Research Article

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Cellular uptake of self-emulsifying

drug-delivery systems: polyethylene glycol
versus polyglycerol surface
Julian David Friedl1 , Christian Steinbring1 , Sergey Zaichik1 , Nguyet-Minh Nguyen Le1,2 &
Andreas Bernkop-Schnürch*,1
1
Department of Pharmaceutical Technology, University of Innsbruck, Institute of Pharmacy, Center for Chemistry & Biomedicine,
Innsbruck, 6020, Austria
2
Department of Industrial Pharmacy, Faculty of Pharmacy, University of Medicine and Pharmacy at Ho Chi Minh City, 700000 Ho
Chi Minh City, Vietnam
*Author for correspondence: Tel.: +43 512 507 58 600 Andreas.Bernkop@uibk.ac.at

Aim: Comparison of the impact of polyethylene glycol (PEG) and polyglycerol (PG) surface decoration
on self-emulsifying drug delivery system (SEDDS)-membrane interaction and cellular uptake. Materials
& methods: PEG-, PEG/PG- and PG-SEDDS were assessed regarding their self-emulsifying properties, sur-
face charge, bile salt fusibility, cellular uptake and interaction with endosome-mimicking membranes.
Results: SEDDS exhibited droplet sizes between 150 and 175 nm, a narrow size distribution and self-
emulsified within 7 min. Higher PEG-surfactant amounts in SEDDS resulted in charge-shielding and thus
in a decrease of ζ potential up to 11 mV. The inert PEG-surface hampered bile salt fusion and interfered
SEDDS–cell interaction. By reducing the PEG-surfactant amount to 10%, cellular uptake increased twofold
compared with PEG-SEDDS containing 40% PEG-surfactant. PG-SEDDS containing no PEG-surfactants
showed a threefold increased cellular uptake. Furthermore, complete replacement of PEG-surfactants by
PG-surfactants led to enhanced cellular interaction and improved disruption endosome-like membranes.
Conclusion: PG-surfactants demonstrated high potential to address PEG-surface associated drawbacks in
SEDDS.

Graphical abstract:

Long-chain
Extracellular site PEG-surface

Cationic surfactant
PG-SEDDS PEG-SEDDS
Short chain
PG-surface

PG-SEDDS
Glycoprotein

Intracellular site

Endosomal Endosomal
PEG-SEDDS
PG-SEDDS escape escape

10.2217/nnm-2020-0127 
C 2020 Future Medicine Ltd Nanomedicine (Lond.) (Epub ahead of print) ISSN 1743-5889
Research Article Friedl, Steinbring, Zaichik, Le & Bernkop-Schnürch

First draft submitted: 27 March 2020; Accepted for publication: 3 June 2020; Published online:
12 August 2020

Keywords: cellular uptake • confocal microscopy • nanoemulsion • PEG-free self-emulsifying drug-delivery system
• polyglycerol surfactants • SEDDS

Nano-sized drug-delivery systems have experienced enormous growth as they were shown to protect their cargo
against enzymatic degradation [1], to improve systemic drug uptake [2] and to provide sustained and even targeted
drug release [3]. Among these nanocarriers in particular lipid-based drug-delivery systems such as self-emulsifying
drug-delivery systems (SEDDS) moved in the limelight of research. The self-emulsifying process of SEDDS usually
requires nonionic surfactants with a HLB >13 in concentrations of more than 25% [4]. In particular, PEGylated
surfactants such as polyethoxylated castor oils (Cremophors) or polysorbates (Tweens) are generally used for
this purpose. Such PEGylated lipids are known to form an inert, hydrophilic polymer layer around the carrier
that is called ‘brush’ or ‘corona’. Beneficial features of this corona were shown in protection against enzymatic
degradation [5], enhanced mucus permeation properties [5,6] and an reduced uptake by the reticuloendothelial
system resulting in a longer circulation half-life [7]. Despite all these advantages, however, PEGylation goes hand
in hand with substantial drawbacks for nanocarriers [8]. Uptake pathways like receptor-mediated endocytosis and
fusion of lipid carriers with the cellular membrane are diminished due to this hydrophilic coating because of a
sterical hindrance. Moreover, the PEG-corona dramatically hinders the endosomal escape of nanocarriers after
internalization leading to an extensive drug degradation in lysosomes [9]. Although formulation scientists are aware
of this so-called PEG-dilemma, so far no adequate alternatives to PEGylated surfactants were tested for SEDDS.
It was therefore the aim of this study to evaluate the potential of an alternative surfactant type for the development
of SEDDS. Since in other fields such as in cosmetic formulations PEGylated surfactants were already successfully
substituted by polyglycerol (PG) surfactants, this surfactant type was chosen for the formulation of PEG-free
SEDDS. Formulations of equal size and polydispersity index (PDI), differing only in the type of surfactant, were
developed to assure high comparability. PEGylated surfactants were exchanged by PG surfactants or by a mixture of
both surfactant types. In addition, a permanent cationic charge was introduced in all formulations by incorporation
of the same amount of dioctadecyldimethylammonium bromide to evaluate the effect of surface decoration on
this charge. The formulations were investigated regarding their capability to form nano-sized emulsions, stability
toward bile salts, cytotoxicity, cellular uptake as well as their interaction with endosome mimicking membranes.

