Poly(Sarcosine) Surface Modification Imparts Stealth-Like Properties to Liposomes

Article abstract of DOI:10.1002/smll.201904716

Circulation lifetime is a crucial parameter for a successful therapy with nanoparticles. Reduction and alteration of opsonization profiles by surface modification of nanoparticles is the main strategy to achieve this objective. In clinical settings, PEGylation is the most relevant strategy to enhance blood circulation, yet it has drawbacks, including hypersensitivity reactions in some patients treated with PEGylated nanoparticles, which fuel the search for alternative strategies. In this work, lipopolysarcosine derivatives (BA-pSar, bisalkyl polysarcosine) with precise chain lengths and low polydispersity indices are synthesized, characterized, and incorporated into the bilayer of preformed liposomes via a post insertion technique. Successful incorporation of BA-pSar can be realized in a clinically relevant liposomal formulation. Furthermore, BA-pSar provides excellent surface charge shielding potential for charged liposomes and renders their surface neutral. Pharmacokinetic investigations in a zebrafish model show enhanced circulation properties and reduction in macrophage recognition, matching the behavior of PEGylated liposomes. Moreover, complement activation, which is a key factor in hypersensitivity reactions caused by PEGylated liposomes, can be reduced by modifying the surface of liposomes with an acetylated BA-pSar derivative. Hence, this study presents an alternative surface modification strategy with similar benefits as the established PEGylation of nanoparticles, but with the potential of reducing its drawbacks.

Full text of DOI:10.1002/smll.201904716

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Poly(Sarcosine) Surface Modification Imparts Stealth-Like
Properties to Liposomes
Stefan Bleher,* Jonas Buck, Christian Muhl, Sandro Sieber, Sabine Barnert,
Dominik Witzigmann, Jörg Huwyler, Matthias Barz, and Regine Süss*
Thereby, one of the main goals of
Circulation lifetime is a crucial parameter for a successful therapy with nanopar-
ticles. Reduction and alteration of opsonization profiles by surface modification
of nanoparticles is the main strategy to achieve this objective. In clinical set-
tings, PEGylation is the most relevant strategy to enhance blood circulation, yet
it has drawbacks, including hypersensitivity reactions in some patients treated
with PEGylated nanoparticles, which fuel the search for alternative strategies.
In this work, lipopolysarcosine derivatives (BA-pSar, bisalkyl polysarcosine)
with precise chain lengths and low polydispersity indices are synthesized,
characterized, and incorporated into the bilayer of preformed liposomes via a
post insertion technique. Successful incorporation of BA-pSar can be realized
in a clinically relevant liposomal formulation. Furthermore, BA-pSar provides
excellent surface charge shielding potential for charged liposomes and renders
their surface neutral. Pharmacokinetic investigations in a zebrafish model show
enhanced circulation properties and reduction in macrophage recognition,
matching the behavior of PEGylated liposomes. Moreover, complement activa-
tion, which is a key factor in hypersensitivity reactions caused by PEGylated
liposomes, can be reduced by modifying the surface of liposomes with an
acetylated BA-pSar derivative. Hence, this study presents an alternative surface
modification strategy with similar benefits as the established PEGylation of
nanoparticles, but with the potential of reducing its drawbacks.
liposomes is to protect the entrapped drug
while also reducing its off-site toxicity, as
shown for multiple formulations.[5–8] To
this end, increasing on-target tissue con-
centration is a key aspect which can be
achieved both by nontargeted and targeted
liposomes.[9–12] In this regard, a crucial point
is the prolongation of systemic circulation
lifetime. It is well-known that upon injec-
tion the majority of conventional (plain)
liposomes are cleared rapidly from the
blood stream by cells of the mononuclear
phagocyte system.[13–15] To enhance circula-
tion times, a variety of approaches to modify
the surface of liposomes emerged,[16–18]
yet ultimately polyethylene glycol (PEG)
established itself as gold standard in the
early 1990s aiming at decreasing opsoniza-
tion.[19–21] PEGylation not only reduces but
also alternates the recognition of nanopar-
ticles by complement factors and opsonins,
resulting in a decreased nanoparticle recog-
nition and clearance by macrophages.[22,23]
Manipulating opsonization, including the
reduction of complement activation thus is
one of the main goals in preventing rapid clearance.
1. Introduction
Today, surface modifications of liposomes are mostly realized
with amphiphilic lipid-polymer conjugates like 1,2-distearoyl-sn-
glycero-3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (DSPE-mPEG2k).[24,25] Despite the benefits and
being considered mostly nonimmunogenic, phospholipid-PEG
conjugates have also demonstrated some drawbacks over the
Nanomedicines have been investigated and used intensely
over the past decades for several biomedical applications.[1]
With numerous products already approved and a variety of
preparations in clinical trials, liposomes are the most advanced
and successful nanomedicine in clinic.[2–4]
S. Bleher, S. Barnert, Prof. R. Süss
J. Buck, Dr. S. Sieber, Dr. D. Witzigmann, Prof. J. Huwyler
Division of Pharmaceutical Technology
Department of Pharmaceutical Sciences
University of Basel
Department of Pharmaceutical Technology and Biopharmacy
and Freiburger Materialforschungszentrum (FMF)
Institute of Pharmaceutical Sciences
Albert Ludwig University of Freiburg
4056 Basel, Switzerland
79104 Freiburg, Germany
E-mail: stefan.bleher@pharmazie.uni-freiburg.de;
regine.suess@pharmazie.uni-freiburg.de
C. Muhl, Dr. M. Barz
Institute of Organic Chemistry
Johannes Gutenberg University Mainz
55128 Mainz, Germany
The ORCID identification number(s) for the author(s) of this article
can be found under https://doi.org/10.1002/smll.201904716.
