Fluorinated lipid constructs permit facile passage of molecular cargo into living cells

Article abstract of DOI:10.1021/ja902777d

(Figure Presented) Fluorinated lipids get rapidly internalized into living cells and are also displayed on the cell surface. The uptake of lipids is energy dependent and is likely via the clathrin-mediated endocytic pathway. Fluorinated lipids are 3-5-fold more efficient in acting as molecular transporters of noncovalently bound proteins than their hydrocarbon counterparts. These materials could serve as efficient molecular transporters for molecules that function in the cytoplasm such as short interfering RNAs (siRNAs).

Full text of DOI:10.1021/ja902777d

Published on Web 08/12/2009
Fluorinated Lipid Constructs Permit Facile Passage of Molecular Cargo into
Living Cells
Laila Dafik,† Venkateshwarlu Kalsani,† Anthony Kar Lun Leung,‡ and Krishna Kumar*,†,§
Department of Chemistry, Tufts UniVersity, Medford, Massachusetts 02155, Koch Institute for IntegratiVe Cancer
Research, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, and Cancer Center,
Tufts Medical Center, Boston, Massachusetts 02110
Received April 14, 2009; E-mail: krishna.kumar@tufts.edu
Scheme 1. Structures of Phospholipids 1-5 and Synthesis from
the Corresponding H-Phosphonates
The plasma membrane enveloping mammalian cells serves a
crucial gatekeeping function by careful regulation of the influx and
exodus of molecules. Barring small (<1 kDa), hydrophobic
molecules that can pass through by passive diffusion, all others
have to confront the impervious and selective membrane barrier to
gain entry.1 Strategies to deliver macromolecules into living cells
have tremendous potential in therapeutic and imaging applications.2,3
Several methods have therefore been devised to achieve this end.4
Conjugates with amphipathic, hydrophobic, or cationic polymers,5-8
and, more recently, carbon nanotubes9-13 have been successfully
deployed. Noncovalent assemblies of lipids and macromolecules,
and liposomes that have the desired molecular consignment on the
inside, have also been used.14,15 While these approaches have
provided a powerful transporter toolkit, the quest for new classes
of molecules remains of high interest. The agents must be nontoxic
and efficient and have reasonable half-lives inside cells to be general
in their applicability.16 Such molecules can potentially be used to
deliver selective imaging probes or be used in chemotherapy and
genetic therapy.
Endocytosis,1,17 a process by which cells internalize mol-
ecules, has been recently exploited for delivering macromolecules
into mammalian cells. This approach has been extremely
effective, and several molecules conjugated to cholesterol have
been shown to traverse the membrane barrier efficiently.18-20
To expand the repertoire of agents that can be used in this
manner, we designed phospholipids that contain a H-phosphonate
handle for further functionalization. Lipid rafts have been
implicated in enhancing the efficiency of endocytosis.21,22 We
have recently shown that fluorinated phospholipids form phase-
separated microdomains of sizes 50-200 nm when mixed with
hydrocarbon lipids with identical headgroup functionalities.23
We envisioned that nanosized domains of fluorinated lipids could
enhance endocytic efficiency and uptake.21-24 Fluorocarbon
lipids are more hydrophobic than their hydrocarbon congeners25-29
and are therefore expected to have higher affinities for mem-
branes.4 Furthermore, fluorinated molecules have been shown
to be biocompatible, and fluorinated “lipoplexes” are significantly
more efficient as DNA transfection reagents compared to their
hydrocarbon counterparts.30
either all hydrocarbon (4 and 5) or partially fluorinated lipid
chains (1-3), and the head groups are adorned with different
linkers attached to biotin (1, 2, and 4) or to the fluorophore
7-nitrobenz-2-oxa-1,3-diazole (NBD, 3 and 5). The agents were
deemed nontoxic to cells in culture, as incubation of HeLa cells
with 2-5 did not show significant difference in counts after 5
days when compared to controls (see Supporting Information).
In addition, the complex of 2 and 4 with avidin conjugated to
fluorescein isothiocyanate (AF, Ka to biotin ∼1015 M-1) also
had little effect on the growth profiles.
