Quantitative evaluation of bioorthogonal chemistries for surface functionalization of nanoparticles

Article abstract of DOI:10.1021/bc3005057

We present here a highly efficient and chemoselective liposome functionalization method based on oxime bond formation between a hydroxylamine and an aldehyde-modified lipid component. We have conducted a systematic and quantitative comparison of this new approach with other state-of-the-art conjugation reactions in the field. Targeted liposomes that recognize overexpressed receptors or antigens on diseased cells have great potential in therapeutic and diagnostic applications. However, chemical modifications of nanoparticle surfaces by postfunctionalization approaches are less effective than in solution and often not high-yielding. In addition, the conjugation efficiency is often challenging to characterize and therefore not addressed in many reports. We present here an investigation of PEGylated liposomes functionalized with a neuroendocrine tumor targeting peptide (TATE), synthesized with a variety of functionalities that have been used for surface conjugation of nanoparticles. The reaction kinetics and overall yield were quantified by HPLC. Reactions were conducted in solution as well as by postfunctionalization of liposomes in order to study the effects of steric hindrance and possible affinity between the peptide and the liposome surface. These studies demonstrate the importance of choosing the correct chemistry in order to obtain a quantitative surface functionalization of liposomes.

Full text of DOI:10.1021/bc3005057

Article
pubs.acs.org/bc
Quantitative Evaluation of Bioorthogonal Chemistries for Surface
Functionalization of Nanoparticles
Lise N. Feldborg,† Rasmus I. Jølck,† and Thomas L. Andresen*
DTU Nanotech, Department of Micro- and Nanotechnology, Technical University of Denmark, Building 423 2800 Lyngby, Denmark
S
* Supporting Information
ABSTRACT: We present here a highly efficient and chemoselective liposome functionalization method based on oxime bond
formation between a hydroxylamine and an aldehyde-modified lipid component. We have conducted a systematic and
quantitative comparison of this new approach with other state-of-the-art conjugation reactions in the field. Targeted liposomes
that recognize overexpressed receptors or antigens on diseased cells have great potential in therapeutic and diagnostic
applications. However, chemical modifications of nanoparticle surfaces by postfunctionalization approaches are less effective than
in solution and often not high-yielding. In addition, the conjugation efficiency is often challenging to characterize and therefore
not addressed in many reports. We present here an investigation of PEGylated liposomes functionalized with a neuroendocrine
tumor targeting peptide (TATE), synthesized with a variety of functionalities that have been used for surface conjugation of
nanoparticles. The reaction kinetics and overall yield were quantified by HPLC. Reactions were conducted in solution as well as
by postfunctionalization of liposomes in order to study the effects of steric hindrance and possible affinity between the peptide
and the liposome surface. These studies demonstrate the importance of choosing the correct chemistry in order to obtain a
quantitative surface functionalization of liposomes.
groups such as maleimides for Michael addition, azides for
copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition22−24
(CuAAC) or Staudinger ligation using triphosphines,25,26
controlled display of surface ligands can be obtained.
Unfortunately, ligand conjugation to the surface of nano-
particles can suffer from low reproducibility and highly varying
yields; e.g., we have previously found that it can be very difficult
to control the Michael addition of a somatostatin receptor
targeting peptide (TATE) to a lipid moiety, even though the
maleimide-based Michael addition is one of the most utilized
conjugation chemistries for peptides and proteins.27,28 As a
consequence hereof, we have conducted a systematic study on a
variety of generally applied conjugation reactions using HPLC
as a method to follow and quantify the reactions both in
solution and by direct conjugation to the liposomal surface.
Reactions conducted directly on liposome surfaces are likely
not to show the same reaction kinetics as ones preformed in
INTRODUCTION
■
Functional nanomaterials have attracted considerable attention
due to their highly interesting properties in relation to
diagnostic,1−3 sensor,4,5 imaging,6,7 vaccine,8 and drug delivery
applications.9,10 Within the drug delivery field, liposomes have
particularly advantageous properties.11 However, despite
positive results obtained by exploiting the EPR-effect,12
numerous methods to improve drug bioavailability in the
diseased tissue, while maintaining stable liposomes during
circulation, is currently being investigated. By coating the
liposome surfaces with targeting ligands such as antibodies,13,14
peptides,15 proteins,16 polysaccharides,17 lectins,18 and vitamin
analogues,19,20 which are recognized by antigens or receptors
selectively, or overexpressed in, e.g., tumor tissue, increased
drug accumulation in tumors can be achieved. Numerous
bioconjugation methods have been established to surface-
modify liposomes with targeting ligands to enhance tissue
specificity. To ensure correct orientation of the targeting ligand,
bioorthogonal and site-specific surface reactions are required.
By the incorporation of unnatural amino acids in peptide or
protein sequences,21 introduction of unnatural functional
Received: September 8, 2012
Revised: October 24, 2012
© XXXX American Chemical Society
A
dx.doi.org/10.1021/bc3005057 | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
solution, and the yield of functionalization will not necessarily
be quantitative and has to be determined case by case.