Dr. D. Witzigmann
Department of Biochemistry and Molecular Biology
University of British Columbia
Health Sciences Mall, Vancouver V6T 1Z3, British Columbia, Canada
© 2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co.
KGaA, Weinheim. This is an open access article under the terms of the
Creative Commons Attribution-NonCommercial License, which permits
use, distribution and reproduction in any medium, provided the original
work is properly cited and is not used for commercial purposes.
DOI: 10.1002/smll.201904716
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last decade. Anti-PEG antibodies are a major concern regarding
the clearance of intravenously administered liposomes modified
with PEG,[26,27] frequently culminating in the rapid clearance of
injected liposomes.[28,29] The occurrence of pre-existing anti-PEG
antibodies even in healthy individuals has increased in everyday
life due to exposure to PEG-containing products.[30] Besides this
so-called accelerated blood clearance phenomenon, it transpired
that both, the anchoring phospholipid and PEG itself are likely
to cause specific and nonspecific[31] immune reactions. Such
reactions are often caused by complement activation[32,33] and
can lead to hypersensitivity reactions (HSRs) possibly triggering
anaphylaxis.[34]
2. Results and Discussion
2.1. Synthesis of Bisalkylated Lipopolysarcosine
One part of the present work was the synthesis of a bisalkylated
polysarcosine. The synthesis (Scheme 1) was adapted and
modified from the literature.[45] The sarcosine-N-carboxyanhy-
dride was synthesized and purified before the polymerization
was started by the addition of the bisalkylamine. In contrast
to monoalkylated pSar lipopolymers, solvent condition had to
be adjusted due to the hydrophobicity of the initiator. Thus,
polymerizations were performed in benzonitrile. After comple-
tion of the reaction, which was assured by Fourier-transform
infrared spectroscopy, the polymer was precipitated in cold
diethylether and purified via dialysis (Figure 1).
The negative charge at the phosphate group of DSPE-
PEG derivatives is predominantly linked to this complement
induction.[35,36]
Conclusively, the development of alternative surface modifica-
tions offers an interesting possibility to circumvent the above-
mentioned limitations of PEG derivatives.[37–39] For this purpose,
a multitude of hydrophilic materials has been proposed.[40–45]
In this work, we report the synthesis of the lipid-like amphi-
phile bisalkyl polysarcosine (BA-pSar) and its successful incor-
poration into the lipid bilayer of preformed liposomes. It was
demonstrated earlier that pSar has properties similar to PEG,
comprising high water-solubility, flexibility, and low immuno-
genicity in vitro,[46,47] making it a promising candidate for sur-
face modification of liposomes.[48] The aim of this study was
to demonstrate that surface modification using pSar-lipopoly-
mers is a valuable strategy to increase circulation half-life of
liposomes while also ensuring good biocompatibility (i.e.,
low complement activation). After synthesis, physicochemical
properties of pSar-modified liposomes were compared with
PEGylated and nonsurface-modified liposomes regarding their
size, polydispersity, and zeta potential. To evaluate the extend of
BA-pSar inserted into the bilayer, we determined the incorpo-
ration efficiency via high performance liquid chromatography
(HPLC) with increasing amounts of BA-pSar added to pre-
formed liposomes using the post-insertion method.
1H NMR experiments demonstrate that the deviation
between obtained and calculated degrees of polymerization is
below 10% (Table 1 and Figure 1A). In line with MALDI-TOF
MS (matrix-assisted laser desorption/ionization-time of flight
1
mass spectrometry) H DOSY (diffusion-ordered spectroscopy)
NMR shows the presence of only one diffusing species con-
firming the absence of free initiator and water-initiated polymer
(Figure 1B). The discrepancy between the molecular weights
determined by HFIP-GPC (hexafluoro-2-isopropanol-gel per-
meation chromatography) and NMR can be explained with the
uncommonly high values of polymethylmethacrylate (PMMA)
in HFIP.[46]
The reported method is a simple way to vary lipopolymer
architectures in a one-step polymerization. Moreover, poly-
sarcosine bears the potential for further end group modifi-
cation, due to the terminal amine end group (Scheme 1). To
maintain the nonionic nature of the polymer, pSar end groups
were acetylated with acetic anhydride in a post-polymerization
manner without any influence on the molecular weight distri-
bution or chemical integrity of the polymer (Figure 2A,B). In
addition, MALDI-ToF analysis of BA-pSar102 was performed
and is displayed in Figure 2C. Despite severe mass discrimi-
nation, the spectrum reveals the full incorporation of the bis-
alkyl amine initiator (dioctadecylamine) without any detectable
side products, i.e., initiation by traces of water or other impuri-
ties. Enlargement of the most intense peaks (Figure 2D) shows
degrees of polymerization of Xn = 19,20 and sub-populations
due to sodium and potassium ionization.
