalyldiimidaz-

ole (ODI) and a set of four fluorescent dyes that emit

light in the presence of ODI in its activated state. ODI

possesses much greater sensitivity than peroxyoxa-

lates and similar chemiluminescent systems (Stig-

brand et al. 1994; Chong et al. 2012; Dasari et al. 2009;

Lee et al. 2007). Its estimated detection limit is as low

as 10-8 M for H2O2 in water (Stigbrand et al. 1994).

H2O2 has been targeted as a biomarker for this study

because its overproduction has been specifically asso-

ciated with skin disease (e.g., cancer) and the progres-

sion of normal wound-healing processes (Niethammer

et al. 2009). The chemical detection scheme using SLNs

for this sensory application has been developed previ-

ously by our group (Patrone et al. 2011). Figure 1 shows

the chemiluminescence of the SLNs as a function of

time for several H2O2 concentrations. A schematic of

the peroxide-detecting SLNs is given in Fig. 2. The

chemical interaction of the particles with H2O2 results

in excitation of the incorporated fluorescent dye. While

this reaction can theoretically be tailored to a specific

application by altering the incorporated dye, and is

easily measured by standard optical detectors, it

requires optimization for topical and therapeutic appli-

cation. For example, particle size and drug-loading

potential are parameters that can greatly impact the

effectiveness and potency of an applied therapeutic. In

this study, a systematic design of experiments (DOE)

approach was used to study the synthesis variables and

properties of SLNs, toward optimization of the PIT

method for topical formulations.

Experimental

Materials

Nonadecane (C19, MP 30–34 �C, Aldrich), Eicosane

(C20, MP 36–38 �C, Aldrich), Heneicosane (C21, MP

39–41 �C, Aldrich), Tetracosane (C24, MP 49–52 �C,

Aldrich), Brij O10 (C18E10, Aldrich), decanol

(Sigma-Aldrich), methanol (Sigma-Aldrich), dode-

cane (Sigma-Aldrich), 1,10-oxalyldiimidazole (ODI,

TCI), Rhodamine B (red, Aldrich), 9,10-diphenylan-

thracene (blue, TCI), bis(N-methylacridinum)nitrate

(blue–green, TCI), 9,10-bis(phenylethynyl)anthra-

cene (green, TCI), and DI Water.

Processing

SLNs were prepared by the phase inversion temperature

(PIT) method Forgiarini et al. (2000, 2001a). The

fluorescent dye (Rhodamine B, 9, 10-diphenylanthracene,

Fig. 1 Chemiluminescence intensity of SLNs as a function

of time and grouped by H2O2 concentration. The aqueous

SLN dispersion in this measurement was composed of

50 mg/mL Brij O10, 40 mg/mL eicosane, 10 mg/mL deca-

nol, 1 mg/mL 1,10-oxalyldiimidazole, and 0.5 mg/mL 9,10-

bis(phenylethynyl)anthracene

H2O H2O2

surfactantlipid dye

diimidazole

Fig. 2 Schematic of H2O2-detecting SLN showing the major

ingredients

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Bis(N-methylacridinum)nitrate, or 9, 10-Bis(phenyl-

ethynyl)anthracene), 1-10-oxalyldiimidazole, metha-

nol, and decanol were combined into a vial. The

amount of methanol added to the mixture varies from

0.76 to 9.5 lg depending on the sample run (Note: Due

to the processing temperature most of the methanol is

expected to evaporate). The following step was the

addition of Brij O10 and the lipid (C19, C20, C21, or

C24). In Table 1, summary of the processing condi-

tions, the concentration of each component is listed.

The resulting mixture was co-melted and stirred. DI

water was then added to the mixture, heated, and

stirred. Under continued stirring, cooling the sample

causes inversion of the water-in-oil emulsion to an oil-

in-water emulsion, creating a nanoemulsion in the

process. A further decrease in temperature below the

melting point of the lipid converts the liquid nano-

emulsion into an aqueous dispersion of SLNs.

Design of experiment (DOE)

Design of Experiments is a statistical method of

conducting a series of experimental runs in which

independent variables (often called factors) are simul-

taneously varied while obtaining a desired effect

(often called response) (Fisher 1935). This method can

explore a large, multivariable parameter space much

more quickly than possible by the traditional method

of varying only one parameter at a time. As such, DOE

has grown in popularity for formulation development.