In this study, we furthermore introduce a novel highly
selective and bioorthogonal conjugation reaction relevant for
the surface functionalization of liposomes. We show a fast and
quantitative coupling of a hydroxylamine functionalized peptide
to an aldehyde-modified liposome, a strategy for coating
liposomes, which to our knowledge has not previously been
reported. In addition, we show a large dependency on the
relative position of the conjugation functionalities. A knowledge
which we believe is highly useful when designing new
functionalized liposomal systems or other functional nanoma-
terials.
at room temperature and aliquots (50 μL) were removed at the
indicated times and analyzed by analytical HPLC. A linear
gradient was used from 100% A (aqueous solution containing
5% MeCN and 0.1% TFA) to 100% B (MeCN containing 0.1%
TFA) over 15 min with a flow rate of 1 mL/min and AUC for
the free TATE was measured relative to AUC for the coupled
product at 280 nm. For the reaction between the maleimide
and thiol functionalities Et3N (50 mM, 6.0 μL, 3.0 equiv.) was
added to facilitate the coupling, while for the copper catalyzed
click reactions CuSO4·5H2O (1.0 M, 2.5 μL, 25 equiv.) and
sodium ascorbate (1.0 M, 5.0 μL, 50 equiv.) were added. Before
injecting the aliquots from the copper catalyzed click reactions
onto the analytical HPLC column 20 μL of a 10% NH4OH
solution was added to ensure the copper ions were in solution
and not interfering with the HPLC signals.29
Conjugation by Post-Functionalization of Liposomes.
Preformed functionalized liposomes ((25 mM, 0.50 mL, 1.1
equiv.) - normalized according to a 45:55 distribution ratio
between the inner- and outer liposomal membrane) were
mixed with the corresponding TATE peptide (4.2 mM, 15 μL,
1.0 equiv.) dissolved in MeOH. The reactions were shaken (not
stirred; to avoid foaming) at room temperature and aliquots
(40 μL) removed for analysis by analytical HPLC. A linear
gradient was used from 100% A (aqueous solution containing
5% MeCN and 0.1% TFA) to 100% B (MeCN containing 0.1%
TFA) over 15 min with a flow rate of 1 mL/min. The AUC for
the free TATE was calculated relative to the AUC of the
phospholipid coupled product at 280 nm to monitor the
conjugation efficiency. For the copper catalyzed click reactions
CuSO4·5H2O (1.0 M, 1.6 μL, 25 equiv.) and sodium ascorbate
(1.0 M, 3.1 μL, 50 equiv.) were added. As for the conjugations
done in solution 20 μL of a 10% NH4OH solution was added
to the copper catalyzed click aliquots prior to HPLC analysis.
EXPERIMENTAL PROCEDURES
■
Synthesis of Functionalized TATE Peptides. The
functionalized TATE peptides 1a−e were synthesized manually
by using a Wang Resin preloaded with NH2-Thr(tBu)-OH by
standard Fmoc methodology. Each coupling was performed by
activating the Fmoc protected amino acid with O-(7-
azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium hexafluor-
ophosphate (HATU) in the presence of 2,4,6-collidine in
DMF. Cleavage of the Fmoc group was carried out using
piperidine in DMF. Completion of each coupling and
deprotection step was monitored by the Kaiser test. Removal
of the acetamidomethyl (Acm) protection groups on the two
cysteines and simultaneous disulfide bond formation was
achieved by addition of Tl(CF3COO)3 in DMF. The resin
containing the cyclized peptide was extensively washed with
DMF and subsequently divided into five solid-phase peptide
synthesis (SPPS) reaction vials in which the N-terminal was
acylated with the desired functionality, cleaved from the solid
support, and purified by HPLC. Full experimental details and
peptide characterization are available as Supporting Informa-
tion.
RESULTS AND DISCUSSION
■
Synthesis of Functionalized DSPE-PEG2000 Phospholi-
pids. Functionalized DSPE-PEG2000 phospholipids 2a−d were
synthesized in a single step from DSPE-PEG2000-NH2 by
acylation of the amino group with the desired functionality in
the presence of either HATU or EDC·HCl. Phospholipid 3 was
synthesized in a single step from DSPE-PEG2000-PDP by
reduction by dithiothreitol (DTT). Full experimental details
and lipid characterization are available as Supporting
Information.
Liposome Formulation. Lipids were dissolved in CH3Cl/
MeOH (9:1) and mixed in the ratio 95:4:1 DSPC/DSPE-
PEG2000/DSPE-PEG2000-R (R being the different functional
groups illustrated in Schemes 2 and 3). The solvent was
removed under a stream of nitrogen and the films placed under
vacuum overnight to remove remaining traces of organic
solvent. The obtained films were hydrated in a Hepes buffer,
(10 mM Hepes, pH 6.5, 145 mM NaCl) at 65 °C for 1 h;
followed by 10 freeze−thaw cycles and extrusion at 65 °C
through a 100 nm polycarbonate filter using an Avanti Polar
Lipids mini-extruder. The size distribution of the liposomes was
analyzed using dynamic light scattering, as well as their zeta
potential using a Brookhaven Zeta PALS analyzer. All sizes
were 100−120 nm and all zeta potentials were slightly negative
(approximately −5 mV).