In order to study the pharmacokinetics of liposomes with
different surface modifications, we selected the zebrafish model
as a validated in vivo tool to assess circulation properties and
macrophage clearance.[49]
Addressing the occurrence of HSR, we also investigated
the complement activation potential of liposomes modified
with BA-pSar and an optimized acetylated derivative of pSar
(pSar102Ac) with net-neutral charge.
The second part of this work was focused on the incorpora-
tion of synthesized pSar derivatives into the lipid bilayer of
Scheme 1. Synthesis of BA-pSar102 based on nucleophilic ring opening polymerization of sarcosine NCA and formation of BA-pSar102Ac via end group
modification (acetylation).
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Figure 1. A) 1H NMR spectrum of BA-pSar102 in CDCl3 with assignment of peaks. B) DOSY NMR spectrum of BA-pSar102 in CDCl3.
liposomes and the comparison with DSPE-mPEG2k. There-
fore, BA-pSar102, BA-pSar102Ac, and DSPE-mPEG2k were used
to modify the surface of preformed 1,2-distearoyl-sn-glycero-
3-phosphocholine (DSPC)/cholesterol (Chol) liposomes. The
lipid ratios were chosen to be similar to the formulation of
an approved PEGylated liposome formulation, namely, Doxil.
Doxil is composed of hydrogenated soya phosphatidylcholine,
Chol, and DSPE-mPEG2k at a molar ratio of 57:38:5.[50]
images are in good agreement with size values obtained by DLS
measurements.
2.3. Zeta Potential of Liposomes
To assess the surface charge shielding potential of the different
surface modifications, an additional set of liposomal formulations
was prepared containing the cationic lipid 1,2-dioleoyl-3-trimethy-
lammonium-propane (DOTAP, 10 mol%) (Figure 4A). Measure-
ments of liposomes without DOTAP (Figure 4B) revealed only
slight differences in zeta potential due to the neutral character
of the formulation. Addition of DOTAP allowed to compare the
influence on surface charge shielding of different formulations
using zeta potential measurements. Notably, size characteris-
tics for all preparations were comparable (Supporting Informa-
tion, Figure S1). Unmodified liposomes showed a relatively high
zeta potential of about +46 mV. In contrast, the zeta potential for
liposomes modified with DSPE-mPEG2k and BA-pSar102 was
reduced drastically, as already reported previously.[51] PEGylation
resulted in a zeta potential of about −6 mV, whereas liposomes
modified with pSar had a zeta potential around +9 mV, suggesting
an effective shielding of charge. The slightly negative zeta poten-
tial for PEGylated liposomes on the one hand is probably due to
the negative charge at the phosphate moiety of DSPE-mPEG2k.
The slightly positive charge of BA-pSar102-modified liposomes on
the other hand can be attributed to the terminal secondary amine
of pSar, which is protonated at physiological pH. Measurements
of liposomes modified with end-capped pSar (BA-pSar102Ac)
displayed a zeta potential of around +2 mV. Since BA-pSar102Ac
bears no charge, this suggests that an effective
2.2. Characterization of Liposomes
The hydrodynamic diameter and polydispersity index (PDI) of
different liposome formulations were determined before and
after purification of liposomes from noninserted lipopolymer
conjugates using dynamic light scattering (DLS). Zeta poten-
tial was measured after purification. Results for size and PDI
before and after post-insertion of lipopolymers followed by
purification are displayed in Table 2. Within the estimated
errors, results obtained for size and PDI were comparable for
all preparations and did not change over the process of post-
insertion. Size was approximately 120 nm and PDI was ≤0.1,
indicating homogenous size distributions for all formulations.
Figure 3 shows Cryo-TEM (cryogenic transmission electron
microscopy) images displaying the morphology of representa-
tive liposomes which were selected for further in vitro and
zebrafish experiments. Unmodified as well as surface-modified
formulations show a narrow size distribution. Neither PEGyla-
tion nor modification with BA-pSar102 induced significant
changes in liposomal morphology or size distribution. Cryo-TEM
shielding of the positive charge introduced by
DOTAP was achieved with this formulation.
Table 1. Synthesized sarcosine-based lipopolymers with an initial monomer to initiator ratio
of M/I = 90, molecular weights and dispersity indices.
Polymer
Initiator/end-group
Mn (Calc)a)
Mn(GPC)b)
Mn(NMR)c)
PDI
[kg mol−1
]
[kg mol−1
]
[kg mol−1
]
2.4. Incorporation Efficiency
BA-pSar102
DODA/H
DODA/Ac
6.9
38.5
7.8
1.2
1.2
For all surface-modified liposomes, DSPE-
BA-pSar102Ac
7.0
38.7
7.8
mPEG2k, BA-pSar102
,
and BA-pSar102Ac
incorporation efficiency into the liposomal
bilayer was monitored via HPLC.
a)Calculated molecular weight (based on M/I ratio); b)Determined by HFIP-GPC using PMMA-standards;
c)Determined by 1H NMR via end-group analysis.