In this study, the DOE processing variables are lipid

composition (which controls lipid melting point), dye

composition, lipid concentration, surfactant concen-

tration, dye concentration, processing temperature,

and thermal mass (which is inversely related to

cooling rate). From Table 1, there are seven factors

(lipid composition, dye composition, oil concentra-

tion, surfactant concentration, dye concentration,

processing temperature, and thermal mass, with levels

of (C19,C20, C21, and C24), (Rhodamine B (red),

9,10-diphenylanthracene (blue), Bis(N-methylacridi-

num)nitrate (blue–green), 9, 10-Bis(phenyleth-

ynyl)anthracene (green)), (2, 6, and 10 %), (2.02,

6.06, and 10.1 %), (0.1, 0.5, and 1.0 mg/mL), (50, 70,

and 90 �C), and (2, 6, and 10 mL), respectively. In this

work, the DOE-Custom Designer platform from the

commercial statistical software JMP� (SAS Institute,

Cary, North Carolina) was used to generate a Reso-

lution V Design, which is capable of estimating not

only the coefficients of the main factors, but also those

Table 1 Summary of the synthesis conditions in this study

Sample

number

Oil Dye Oil

concentration

(%)

Surfactant

concentration

(%)

Dye

concentration

(mg/mL)

Processing

temperature

(�C)

Thermal

mass

(mL)

1 C20 Rhodamine B 10 10.1 0.1 50 2

2 C19 9,10-diphenylanthracene 10 10.1 1.0 90 2

3 C19 Bis(N-methylacridinum)nitrate 6 6.06 0.5 70 6

4 C21 9, 10-Bis(phenylethynyl)anthracene 10 10.1 0.1 90 10

5 C19 Rhodamine B 2 2.02 1.0 90 10






11 C19 Rhodamine B 6 6.06 0.5 70 6


13 C24 Rhodamine B 10 10.1 1.0 90 2





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two-factor interactions. The regression model that was

utilized is:

Y ¼ I þ b1X1 þ b2X2 þ b3X3 þ b1;2X1X2 þ b1;3X1X3

þ b2;3X2X3. . .

where Y is the responses to be measure are particle size,

and polydispersity, bi is the coefficients of the factors,

X1 is the lipid composition (C19, C20, C21, or C24),

X2 is the dye composition (Rhodamine B, 10-diphen-

ylanthracene, bis(N-methylacridinum)nitrate, or 9,

10-bis(phenylethynyl)anthracene), X3 is the values of

the lipid concentration (2, 6, or 10 %), X4 is the values

of the surfactant concentration (2.02, 6.06, 2, or

10.1 %), X5 is the values of the processing temperature

(50, 70, or 90 �C), X6 is the values of the thermal mass

(2, 6, or 10 mL).

The design calls for 17 runs, which are listed in

Table 1.

Dye solubility

The determination of the maximum amount of dye that

can be dissolved in the lipid (C19, C20, C21, and C24)

for each dye was performed by first adding an excess

of the dye into the lipid. The mixture was melted

together and then a known volume of the supernatant

dye–lipid solution was diluted into a 25 wt/wt%

decanol in dodecane. A series of standards were

prepared and used to construct a calibration curve

(e.g., absorbance as function of dye concentration) to

determine the dye solubility (e.g., concentration) for

each lipid. The dye concentration dissolved into the

lipid was calculated using the calibration curve. The

absorbance of three replicates was measured.

Phase inversion temperature

The phase inversion temperature was determined

utilizing a water bath. The samples were slowly

heated and gently agitated until the solution appeared

clear, at which time the temperature was recorded

Forgiarini et al. (2000, 2001a, b).

Particle size and polydispersity

The particle size and the polydispersity of the SLNs

were measured using dynamic light scattering (DLS).

In this technique, the uses frequency shifted light to

measure nano-size particles. The samples were mea-

sured as prepared, using Nanotrac Ultra (serial number

U1985IS). The particle diameter and polydispersity of

particle size distribution are reported.