Materials & methods

Materials
Bile salt mixture (sodium cholate, sodium deoxycholate; 1:1), sodium taurocholate, dioctadecyldimethylammonium
bromide (DODAB), Triton X100 and Tween 80 (TW80) were purchased from Sigma-Aldrich (Vienna, Austria).
Tegosoft PC41 was a gift from Evonik (Hamburg, Germany). Natragem SP140 NP was supplied by Croda (Nettetal,
Germany). Peceol was supplied as free sample by Gattefossé (Lyon, France). Captex 355 was a gift from Abitec
(WI, USA). Lumogen yellow (LGY) was supplied by Kremer pigmente (Aichstetten, Germany). Lipoid S 100 (soy
phosphatidylcholine) was obtained by Lipoid (Ludwigshafen, Germany). All other excipients were of high quality
and purchased from MERCK (Vienna, Austria)

Preparation & characterization of SEDDS

SEDDS preconcentrates were prepared by mixing oil, emulsifier and co-solvents in ratios listed in Table 1. The
cationic charge was introduced by dissolving dioctadecyldimethylammonium bromide (DODAB) in the oil phase
under heating the oil/cationic surfactant mixture up to 70◦ C and vortexing. Soy lecithin was dissolved in pure
ethanol in a final concentration of 300 mg/ml and added to the oil phase. After addition of surfactants and
co-solvents the mixture was vigorously shaken at 800 rpm and 37◦ C for 1 h in a thermomixer (Eppendorf,
Hamburg, Germany). The final preconcentrate was visually investigated for turbidity or phase separation. In order
to determine size and ζ potential preconcentrates were diluted 1:100 in demineralized water by slightly inverting
each mixture followed by incubation for 5 min at 37◦ C in a thermomixer (Eppendorf ). Size distribution of the
oily droplets was determined via dynamic light scattering technique (Malvern Zetasizer ZSP, Worcestershire, UK).
The experiments were conducted at 37◦ C in triplicate. ζ potential was measured in triplicate using a palladium
electrode equipped dip cell (Malvern Universal Dip Cell, Worcestershire, UK).

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 Cellular uptake of self-emulsifying drug-delivery systems: PEG versus PG surface Research Article

Table 1. Composition of self-emulsifying drug-delivery system preconcentrate in % (v/v).

Formulation Tegosoft PC41 Natragem Tween 80 Captex 355 Phospathidylcholine in Peceol Captex 355
S140 NP (120 mg/ml DODAB) ethanol (300 mg/ml)
PG 35% 20% 25% 10% 10%
PEG/PG 25% 20% 10% 25% 10% 10%
PEG 40% 25% 10% 10% 15%
DODAB: Dioctadecyldimethylammonium bromide; NP: No preservative; PEG: Polyethylene glycol; PG: Polyglycerol.

Emulsification time
The emulsification time of the different formulations was examined using a standard USP dissolution apparatus type
II (Erweka DT 600, Heusenstamm, Germany). One milliliter of preconcentrate labelled with LGY (0.2 mg/ml)
as lipophilic fluorescence marker was diluted in 900 ml of demineralized water at 37 ± 0.5◦ C. Gentle stirring at
50 rpm was provided by a standard stainless steel paddle [10]. The time needed for complete emulsification and
dispersion of the dye was assessed visually. Subsequently, the transmittance was measured at room temperature
in a 700 μl quartz cuvette at a wavelength of 600 nm using a UV–Vis spectrophotometer (Shimadzu UV Mini,
Korneuburg, Austria). Demineralized water served as 100% value.

Stability studies
Stability over time
SEDDS, emulsified in a concentration of 1%, were incubated at 37◦ C for over 24 h in a thermomixer. Samples
were withdrawn at 4 and 24 h to determine the size alteration via dynamic light scattering technique (Malvern
Zetasizer ZSP), thus indicating the kinetic stability of the system.
In order to assure the storage stability of the different preconcentrates PG- and PEG-SEDDS were stored at
room temperature over 5 months and subsequently emulsified in water obtaining a concentration of 1% (v/v).
Samples were analyzed by visual examination of the preconcentrates and the resulting emulsions as well as by size
determinations as described above.

Thermodynamic stability studies

The thermodynamic stability of SEDDS was evaluated according to Gulam et al. [11]. After centrifugation for
15 min at 10,000 rpm/6708×g (Eppendorf Minispin, Hamburg, Germany) 1% (v/v) SEDDS were analyzed
for stability in terms of phase separation, creaming, cracking and changes in droplet size. SEDDS were further
subjected to three freeze thaw cycles at -20◦ C and 20◦ C for a storage period of 12 h at each temperature level. After
each cycle the droplet size of each formulation was determined as indicator of thermodynamic stability.

Stability toward dilution

The robustness against dilution was determined by an adapted method previously described by Abdallah et al. [12].
Briefly, preconcentrates were diluted in increasing amounts of demineralized water from 1:50 up to 1:10,000. After
incubation for 30 min at 37◦ C and under gentle agitation at 500 rpm utilizing a thermomixer (Eppendorf ), the
size of oily droplets was examined at each dilution step by dynamic light scattering as described above.

Stability against cholic acids

Cholic acid, deoxycholic acid and taurocholic acid were chosen in a molar ratio of 1:1:1 for simulating fasted
intestinal bile salts conditions. SEDDS were emulsified in a concentration of 1% in a solution containing 10 mM
NaCl and 3 mM mixture of bile salts dissolved in demineralized water. Additionally, pH was adjusted to 6.8 using
0.01 M HCl or 0.01 M NaOH. The alteration of droplet size and ζ potential was measured after 4 h of incubation
at 37◦ C and 300 rpm (Thermomixer, Eppendorf ).