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Figure 2. A) Normalized HFIP-GPC traces of BA-pSar102 and BA-pSar102Ac. B) Enlarged HFIP-GPC traces of BA-pSar102 and BA-pSar102Ac for a better
comparison. C) Normalized MALDI-ToF spectrum of BA-pSar102. D) Enlargement of the most intensive peaks of MALDI-ToF spectrum of BA-pSar102
.
In preliminary tests, different amounts of BA-pSar102
(1–15 mol%) were dried from stock solutions, hydrated with
preformed liposomes, and post-insertion into the liposomal
bilayer was analyzed. Thereby, incorporation of at least
4.5 mol% was achieved by hydrating 7.5 mol% dried BA-pSar102
or BA-pSar102Ac with preformed liposomes. Excess conjugate
was removed by ultrafiltration. These results correspond to an
incorporation efficiency of approximately 60% of added conju-
gate. All further experiments therefore were conducted using
the same setup, adding preformed liposomes to 7.5 mol% of
dried BA-pSar102 or BA-pSar102Ac, respectively. When hydrating
5 mol% of dried DSPE-mPEG2k in an analogous manner,
complete incorporation was observed. Incorporation efficiency
results obtained after purification are shown in Table 3.
In summary, BA-pSar102 and BA-pSar102Ac were successfully
incorporated into the lipid bilayer, resulting in liposomes with
a narrow size distribution. Liposomes modified with lipopoly-
sarcosines also showed effective surface charge shielding,
comparable with findings for PEGylated liposomes. To further
evaluate the potential use of BA-pSar102-modified liposomes,
clearance by macrophages and circulation properties were
investigated in vivo in the zebrafish model.
including malformations or increased mortality were observed
(data not shown), indicating the biocompatibility of all lipo-
some formulations including both surface modifications.
At 3 h post injection (hpi), all liposome formulations demon-
strated a diffusive fluorescence staining without accumulations
in the posterior caudal vein (PCV) region (Figure 5). Recently,
it has been shown that endothelial cells in the PCV express
stabilin receptors which can scavenge liposomes and thereby
prevent blood circulation.[52] However, an increased colocaliza-
tion (yellow) of conventional liposomes (red) and macrophages
(green) was already detected at this time point indicating mac-
rophage clearance (white arrows, Figure 5).
A clear difference in circulation characteristics of different
liposome formulations was observed 24 hpi (Figure 5). Unmod-
ified DSPC/Chol liposomes demonstrated decreased circulation
properties indicated by a dotted staining pattern (i.e., clear-
ance by stabilin scavenger receptors). In contrast, both surface-
modified liposomes (i.e., DSPE-mPEG2k and BA-pSar102) still
demonstrated a prolonged circulation lifetime.[53] Strikingly,
macrophage clearance of unmodified liposomes increased sig-
nificantly as compared to surface-modified liposomes (indicated
by yellow color of colocalization).[54] Both PEG and pSar pre-
vented macrophage clearance signifying their ability to confer a
stealth effect to the liposomes (Figure 5). This can be explained
by the advantageous properties of lipopolymer surface modi-
fication including decreased opsonization and steric stabiliza-
tion. In addition, this prevents time-dependent formation of
aggregates via steric stabilization preferentially sequestered by
macrophages. Conclusively, BA-pSar102-modified liposomes
outperformed unmodified control liposomes (i.e., DSPC/Chol)
with favorable pharmacokinetic characteristics—an improved
blood circulation lifetime and a decreased macrophage clear-
ance. Moreover, BA-pSar102-modification of liposomes resulted
in similar in vivo properties as DSPE-mPEG2k liposomes,
which are considered as the gold standard for long circulating,
stealth liposomes.
2.5. Assessment of Liposome Pharmacokinetics In Vivo
Fluorescently labeled liposomes with different surface modifi-
cations (i.e., DSPE-mPEG2k as positive control for stealth prop-
erties or BA-pSar102) and unmodified (conventional) liposomes
(negative control) were injected into the blood circulation of
zebrafish larvae. No acute signs of toxicity (i.e., denaturation
of tissue fluids or yolk, heart failure) nor long-term effects
Table 2. Physicochemical characterization of liposomal formulations
after purification. Values are means SD, n = 3.
Formulation
Hydrodynamic diameter [nm]
107 10
PDI
DSPC/Chol/DiI
0.05 0.02
0.06 0.02
0.09 0.02
0.05 0.02
2.6. In Vitro Complement Activation
DSPC/Chol/DiI/DSPE-mPEG2k
DSPC/Chol/DiI/BA-pSar102
DSPC/Chol/DiI/BA-pSar102Ac
113
123 10
130
9
Figure 6 shows the elevated sC5b-9 levels after incubation of
liposomes and controls with three individual serum samples.