Synthesis and Characterization. The model peptides
1a−1e for somatostatin receptor targeting30 were prepared
using a SPPS strategy as shown in Scheme 1.31 The N-terminal
functionalized TATE peptides 1a−e were synthesized by SPPS
on H-Thr(tBu)-OH preloaded Wang Resin. Acylation of the N-
terminal of the peptide was achieved by activating the Fmoc-
protected amino acid with HATU to ensure high conversion
with minimum epimerization. After assembly of the resin
immobilized octapeptide, H-D-Phe-Cys(Acm)-Tyr(tBu)-D-Trp-
(Boc)-Lys(Boc)-Thr(tBu)-Cys(Acm)-Thr(tBu)-resin, the pep-
tide was treated with Tl(CF3COO)3, which in a single step
removed the two Acm-protection groups present on the
cysteine residues and formed the intramolecular disulfide bond.
The reaction was followed by cleavage of a small amount of
peptide from the resin with TFA and analyzing the crude
peptide by MALDI-TOF MS. Complete Acm-removal and
disulfide formation were observed after 75 min with no sign of
dimerization. After extensive washing of the resin, the N-
terminal was acylated with the desired carboxylic acid
functionalized linker derivatives and cleaved from the solid
support by treatment with a mixture of TFA/H2O/TIS
(95:2.5:2.5) to give the desired functionalized TATE peptides
1a−e. Final purification of the peptides was accomplished by
HPLC and the obtained products 1a−e characterized by
MALDI-TOF MS (Table 1). In order to formulate liposomes
exposing the appropriate chemical functionalities at the distal
end of the PEG-corona, PEGylated phospholipids with the
desired functionalities were synthesized from either DSPE-
Conjugation in Solution. DSPE-PEG2000 functionalized
lipid (0.2 mM, 0.55 mL, 1.1 equiv.) was dissolved in MeOH
and mixed with the corresponding functionalized TATE in
MeOH (0.2 mM, 0.50 mL, 1.0 equiv.). The reaction was stirred
B
dx.doi.org/10.1021/bc3005057 | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
Scheme 1. Synthesis of the TATE-Derivatives 1a−1e
Scheme 2. Synthesis of the DSPE-PEG2000-NH2 Modified
Phospholipids 2a−d
Scheme 3. Synthesis of the DSPE-PEG2000-SH (3)
Functionalized Phospholipid
Table 1. Chemical Structure of R-COOH Used for Synthesis
of TATE Derivatives 1a−1e and Corresponding
Characterization by MALDI TOF MS
free thiol (Scheme 3). The DSPE-PEG2000-maleimide (4)
functionalized phospholipid was purchased from Avanti Polar
Lipids, Inc., Alabama.
In order to evaluate the efficiency of the different coupling
strategies and make a valid comparison, a standardized protocol
was used to minimize the influence of other parameters.
Functionalized liposomes composed of DSPC/DSPE-PEG2000
/
DSPE-PEG2000-R (95:4:1) (R = alkyne (2a), cycloalkyne (2b),
aldehyde (2c), azide (2d), thiol (3), or maleimide (4)) were
prepared by the method described by Bangham and co-
workers.34 The lipid components were dissolved in a mixture of
CHCl3/MeOH (9:1), and the solvent was removed to form a
transparent lipid film. The lipid film was hydrated in HEPES-
buffer (pH 6.5) at 65 °C and extruded through 100 nm
polycarbonate filters to form liposomes in the 100 nm range.
The liposomes were characterized by dynamic light scattering
(DLS), and the ζ-potentials were measured to ensure that
liposomes were formed. All liposomes had a diameter of
approximately 100 nm and slightly negative ζ-potential due to
the presence of the DSPE-PEG2000 lipids. Postfunctionalization
was carried out by adding the required components for the
individual reactions (e.g., CuSO4·5H2O and sodium ascorbate
for the CuAAC reaction), followed by addition of the required
functionalized TATE peptide 1a−e (1 equiv peptide to 1.1
equiv of the functionalized lipid component) in MeOH.
Postfunctionalization reactions were carried out at room
temperature and carefully mixed using a plate shaker.
PEG2000-NH2 or DSPE-PEG2000-PDP as illustrated in Schemes
2 and 3. PEGylated phospholipids exposing a terminal alkyne, a
cyclic alkyne, an aldehyde, or an azide functionality at the distal
end of the polymer (2a−d) were synthesized by acylating
DSPE-PEG2000-NH2 with 4-pentynoic acid, 1-fluorocyclooct-2-
ynecarboxylic acid, 4-carboxybenzaldehyde, or 5-azidopentanoic
acid, respectively. 1-Fluorocyclooct-2-ynecarboxylic acid was
synthesized as described elsewhere.32 DSPE-PEG2000-SH (3)
was synthesized in a single step from DSPE-PEG2000-PDP by
reduction of the pyridyldithiopropionate (PDP) group by DTT
applying standard conditions33 which resulted in the desired
C
dx.doi.org/10.1021/bc3005057 | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
For the conjugation reactions in solution, functionalized
phospholipids (1.1 equiv) and functionalized TATE peptides
(1 equiv) were dissolved in methanol, mixed with the required
additives to facilitate reaction, as described in the Experimental
Procedures, and stirred at room temperature. Triplicates of all
reactions were carried out to ensure reproducibility and
elucidate the heterogeneity of liposomal postfunctionalization.