Cloud point determination

Preconcentrates were diluted in a ratio of 1:200 and gently shaken at 300 rpm. Once emulsified, the samples were
gradually heated in a thermomixer (Eppendorf ) with gentle agitation at 500 rpm from room temperature to 100◦ C
in steps of 5◦ C and maintenance at each step for 10 min. After having reached the cloud point samples were cooled
down to room temperature and droplet size was determined. For comparison, pure surfactants were diluted in the
same concentration as used in SEDDS and cloud point was determined as described above.

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Cell viability – resazurin assay

The potential cytotoxic effect of 0.05, 0.1, 0.5 and 1% (v/v) SEDDS was investigated by a slightly modified
resazurin assay as described earlier [13]. In brief, a 96-well plate was seeded in a density of 5 × 105 Caco-2 cells
per well. The cells were incubated for 3 days in minimal essential media (MEM) supplemented with 10% (v/v)
heat inactivated fetal calf serum and penicillin/streptomycin solution (100 units/0.1 mg/l) at 95% humidity and
37◦ C in an atmosphere of 5% CO2 to obtain a monolayer. Then 100 μl of SEDDS test samples were added to
each well in the indicated concentrations and incubated for 4 h at 37◦ C on the Caco-2 monolayer. Test samples
were subsequently removed and cells were washed with 100 μl of phosphate-buffered saline in order to dispose the
phenol red in the MEM as well as remaining SEDDS from the cells. Afterward, 100 μl of resazurin solution in a
concentration of 0.1 % (m/v) were added to each well and incubated for 2 h. Resazurin medium was prepared by
diluting a 10% (m/v) stock solution 1:100 with white minimal essential medium (MEM omitting the indicator
phenol red). After 2 h, 100 μl of the supernatant were withdrawn and measured after background correction at
an excitation wavelength of 540 nm and an emission wavelength of 590 nm using a microplate reader (Tecan,
Salzburg, Austria). Pure white MEM and Triton X-100 in concentration of 0.5% served as negative and positive
control, respectively.

Cellular uptake studies

For flow cytometry studies Caco-2 cells were seeded in a 12-well plate until they reached 100% confluency. SEDDS
were labeled with 0.15% (m/m) LGY and equality of fluorescence intensity among all formulations was confirmed
using a spectrophotometer. As LGY exhibits a log p-value of 7.48 the marker remains in the oil droplets formed
by SEDDS. Cells were incubated with 0.05% SEDDS dispersed in sterile glucose-HEPES buffer pH 7.4 for
4 h. Afterward cells were detached by accutase treatment, rinsed with ice cold phosphate-buffered saline (PBS)
three-times and analyzed by flow cytometry (Attune NxT Flowcytometer, Thermo Fisher Scientific, MA, USA).
The data were analyzed using a custom-written MatLab program and fluorescence intensity distribution data
were represented using the logicle display developed by Parks et al. [14]. The same gating strategy was used for all
SEDDS tested and >10,000 cells of each sample were analyzed. Surface absorbed SEDDS were quenched using
Trypan Blue treatment prior to flow cytometric analysis as described by Srivastava et al. [15]. All experiments were
performed in triplicate. The relative mean fluorescence intensity values (RMFI) quantify the fluorescent intensity
of LGY-labeled SEDDS being proportional to their average concentration per cell. RFMI was calculated from the
mean fluorescence intensity values (MFI). In this calculation, the RMFI value of the control sample (Caco-2 cells
only, treated with Trypan Blue) is always zero: RMFI = MFI(S)/MFI(C) −1; MFI(S) = MFI of the treated sample;
MFI(C) = MFI of the control; results are presented as mean ± SD, n = 3.

Membrane interaction – endosomal escape study

The cellular membrane of red blood cells is frequently used to mimic the in vivo conditions after endosomal
internalization of nanocarriers [16]. Erythrocytes concentrate, kindly donated by Tirol Kliniken GmbH (Innsbruck,
Austria), was used to evaluate the in vitro hemolysis of SEDDS. Prior to the assay, RBC concentrate was diluted 1:100
(v/v) with sterile glucose-HEPES buffer pH 7.4. To 0.5 ml of this suspension 0.5 ml of SEDDS in glucose-HEPES
buffer were added resulting in a final SEDDS concentration of 0.05, 0.1, 0.5 and 1% (v/v). Samples were incubated
on thermomixer (Eppendorf ) at 300 rpm and 37◦ C for 4 h followed by centrifugation at 2730 rpm or 500×g for
10 min (Eppendorf Minispin). The released hemoglobin was quantified in the supernatant via UV-spectrometry
at a wavelength of 415 nm (Tecan, Salzburg, Austria). A 0.5% Triton X-100 solution containing glucose-HEPES
buffer served as 100% reference value hemolysis (positive control) and buffer mixed with erythrocyte suspension
served as negative control [17].
The following equation was used to calculate the extent of hemolysis in percentage:

 
Abs (T) − Abs neg
% Hemolysis =    
Abs pos − abs neg

where Abs (T) is absorbance of test sample, Abs (neg) is absorbance of the negative control and Abs (Pos) is
absorbance of the positive control.

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 Cellular uptake of self-emulsifying drug-delivery systems: PEG versus PG surface Research Article

Table 2. Size, PDI, ␨ potential and cloud point of polyglycerol, PEG and PEG/polyglycerol formulations.
Formulation Size (nm) PDI ␨ potential (mV) Cloudpoint (◦ C)
PG 168.25 ± 3.79 0.169 +58.4 ± 4.83 ⬎100
PEG/PG 172.61 ± 3.77 0.179 +52.5 ± 2.02 ⬎100
PEG 152.61 ± 5.91 0.174 +46.8 ± 1.08 ⬎100
Data are shown as mean ±SD (n = 3).
PDI: Polydispersity index; PEG:Polyethylene gycol; PG: Polyglycerol.