2
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Figure 3. Cryo-TEM images showing the morphology of different liposomal formulations. A) DSPC/Chol/DiI, B) DSPC/Chol/DiI/DSPE-mPEG2k, and
C) DSPC/Chol/DiI/BA-pSar102
.
As expected there are variations in sC5b-9 levels in the sera
of different donors, as can be seen for the negative control
(HBS, HEPES buffered saline). Compared to HBS, PEGylated
liposomes induced a 1.5- to 2.5-fold increase in comple-
ment activation (C activation) in all tested sera, which is in
good accordance with values obtained from literature.[36] For
BA-pSar102-modified liposomes, an increase in C activation
was observed, resulting in 2.5- to 5-fold higher sC5b-9 levels
as compared to HBS. Charged liposomes are known to elicit
the complement system and cationic liposomes are known
to do so via the alternative pathway.[55] Thus, C activation
enhancement by BA-pSar102 compared to DSPE-mPEG2k
comes as no surprise as the N-terminus of the polymeric chain
bears a secondary amine, which is present in its ammonium
salt form under physiological conditions. In a similar manner,
the negative charge at the phosphate moiety of DSPE-PEG is
well known to stimulate C activation.[35] In order to exploit the
advantageous synthetic features of lipopolysarcosine, the opti-
mized end-capped BA-pSar102Ac derivative was incorporated
into liposomes. Interestingly, BA-pSar102Ac-modified liposomes
demonstrated improved properties with decreased complement
activation. Compared to HBS only a 2.5-fold increase in sC5b-9
levels in serum C, and only 1.5-fold increase in serum A and
strikingly no increase in serum B was observed.
Our findings show that replacing the anchoring DSPE with
dioctadecylamine and subsequent end-capping of pSar with a
neutral acetyl moiety results in a significantly decreased C acti-
vation in two of the three tested sera when comparing DSPE-
mPEG2kwithBA-pSar102Ac. Thismostlikelyisaresultofomitting
the charges both at the anchor (hydrophilic–hydrophobic
interface) and the polymeric chain, as it was previously demon-
strated for free (nonliposomal incorporated) polymers.[47]
3. Conclusion
In this study, we present the one step synthesis of a lipopoly-
sarcosine derivative enabling controlled modification of
liposomes to enhance systemic circulation. The controlled
polymerization conditions allow for an accurate degree of
polymerization resulting in the precise tailoring of pSar chain
length while keeping polydispersity low. Due to the reactive
Table 3. Efficiency of conjugate insertion. Percent of incorporated
polymer-conjugates determined by HPLC analysis after purification.
Values are means SD, n = 3.
Figure 4. Surface charge shielding of liposomes. Zeta potential of dif-
ferent liposomal formulations containing A) 10 mol% DOTAP or B) no
Formulation
Incorporation efficiency [%]
DSPC/Chol/DiI/BA-pSar102
DSPC/Chol/DiI/BA-pSar102Ac
DSPC/Chol/DiI/DSPE-mPEG2k
63
57
96
6
4
6
●
■
▼
DOTAP. nonsurface-modified liposomes, PEGylated liposomes,
▲
liposomes modified with BA-pSar102Ac, and liposomes modified with
BA-pSar102. Symbols depict measured values, dashed lines show mean
values SD, n = 3.
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Figure 5. Assessment of liposome pharmacokinetics in transgenic zebrafish. Conventional liposomes and liposomes surface-modified with DSPE-
mPEG2k or BA-pSar102 were injected into the blood circulation of zebrafish (2 days post fertilization). Confocal images of tail region were acquired
3 and 24 hpi. White arrows indicate macrophage clearance of liposomes assessed by colocalization (yellow color) of green fluorescent macrophages
(KAEDE) and fluorescently labeled liposomes (DiI, red). Quantitative analysis of macrophage clearance was performed using PCC. Values are
means SD, n = 6. *p < 0.05 or **p < 0.01 (one-way analysis of variance (ANOVA) followed by Bonferroni post hoc test) as compared to conventional
liposomes composed of DSPC/Chol/DiI.
amino group, further diversification can be achieved in a
simple post-polymerization modification step without altering
the polydispersity of the precursor polymer. Using this approach,
the positive net charge of BA-pSar102 was omitted by acetylation
of the terminal amine. Subsequently, we were able to success-
fully incorporate BA-pSar102 and BA-pSar102Ac into the lipid
bilayer of preformed liposomes using post-insertion technique.
Liposome characterization including the determination of
size and morphology revealed a monodisperse size distribution
with mostly unilamellar vesicles. Zeta potential measurements
of surface-modified liposomes demonstrated an effective
shielding of surface charge. In contrary to modification with
DSPE-mPEG2k and BA-pSar102, which still resulted in a slightly
negative or positive zeta potential, BA-pSar102Ac-modified
liposomes unveiled an almost net-neutral surface charge.
Furthermore, the alternative hydrophobic anchor not only
circumvents the negative charge that comes with the widely
used DSPE-PEG derivatives but also provides sufficiently firm
binding for the relatively large pSar chain into the bilayer.
