Charge-reversal amphiphiles for gene delivery

Article abstract of DOI:10.1021/ja0474906

Delivering a missing gene or a functional substitute of a defective gene has the potential to revolutionize current medical care. Of the two gene delivery approaches, viral and synthetic vectors, synthetic cationic vectors possess several practical advant

Full text of DOI:10.1021/ja0474906

Published on Web 09/08/2004
Charge-Reversal Amphiphiles for Gene Delivery
Carla A. H. Prata,† Yuxing Zhao,† Philippe Barthelemy,‡ Yougen Li,§ Dan Luo,§ Thomas J. McIntosh,|
Stephen J. Lee, and Mark W. Grinstaff*,†
Departments of Biomedical Engineering and Chemistry, Boston UniVersity, Boston, Massachusetts 02215,
Departement de Chimie, UniVersite´ d’AVignon, F-84000 AVignon, France, Department of Cell Biology,
Duke UniVersity Medical Center, Durham, North Carolina 27710, Department of Biological and EnVironmental
Engineering, Cornell UniVersity, Ithaca, New York 14853, and Army Research Office, RTP, North Carolina 27709
Received April 29, 2004; E-mail: mgrin@bu.edu
Gene therapy offers the potential to cure a wide range of diseases
by delivering a missing gene or a functional substitute of a defective
gene.1-6 The two most common methods for gene delivery use
either viral7,8 or synthetic vectors.1-4,9,10 Viruses are efficient carriers
of genes, but there are risks associated with their clinical applica-
tion.8 Consequently, there is intense activity in developing and
evaluatingsyntheticnonviralvectors,includingcationicamphiphiles,10-16
Figure 1. Proposed mechanism for release of DNA from the charge-reversal
amphiphiles. A supramolecular assembly is formed between the DNA and
the multiwalled vesicles of the amphiphile. Upon enzymatic hydrolysis of
linear polymers,17-19 and dendrimers.20-22 Since the pioneering
research of Felgner, MacDonald, and Magee, cationic amphiphiles
have been investigated because of their low toxicity, nonimmuno-
genicity, and ease of synthesis; today, these amphiphile vectors are
in clinical trials. Yet with these amphiphiles, the gene transfection
activity is low, reflecting inefficiencies in the overall transfection
pathway that includes DNA-synthetic vector complexation, en-
docytosis, endosomal escape, nuclear entry, and finally, expression.
Our research effort is focused on improving the release of DNA
from the DNA-amphiphile supramolecular complex. Herein we
describe and characterize a functional amphiphile for gene delivery
that undergoes an electrostatic transition intracellularly from cationic
to anionic and shows enhanced gene transfection efficiency.
This charge-reversal amphiphile performs two roles: first, it binds
and then releases DNA, and second, as an anionic multicharged
amphiphile, it destabilizes bilayers. Thus, the amphiphile undergoes
the following reactions: it complexes plasmid DNA and forms a
supramolecular DNA-cationic amphiphile assembly; upon entering
the cell (e.g., via endocytosis) esterases hydrolyze the terminal ester
linkages to afford anionic amphiphiles; and finally, the anionic
amphiphiles repel DNA and disrupt the lipid bilayer of the
supramolecular complex releasing the plasmid DNA for subsequent
transcription (Figure 1). This approach, which benefits from a
change in electrostatic forces to release DNA, departs from previous
functional vectors.23-32 The aforementioned sequential steps require
a cationic amphiphile possessing three distinct structural compo-
nents: a cationic headgroup, hydrophobic chains, and terminal ester
linkages. The prototype amphiphile, 1, has a cationic ammonium
headgroup to bind DNA, lipophilic acyl chains to form a bilayer,
and benzyl esters at the terminus of the acyl chains for enzymatic
hydrolysis (Figure 2). To assess the role of each structural
component, we prepared compounds 2-4. Compounds 1 through
4 were synthesized as described in the Supporting Information.
To determine whether the amphiphiles bind DNA, we performed
a standard ethidium bromide-DNA fluorescence quenching exclu-
sion assay. DNA binding is observed for 1, 3, and 1,2-dioleoyloxy-
3-(trimethylammonio)-propane (DOTAP, 5) as the fluorescence
intensity decreases rapidly, but not for 2 or 4 (Figure 3A).
the terminal esters of this amphiphile, the DNA is released from the
assembly by the newly formed anionic amphiphiles. Not drawn to scale.
Figure 2. Amphiphiles under investigation for gene delivery.
Figure 3. (A) Ethidium bromide displacement assay showing the fluores-
cence intensity as a function of synthetic vector/DNA charge ratio for
compounds 1-4 and DOTAP. (B) Ethidium bromide displacement assay
showing the fluorescence intensity as a function of time in the presence of
a porcine liver esterase (300 units/mL).
Amphiphile 1 forms a 1:1 complex with a binding constant of
∼10-7 M,-1 similar in magnitude to DOTAP.33 Amphiphile 2 is
anionic and does not bind DNA as a consequence of unfavorable
electrostatic interactions. Compound 4 possesses a cationic charge
but lacks the hydrophobic acyl chains also required for formation
of a strong interaction with DNA. Next, the above DNA/EtBr/
amphiphile (1, 3, or DOTAP) solution was incubated with an
esterase at pH 7.4 and 37 °C (100 mM NaCl, 100 mM Tris buffer).
† Boston University.
‡ Universite´ d’Avignon.
§ Cornell University.
| Duke University Medical Center.
Army Research Office.
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10.1021/ja0474906 CCC: $27.50 © 2004 American Chemical Society