600 100
Emulsification time (s)

Transmittance (%)
80
400
60

40 Figure 1. Emulsification time (gray bars) and resulting

200 transmittance (black bars) of emulsified polyglycerol-,
20 poly(ethylene glyol)/polyglycerol- and poly(ethylene
glyol)-self-emulsifying drug-delivery system. Data are shown
0 0 as mean ± SD (n = 3).
PG PEG/PG PEG PEG: Poly(ethylene glyol); PG: Polyglycerol.

Life cell imaging using confocal microscopy

Cellular uptake of SEDDS was further confirmed utilizing confocal laser scanning microscopy (Leica TCS SP8)
with the appropriate filter sets. In brief, Caco-2 cells were seeded in a eight-well chamber (μ-slide, ibidi). After
reaching confluency, the Caco-2 cells were incubated with 0.05% SEDDS dispersed in sterile glucose-HEPES
buffer pH 7.4 for 4 h. Subsequently, cells were washed with prewarmed PBS thrice. The nucleus was stained
using Hoechst 33528 (1 μg/ml). All fluorescence images were recorded under equal confocal settings. Image post
processing was performed utilizing the open source image processing and analysis platform ImageJ: the yz- and
xz-projections were prepared from 5 xy-images of an image stack taken at 0.2 μm z-step length. Moreover, in order
to delete fluorescence bleed-through between detection channels due to overlapping emission spectra, spectral
unmixing was applied. Furthermore 2D image filtering was conducted using a Gaussian filter.

Statistical data analysis

Statistical data analysis was performed on GraphPad Prism (version 5.01) using the student t-test and the analysis
of variance (ANOVA) followed by Bonferroni correction with p < 0.01 as the minimal level of significance. All
values are expressed as means ± SD.

Results
Preparation & characterization of SEDDS
Three SEDDS formulations with a final composition as listed in Table 1 were developed. PEG-SEDDS contained
Tween 80 as surfactant whereas Natragem S140 NP and Tegosoft PC41 were used as surfactants in PG-SEDDS.
PEG/PG-SEDDS contained 10% of PEG-surfactants and 45% PG-surfactants.
As in particular droplet size and surface charge have a significant influence on cellular uptake [18,19], these
parameters were unified for all tested formulations. In a 1% (v/v) dispersion PG-SEDDS exhibited a droplet size of
168.25 ± 3.79 nm with a PDI of 0,169. The replacement of 10% Polyglyceryl-4 caprat with 10% Tween 80 caused
no severe change in droplet size and PDI. Whereas in PEG-SEDDS the amount of triglyceride (Captex 355) had
to be adjusted to achieve comparable size and PDI values as shown in Table 2. Dioctadecyldimethylammonium
bromide (DODAB), a permanently charged cationic lipid, was incorporated in all formulations to compare the
effect of SEDDS surface decoration on surface charge. Although all formulations contained the same amount of
DODAB, significant differences in the surface charge of the droplets were obtained, as listed in Table 2. In case of
PEG-SEDDS exhibited a ζ potential of 46.8 ± 1.08 mV. Compared with PG-SEDDS a in ζ potential of 11.6 mV
could be detected and a decline of 5.9 mV when compared with PEG/PG-SEDDS.

Emulsification time
The time required for total emulsification is displayed in Figure 1. All three formulations needed more than 3

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Research Article Friedl, Steinbring, Zaichik, Le & Bernkop-Schnürch

1.0

200
0.8

Figure 2. Size (bars) and PDI (lined bars) of

150
Size (nm)

0.6 polyglycerol-, poly(ethylene glyol)/polyglycerol-

PDI
and poly(ethylene glyol)-self-emulsifying
100 0.4 drug-delivery system (1 % v/v) emulsified in
25 mM HEPES buffer pH 6.8. Samples were
analyzed at time 0 h (black bars), 4 h (gray bars)
50 0.2
and after 24 h (light gray bars) of incubation at
37◦ C. Data are shown as mean ± SD (n = 3).
0 0.0 PDI: Polydispersity index; PEG: Poly(ethylene
PG PEG/PG PEG glyol); PG: Polyglycerol.

200
80

60 Figure 3. Bile salt interaction of

150
polyglycerol-, poly(ethylene

ζ potential (mV)
40 glycol)/polyglycerol- and poly(ethylene
Size (nm)

glycol)-self-emulsifying drug-delivery
20
100 system. Size before (white bars) and
0 after 4 h (gray bars) of incubation with
3 mM bile salt solution. ζ potential before
-20 (• connected with dotted line) and after
50
(• connected with line) bile salt
-40 incubation. Data are shown as mean ± SD
(n = 3).
0 -60 PEG: Poly(ethylene glyol); PG:
PG PEG/PG PEG Polyglycerol.

min to emulsify and resulted in transparent emulsions. PEG-SEDDS exhibited with 388 s the longest time to
completely emulsify, twice as long as PG-SEDDS. PEG/PG-SEDDS emulsified in less than 250 s comparably
faster than PEG-SEDDS and slower than SEDDS without PEG-surfactants.