Moreover, BA-pSar-modified liposomes also featured pro-
longed circulation properties and decreased recognition by
macrophages in the zebrafish model similar to PEGylated
liposomes. Finally, liposome-mediated C activation could be
reduced by modification of liposomes with an end-capped pSar
derivative. All these results confirm that surface modification of
liposomes with pSar or its derivatives is a promising alternative
to well-established PEGylation strategies, especially in regard
of reducing potential HSR. Equipped with an easily modifiable
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Figure 6. Complement activation of different liposomal formulations and controls. A–C) Samples were incubated for 1 h with human serum of three
individuals. Values are means SD, n = 2–3. ns: not significant, **p < 0.01 or ***p < 0.001 (one-way ANOVA followed by Bonferroni post hoc test) as
compared to liposomes modified with BA-pSar102Ac.
and precipitated with 300 mL of dry n-hexane. The solution was cooled
to −18 °C and stored for 18 h to complete precipitation. The solid was
filtered under dry nitrogen atmosphere and dried in a stream of dry
nitrogen for 60–90 min and afterward under high vacuum for 2 h in the
sublimation apparatus. The crude product was sublimated at 85 °C and
1 × 10−3 mbar. The product was collected from the sublimation apparatus
in a glovebox on the same day. The purified product (110 mmol, 65%
yield, colorless crystallites; melting point: 102–104 °C (lit: 102–105 °C))
was stored in a Schlenk tube at −80 °C.
end-group, incorporation of lipopolysarcosine and further func-
tionalization is also a promising option for the exploitation of
active drug targeting or combination therapies.
4. Experimental Section
Materials: n-Hexane (Fischer Scientific, Waltham, USA) was
distilled from Na/K. Benzonitrile was purchased from Sigma Aldrich
(Steinheim, Germany), dried over CaH2, and freshly distilled prior to
use. Tetrahydrofuran (THF) and toluene were purchased from Fischer
Scientific (Waltham, USA), dried over Na, and freshly distilled prior to
use. HFIP was purchased from Fluorochem (Hadfield Derbyshire, UK).
Dioctadecylamine was purchased from Fluka (St. Gallen, Switzerland)
and dried at 40 °C under vacuum (1 × 10−3 mbar) for 24 h. Diphosgene
was purchased from Alfa Aesar (Ward Hill, MA, USA) and deuterated
solvents from Deutero GmbH (Kastellaun, Germany).
1H NMR (300 MHz, CDCl3): δ/ppm = 4.22 (2H, s, –CH2–CO–), 2.86
(3H, s, –CH3).
Synthesis of Polysarcosine: Sarcosine NCA was transferred into a pre-
dried Schlenk tube equipped with a stir bar under nitrogen counter flow
and again flame-dried under high vacuum for 1 h. Subsequently, the NCA
was dissolved in dry benzonitrile to yield a solution of 100 mg mL−1
with
respect to the NCA. 1/n equivalent of dioctadecylamine was dispersed
in pre-dried toluene and added to the NCA solution. The solution was
stirred at room temperature and kept at a constant pressure of 1.25 bar
of dry nitrogen via a schlenk line to prevent impurities from entering the
DSPC and DSPE-mPEG2k were generously donated by Lipoid GmbH
(Ludwigshafen, Germany). DOTAP was obtained from Merck (Darmstadt,
reaction vessel while allowing CO
2 to escape. Completion of the reaction
Germany).
1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine
was confirmed by Fourier transform infrared (FT-IR) spectroscopy
(disappearance of the NCA related peaks (1853 and 1786 cm−1)).
After completion of the reaction, the polymer was precipitated in
cold diethylether and centrifuged (4500 rcf at 4 °C for 15 min). After
discarding the liquid fraction, new ether was added and the polymer was
resuspended in a sonication bath. The suspension was centrifuged again
and the procedure was repeated. The polymer was dissolved in water,
dialyzed against MilliQ water and lyophilized, obtaining a colorless, stiff
and porous solid. The yield after purification was 91% of the theoretically
achievable mass of the polymer.
perchlorate (DiI) was purchased from Invitrogen (Thermo Fisher
Scientific, Waltham, USA). Chol was purchased from Sigma Aldrich
(Steinheim, Germany). Other chemicals were purchased from Sigma-
Aldrich (Steinheim, Germany) and used as received unless stated
otherwise. Human serum samples of ten healthy donors were obtained
from the University Medical Center Freiburg. Sera were stored at a
temperature of −80 °C until used.
Synthesis of Sarcosine N-Carboxyanhydride:
A total of 14.92 g
(167.4 mmol) sarcosine, dried under vacuum for 1 h, was weighed into a
pre-dried, three-neck, round-bottom flask. A total of 300 mL of absolute
THF was added under a steady flow of nitrogen, 16.2 mL (134 mmol)
of diphosgene was added slowly via syringe, and the nitrogen stream
was reduced. The colorless suspension was mildly refluxed for 3 h,
yielding a clear solution. Afterward, a steady flow of dry nitrogen was led
through the solution for another 3 h while the outlet was connected to
two gas washing bottles filled with aqueous NaOH solution to neutralize
phosgene. The solvent was evaporated under reduced pressure, yielding
a brownish oil as a crude reaction product. The oil was dried under
reduced pressure (1 × 10−3 mbar for 2 h) to obtain an amorphous
solid, free of phosgene and HCl, confirmed by testing against a silver
nitrate solution. The crude product was redissolved in 40 mL of THF
1H NMR (400 MHz; CDCl3): δ/ppm: 4.51–3.80 (204H; br;
(2n)–CO–CH2–NMe–); 3.12–2.62 (306H; br; (3n)–N–CH3–); 1.83–1.00
(64H; br; (–CH2–(CH2)16–CH3)2); 0.87 (6H; t; (–CH2–(CH2)16–CH3)2).