The amount of functionalized TATE added in the two
conjugation approaches was carefully controlled by measuring
the concentration of the peptide stock solution by UV−vis, thus
ensuring exactly the same peptide concentration in all reactions.
To ensure the same total concentration of phospholipids in all
reactions, phosphorus content measurement was carried out by
ICP-AES. All reactions were monitored for 24 h by HPLC
using UV-detection (λ = 280 nm), and up to 72 h in selected
cases, by removing small aliquots from the reaction vial, and the
conjugation efficiency was calculated based on the area under
Figure 2. Conjugation of DSPE-PEG2000 and TATE by oxime bond
formation. Reactions carried out in solution and directly on the
the curve (AUC) for the free peptide and the DSPE-PEG2000
-
liposome surface. TATE-hydroxylamine (1e) + DSPE-PEG2000
-
○
TATE conjugate, respectively (Figure 1). MALDI-TOF MS
was used to verify that an excess of the functionalized
phospholipids reagent existed in all reactions that did not go
to completion within 72 h.
benzaldehyde (2c) in solution ( ), TATE-hydroxylamine (1e) +
□
DSPE-PEG2000-benzaldehyde (2c) on liposomes ( ). All values are
means SEM (n = 3).
conditions, the conjugation yield reached 78% within 5 min.
Without the presence of the catalyst, 30% conversion was
observed within the same time frame. The oxime bond
formation is a reversible process; however, HPLC and
MALDI TOF MS did not show any reversible formation of
the starting materials within one week in aqueous medium,
which supports the reporting of oxime bonds to be more
hydrolytically stable than, e.g., its analogous hydrazine bond at
physiologically relevant pH.39 The position of these two
functional groups is not interchangeable, as placing the
aldehyde functionality on TATE will induce the risk of
intramolecular imine formation.
Conjugation of TATE and DSPE-PEG2000 by CuAAC. An
often applied procedure for nanoparticle functionalization is the
CuAAC reactioncommonly referred to as the Click
Reaction.22−24 Conjugation of TATE and DSPE-PEG2000 by
the CuAAC reaction was carried out using sodium ascorbate to
generate CuI from CuSO4·5H2O in situ. Reactions were carried
out with the azide present on the TATE peptide, the alkyne at
the PEG terminal of DSPE-PEG2000, and vice versa. The relative
position of the functional moieties was found to have no
significant effect on the reaction efficacy under the solution
conditions, as both orientations result in approximately 98%
yield after 12 h (Figure 3). A different result was obtained when
monitoring the postfunctionalization reaction on the liposome
surface. The cycloaddition between the azide-functionalized
TATE (1c) and the alkyne-modified phospholipid (2a)
resulted in about 75% yield after 2 h, while the opposite
position having the azide functionality present on the liposomes
and the alkyne at the N-terminal of the peptides leveled out at
43% (Figure 3).
Functionalization of azide-modified liposomes by the
CuAAC reaction has not previously been reported elsewhere
to our knowledge. Functionalization of liposomes by the
CuAAC reaction has consistently been carried out with alkyne-
modified liposomes and azide-functionalized ligands; thus,
these reports have not observed the clear negative trend
observed here with the azide-functionalized liposomes.
Furthermore, removing the copper fully from the final product
to avoid copper induced cytotoxicity can be challenging, which
Figure 1. Example of the monitoring of a conjugation reaction by
HPLC over time at 280 nm. The decreasing peak at 8.5 min belongs to
the free TATE-maleimide (1a), while the peak increasing at 15.6 min
is the product from the Michael addition between TATE-maleimide
(1a) and DSPE-PEG2000-SH (3).
Conjugation of TATE and DSPE-PEG2000 by Oxime
Formation. This method, which has not previously been
reported for the surface functionalization of liposomes, is
chemoselective and bioorthogonal to functional groups in most
natural occurring ligands and could serve as an excellent
alternative to existing liposome postfunctionalization meth-
ods.35 TATE-hydroxylamine (1e) and DSPE-PEG2000-benzal-
dehyde (2c) were conjugated, both in solution and on the
liposome surface, by simply mixing the two components
without any additives. Oxime bond formation in solution was
found to be quantitative within one hour, whereas eight hours
of incubation was needed once performed directly on the
liposome surface (Figure 2). Both procedures resulted in
complete conversion to the phospholipid coupled TATE within
24 h under mild conditions.