Incubation of HeLa cells with 3 at 37 °C resulted in intensely
fluorescent cells as evinced by counting on a fluorescence plate
reader (Figure 1). The increase in fluorescence was concentration
dependent (see Supporting Information, Figure S9). Further
inspection by fluorescence microscopy revealed that the emanat-
ing luminescence was distributed both on the cellular surface
and in the interior. Similar experiments with 8 did not result in
any cellular fluorescence. Jurkat and HL60 cells when treated
similarly with 3 exhibited comparable levels of fluorescence
To investigate the ability of fluorinated lipids to act as
macromolecule transport agents, compounds 1-5 were designed
and synthesized (Scheme 1).31,32 Agents 1, 3, and 5 are
derivatives of 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine
(DPPE), while 2 and 4 are related phosphodiesters. Preparation
of conjugates from the respective H-phosphonates followed
previously established procedures.33 These molecules contain
† Tufts University.
‡ Massachusetts Institute of Technology.
§ Tufts Medical Center.
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Figure 1. Relative fluorescence from HeLa cells treated with 3 (100 µM)
or the complex 2:AF (100 µM) under various conditions.
suggesting that the process is general across many cell lines.
Localization of 3 was further probed in HeLa cells using
fluorescence microscopy.
Internalization of lipids was energy dependent suggesting that
it is facilitated by endocytosis.19 When incubations of HeLa cells
with 3 or the complex 2:AF were carried out at 4 °C,
fluorescence from the cells decreased significantly. Indeed, only
39% and 57% of fluorescence was detectable when compared
to the experiment at 37 °C for 3 and the 2:AF complex,
respectively (Figure 1). Furthermore, addition of sodium azide,
an ATP depleting poison, and a known inhibitor of endocytosis,
also resulted in a 21% decrease in fluorescence. These experi-
ments implicate endocytosis as the primary mechanism of
transport of the exogenous materials.34,35 We further interrogated
whether endocytosis was orchestrated via a particular pathway.
Participation of clathrin coated pits is frequently invoked in
endocytic events, and it can be disrupted by incubation under
hypertonic conditions (400 mM sucrose).36 Indeed, when HeLa
cells were treated with 3 or the 2:AF complex under 400 mM
sucrose, the fluorescence from the cells was diminished by 61%
and 55%, respectively. These results were further corroborated
with fluorescence microscopy of the resultant cells (see Sup-
porting Information).
The ability of the transport agents to deliver macromolecules
across membranes was investigated by incubation of HeLa cells
with each of the preformed complexes 1:AF, 2:AF, and 4:AF
in separate experiments. The cells were pelleted by centrifugation
twice and washed with PBS, resuspended in buffer, and then
examined by microscopy or fluorescence counting. All agents
were effective at ferrying AF into the cell. In contrast, neither
AF nor the AF:biotin complex were by themselves able to
traverse the membrane, resulting in cells that were minimally
fluorescent. It has been previously established that the binding
affinity of lipid-biotin conjugates to AF is several orders of
magnitude less tighter than biotin.37,38 When free biotin (g5
equiv, 1 h) was allowed to equilibrate with 2:AF prior to
incubation with HeLa cells, the cells only exhibited background
levels of fluorescence.
Figure 2. Fluorescence microscopy images of HeLa cells treated with
2:AF and 3 at 0 µM (a,d), 10 µM (b,e), and 100 µM (c,f), respectively.
Cells were fixed with paraformaldehyde (4% w/v) and stained with DAPI
(nucleus, blue channel; a-f). Overlay images clearly show that material
is excluded from the nucleus. Images were taken at the same exposure
for different concentrations of the lipid constructs. Bar, 10 µm (see panel
a, bottom right).
reduction with 5 mM sodium dithionite, a reagent known to
extinguish NBD fluorescence, 59% of the fluorescence from the
cells was lost. Biotin exchange for 1 h resulted in a similar (73%)
loss of fluorescence in the case of the 2:AF treated cells.
Assuming NBD fluorescence is not affected by the environment
and accounting for self-quenching in 100 µM solutions of 3,
treatment with a 100 µM solution resulted in g106 molecules
on the surface of each cell, available for reduction. The
localization of synthetic constructs and macromolecular cargo
inside cells was investigated using microscopy done in the
presence of a nucleus specific dye, 4′,6-diamidino-2-phenylindole
(DAPI). As the overlay images show in Figure 2, light emitted
by 3 and 2:AF originates from the cytoplasmic region and the
agents are excluded entirely from the nucleus.
In general, the fluorinated lipid constructs 2 and 3 were more
efficient in transport and uptake as compared to their hydrocarbon
counterparts 4 and 5. As judged from fluorescence counting, this
difference was 2.6-fold. This difference in efficiency of uptake was
also confirmed using flow cytometry (Figure 3). While the
hydrocarbon lipids conferred a 13-fold increase in mean fluores-
cence intensity of cells over background, the cells treated with the
fluorinated congeners were intensely fluorescent with a 63-fold
increase in mean fluorescence (a difference of 4.8-fold over the
To assess what fraction of 3 or the complex 2:AF resides on
the outer leaflet of the plasma membrane, we subjected cells to
reduction by sodium dithionite (in the case of 3) or to exchange
with free biotin (in the case of the 2:AF complex).19 Upon
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focal planes in the z-axes of Figure 2. This material is available free of
charge via the Internet at http://pubs.acs.org.
