Liposomes from novel photolabile phospholipids: Light-induced unloading of small molecules as monitored by PFG NMR

Article abstract of DOI:10.1021/ja016874i

A molecular dithiane-based approach to synthesis of novel photolabile phospholipids is developed. These lipids are used in formulations with egg-POPC and cholesterol to prepare light-sensitive liposomes. Irradiation of such liposomes in PBS buffer (medium

Full text of DOI:10.1021/ja016874i

Published on Web 04/24/2002
Liposomes from Novel Photolabile Phospholipids: Light-Induced Unloading
of Small Molecules As Monitored by PFG NMR
Yongqin Wan,† Joseph K. Angleson,‡ and Andrei G. Kutateladze*†
Contribution from the Department of Chemistry and Biochemistry, and Department of Biological Sciences,
UniVersity of DenVer, DenVer, Colorado 80208
Received August 17, 2001
Scheme 1
Light-triggered unloading of liposomes offers an attractive
alternative to the temperature or pH-driven modulation of membrane
permeability. The known approaches to photoresponsive vesicles
are based on disrupting the integrity of the lipid bilayer through (i)
photoinduced polymerization of, e.g., sorbate-based lipids causing
phase changes in the bilayer and enhanced leakage at the boundaries
of polymerized domains,1 (ii) dramatic conformational changes due
to photoisomerization in the incorporated azobenzene or alkene
fragment,2 (iii) photoinduced release of a masked hydrophilic group
in the hydrophobic region of the bilayer membrane3 or, finally,
(iv) phototransformation of lipid headgroups, e.g via oxygenation
of a double bond.4 While severing hydrophilic headgroups appears
to be a simple and straightforward way to disrupt the lipid bilayer,
this approach did not receive as much attention.
Recently we have developed a novel strategy for the assembly
of photolabile molecular systems based on carbonyl additions of
Scheme 2
substituted di- and trithianes. This methodology allows us to link
various molecular blocks with photolabile latches that can be unfast-
ened on demand via photoinduced electron transfer.5 In this com-
munication we report on the synthesis of novel phosphatidylcholine-
like lipids, in which the hydrophilic phosphocholine headgroups
are connected to the hydrophobic tails via a photolabile, dithiane-
based tether. The photocleavable unit can also be outfitted with
the dual-purpose nitropyridinamino group serving as an internal
ET-sensitizer and as a model element of molecular recognition.5d
Here we show that such photolabile lipids can be used in formu-
lations with egg palmitoyl-oleoyl phosphatidylcholine (POPC) and
cycles to disrupt large multilamellar vesicles (LMV) and extruded
21 times at 55 °C through a polycarbonate filter with 100 nm pores
to form large unilamellar vesicles (LUV). Subsequent gel filtration
cholesterol to form vesicles capable of unloading their content upon
on Sephadex was carried out to remove untrapped probes.
irradiation.
Conventional assays of liposome leakage are based on fluores-
Synthetic approaches to the photolabile phospholipids are shown
cence recovery derived from reduced efficiency of collisional
in Schemes 1 and 2. 5,5-Bis(hydroxymethyl)-1,3-dithiane5e is alkyl-
quenching in the bulk solution as opposed to nearly total quenching
ated to attach two hydrophobic hydrocarbon tails, lithiated and ad-
inside the vesicle due to the higher internal concentration of
ded to a solution of in situ generated N-silylimine 2. Amine 4 is
fluorophore quencher.1b,c,g While these are very simple assays for
reacted with 2-fluoro-5-nitropyridine, the phenol is deprotected, and
studying liposome leakage induced by changes in pH, temperature,
phosphocholine is introduced via a standard procedure6 involving
and other nonradiative chemical processes, we detected considerable
1-chloro-phosphadioxolane to furnish photolabile lipid 6, Scheme 1.
photobleaching of several fluorophores during the photochemical
experiments, rendering fluorescent assays less suitable for quantita-
Alternatively, 5-(4-hydroxyphenyl)-1,3-dithiane7 (7) can be util-
ized to carry the hydrophilic headgroup (Scheme 2). In this example
tive monitoring of photoinitiated release. Also, the light absorption
the hydrophobic tails are attached to the carbonyl component, e.g.
by the probe molecule may interfere with the desired photochem-
3,4-dihydroxybenzaldehyde (8), and thus hydroxyalkyl dithiane 10
istry, especially when accurate quantum yield measurements are
is produced. This lipid requires external sensitization for cleavage.
required. We suggest that these shortcomings in photochemical
The liposomes were then prepared from a three-component
experiments can be overcome by utilizing the Pulse Field Gradients
mixture of POPC(egg)-cholesterol-photolipid in the ratio 5:3:2.
First, a thoroughly dried lipid film was hydrated with a 0.15 M
phosphate buffered saline solution (PBS, pH 7.0) containing the
probe molecule. The suspension was subjected to 4 freeze-thaw
(PFG) NMR technique8 for determining the self-diffusion coef-
ficients of small probe molecules, which do not have any interfering
UV absorption. When the probe is confined inside the vesicle its
apparent diffusion coefficient is equal to that of the carrier liposome.
It changes by orders of magnitude, depending on the hydrodynamic
size ratio, when the probe is released into the bulk solution. We
further suggest using probes that carry fluorine-containing groups,
* Corresponding author. E-mail: akutatel@du.edu.
†Department of Chemistry and Biochemistry.
‡Department of Biological Sciences.
9
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10.1021/ja016874i CCC: $22.00 © 2002 American Chemical Society