C O M M U N I C A T I O N S
results also highlight the importance and sensitivity of noncovalent
interactions in the formation and dissolution of supramolecular
DNA-amphiphile assemblies. Current studies are focused on
optimizing the structure of the charge-reversal amphiphile and the
formulation conditions to further increase the gene transfection
efficiency. Importantly, these amphiphiles represent a conceptual
departure from the current cationic molecules under investigation,
and these results are likely to facilitate the design, development,
and evaluation of new synthetic nonviral vectors for the delivery
of therapeutic DNA.
Figure 4. Gene transfection results after 48 h in CHO cells.
Acknowledgment. This work was supported by the Army
As shown in Figure 3B, an increase in fluorescence over time is
seen for amphiphile 1, consistent with hydrolysis of the terminal
benzyl esters and disruption of the DNA-amphiphile supramolecu-
lar complex followed by re-intercalation of EtBr in DNA.34 No
increase in fluorescence over time is observed with amphiphiles 3 or
DOTAP, which possess a terminal amide linkage or methyl group,
respectively. The fluorescence data indicate that release of DNA
from the supramolecular assembly does not occur with amphiphiles
lacking a terminal hydrolyzable ester linkage and those linkages near
the cationic headgroup are less accessible to enzymatic hydrolysis.
Given the polar headgroup and long hydrophobic acyl chains
present in the structure of 1, this amphiphile is likely to form bilayer
vesicles in aqueous solution. Dispersion of 1 in water by sonication
leads to vesicles in the presence and absence of DNA. Vesicles
are not observed with the anionic amphiphile 2 (multicharged
surfactant) or cationic compound 4, which lacks the necessary
hydrophobic chains necessary for bilayer formation. A differential
scanning calorimetry (DSC) trace of hydrated amphiphile 1 shows
a phase-transition temperature at approximately 55 °C.
The X-ray diffraction spectrum at 25 °C of the hydrated vesicle
pellet of 1 shows three diffraction orders of a lamellar repeat period
of 5.22 ( 0.03 nm with a sharp wide-angle spacing of 0.46 ( 0.01
nm, which is characteristic of an ordered bilayer phase. Upon
addition of DNA, the lamellar repeat period (d ) 5.31 ( 0.14 nm)
and wide-angle spacing (0.46 ( 0.01 nm) do not significantly
change. This suggests a model, like that shown in Figure 1, where
the DNA is entrapped at the surface or at the interface between
multiwalled vesicles in solution. This is a different structural model
than for complexes of DNA with either DOTAP35 or cationic
triesters of phosphatidylcholine,16 where a smectic phase is formed
with the DNA chains located between the adjacent lipid bilayers
within the multilamellar liposome. Moreover, adding 1% w/w of
the multicharge anionic amphiphile 2 to DOPE bilayers affords a
broader phase-transition temperature compared to pure DOPE. This
result is consistent with the anionic surfactant, 2, formed in the
hydrolysis reaction of 1, destabilizing lipid bilayers.
Transfections experiments using the reporter gene, â-galactosi-
dase (â-gal, pVax-LacZ1, invitrogen) were performed with chinese
hamster ovarian (CHO) cells (see Supporting Information). As
shown in Figure 4, cationic amphiphile 1 was the most effective
vector for transfecting the â-galactosidase gene. Significantly,
compounds 2 through 4 showed minimal transfection activity
comparable to the negative control and naked DNA. DOTAP and
TransFast reagent both transfect DNA, but at lower levels. The
results observed with anionic amphiphile, 2, and cationic compound,
4, are consistent with the poor affinity of these compounds to bind
DNA. The lack of transfection with amphiphile 3 conveys the
important role the cleavable terminal ester linkages perform in these
amphiphiles. Preliminary screening on additional cells lines showed
that 1 can also facilitate the transport of DNA in human embryonic
kidney (HEK293) and erythroleukemic (K562) cell lines.
Research Office (M.W.G.) and Grant GM27278 from the National
Institutes of Health (T.J.M.). D.L. acknowledges Erin King for
technical assistance and the USDA and Cornell Advanced Center
for Biotechnology for financial support. We also thank Drs. Jun-
lin Guan and Zara Melkoumian for providing the cell lines.
Supporting Information Available: Complete experimental details,
characterization data, and cytotoxity experiments. This material is
available free of charge via the Internet at http://pubs.acs.org.
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In summary, a charge-reversal or charge-switchable amphiphile
is shown to be an effective nonviral vector for gene delivery. These
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