References
(1) Conner, S. D.; Schmid, S. L. Nature 2003, 422, 37–44.
(2) Smith, D. A.; van de Waterbeemd, H. Curr. Opin. Chem. Biol. 1999, 3,
373–378.
(3) Doerr, A. Nat. Methods 2006, 3, 770–771.
(4) Kam, N. W. S.; Liu, Z. A.; Dai, H. J. Angew. Chem., Int. Ed. 2006, 45,
577–581.
(5) Morris, M. C.; Depollier, J.; Mery, J.; Heitz, F.; Divita, G. Nat. Biotechnol.
2001, 19, 1173–1176.
(6) Yan, X. H.; He, Q.; Wang, K. W.; Duan, L.; Cui, Y.; Li, J. B. Angew.
Chem., Int. Ed. 2007, 46, 2431–2434.
(7) Rothbard, J. B.; Garlington, S.; Lin, Q.; Kirschberg, T.; Kreider, E.;
McGrane, P. L.; Wender, P. A.; Khavari, P. A. Nat. Med. 2000, 6, 1253–
1257.
(8) Okuyama, M.; Laman, H.; Kingsbury, S. R.; Visintin, C.; Leo, E.; Eward,
K. L.; Stoeber, K.; Boshoff, C.; Williams, G. H.; Selwood, D. L. Nat.
Methods 2007, 4, 153–159.
(9) Feazell, R. P.; Nakayama-Ratchford, N.; Dai, H.; Lippard, S. J. J. Am.
Chem. Soc. 2007, 129, 8438.
(10) Kostarelos, K.; Lacerda, L.; Pastorin, G.; Wu, W.; Wieckowski, S.;
Luangsivilay, J.; Godefroy, S.; Pantarotto, D.; Briand, J. P.; Muller, S.;
Prato, M.; Bianco, A. Nat. Nanotechnol. 2007, 2, 108–113.
(11) Chen, X.; Tam, U. C.; Czlapinski, J. L.; Lee, G. S.; Rabuka, D.; Zettl, A.;
Bertozzi, C. R. J. Am. Chem. Soc. 2006, 128, 6292–6293.
(12) Kam, N. W. S.; O’Connell, M.; Wisdom, J. A.; Dai, H. J. Proc. Natl. Acad.
Sci. U.S.A. 2005, 102, 11600–11605.
Figure 3. Flow cytometry of HeLa cells (typically 3 × 104) suspended in
phosphate buffered saline treated with 8 and 3 (panel a) and with 3 and 5
(panel b). Cells treated with the fluorinated construct 3 were 4.8-fold more
intensely fluorescent than those reated with the hydrocarbon analogue 5.
(13) Liu, Z.; Cai, W. B.; He, L. N.; Nakayama, N.; Chen, K.; Sun, X. M.; Chen,
X. Y.; Dai, H. J. Nat. Nanotechnol. 2007, 2, 47–52.
hydrocarbon version). These results demonstrate the superior ability
of the fluorinated lipids to trigger and act as participants in the
endocytic events.
(14) Jones, M. N. Liposomes, Pt E; Elsevier Academic Press Inc: San Diego,
CA, 2005; Vol. 391, pp 211-228.
(15) Torchilin, V. P. Nat. ReV. Drug DiscoVery 2005, 4, 145–160.
(16) Ng, P. P.; Dela Cruz, J. S.; Sorour, D. N.; Stinebaugh, J. M.; Shin, S. U.;
Shin, D. S.; Morrison, S. L.; Penichet, M. L. Proc. Natl. Acad. Sci. U.S.A.
2002, 99, 10706–10711.
In summary, we report a new class of macromolecular transport
agents capable of entering living cells through endocytosis. They
are furthermore able to facilitate the entry of noncovalently bound
proteins. The delivery agents were nontoxic and were distributed
both on the surface and in the cytoplasm of cells but not in the
nucleus. Fluorinated lipids were found to be superior in facilitating
transport in this manner. A large number of molecules can be
displayed on the cell surface using this methodology (∼106). This
study paves the way for the clustered display of ligands on cell
surfaces and intracellular delivery of macromolecules for imaging
and therapeutic applications. These constructs may be especially
useful for delivering short interfering RNAs (siRNAs), as the main
events in RNAi are localized in the cytoplasm. Studies along these
lines are in progress in our laboratories.