C O M M U N I C A T I O N S
liposomes in formulations with POPC and cholesterol. The lipids
can be equipped with hydrogen bond-based elements of molecular
recognition offering a possibility to rationally modify the surface
of vesicles. Obviously, before this chemistry could be used to solve
“real” problems of drug delivery, the efficiency of the photofrag-
mentation and subsequent release will have to be improved.
We also have developed a simple assay to monitor the release
of small organic molecules based on PFG NMR. The potential
advantage of 19F monitoring is that many biomedically relevant
compounds of interest can be labeled via, for example, trifluoro-
acetylation and their unloading from liposomes or other delivery
vehicles can then be followed easily.
Figure 1. (a) Relative size and distances traveled in 0.15 s and (b) release
of 11 from POPC-cholesterol-6 in the dark (4) and irradiated ([).
Work is in progress in our laboratories to improve the dark
stability of photosensitive liposomes and the efficiency of photo-
release, and to model recognition events based on vesicle surface
modification.
Figure 2. TEM images of photolabile liposomes before (A) and after
irradiation (B)-same scale.
e.g. trifluoromethyl, for easy detection with 19F PFG NMR.9 In
such a case, the monitoring is reduced to observing one signal with
no interference from other peaks and no need for water suppression.
Acknowledgment. Support of this research by the National
Science Foundation (Career Award CHE-9876389) is gratefully
acknowledged. We thank Janet Lieber for assistance with electron
microscopy.
1
We tested several probe molecules for 19F and H PFG NMR
monitoring of membrane permeability. Small inorganic anions
showed unacceptably high rates of dark leakage, even when control
experiments were run with liposomes made of well-documented
stable POPC-cholesterol formulations: at room temperature the life-
times of leakage for salts KF (DS ) 1.33 × 10-5 cm2 s-1), KPF6
(DS ) 1.14 × 10-5 cm2 s-1), and KOTf (DS ) 9.08 × 10-6 cm2
s-1) were less than 0.5 h, making them impractical for monitoring.
Larger cations were escaping much slower, for example, the lifetime
of dark leakage for N-methyl-4-trifluoromethylpyridinium (11) ex-
ceeded 18 h. While pyridine 11 was our probe of choice for this
study, we note that sodium 2-(trimethylsilyl)ethanesulfonate (Me3-
SiCH2CH2SO3Na, DS ) 3.9 × 10-6 cm2 s-1), commonly used as
Supporting Information Available: Experimental details (PDF).
This material is available free of charge via the Internet at http://
pubs.acs.org.
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