Oxime bond formation is known to be accelerated by the
presence of aniline,36 acetic acid,37 or Lewis acids.38 In order to
verify this, acetic acid (0.1% v/v) was added to a liposome
batch containing TATE-hydroxylamine (1e). Under these
D
dx.doi.org/10.1021/bc3005057 | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
solution. This is presumably due to favorable interactions
between the liposome surface and TATE-azide (1c). As TATE-
azide (1c) is not highly soluble in the buffer, it prefers to bind
to the PEG layer, bringing it closer to the reaction sites on the
surface, which leads to a faster reaction. Choosing a more
reactive cyclooctyne could not only increase the reaction rate in
solution, but also increase the risk of side product
formation.42,43 However, a relatively long synthetic procedure
to obtain the cyclooctyne moiety limits the use of this type of
copper-free click reaction. Furthermore, it has been shown that
thiols can add spontaneously to cyclooctynes limiting the
bioorthogonality, as some degree of nonspecific reactions can
occur.44
Conjugation of TATE and DSPE-PEG2000 by Michael
Addition. The final approach tested to conjugate TATE and
DSPE-PEG2000 was using the Michael addition procedure. In
general, the Michael addition proceeded rapidly within the first
four hours until reaching a plateau, and reactions carried out in
solution were slightly faster compared to the liposomal
counterpart. The orientation of the maleimide and the thiol
was found to have a decisive impact on the conjugation
efficiency. Michael addition in solution between DSPE-
PEG2000-SH (3) and TATE-maleimide (1a) resulted in 97%
conversion, whereas when performed on the liposome surface,
92% conversion was observed. The opposite orientation of
Figure 3. Conjugation of DSPE-PEG2000 and TATE by the CuAAC
reaction. Reactions carried out in solution and directly on the liposome
surface. TATE-azide (1c) + DSPE-PEG2000-alkyne (2a) in solution
○
( ), TATE-azide (1c) + DSPE-PEG2000-alkyne (2a) on liposomes
◊
□
( ), TATE-alkyne (1d) + DSPE-PEG2000-azide (2d) in solution ( ),
TATE-alkyne (1d) + DSPE-PEG2000-azide (2d) on liposomes (Δ). All
values are means SEM (n = 3).
to some degree reduces the usability of this reaction for
biological purposes.
functionalities expressed in the form of DSPE-PEG2000
-
Conjugation of TATE and DSPE-PEG2000 by Cu-Free
Click Chemistry. A method to avoid addition of toxic copper
salts as catalyst in the triazole formation is to use strained or
electron-deficient alkynes.40,41 This strategy was studied using
the DSPE-PEG2000-cyclooctyne (2b) and azide-functionalized
TATE (1c). In solution, the conjugation was remarkably slow,
yet highly reliable and reproducible with no form of
decomposition or side product formation. As the reaction
was terminated after 50 days, a product yield of 86% was
obtained without reaching a plateau. Applying the post-
functionalization protocol resulted in a remarkably faster initial
reaction (Figure 4), and a level of 84% yield was reached after
72 h.
maleimide (4) and TATE-thiol (1b) only resulted in 53%
product formation when performed in solution and 40% for the
postfunctionalization of liposomes (Figure 5), which was
Interestingly, triazole formation was significantly faster when
performed on liposome surfaces compared to the reaction in
Figure 5. Conjugation of DSPE-PEG2000 and TATE by Michael
addition. Reactions carried out in solution and directly on the
liposome surface. TATE-maleimide (1a) + DSPE-PEG2000-SH (3) in
○
solution ( ), TATE-maleimide (1a) + DSPE-PEG2000-SH (3) on
□
liposomes ( ), TATE-thiol (1b) + DSPE-PEG2000-maleimide (4) in
◊
solution ( ), TATE-thiol (1b) + DSPE-PEG2000-maleimide (4) on
liposomes (Δ). All values are means SEM (n = 3).
surprising. The origin for this observation is not clear, however;
multiple factors could have caused this drop in conversion.
Hydrolysis of the maleimide moiety to the nonreactive
maleamic acid at pH > 8.5 has been reported in the literature,45
but at pH 6.5 where our reactions are carried out, this should be
minimized.46,47 This was confirmed by MALDI-TOF MS where
intact DSPE-PEG2000-maleimide as well as unreacted TATE-SH
was observed. However, a minor amount of methoxy-
substituted maleimide was also observed by MALDI-TOF
MS indicating that the oxa-Michael addition is a competing side
Figure 4. Conjugation of DSPE-PEG2000 and TATE by strain-
promoted Click reaction. Reactions were carried out in solution and
directly on the liposome surface. TATE-azide (1c) + DSPE-PEG2000
-
○
cyclooctyne (2b) in solution ( ), TATE-azide (1c) + DSPE-PEG2000
-
□
cyclooctyne (2b) on liposomes ( ). All values are means SEM (n =
3).
E
dx.doi.org/10.1021/bc3005057 | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
reaction.48 As poor conversion was observed for the reaction
carried out in solution as well as on the surface, we speculated
that the TATE-thiol (1b) peptide was the limiting factor in this
reaction. TATE-thiol (1b) is highly prone to oxidation resulting
in nonreactive TATE-dimers; however, dimerization was not
observed by HPLC or by MALDI-TOF MS. As a positive
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: Thomas.andresen@nanotech.dtu.dk.