(17) Mayor, S.; Pagano, R. E. Nat. ReV. Mol. Cell Biol. 2007, 8, 603–612.
(18) Peterson, B. R. Org. Biomol. Chem. 2005, 3, 3607–3612.
(19) Boonyarattanakalin, S.; Martin, S. E.; Dykstra, S. A.; Peterson, B. R. J. Am.
Chem. Soc. 2004, 126, 16379–16386.
(20) Sun, Q.; Cai, S.; Peterson, B. R. J. Am. Chem. Soc. 2008, 130, 10064–
10065.
(21) Nabi, I. R.; Le, P. U. J. Cell Biol. 2003, 161, 673–677.
(22) Pelkmans, L.; Fava, E.; Grabner, H.; Hannus, M.; Habermann, B.; Krausz,
E.; Zerial, M. Nature 2005, 436, 78–86.
(23) (a) Yoder, N. C.; Kalsani, V.; Schuy, S.; Vogel, R.; Janshoff, A.; Kumar,
K. J. Am. Chem. Soc. 2007, 129, 9037–9043. (b) Yoder, N. C.; Yüksel,
D.; Dafik, L.; Kumar, K. Curr. Opin. Chem. Biol. 2006, 10, 576–583. (c)
Schuy, S.; Faiss, S.; Yoder, N. C.; Kalsani, V.; Kumar, K.; Janshoff, A.;
Vogel, R. J. Phys. Chem. B 2008, 112, 8250–8256.
(24) Schuy, S.; Faiss, S.; Yoder, N. C.; Kalsani, V.; Kumar, K.; Janshoff, A.;
Vogel, R. J. Phys. Chem. B 2008, 112, 8250–8256.
(25) Bilgic¸er, B.; Fichera, A.; Kumar, K. J. Am. Chem. Soc. 2001, 123, 4393–
4399.
(26) Bilgic¸er, B.; Kumar, K. Tetrahedron 2002, 58, 4105–4112.
(27) Bilgic¸er, B.; Kumar, K. Proc. Natl. Acad. Sci. U.S.A. 2004, 101, 15324–
15329.
Acknowledgment. We thank Profs. David Lee, David Walt,
and Kyongbum Lee for the generous use of their facilities and
Stephen Kwok, Dr. Alenka Lovy-Wheeler, and Dr. Raghnild
Whitaker for their help with flow cytometry and microscopy.
We thank Eliza Vasile for maintaining the MIT/CCR Microscopy
and Imaging core facility. A.K.L.L. is a special fellow of the
Leukemia and Lymphoma Society. This work was supported in
part by the NIH (CA125033 and GM65500). The ESI-MS and
NMR facilities at Tufts are supported by the NSF (0320783 and
0821508). This paper is dedicated to Prof. Matthew Zimmt
(Brown University) on the occasion of his 50th birthday.
(28) Bilgic¸er, B.; Xing, X.; Kumar, K. J. Am. Chem. Soc. 2001, 123, 11815–
11816.
(29) Myers, K. E.; Kumar, K. J. Am. Chem. Soc. 2000, 122, 12025–12026.
(30) Krafft, M. P. AdV. Drug DeliVery ReV. 2001, 47, 209–228.
(31) Grant, A. M.; Hanson, P. K.; Nichols, J. W. Traffic 2008, 2, 37–50.
(32) Hanson, P. K.; Grant, A. M.; Nichols, J. W. J. Cell Sci. 2002, 115, 2725–
2733.
(33) Liu, X. Y.; Stocker, B. L.; Seeberger, P. H. J. Am. Chem. Soc. 2006, 128,
3638–3648.
(34) Smythe, E.; Warren, G. Eur. J. Biochem. 1991, 202, 689–699.
(35) Schmid, S. L.; Carter, L. L. J. Cell Biol. 1990, 111, 2307–2318.
(36) Heuser, J. E.; Anderson, R. G. W. J. Cell Biol. 1989, 108, 389–400.
(37) Hussey, S. L.; Peterson, B. R. J. Am. Chem. Soc. 2002, 124, 6265–6273.
(38) Powers, D. D.; Willard, B. L.; Carbonell, R. G.; Kilpatrick, P. K. Biotechnol.
Prog. 1992, 8, 436–453.
Supporting Information Available: Experimental procedures,
synthesis and analytical data for compounds, and movies of different
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