Acetylation of Polysarcosine: 100 mg of polysarcosine (BA-pSar102
)
was dissolved in 0.5 mL dimethylformamide (DMF), 10 equivalents
(in respect to the calculated molecular mass of the polymer) of
diisopropylamine were added and the solution was stirred for 30 min
at room temperature. Afterward, 5 equivalents of acetic acid anhydride
were added and the solution was stirred overnight at room temperature.
The polymer was precipitated in cold ether and centrifuged (4500 rcf
at 4 °C for 15 min). After discarding the liquid fraction, fresh ether
was added, the polymer was resuspended in a sonication bath and
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Table 4. Lipid compositions of prepared formulations.
Table 5. RP-8 column gradient. A: MeOH, B: NH4OAc buffer pH 4.0,
C: ACN. A methanol/water/acetonitrile (7:2:1) to methanol/acetonitrile
(9:1) gradient was used to separate PEGylated samples on a C8 reversed
phase column. Solvents contained 4 mmol L−1 ammonium acetate.
Formulation
Initial molar ratio
60:39:1
Ratio after purification
DSPC/Chol/DiI
–
DSPC/Chol/DiI/DSPE-mPEG2k
DSPC/Chol/DiI/BA-pSar102
DSPC/Chol/DiI/BA-pSar102Ac
DSPC/Chol/DOTAP
55:39:1:5
55:39:1:4.8
57:39:1:3.2
57:39:1:3.4
–
Time
0
Flow [mL]
0.75
%A
70
70
90
90
70
%B
20
20
0
%C
10
10
10
10
10
57:39:1:7.5
57:39:1:7.5
50:40:10
5
0.75
15
25
30
0.75
DSPC/Chol/DOTAP/
DSPE-mPEG2k
45:40:10:5
45:40:10:4.9
0.75
0
0.75
20
DSPC/Chol/DOTAP/BA-pSar102
DSPC/Chol/DOTAP/BA-pSar102Ac
47:40:10:7.5
47:40:10:7.5
47:40:10:3.1
47:40:10:3.3
instrument (Brookhaven Instruments Corp., Holtsville, USA). For size
measurements, samples were diluted with HBS to 0.1 mmol L−1 in
disposable plastic cuvettes. Zeta potential was measured using a high
concentration cell (Malvern Instruments, Malvern, UK) as described
earlier.[59] Liposomes were diluted to 1 mmol L−1 with 10 mmol L−1 HBS
and measured immediately after dilution.
Cryo-TEM: Liposomes were diluted with HBS to achieve a final
concentration of 5 to 10 mmol L−1 lipid. About 3 µL were applied on
a 400 × 100 mesh Quantifoil S 7/2 holey carbon film on a copper grid
(Quantifoil Micro Tools GmbH, Jena, Germany). Excess liquid on the
grid was removed with filter paper. The sample then was flash frozen
by injection into liquid ethane. All sample preparation steps were
conducted in a climate-controlled room using a CryoBox 340719 (Carl
Zeiss, Oberkochen, Germany). Subsequent fixation of the grid on the
sample rod (626-DH, Gatan, Warrendale, USA) and transfer of the rod
into the TEM (Leo 912 Ω-mega, Carl Zeiss, Oberkochen, Germany)
were conducted under nitrogen atmosphere at −183 °C. The instrument
was operated at 120 kV and camera pictures (Proscan HSC 2, Oxford
Instruments, Abingdon, USA) were taken with a 6300- to 12 500-fold
magnification from different positions of the grid.[60]
Quantification of Conjugate Incorporation: Using a Waters Alliance 2695
separation module (Waters, Milford, USA), mounted with RP-8 (Kinetex
5 µm C8 100 Å, 250 × 4.6 mm) and HILIC (hydrophilic interaction
liquid chromatography, Luna 5 µm HILIC 200 Å, 250 × 4.6 mm; both
Phenomenex, Torrance, USA) columns, an HPLC method was developed
to separate polymer-conjugates from other lipid components. Standards
were dissolved in MeOH, samples were diluted to approximately
0.5 mmol L−1 with MeOH. DSPE-mPEG2k content was analyzed on an
RP-8 column at 45 °C with the gradient shown in Table 5. BA-pSar102
and BA-pSar102Ac contents were analyzed on an HILIC column at 25 °C
running the gradient as depicted in Table 6. The incorporation efficiency
centrifuged afterward. This procedure was repeated. The polymer was
dissolved in water, dialyzed against MilliQ water to remove excess of
acetic acid and residual traces of DMF. After lyophilization, a colorless,
stiff, and porous solid was obtained.