Melting point and latent heat of melting

The thermal behavior of SLNs was studied using a

Mettler-Toledo differential scanning calorimeter

(DSC) equipped with an auto-sampler and liquid

nitrogen as the cooling source. The as-prepared SLNs

were pipetted into an aluminum pan with mass of

approximately 21–25 mg. SLNs were then hermeti-

cally sealed inside 40 lL aluminum pans to minimize

moisture loss during the DSC scan. The latent heat of

melting was calculated using the integral of the area

under the curve in the DSC plot (amount of energy)

divided by the amount of material. The melting point

and the latent heat of melting are reported.

Results

Melting point and latent heat of melting

Table 2 reports that the synthesized SLNs have a

melting point between 24 and 46 �C and a latent heat

of melting from 0.59 to 6.58 J/g, depending on the

synthesis conditions. While the melting points of the

SLNs were expected to vary with the lipid composi-

tion, we also observed a large variation in the latent

heat of melting in the SLNs synthesized by this

method. The crystallinity (e.g., degree of structural

order) of a solid can be quantified using the latent heat

of melting. The various SLN populations were char-

acterized as having different levels of crystallinity,

correlating with the concentration of lipids. Increasing

the concentration of solute was shown to cause a

decrease in the SLN latent heat of melting, and, hence,

crystallinity. As expected, the addition of solute

molecules decreased the overall crystallinity by dis-

rupting the packing of lipid molecules.

The results showed a melting temperature of

32.69 �C for SLNs using eicosane (C20) as lipid,

which is ideal for skin applications where the mean

skin surface temperature is 32–35 �C (Freitas 1999).

We also explored the utilization of several other lipids,

which resulted in a variation in the melting

J Nanopart Res (2014) 16:2252 of 10 2252

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temperature of the SLNs. In Fig. 3a, the melting

temperatures for SLNs using nonadecane (C19) and

triacontane (C30) are 27.7 and 61.8 �C, respectively.

Note the melting point depression relative to the bulk

lipid in Fig. 3a. Here, the confinement to small

dimensions and the high surface area-to-volume ratio

depress the nanoparticle melting point below the value

of the bulk lipid. Increasing concentrations of decanol

were also shown to cause a decrease in the SLN

melting point. Figure 3b shows a polynomial

relationship.

Varying the aliphatic chain length of the lipid from

19 to 30 gives a 34.2 �C range in melting temperature

for the SLNs. This control allows one to tailor the

melting temperature to a specific application (e.g., drug

delivery). In the case of hydrogen peroxide sensors, the

chemiluminescence becomes inactive below the melt-

ing point. The solidified SLN traps the ODI and

fluorophore within its hydrophobic core. Trapped

within the frozen lipid, ODI is unable to diffuse to

the SLN surface, where it can react with available

H2O2. Similarly, the fluorophore is unable to diffuse

toward the activated ODI, where it exchanges energy to

excite fluorescence. One can exploit this phenomenon

to prevent premature chemiluminescence when not in

use, to provide longer shelf life, or to use the increased

temperature of inflammation or illness as a trigger for

the H2O2 sensory function.

Particle size and polydispersity

The particle size and polydispersity of the SLNs are

summarized in Table 2. In this study, the SLNs

synthesized using the PIT method had a particle size

ranging from 7.8 to 44.9 nm, with a single outlier at

214.2 nm. The polydispersity was narrow, ranging

from 9.2 to 0.69 %. Figure 4 shows the linear regres-

sion fit of the particle size data as a function the relevant

parameters. No correlation had a level of confidence

greater than 85 %; the results for polydispersity were

similar. Although no positive correlations were iden-

tified, this finding actually indicates that small particle

size and low polydispersity can be achieved almost

regardless of synthesis conditions or payload. Provided

that the system possesses a well-defined phase inver-

sion temperature, these data suggest that it is possible

to maintain similar physical properties for SLN carriers

across a broad range of payloads.