Stability studies
Stability studies of SEDDS showed no significant change in size within in 24 h and all formulations exhibit
a narrow size distribution (PDI <0.25). These results are summarized in Figure 2. Further stability studies via
centrifugation (Supplementary Table 1) and repeated freeze-thaw cycles (Supplementary Figure 1) confirmed the
stability of PEG-SEDDS, PEG/PG-SEDDS and PG-SEDDS. Neither phase separation, nor creaming nor major
changes in size and PDI were observed after storage at room temperature for over 5 months (Supplementary Figure
2).
As the self-emulsification process is strongly affected by the available aqueous phase, the robustness toward
dilution was evaluated to verify the kinetic stability of SEDDS [18–20]. As shown in Supplementary Figure 3, a
minor decline in droplet size of about 30 nm was observed in case of PG- and PEG/PG-SEDDS, whereas
PEG-SEDDS exhibited with up to 60 nm a more pronounced decline.
Incubation of SEDDS with bile salts showed no phase separation, agglomeration or precipitation, confirming
the stability of all formulations. As illustrated in Figure 3, PEG-, PEG/PG- and PG-SEDDS decreased in their ζ
potential and size. The decline in droplet size was comparable in all three formulations within a range of 35–
50 nm. Furthermore, the ζ potential of PEG-SEDDS decreased about 63 mV within 4 h of incubation. An even
more pronounced decrease of 77 mV was observed for PEG/PG-SEDDS while the ζ potential of PG-SEDDS
declined about 92 mV.
The examination of cloud points showed no differences between the formulations. Cloud points were over the
heating maximum of 100◦ C (Table 2) for all three formulations. This indicates high resistance against thermal
fluctuation.

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 Cellular uptake of self-emulsifying drug-delivery systems: PEG versus PG surface Research Article

100

80
Viability (%)

Figure 4. Cell viability (in [%]) of Caco-2 cells after 4-h

60 incubation with polyglycerol- (black bars), and poly(ethylene
glyol)/polyglycerol- (gray bars) and poly(ethylene
40 glyol)-self-emulsifying drug-delivery system (light gray bars)
** in indicated concentrations. Triton X 100 served as positive
***
20 control and red MEM as negative control. Data are shown as
mean ± SD (n = 3).
0 **p < 0.01; ***p < 0.001 in comparison with PEG-SEDDS in
0.05 0.1 0.5 1.0 the respective concentration.
SEDDS concentration (v/v) (%) SEDDS: Self-emulsifying drug-delivery system.

70

***
60

***
50

40 ***
RMFI

***
30

20 ***
***

10

0
ControlTB

Control

PGTB

PG

PEG/PGTB

PEG/PG

PEGTB

PEG
10,000 100,000
Fluorescence intensity

Figure 5. Cellular uptake of polyglycerol-, poly(ethylene glyol)/polyglycerol- and poly(ethylene

glyol)-self-emulsifying drug-delivery system after 4-h incubation on a Caco-2 cell line at a concentration of 0.05 %
(v/v) by flow cytometry. Lumogen Yellow (LGY) was used as lipophilic fluorescence marker embedded in
self-emulsifying drug-delivery system (SEDDS). (A) shows the shift in fluorescence intensity of PG- (green), PEG/PG-
(blue) and PEG- (red) SEDDS in contrast to the control (black). (B) Displays the RFMI per formulation PEG- (light gray
bar), PEG/PG- (gray bar) and PG- (black bar) SEDDS. Lined bar shows the fluorescence intensity without quenching of
surface adhered SEDDS by trypan blue. Relative mean fluorescence intensity values quantify the fluorescent intensity
of LGY-labeled SEDDS per cell. Data are shown as mean ± SD (n = 3).
***p < 0.001 compared with each formulation.
PEG: Poly(ethylene glycol); PG: Polyglycerol; RMFI: Relative mean fluorescence intensity.

Cell viability
In a concentration range of 0.05–0.5% (v/v) all types of SEDDS showed a cell survival ≥75%. Declined cell
viability could only be observed in the highest tested concentration of 1% (v/v). As illustrated in Figure 4, only
at this concentration, PEG-SEDDS still exhibit with approximately 75% a significantly (p < 0.01) higher cell
viability compared with PEG/PG- or PG-SEDDS with viabilities of approximately 15% and 28%, respectively.
Incubation with 1% (v/v) PEG/PG-.SEDDS compared with PG-SEDDS showed, contrastingly, no significant
(p < 0.01) lower cytotoxicity. Since a in the concentration of 0.05% (v/v) no notable cytotoxicity was observed
for all tested SEDDS, this concentration was applied for cellular uptake studies.

Cellular uptake studies

The results shown in Figure 5A denoting a shift of the fluorescence intensity toward higher values for all three
formulations compared with the control signal confirmed a cellular uptake of these SEDDS. In Figure 5B the uptake
of SEDDS is shown. PG-SEDDS were taken up by 89% and exhibit with a threefold increase in RFMI value a

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Research Article Friedl, Steinbring, Zaichik, Le & Bernkop-Schnürch

**
100 ** ** Figure 6. In vitro endosomal escape study using fresh
**
Hemolysis (%) human erythrocytes. Hemolysis of PG, PEG/PG-and
80 PEG-self-emulsifying drug-delivery system in a concentration
of 1% (black bars), 0.5% (gray bars), 0.1% (light gray bars)
60
and 0.05% (white bars) at 37◦ C for 4 h. Triton X 100 served as
*** *** positive control and PBS pH 7.4 as negative control. Data are
40
shown as mean ± SD (n = 3).
20 **p < 0.01; ***p < 0.001 in comparison with
PEG-self-emulsifying drug-delivery system in the respective
0 concentration.
PG PEG/PG PEG PEG: Poly(ethylene glycol); PG: Polyglycerol.
PG

Figure 7. Cellular uptake visualized by

confocal microscopy. PG-,PEG/PG- and
PEG-self-emulsifying drug-delivery system
PEG/PG

were incubated for 4 h on a Caco-2 cell line.