Author Contributions
†Authors contributed equally.
control of the reactivity of the commercial DSPE-PEG2000
-
Notes
maleimide (4), a test reaction using 2-mercaptoethanol was
conducted. Using this small and highly reactive thiol, complete
product formation was observed within two hours as confirmed
by MALDI-TOF MS, confirming excellent reactivity of DSPE-
PEG2000-maleimide (4). Hence, the reason for the low coupling
efficiency between DSPE-PEG2000-maleimide (4) and TATE-
SH (1b) is not clear.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The Danish Strategic Research Council (NABIIT) grant 2106-
07-0033 and 2106-08-0081 is gratefully acknowledged for
funding.
ABBREVIATIONS
■
CONCLUSION
■
EPR, enhanced permeability and retention effect; CuAAC,
copper catalyzed azide−alkyne cycloaddition; TATE,
[Tyr3,Thr8]-octreotate; DSPC, 1,2-distearoyl-sn-glycero-3-
phosphocholine; DSPE, 1,2-distearoyl-sn-glycero-3-phosphoe-
thanolamine; HPLC, high-performance liquid chromatography;
SPPS, solid-phase peptide synthesis; MALDI TOF MS, matrix-
assisted laser desorption/ionization time-of-flight mass spec-
troscopy; DLS, dynamic light scattering; ICP-AES, inductively
coupled plasma atomic emission spectroscopy.
In conclusion, a novel highly effective method to functionalize
liposomes by postfunctionalization as well as under solution
conditions has been introduced in the form of an oxime bond
formation between an aldehyde and a hydroxylamine. This
conjugation is chemoselective and bioorthogonal to functional
groups in most naturally occurring relevant ligands, and based
on our investigations, it could serve as an excellent alternative
to existing liposome postfunctionalization methods.
In addition, a systematic study has been conducted to
elucidate the optimal postfunctionalization chemistry with
focus on the relative position of the reactive functionalities.
In general, the reactions carried out directly on the surface of
the functionalized liposomes were slower than the solution-
phase counterpart, except for the strain-promoted Click
reaction, which surprisingly showed the opposite trend. Despite
of possible heterogeneities of liposomal formulations, surface
conjugation reactions were, in general, highly reproducible. The
relative position of the reactive functionalities was found to
have a significant impact on the degree of conversion for the
Michael addition and the CuAAC reaction. The Michael
addition was found to be most effective when the nucleophilic
thiol was present at the distal end of the PEG polymer
compared to the N-terminal of TATE. A similar observation
was observed for the CuAAC reaction. The relative positions of
the azide and the alkyne did not influence the degree of
conversion in solution; however, once performed on the
liposome surface it had a decisive impact. Having the alkyne
functionality present at the liposome surface resulted in 75%
conversion, whereas the azide functionalized liposomes only
resulted in 43% product formation.
REFERENCES
■
(1) Petersen, A. L., Hansen, A. E., Gabizon, A., and Andresen, T. L.
(2012) Liposome imaging agents in personalized medicine. Adv. Drug
Delivery Rev. 64, 1417−1435.
(2) Phillips, M. A., Gran, M. L., and Peppas, N. A. (2010) Targeted
nanodelivery of drugs and diagnostics. Nano Today 5, 143−159.
(3) Petersen, A. L., Binderup, T., Jølck, R. I., Rasmussen, P.,
Henriksen, J. R., Pfeifer, A. K., Kjaer, A., and Andresen, T. L. (2012)
Positron emission tomography evaluation of somatostatin receptor
targeted 64Cu-TATE-liposomes in a human neuroendocrine carcino-
ma mouse model. J. Controlled Release 160, 254−263.
(4) Pavlov, V., Xiao, Y., Shlyahovsky, B., and Willner, I. (2004)
Aptamer-functionalized Au nanoparticles for the amplified optical
detection of thrombin. J. Am. Chem. Soc. 126, 11768−11769.
(5) Liu, J., and Lu, Y. (2006) Preparation of aptamer-linked gold
nanoparticle purple aggregates for colorimetric sensing of analytes.
Nat. Protoc. 1, 246−252.
(6) Taylor, K. M. L., Kim, J. S., Rieter, W. J., An, H., Lin, W., and Lin,
W. (2008) Mesoporous silica nanospheres as highly efficient MRI
contrast agents. J. Am. Chem. Soc. 130, 2154−2155.
(7) Sun, I. C., Eun, D. K., Koo, H., Ko, C. Y., Kim, H. S., Yi, D. K.,
Choi, K., Kwon, I. C., Kim, K., and Ahn, C. H. (2011) Tumor-
targeting gold particles for dual computed tomography/optical cancer
imaging. Angew. Chem., Int. Ed. 50, 9348−9351.
(8) Muhs, A., Hickman, D. T., Pihlgren, M., Chuard, N., Giriens, V.,
Meerschman, C., van der Auwera, I., van Leuven, F., Sugawara, M.,
Weingertner, M. C., Bechinger, B., Greferath, R., Kolonko, N., Nagel-
Steger, L., Reisner, D., Brady, R. O., Pfeifer, A., and Nicolau, C. (2007)
Liposomal vaccines with conformation-specific amyloid peptide
antigens define immune response and efficacy in APP transgenic
mice. Proc. Natl. Acad. Sci. U.S.A. 104, 9810−9815.
(9) Davis, M. E., Chen, Z. G., and Shin, D. M. (2008) Nanoparticle
therapeutics: An emerging treatment modality for cancer. Nat. Rev.
