Design of photocleavable lipids and their application in liposomal "uncorking"

Article abstract of DOI:10.1039/b503423j

The design of o-nitrobenzyl containing photocleavable lipid-amino acid conjugates, and their application in liposomal uncorking are described. The Royal Society of Chemistry 2005.

Full text of DOI:10.1039/b503423j

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Design of photocleavable lipids and their application in liposomal
‘‘uncorking’’{
Binita Chandra, Sanku Mallik* and D. K. Srivastava*
Received (in Cambridge, MA, USA) 9th March 2005, Accepted 8th April 2005
First published as an Advance Article on the web 11th May 2005
DOI: 10.1039/b503423j
developing a more general ‘‘triggered’’ release methodology, we
noted that o-nitrobenzyl substituted compounds are easily cleaved
by near-UV radiation,13 and they can be derivatized to function as
photocleavable lipids. There are a few reports of the design of
photocleavable lipids,9,13 but their syntheses are rather elaborate
and often time consuming. Toward this end, we developed a
synthetic scheme for conjugating a C18-amine and selected
negatively-charged polar amino acids via the o-nitrobenzyl group
as a spacer (lipids 1 and 2, Scheme 1).
The design of o-nitrobenzyl containing photocleavable lipid–
amino acid conjugates, and their application in liposomal
uncorking are described.
The drug design/discovery endeavour of biomedical research often
produces compounds that are not easily targetable to the desired
site in the human body to exhibit their effects. Aside from the
problems associated with solubility, biodegradation, membrane
permeability, and cytotoxicity of putative drugs, their target
specific ‘‘delivery’’ has been one of the major challenges of
pharmaceutical research. Various drug carriers (e.g., liposomes,
polymers, micro-spheres, antibody–drug conjugates) have been
developed to alter the bio-distribution and pharmacokinetic
properties of drug molecules. Among such carriers, liposomes
offer several advantages as clinical drug delivery vehicles, and at
present, there are 13 liposome-mediated drug delivery systems
approved for the treatment of a variety of human diseases (e.g.,
breast cancer, ovarian cancer, meningitis, fungal infections,
leukaemia, etc.).1 In addition, the liposome mediated delivery of
about 30 other small molecule drugs, DNA fragments, and
diagnostic compounds are currently at different stages of clinical
trials.2
The overall synthesis was accomplished via four easy steps: (i)
selective nitration at the o-position of the aminomethyl group of
p-aminomethyl benzoic acid, (ii) conjugation of stearylamine at the
carboxyl group of compound 3, (iii) removal of the amine
protecting group and attachment of the selected amino acids via
the a-carboxyl group, (iv) final removal of the protecting groups.
A detailed account of the syntheses are given in the ESI.{
Based on the literature precedent,13 it was anticipated that the
o-nitrobenzyl group of the lipids 1 and 2 would be cleaved upon
irradiation by UV/visible light in the 320–400 nm region. The
In liposome mediated target-specific drug delivery systems, three
features need to be taken into consideration: (i) appropriate
coating of liposomes to circumvent their clearance by the
reticuloendothelial system (RES),3 (ii) attachment of the target-
specific recognition moiety (e.g., antibodies, receptor agonist/
antagonist), and (iii) incorporation of the triggering mechanism.1
Attachment of receptor-specific ligands ensures their adhesions to
target cell surfaces.1,4 Although the surface-attached liposomes can
be passively internalized inside the cells and release their contents,
the overall process is usually slow, unless they are subjected to
external triggers. Such triggering agents include change in pH,5
mechanical stress,6 metal ions,7 temperature,8 light9 and enzymes.10
We recently formulated liposomes with collagen mimetic triple
helical peptides, and demonstrated the trigger release of their
contents by a matrix metalloproteinase, MMP-9.11 Since MMP-9
is overexpressed in a variety of cancerous tissues,12 the overall
methodology is likely to find applications in drug delivery for
suppression/attenuation of invasion and metastasis. However, the
above approach has the limitation of uncorking liposomes at
cancerous tissues where MMP-9 is not overexpressed. In pursuit of
{ Electronic supplementary information (ESI) available: experimental
details for the synthesis of lipids 1 and 2; liposome formation and release
conditions. See http://www.rsc.org/suppdata/cc/b5/b503423j/
*sanku.mallik@ndsu.edu (SM) (Sanku Mallik)
Scheme 1 The structures of the lipids incorporating the o-nitrobenzyl
group and their syntheses are shown.