Cells incubated with Opti MEM served as
control. Nuclei were stained with Hoechst
33528 (A), the membrane was stained with
Nile red (C) and Lumogen Yellow was
incorporated in self-emulsifying
PEG

drug-delivery system as fluorescence marker

(B). Section (D) shows the merged pictures
of all three stains.
PEG: Poly(ethylene glycol); PG: Polyglycerol.

significantly improved cellular uptake (p < 0.001) compared with PEG-SEDDS. Intriguingly, PEG/PG-SEDDS,
showing no significant (p < 0.01) difference to PG-SEDDS in cytotoxicity experiments exhibited a comparatively
higher uptake of 13% to PEG-SEDDS and an approximately twofold higher RFMI value. The addition of just
10% of the long-chain PEG-surfactant to SEDDS diminished cell attachment and/or cellular uptake by one third
compared with the entirely short-chain PG surface decoration.

Membrane interaction – endosomal escape study

The interaction of nanocarriers with erythrocyte membranes leading to the release of hemoglobin is frequently
assessed as their hemolytic potential corresponds to their endosomal escape properties [16,21]. Results shown in
Figure 6 demonstrate substantial differences in the membrane interactions of PEG- versus PG-surface. After 4 h,
PEG-SEDDS showed significantly (p < 0.01) less hemolysis than PEG/PG- and PG-SEDDS in the concentration
range of 1–0.1% (v/v). In low concentrations of 0.05 and 0.1% the hemolysis caused by PEG-SEDDS was almost
negligible, whereas the other two formulations still caused hemolysis in the range of 20 and 40%, respectively.

Life cell imaging using confocal microscopy

As illustrated in Figure 7, cells incubated with PG-SEDDS expose increased fluorescence compared with PEG
and PEG/PG-SEDDS. PEG-SEDDS fluorescence was least pronounced. This supports the uptake data obtained
by flow cytometry. Furthermore, fluorescence could be detected in the nucleus of Caco-2 cells, confirming an
endosomal escape of SEDDS supporting the data obtained by RBC interaction study.

Discussion
Preparation & characterization of SEDDS
We compared the surface decoration of SEDDS based on long-chain PEG-surfactants with PEG-free SEDDS
containing short-chain PG surfactants. PG surfactants have already been incorporated in SEDDS by Zahir-Jouzdani
et al. [22] and even an oral cyclosporine SEDDS formulation containing PG-surfactants was commercialized in
2010 [23]. In all so far established formulations, however, a high amount of PEGylated surfactants such as polyoxyl-
35 castor oil (Cremophor EL), polyoxyl-40 hydrogenated castor oil (Cremophor Rh40) or PEGylated sorbitan

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 Cellular uptake of self-emulsifying drug-delivery systems: PEG versus PG surface Research Article

esters (Tween) were a prerequisite for the self-emulsification process. In order to achieve an HLB >11 necessary
for self-emulsification, the PEG-moieties of these surfactants have generally to exceed ten ethylene glycol subunits.
Within this study, we used polysorbate 80 with HLB of 15. Assuming that all 20 ethylene glycol subunits of
this surfactant are immobilized on just one binding site of the sorbitan structure, the PEG-chain length would
still not exceed 900 Da. In contrast to solid nanocarriers, however, the use of PEG-surfactants with even shorter
PEG-chain length, is in case of as self-assembling liquid nanocarriers such as SEDDS not feasible. As the physico-
chemical properties of surfactants determine the self-emulsifying process, size, PDI and, in particular, stability of
droplets formed by SEDDS, even a minor change in PEG-chain length has a fundamental impact. A shortening
of the PEG-chain length is associated with a lower hydrophilicity of the head group and a consequent decrease
in HLB. PEG23 laurate (HLB of 16), for instance, cannot be simply substituted by PEG-4 laurate (HLB of 9)
as its emulsifying properties are insufficient to reduce the interfacial tension to the required minimum to form
of stable, unimodal, nanosized droplets. In contrast, the PG head group of polyglyceryl-4-laurate displays due to
free hydroxyl functions a higher hydrophilicity, an HLB of 11 and thus better emulsifying properties at an equal
number of hydrophilic subunits. In preliminary studies, short glycerol chain surfactants such as polyglyceryl-4
caprate (Tegosoft PC41) and a mixture of polyglyceryl-4 laurate/sebacate and polyglyceryl-6 caprylate/caprate
(Natragem S140 NP) demonstrated emulsifying properties that can be compared with long-chain PEG-surfactants
(data not shown) and were therefore selected to formulate PEG-free SEDDS.
A correlation between SEDDS-surface and cell interaction or uptake can only be established by excluding other
strongly influencing parameters such as varying droplet size or excipients. In order to formulate SEDDS with
varying surfactant types, keeping the droplet size and size distribution for all formulations in the same range, the
adjustment of surfactant to oil ratio (s/o) is essential. Therefore, only surfactant types/amounts were adjusted to
the amount of Captex 355. All other components were incorporated in the same quantity to avoid excipient-related
impact on cellular uptake. Three SEDDS formulations meeting the above mentioned criteria were developed.
PG-SEDDS containing only PG-surfactants were compared with PEG-SEDDS containing only PEG-surfactant
and PEG/PG-SEDDS containing a mixture of both surfactant types. Size characterization demonstrated that all
formulations formed translucent nanosized O/W emulsions with narrow and comparable size distribution. By
investigating the ζ potential all SEDDS exhibited a highly cationic ζ potential, related to the incorporation of
DODAB. The varying surface decoration, however, affected the surface charge of oil droplets. The surface charge
decreased with increasing PEG-surfactant content, supporting the assumption of a charge shielding by the non-
ionic hydrophilic corona. These results are in agreement with other studies were an increased PEG density caused
pronounced charge shielding effect [24–26]. Kumar et al., for instance, reported a decrease in ζ potential of lipid
nanocarriers by 14 mV, when the PEG-surfactant concentration was raised from 1.5 to 10% [26].