Table 2 Summary of DOE results, including values for each of the responses dye solubility, phase inversion temperature, particle

size, polydispersity, melting point, and latent heat of melting

Sample

number

Dye solubility

(mg/mL)

Phase inversion

temperature (�C)

Particle

size (nm)

Polydispersity

(%)

Melting point

(�C)

Latent heat

of melting (J/g)

1 5.73 36 8.95 9.18 30.67 4.43

2 1.4 32 214.2 0.686 24.67 3.3

3 0.014 36 44.9 0.2251 24.33 2.39

4 1.71 48 12.2 2.11 37.67 6.18

5 3.37 40 7.75 1.051 24 0.59

6 1.25 31 8.41 1.297 24.33 2.26

7 0.014 32 8.63 9.2 24.67 3.71

8 0.036 45 11.74 1.577 32.67 0.94

9 0.011 27 16.67 2.717 30.33 4.52

10 2.92 60 7.59 1.644 45.67 1.38

11 3.37 38 8.11 3.41 24.33 2.49

12 4.03 45 13.79 1.772 37.33 5.87

13 5.92 55 7.86 8.22 29.33 1.31

14 1.4 36 8.05 2.799 24.33 2.59

15 0.011 31 18.34 0.904 27.67 1

16 1.83 13.05 3.72 27 0.94

17 1.76 55 10.51 1.689 29.67 1.16

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Dye solubility

The dye solubility of the SLNs prepared under

different synthesis conditions is summarized in

Table 2. The solubility of the dye in the oil phase

ranged from 0.011 to 5.92 mg/mL, depending on the

dye. Bis(N-methylacridinium)nitrate, which is water

soluble, showed the lowest solubility, whereas rhoda-

mine B had the highest solubility (Fig. 5). 9,10-

Bis(phenylethynyl)anthracene and 9,10-diphenylan-

thracene, which are actually the two most hydrophobic

fluorophores included in this study, displayed inter-

mediate solubilities. Rhodamine B was roughly twice

as soluble in the oil phase as these two dyes despite

having significant water solubility.

Design of experiments

Table 2 summarizes the responses of the each DOE

run. The responses include dye solubility, phase

inversion temperature, particle size, polydispersity,

melting point, and latent heat of melting.

Discussion

In this study we explored the synthesis of SLNs

appropriate for the delivery of sensory compounds to

the inner layers of the skin. As described previously,

the PIT method was used to study the effects of

synthesis variables on the resulting SLNs properties

(phase inversion temperature, particle size, polydis-

persity, melting point, and latent heat of melting). The

outcomes of this study demonstrate that: (1) PIT

method allows the synthesis of SLNs with ultra-small

size and low polydispersity, (2) the latent heat of

melting and melting point of the SLNs can be

controlled, and (3) the dye (drug) chemistry will

impact the payload and thereby the therapeutic and

detection potential. Statistical analysis revealed that

the PIT method, while allowing total control over the

thermal properties, resulted in a well-controlled syn-

thesis of ultra-small particles, while allowing great

flexibility in the processing conditions and incorpo-

rated compounds.

Particle size analysis revealed that 47.1 and 88.2 %

of the SLNs synthesized in this study has a particle size

between 0–10 nm and 0–20 nm, respectively. The

results suggest that the PIT method offers limited

control over particle size, but perhaps, more impor-

tantly, it can consistently produce SLNs with ultra-

small sizes regardless of the synthesis conditions or

payload. Interestingly, we found that the processing

conditions did not have any statistically significant

effects on particle size and polydispersity. This weak

Fig. 3 a Melting points as a function of bulk lipid and plain

SLNs. b Eicosane (C20) with different percentages of decanol.

Sample means were determined to be significant (P \ 0.05) by

one-way analysis of variance (P = 0.0074). c Latent heat of

melting as function of lipid concentration

J Nanopart Res (2014) 16:2252 of 10 2252

123

dependence on process parameters indicates a robust-

ness of the PIT method versus relatively small changes

in conditions. We note here that the conditions were

limited to those that produced a phase inversion

between room temperature and 100 �C. For example,

the lipid to surfactant ratio was held constant in order

to meet this requirement. The more accurate interpre-

tation of this finding, then, is that controlling the phase

inversion temperature supersedes the control of par-

ticle size. Precise control of particle size and size

distribution may still be possible, but it must be done

within the constraints of maintaining an appropriate

phase inversion temperature for each specific payload.

The total control over the thermal properties (e.g.

melting point) of the SLNs increases the potential

applications that can be envisioned for this technol-

ogy. In addition, this information serves as guidance

for the determination of the storage conditions, which

are important to extend the shelf life of the particles.