dk.srivastava@ndsu.edu (DKS) (D. K. Srivastava)
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photocleavage of the o-nitrobenzyl group proceeds via abstraction
of a benzylic hydrogen by the photo-activated nitro group. This is
followed by an electron-redistribution to form an aci-nitro form,
which finally rearranges to form the o-nitroso benzaldehyde
product.13,14 It is known that the precursor–nitro conjugates
exhibit absorption maxima in the range of 250–270 nm,13,14 the
intermediate and final nitroso-derivatives are characterized by the
red shift in the corresponding aromatic absorption band by 50–
80 nm.14,15 Hence, the time course of the overall cleavage process
of the amphiphilic lipid–amino acid conjugates could be easily
probed spectrophotometrically. Since lipids 1 and 2 exhibited
similar spectral features, the results are discussed only for lipid 1.
Fig. 1 shows the time dependent spectral changes upon irradiation
of an ethanolic solution of lipid 1 at 365 nm.
Based on literature reports,13,14 the structures of the photolysis
products for lipid 1 are shown in Scheme 2.
The liposomes were prepared with 1,2-distearoyl-glycero-3-
phosphocholine (DSPC, 95% by weight) and 5% of the
photocleavable lipid 1 in 50 mM HEPES buffer (pH 5 7.0).
The liposomes were characterized by transmission electron
microscopy (see ESI{) and the average size of the liposomes was
found to be 60–70 nm. A self-quenching hydrophilic dye,
6-carboxyfluorescein, was encapsulated in the liposomes.16 The
rate of content release will depend on the structures of the
encapsulated molecules. To facilitate the release, a hydrophilic dye
was selected for these studies. The excitation and emission maxima
of 6-carboxyfluorescein were determined to be 495 and 518 nm,
respectively (see ESI{). Due to the self quenching effect of the
above fluorophore at high concentration17 (the condition which
prevails in the lumen of the liposomes due to the local
concentration effect), the release of the dye from liposomes (upon
uncorking) was expected to proceed in concomitance with the
increase in the fluorescence intensity at 518 nm (lex 5 495 nm).
Hence, we could irradiate the 6-carboxyfluorescein encapsulated
liposomes at 365 nm (for photocleavage), and monitor their
uncorking by measuring the release of the fluorophore at 518 nm
(see ESI{).
The spectral data of Fig. 1 indicate that the irradiated
o-nitrobenzyl group of lipid 1 shows a pronounced absorption
peak at 247 nm, with a broad shoulder at 300 nm, and a minor
shoulder at 220 nm. As the time of irradiation increases, the
intensities of all these peaks increase. However, the shoulder peak
of the original (uncleaved) lipid at 300 nm is split into two peaks
with absorption maxima at 290 and 315 nm respectively. Of these
peaks, the latter is characterized by the formation of a ‘‘nitroso’’
derivative of the cleaved product.14 Since the overall spectral
changes conformed to clean isosbestic points at 218, 260, and
385 nm, it implied that there were no spectrally distinct
intermediates during the course of the overall cleavage process.
However, to further probe whether some kinetically significant
(albeit spectroscopically undetectable) intermediate was produced
during the course of the photocleavage reaction, we analyzed the
time slice of the absorption changes at 315 nm. As shown in the
inset of Fig. 1, the kinetic profile was best fitted by a single
Fig. 2 shows the plot of the increase in the fluorescence intensity
at 518 nm as a function of the irradiation (at 365 nm) time. A
control experiment was also performed, in which the liposomes
were not irradiated (solid squares).