Emulsification time
In terms of self-emulsification PG-SEDDS exhibited a faster dispersion compared with PEG-SEDDS. Wei et al.
postulated two mechanisms that are likely influencing self-emulsification process [27]. The viscosity of the formu-
lation on the one hand and the free enthalpy of the system depended on the surfactant concentration on the
other hand are decisive for successful self-emulsification [4,28]. PEG-SEDDS exhibited the lowest viscosity (data not
shown) but with 0.8 also the lowest surfactant to oil (S/O) ratio. Other studies report that SEDDS containing more
than 40% long-chain PEG-surfactants tend to form gel layers delaying the penetration of water [29]. This effect
might explain the longest emulsification time determined for PEG-SEDDS. Although PEG/PG- and PG-SEDDS
exhibited the same (S/O) ratio of 1.6, emulsification time in case of PEG/PG-SEDDS was longer. This led to
the assumption that the different physico-chemical properties of PG- and PEG-surfactants could individually in-
fluence the preconcentrate-water interface. A study of mixed micelles with hydrated phosphatidylcholine provided
insights into different geometries of PG-surfactants compared with polysorbate 80. Rupp et al. [30] noticed that
the shape of the PG-surfactant has a conical geometry that is strong enough to form mixed micelles. Polysorbate
80, however, was not able to form unimodal micelles. It was suspected that the PEG-head groups cannot provide
sufficient hydrophilicity to form a pronounced conical shape required to generate unimodal micelles. In SEDDS, a
different molecular arrangement of the PG-surfactants at the interface, as seen in mixed micelles, as well as a more
pronounced hydration of the preconcentrates related to the PG-head group would be conceivable. As this study
shows one of the first reports on PG-surfactants in SEDDS, no data on the interfacial behavior are available so far.

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Research Article Friedl, Steinbring, Zaichik, Le & Bernkop-Schnürch

Stability studies
After emulsification all SEDDS maintained their intrinsic properties over 24 h guaranteeing a stable system over
the intestinal transit period. Based on the observed long term storage stability, preconcentrates containing PG- or
mixtures of PEG/PG-surfactants can, likewise to common SEDDS preconcentrates, be stored at room tempera-
ture. In freeze-thaw cycle, high centrifugation and dilution experiments PEG-free SEDDS proved a comparable
thermodynamic stability as PEG-SEDDS. However, PEG-SEDDS were found to be less susceptible to bile salt
fusion as non-PEGylated SEDDS. Increasing amounts of long PEG chains on the surface of the oily droplets
are likely increasing resistance toward the incorporation of bile salts. These data support findings of Iwanaga
et al., who observed a protective effect for liposomes against bile salts in the GIT due to the incorporation of
distearoylphosphatidylethanolamine-polyethyleneglycol 2000 in the formulation. They postulated that bile salts
diffusion through the hydrated PEG corona is hindered resulting in lower interactions with the lipid membrane of
liposomes [1].
The cloud point of SEDDS is likely more affected by the incorporation of ionic lipids as by the type of
nonionic surfactant selected for SEDDS preparation. Nonionic surfactants bind water at room temperature through
their hydrophilic head group and are thus stabilized by this hydrated layer. At higher temperatures the head
group dehydrates resulting in increased lipophilicity and thus aggregation that appears cloudy. On contrary, ionic
surfactants show the opposite effect at higher temperatures due to the increased degree of dissociation of the counter
ion [31]. The incorporation of ionic surfactants such as DODAB likely increased the cloud point and stabilized
SEDDS.

Cell viability
Although Caco-2 cells are known to be more challenging to transfect than nonpolar undifferentiated cells such as
COS-7 or HEK-293T [32], they were nevertheless chosen for cytotoxicity and uptake studies as they simulate in
vivo conditions more closely.
The lower cytotoxic effect of PEG-SEDDS, taking the negative surface charge of Caco-2 cells into account, is likely
related to the charge shielding effect of PEG corona resulting in a minor ionic attraction due to the lowered surface
charge. Short-chain PG surface decoration in PG-SEDDS, in contrary, facilitated an enhanced ionic interaction
and thus an intensified membrane depolarization. PEG/PG-SEDDS containing 10% long-chain PEG-surfactant
could not provide a dense surface coating in order to shield against interactions with the cell membrane. Yuan et al.
observed the same effect regarding the PEGylation of SLN on viability of a Caco-2 cell layer. The higher the extent
and molecular mass on PEG moieties at the SLN surface increased, the lower was their cytotoxic effect [33].

Cellular uptake studies

The attraction of cationic nanocarriers to anionic cell membrane substructures such as glycoproteins is likewise
postulated as a mechanism for their cell attachment and subsequent uptake. Cells lacking these anionic substructures
cannot efficiently bind nanocarriers and showed barely no uptake efficiency compared with normal cells [34].
Moreover, a notable increase in cellular uptake was already observed by reducing the amount of polysorbate 80–
10%. Therefore, charge shielding and sterical hindrance caused by the long-chain PEG corona is likely the main
reason for diminished electrostatic attractions between cells and PEG-SEDDS leading to a decrease in cellular
uptake. In a recent study of Zhao et al. [35] shortening of PEG-length from PEG5000 to PEG3350, PEG 2000 or
PEG1000 resulted in an enhanced cellular uptake of modified PLGA nanoparticles going hand in hand with an
increase of cytotoxicity, indicated by significantly reduced HC50 values, due to the incorporated anticancer drug.
These observations are in good agreement with our results and support the hypothesis that the replacement of
PEG-moieties on SEDDS surface represents a rational concept for improving cellular uptake of SEDDS.