The melting point can easily be tailored by changing

the aliphatic chain length of the lipid, which represents

a minimal change in the synthesis of the SLNs. On the

other hand, the different levels of crystallinity, which

correlate well with the concentration of lipids, will

impact the interaction with the skin (Schafer-Korting

et al. 2007). The latent heat of melting, which is

proportional to the amount of crystalline lipid, reflects

both the quality of crystallinity and the fraction of

solid within the SLNs. Overall, we observed that the

latent heat of melting decreased due to the addition of

impurities (dye, decanol, etc.) and due to the ultra-

small diameter of the nanoparticle. The occlusivity of

lipid nanoparticle dispersions is impacted not only by

particle size and lipid concentration but also with the

degree of crystallinity of the lipid matrix. These

factors, therefore, can directly impact the interaction

of particles with skin (Schafer-Korting et al. 2007;

Wissing and Muller 2002). The chemistry of the

loading compounds will impact the payload, which

will translate to a potential impact of the therapeutic

and detection capabilities. The payload of the SLNs

will determine the therapeutic regimen and the

detection lifetime when used as a biosensor.

Fig. 4 Linear regression of the particle size as a function of relevant parameters

Fig. 5 Dye solubility as a function of dye composition.

Rhodamine B = red, 9,10-diphenylanthracene = blue, Bis

(N-methylacridinum)nitrate = blue–green, and 9, 10-bis

(phenylethynyl)anthracene = green

2252 of 10 J Nanopart Res (2014) 16:2252

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Conclusions

In this study, we employed the PIT method and a

systematic DOE approach to synthesize and charac-

terize ultra-small SLNs. We additionally examined the

role of the synthesis parameters on SLN. The main

conclusions of this study are: (1) the PIT method is a

versatile method with which to synthesize SLNs with

ultra-small sizes and low polydispersity, (2) the latent

heat of melting and melting point of the SLNs can be

controlled, and (3) the loading compound chemistry

will impact the payload as well the therapeutic and

detection applications. In comparison with other

studies using the PIT method (Forgiarini et al. 2000,

2001a, b; Carbone et al. 2012), this study showed that

the synthesis of SLNs with small particle size can be

achieved almost regardless of synthesis conditions or

payload. Indicating that using the synthesis conditions

in this study will be possible to maintain similar

physical properties. More importantly, if the system

possesses a well-defined phase inversion temperature,

ultra-small particle size SLNs can be obtained.

Furthermore, the particle sizes of the SLNs in this

study are significantly smaller than that in other

studies using the PIT method (Forgiarini et al. 2000,

2001a, b; Carbone et al. 2012).

SLNs with 7.6 nm particle diameter and polydis-

persity of 1.6 % were synthesized by the phase

inversion temperature method using tetracosane

(C24) as a lipid, 2.0 % oil concentration, 2.02 %

surfactant concentration, a processing temperature of

50 ˚C, and thermal mass of 10 mL. Overall, we

conclude that when using this method, it is possible

to consistently produce SLNs with particle diameters

less than 20 nm and low polydispersity, almost

regardless of synthesis conditions or payload. Addi-

tionally, this method allows the use of a large number

of lipids giving total control over the thermal proper-

ties of SLNs and expanding their applications.

Finally, the maximum payload is controlled by the

chemistry of the loading compound. However, the

solubility can be modified somewhat through the

addition of co-solvents in the lipid phase such as

decanol and menthol to increase the hydrophilicity in

the oil phase. Although co-solvents will tend to

decrease the melting point and crystallinity, their

concentration can be varied over a considerable range

without completely disrupting the phase inversion or

completely suppressing the freezing phase transition.

Ultimately, the dye solubility is a critical aspect for the

therapeutic and detection applications, since low

solubility could limit the signal strength or the lifetime

of the particle sensor in vivo. Dye solubility will also

affect the partitioning of the active ingredients

between the lipid and water phases. SLNs are an

effective vehicle for solubilizing lipophilic com-

pounds in water or water-based lotions. Transparent

in color and low in viscosity, the SLN suspensions

prepared in this study are nearly indistinguishable

from an aqueous solution, even for very hydrophobic

payloads. SLNs will be less effective, however, for

compounds that partition strongly into water.

Acknowledgments This research was sponsored by the

Research and Exploratory Development Department, The

Johns Hopkins University Applied Physics Laboratory, 11100

Johns Hopkins Road, Laurel, Maryland 20723.

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synthesis of sub-10 nm solid lipid nanoparticles for topical and biomarker detection applications

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