Drug Discovery 7, 771−782.
(10) Petros, R. A., and DeSimone, J. M. (2010) Strategies in the
design of nanoparticles for therapeutic applications. Nat. Rev. Drug
Discovery 9, 615−627.
The findings described above will be useful for future
postfunctionalization designs and clearly illustrate that care
should be taken to select the most appropriate chemistry for
the desired chemical manipulation. Furthermore, it is also
evident that postfunctionalized nanoparticles in general should
always be analyzed in order to ensure the desired composition
of the nanoparticle constructs.
ASSOCIATED CONTENT
■
S
* Supporting Information
Detailed description of the synthetic approach to compounds
1a−e, 2a−d, 3 and their characterization (1H NMR, FT-IR,
HPLC, and MALDI-TOF MS). This material is available free of
charge via the Internet at http://pubs.acs.org.
(11) Gregoriadis, G., Wills, E. J., Swain, C. P., and Tavill, A. S. (1974)
Drug-carrier potential of liposomes in cancer chemotherapy. Lancet 1,
1313−1316.
F
dx.doi.org/10.1021/bc3005057 | Bioconjugate Chem. XXXX, XXX, XXX−XXX

Bioconjugate Chemistry
Article
(12) Maeda, H., Wu, J., Sawa, T., Matsumura, Y., and Hori, K. (2000)
Tumor vascular permeability and the EPR effect in macromolecular
therapeutics. A review. J. Controlled Release 65, 271−284.
(13) Koning, G. A., Gorter, A., Scherphof, G. L., and Kamps, J. A.
(1999) Antiproliferative effect of immunoliposomes containing 5-
fluorodeoxyuridine dipalmitate on colon cancer cells. Br. J. Cancer 80,
1718−1725.
(34) Bangham, A. D., and Horne, R. W. (1964) Negative staining of
phospholipids and their structural modification by surface-active
agents as observed in the electron microscope. J. Mol. Biol. 8, 660−668.
(35) Richard, A., and Bourel-Bonnet, L. (2005) Internalization of a
peptide into multilamellar vesicles assisted by the formation of an
alpha-oxo oxime bond. Chem.Eur. J. 11, 7315−7321.
(36) Dirksen, A., and Dawson, P. E. (2008) Rapid oxime and
hydrazone ligations with aromatic aldehydes for biomolecular labeling.
Bioconjugate Chem. 19, 2543−2548.
(37) Gustafsson, M., Olsson, R., and Andersson, C.-M. (2001)
General combinatorial synthesis of tertiary amines on solid support. A
novel conditional release strategy based on traceless linking at
nitrogen. Tetrahedron Lett. 42, 133−136.
(38) Sridhar, M., Narsaiah, C., Raveendra, J., Reddy, G. K., Reddy,
M., Reddy, M. K. K., and Ramanaiah, B. C. (2011) Efficient
microwave-assisted synthesis of oximes from acetohydroxamic acid
and carbonyl compounds using BF3·OEt2 as the catalyst. Tetrahedron
Lett. 52, 4701−4704.
(39) Rose, K., Zeng, W., Regamey, P. O., Chernushevich, I. V.,
Standing, K. G., and Gaertner, H. F. (1996) Natural peptides as
building blocks for the synthesis of large protein-like molecules with
hydrazone and oxime linkages. Bioconjugate Chem. 7, 552−556.
(40) Agard, N. J., Prescher, J. A., and Bertozzi, C. R. (2004) A strain-
promoted [3 + 2] azide-alkyne cycloaddition for covalent modification
of biomolecules in living systems. J. Am. Chem. Soc. 126, 15046−
15047.
(41) Li, Z., Seo, T. S., and Ju, J. (2004) 1,3-dipolar cycloaddition of
azides with electron-deficient alkynes under mild condition in water.
Tetrahedron Lett. 45, 3143−3146.
(42) Chenoweth, K., Chenoweth, D., and Goddard, W. A., III. (2009)
Cyclooctyne-based reagents for uncatalyzed click chemistry: A
computational survey. Org. Biomol. Chem. 7, 5255−5258.
(43) Codelli, J. A., Baskin, J. M., Agard, N. J., and Bertozzi, C. R.
(2008) Second-generation difluorinated cyclooctynes for copper-free
click chemistry. J. Am. Chem. Soc. 130, 11486−11493.
(44) Fairbanks, B. D., Sims, E. A., Anseth, K. S., and Bowman, C. N.
(2010) Reaction rates and mechanisms for radical, photoinitated
addition of thiols to alkynes, and implications for thiol-yne
photopolymerizations and click reactions. Macromolecules 43, 4113−
4119.
(14) Koning, G. A., Morselt, H. W., Gorter, A., Allen, T. M., Zalipsky,
S., Scherphof, G. L., and Kamps, J. A. (2003) Interaction of differently
designed immunoliposomes with colon cancer cells and Kupffer cells.
An in vitro comparison. Pharm. Res. 20, 1249−1257.