1H NMR (400 MHz; CDCl3): δ/ppm: 4.51–3.80 (204H; br; (2n)–
CO–CH2–NH–); 3.12–2.62 (306H; br; (3n)–N–CH3–); 1.83–1.00 (64H;
br; –CH2–(CH2)16–CH3); 0.87 (6H; t; –CH2–CH3).
1H NMR spectra were recorded on a Bruker (Billerica, MA, USA)
AC 400 at a frequency of 400 MHz, respectively. 2D NMR spectra as
1H DOSY were recorded on a Bruker Avance III HD 400 at 400 MHz.
All spectra were recorded at room temperature (25 °C) and calibrated
using the solvent signals. Melting points were measured using a Mettler
FP62 melting point apparatus at a heating rate of 2.5 °C min−1. GPC was
performed with HFIP containing 3 g L−1 potassium trifluoroacetate as
the eluent at 40 °C and a flow rate of 0.8 mL min−1. The columns were
packed with modified silica (PFG column particle size: 7 µm, porosity:
100 and 1000 Å). PMMA standards (Polymer Standards Services GmbH,
Mainz, Germany) were used for calibration and toluene was used as
the internal standard. A refractive index detector (G1362A RID) and
an UV-vis detector (at 230 nm unless otherwise stated; Jasco, Gross-
Umstadt, Germany, UV-2075 Plus) were used for polymer detection.
Preparation of Liposomes: Liposomes were prepared using the thin-
film hydration method. For zebrafish experiments, 1 mol% DiI was
added as a fluorescent label. Briefly, stock solutions of all components
were prepared by dissolving dry powders in CHCl3 and/or MeOH. Stock
solutions were mixed in molar ratios according to Table 4. Organic
stocks were removed using a rotary evaporator (Rotavapor R, Büchi,
Essen, Germany) and lipid films were dried for at least 2 h to ensure
complete removal of organic solvents. Lipid films were hydrated with
HBS (140 mmol L−1 NaCl, 10 mmol L−1 Hepes (4-(2-hydroxyethyl)-1-
piperazineethanesulfonic acid), pH 7.4) resulting in a lipid concentration
of 20 mmol L−1. Dispersions were extruded 41 times through an
80 nm polycarbonate membrane. All steps were conducted at 65 °C.
Phospholipid content was determined using Bartlett’s Assay.[56] For exact
compositions of liposomes, see Table 4.
Table 6. HILIC column gradient. A: MeOH, B: H2O, C: NH4OAc buffer
(100 mmol L−1 ammonium acetate buffer at pH 5.0), D: ACN. A complex
gradient containing 5 mmol L−1 ammonium acetate buffer was used to
separate samples containing lipopolysarcosines.
Post-Insertion of PEG- and pSar-Conjugates: DSPE-mPEG2k,
BA-pSar102, and BA-pSar102Ac were inserted into preformed liposomes
using the post-insertion technique.[57,58] Briefly, organic stock solutions
Time
0
Flow [mL]
%A
0
%B
0
%C
5
%D
95
95
82
82
30
30
5
1
1
1
1
1
1
1
1
1
1
containing defined amounts of DSPE-mPEG2k, BA-pSar102
, and
4
0
0
5
BA-pSar102Ac were evaporated for several hours using an evaporation
centrifuge (Concentrator 5301, Eppendorf, Hamburg, Germany).
Dispersions of preformed liposomes then were added to the residue,
vortex mixed, and incubated at 65 °C for 1 h under constant agitation
(Thermomixer comfort, Eppendorf, Hamburg, Germany). To remove
any noninserted conjugates, liposomes were washed three times with
HBS using Vivaspin Turbo 4 ultrafiltration devices (100 kDa MWCO PES
membranes, Sartorius, Göttingen, Germany) at 3000 rcf for 30 min. For
molar ratios after purification see Table 4.
7
10
10
40
40
40
40
0
3
5
10
14
17
19
21
22
25
3
5
25
25
50
50
0
5
5
5
5
5
5
95
95
Particle Size and Zeta Potential Measurements: Mean hydrodynamic
diameter, size distribution, and zeta potential were assessed using a
Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) and ZetaPals
0
0
5
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of both polymer-conjugates was quantified with a Corona charged
aerosol detector (Corona CAD ESA 542, Dionex, Sunnyvale, USA) after
performing the post-insertion technique followed by a purification step.
Data were analyzed with Empower 3.0 software (Waters, Milford, USA).
Incorporation efficiency was calculated using the formula shown in
Equation (1).
Keywords
complement activation, liposomes, pharmacokinetics, polysarcosine,
surface modification
Received: August 21, 2019
Revised: October 23, 2019
Published online:
Equation (1). Incorporation efficiency of polymer-conjugates
ccp × clt
cct × clp
(1)
IE =
× 100%
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The research project was supported by the Swiss National Science
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Conflict of Interest
The authors declare no conflict of interest.
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2019 The Authors. Published by WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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