Membrane interaction – endosomal escape study

The results of the in vitro hemolysis assay, imitating endosomal escape, support the hypothesis that the hydrated
inert PEG corona shields PEG-SEDDS not only from interacting with cellular membranes but also with endosomal
membranes. As already noted in the cytotoxicity test, the addition of 10% long-chain PEG surfactants to PG-
SEDDS is not sufficient to attenuate the interaction with the endosome mimicking membrane as no significant
differences between these formulations were observed. This may be related to the structural arrangement of PEG-
chains on the SEDDS surface. In case of a low PEG-surface concentration, the molecular arrangement is referred
to as ‘mushroom’ conformation, that often lacks efficient shielding properties [36]. In high concentrations PEG

10.2217/nnm-2020-0127 Nanomedicine (Lond.) (Epub ahead of print) future science group

 Cellular uptake of self-emulsifying drug-delivery systems: PEG versus PG surface Research Article

chains change their molecular arrangement forming a ‘corona’ or a ‘dense brush’, capable of supressing interactions
with erythrocytes or target cells [6,37,38]. According to these results, PG-SEDDS could be promising candidates for
intracellular delivery of drugs. In particular, nucleic acid-based therapeutics, where cellular uptake and lysosomal
degradation are major hurdles, will take advantage of such delivery systems.

Life cell imaging using confocal microscopy

The results of cellular uptake studies combined with confocal imaging imply a correlation between the long-chain
PEGylated surfactant concentration and SEDDS uptake. The higher the PEG concentration in SEDDS the lower
the cell–droplet interaction and the lower the cellular uptake of SEDDS. In a study of Chan et al. identified
liposomes containing 5 mol% PEG2000-lipid as the best compromise to provide sterical stabilization and to
guarantee nonetheless high cellular uptake efficiency. Already 10 mol% of PEG-lipid in liposomes led to a sharp
decrease of transfection efficacy in one order of magnitude [39]. In order to achieve both an enhanced cellular
uptake and a shielding effect it is necessary to balance the PEG concentration likely varying from formulation to
formulation.

Conclusion
In this study long-chain PEG-surfactant were found to form an inert hydrophilic corona at SEDDS surface
that shielded the cationic surface charge thus restraining cell-SEDDS interactions. On the contrary, PG-SEDDS
displaying short-chain PGs on the surface, exhibited not only intensified SEDDS–cell interaction but also enhanced
membrane disruption. PG-surfactants, therefore, can be considered as promising PEG-alternative, especially for the
development of advanced self-emulsifying drug-delivery formulations addressing cytosolic targets. Moreover, PGs
might be a promising choice to tackle PEG associated drawbacks in the future, not only for SEDDS but also for a
variety of nanocarriers like liposomes, solid lipid nanoparticles and many more. Since this is one of the first studies
exploring SEDDS surface – cell interactions, we are just at the beginning. Further research focusing on the impact
of different head groups, their chain length dependent charge shielding or stealth characteristics is mandatory to
improve our understanding regarding the impact of surface modification of SEDDS on cellular uptake.

Summary points
• Self-emulsifying drug-delivery systems (SEDDS) were developed and polyethylene glycol (PEG)-based surfactant
where stepwise replaced by short-chain polyglycerol (PG) surfactants. Formulations exhibited a droplet size of
150–175 nm, a low polydispersity index.
• Thermodynamic and kinetic stability studies like stability over time, freeze-thaw cycling or robustness to dilution
proofed the suitability of PG-surfactants for SEDDS development.
• The dense, inert PEG-layer in PEG-SEDDS facilitated a charge-shielding of the incorporated
dioctadecydimethylammonium bromide (DODAB). Although equal amounts of DODAB were incorporated, ζ
potential of SEDDS declined from +58.4 to +46.8 mV by increasing amounts of PEG.
• Bile salts fusion with PG-SEDDS was enhanced while the hydrated PEG corona hampered the incorporation and
thus PEG-SEDDS ζ potential decrease of 63 mV was less intense compared to 77 and 92 mV of PEG/PG-SEDDS
and PG-SEDDS, respectively.
• Reduced interaction of PEG-SEDDS with Caco-2 cells was seen in cytotoxicity experiments and flow cytometric
measurements of SEDDS cellular uptake. By reducing the PEG-surfactant amount to 10%, cellular uptake
increased twofold compared with PEG-SEDDS. SEDDS lacking PEG-surfactants showed a threefold increased
uptake which was confirmed by confocal microscopic imaging of cellular uptake.
• Even at low concentrations incubation with PG-SEDDS led to increased disruption of endosome-like membranes.
Whereas in concentration of 0.1–0.05% (v/v) PEG-SEDDS displayed negligible membrane disruption.

Supplementary data
To view the supplementary data that accompany this paper please visit the journal website at:
www.futuremedicine.com/doi/suppl/10.2217/nnm-2020-0127

Financial & competing interests disclosure

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or finan-
cial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria,
stock ownership or options, expert testimony, grants or patents received or pending, or royalties.
No writing assistance was utilized in the production of this manuscript.

future science group 10.2217/nnm-2020-0127

Research Article Friedl, Steinbring, Zaichik, Le & Bernkop-Schnürch

Ethical conduct of research

The authors state that they have obtained appropriate institutional review board approval or have followed the principles outlined
in the Declaration of Helsinki for all human or animal experimental investigations.

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