̀
(15) Nardin, A., Lefebvre, M. L., Labroquere, K., Faure, O., and
Abastado., J. P. (2006) Liposomal muramyl tripeptide phosphatidy-
lethanolamine: Targeting and activating macrophages for adjuvant
treatment of osteosarcoma. Curr. Cancer Drug Targets 6, 123−133.
(16) Chiu, S. J., Liu, S., Perrotti, D., Marcucci, G., and Lee, R. J.
(2006) Efficient delivery of a Bcl-2-specific antisense oligodeoxyr-
ibonucleotide (G3139) via transferrin receptor-targeted liposomes. J.
Controlled Release 112, 199−207.
(17) Zhu, J., Xue, J., Guo, Z., Zhang, L., and Marchant, R. E. (2007)
Biomimetic glycoliposomes as nanocarries for targeting P-selectin on
activated platelets. Bioconjugate Chem. 18, 1366−1369.
(18) Matsui, M., Shimizu, Y., Kodera, Y., Kondo, E., Ikehara, Y., and
Nakanishi, H. (2010) Targeted delivery of oligomannose-coated
liposome to the omental micrometastasis by peritoneal macrophages
from patients with gastric cancer. Cancer Sci. 101, 1670−1677.
(19) Lee, R. J., and Low, P. S. (1994) Delivery of liposomes into
cultured KB cells via folate receptor-mediated endocytosis. J. Biol.
Chem. 269, 3198−3204.
(20) Sudimack, J., and Lee, R. J. (2000) Targeted drug delivery via
the folate receptor. Adv. Drug Delivery Rev. 41, 147−162.
(21) O’Donnell, M. J., Zhou, C., and Scott, W. L. (1996) Solid-phase
unnatural peptide synthesis. J. Am. Chem. Soc. 118, 6070−6071.
(22) Tornøe, C. W., Christensen, C., and Meldal, M. (2002)
Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific
copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to
azides. J. Org. Chem. 67, 3057−3064.
(23) Meldal, M., and Tornøe, C. W. (2008) Cu-catalyzed azide-
alkyne cycloaddition. Chem. Rev. 108, 2952−3015.
(24) Rostovtsev, V. V., Green, L. G., Fokin, V. V., and Sharpless, K. B.
(2002) A stepwise huisgen cycloaddition process: Copper(I)-catalyzed
regioselective ″ligation″ of azides and terminal alkynes. Angew. Chem.,
Int. Ed. 41, 2596−2599.
(45) Gregory, J. D. (1955) The stability of N-ethylmaleimide and its
reaction with sulfhydryl groups. J. Am. Chem. Soc. 77, 3922−3923.
(46) Barradas, R. G., Fletcher, S., and Porter, J. D. (1976) The
hydrolysis of maleimide in alkaline solution. Can. J. Chem. 54, 1400−
1404.
(47) Matsui, S., and Aida, H. (1978) Hydrolysis of some N-
alkylmaleimides. J. Chem. Soc., Perkin Trans. 2, 1277−1280.
(48) Finnegan, R. A., and Mueller, W. H. (1965) Base-catalyzed
addition and solvolysis reactions of N-phenyl-maleimide in methanol.
J. Pharm. Sci. 54, 1257−1260.
(25) Staudinger, H., and Meyer, J. (1919) New organic compounds
of phosphorus. III. phosphinemethylene derivatives and phosphini-
mines. Helv. Chim. Acta 2, 635−646.
(26) Saxon, E., and Bertozzi, C. R. (2000) Cell surface engineering by
a modified Staudinger reaction. Science 287, 2007−2010.
(27) Reubi, J. C., and Maecke, H. R. (2008) Peptide-based probes for
cancer imaging. J. Nucl. Med. 49, 1735−1738.
(28) Jølck, R. I., Feldborg, L. N., Andersen, S., Moghimi, S. M., and
Andresen, T. L. (2011) Engineering liposomes and nanoparticles for
biological targeting. Adv. Biochem. Eng. Biotechnol. 125, 251−280.
(29) Sharpless, W. D., Wu, P., Hansen, T. V., and Lindberg, J. G.
(2005) Just click it: Undergraduate procedures for the copper(I)-
catalyzed formation of 1,2,3-triazoles from azides and terminal
acetylenes. J. Chem. Educ. 82, 1833−1836.
(30) Bauer, W., Briner, U., Doepfner, W., Haller, R., Huguenin, R.,
Marbach, P., Petcher, T. J., and Pless, J. (1982) SMS 201−995: A very
potent and selective octapeptide analog of somatostatin with
prolonged action. Life Sci. 31, 1133−1140.
(31) Merrifield, R. B. (1963) Solid phase peptide synthesis. I. The
synthesis of a tetrapeptide. J. Am. Chem. Soc. 85, 2149−2154.
(32) Schultz, M. K., Parameswarappa, S. G., and Pigge, F. C. (2010)
Synthesis of a DOTA-biotin conjugate for radionuclide chelation via
Cu-free click chemistry. Org. Lett. 12, 2398−2401.
(33) Cleland, W. W. (1964) Dithiothreitol, a new protective reagent
for SH groups. Biochemistry 3, 480−482.
G
dx.doi.org/10.1021/bc3005057 | Bioconjugate Chem. XXXX, XXX, XXX−XXX