When we attempted to analyze the cleavage data by a single
exponential rate equation, the fit was not good. This was not
unexpected since the time course of fluorescence increase involves a
finite lag phase. Such a kinetic profile could emerge if the release of
the liposome encapsulated fluorophore required some structural
adjustments in the liposomal lipid domains. The kinetic data of
Fig. 3 could be best fitted by a sequential two step kinetic
equation18 in the following form [eqn. (1)], with k1 and k2 values of
0.246 and 0.039 min21, respectively.
exponential rate equation, with a rate constant of 0.43 min21
,
suggesting that the overall cleavage reaction indeed involved a
single step. This rate is comparable to reported cleavage rates for
the o-nitrobenzyl group under similar irradiation conditions.15
L ꢀ F DCkC1 A Lꢁ ꢀ F DCkC2 A LꢁzF
ꢂ
ꢀ
ꢁ
ꢃ
À
Á
(1)
1
k2e{k tꢀk1e{k t
1
2
F~½L ꢀ Fꢂ 1z
k1ꢀk2
In eqn. (1), L and F represent liposome and 6-carboxyfluor-
escein (fluorophore), respectively. L* represents the ‘‘intermediary’’
structure of the liposome, which still harbors the fluorophore in its
lumen. The fluorophore is released during the second step.
Alternatively, the model mechanism of eqn. (1) can be explained
on the basis that the fluorophore exists in the ‘‘self-quenched’’ and
‘‘free’’ states, and the biphasic kinetic profile of Fig. 2 is a result of
the transition between such states. Irrespective of the nature of the
Fig. 1 Time dependent spectral changes upon irradiation of lipid 1 at
365 nm. The spectra were recorded during 5 minutes of irradiation in 30 s
intervals. The inset shows the time slice of the spectral changes at 315 nm.
The solid smooth line is the best fit of the data for a single exponential rate
Scheme 2 The structures of the products after photolysis of lipid 1 are
equation, with a rate constant of 0.43 min21
.
shown.
3022 | Chem. Commun., 2005, 3021–3023
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photo-induced uncorking of liposomes and release of their
contents. The liposomes were found to be stable (in the absence
of light) for more than two weeks at 4 uC. The rate of contents
release is useful for in vivo applications.10,19 Thus, our overall
methodology has the potential to find applications in the area of
‘‘drug delivery’’ in biomedical research.1{
Binita Chandra, Sanku Mallik* and D. K. Srivastava*
Department of Chemistry, Biochemistry and Molecular Biology, North
Dakota State University, Fargo, ND-58105.
E-mail: sanku.mallik@ndsu.edu (SM); dk.srivastava@ndsu.edu (DKS);
Fax: +1 701-231-8831 (SM); Fax: +1 701-231-8324 (DKS);
Tel: +1 701-231-8829 (SM) Tel: +1 701-231-7831 (DKS)
Notes and references
{ Research was supported by the NIH grants 1R15 HL077201-01 to DKS
and 1R01 GM 63204-01A1 and 1P20 RR 15566-01 to SM.
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Fig. 2 Kinetics of release of 6-carboxyfluorescein upon irradiation of
liposomes incorporating lipid 1 at 365 nm (solid circle). The solid squares
represent the control experiment in which the liposomes were not
irradiated. The solid smooth line is the best fit of the data for a ‘‘two-
step’’ liposomal uncorking according to eqn. (1), for the k1 and k2 values
of 0.246 and 0.039 min21, respectively.
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Fig. 3 The photorelease of the liposomal contents is illustrated.
‘‘species’’ involved in the overall microscopic pathway, it is clear
that the rate constant of photocleavage of lipid 1 (0.43 min21
;
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Fig. 1) is comparable to that of the first step in eqn. (1).
The similarity of the two rate constants suggests that the first
step in the release process is the loss of the hydrophilic head group
of the lipid 1. The resultant nitroso benzaldehyde compound
(Scheme 2) destabilizes the liposome bilayer. It is possible that lipid
reorganization also takes places before the encapsulated dye is
released. We are currently in the process of deciphering the kinetic
mechanism of the overall process, and we will report these findings
subsequently. The contemplated sequence of steps intrinsic to the
photocleavage of lipid
1 (incorporated in the liposomes),
uncorking of the liposomes, and release of their contents is shown
in the cartoon of Fig. 3.
Due to ease of the syntheses of o-nitrobenzyl conjugated
photocleavable lipids and their abilities to become incorporated
in the liposomes, we could demonstrate the feasibility of the
19 A. S. L. Derycke and P. A. M. de Witte, Adv. Drug Delivery Rev., 2004,
56, 17–30.
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