Cationic amphiphile with shikimic acid headgroup shows more systemic promise than its mannosyl analogue as DNA vaccine carrier in dendritic cell based genetic immunization

Article abstract of DOI:10.1021/jm901295s

Mannosylated cationic vectors have been previously used for delivering DNA vaccines to antigen presenting cells (APCs) via mannose receptors expressed on the cell surface of APCs. Here we show that cationic amphiphiles containing mannose-mimicking quinic

Full text of DOI:10.1021/jm901295s

J. Med. Chem. 2010, 53, 1387–1391 1387
DOI: 10.1021/jm901295s
Cationic Amphiphile with Shikimic Acid Headgroup Shows More Systemic Promise Than Its Mannosyl
Analogue as DNA Vaccine Carrier in Dendritic Cell Based Genetic Immunization
Ramishetti Srinivas,†, Priya P. Karmali,†,^ Dipankar Pramanik,†,# Arup Garu,† Yenugonda Venkata Mahidhar,†,¥
Bharat K. Majeti,†,ꢀ Sistla Ramakrishna,‡ Gunda Srinivas,§ and Arabinda Chaudhuri*,†
†
‡
Division of Lipid Science and Technology, and Pharmacology Division, Indian Institute of Chemical Technology, Hyderabad 500 007, India, and
§Centre for Cellular and Molecular Biology, Hyderabad, 500 607, India. Present address: School of Pharmacy, University of North Carolina,
Chapel Hill, NC 27599-7360. ^Present address: Cancer Research Center, The Burnham Institute, La Jolla, CA 92037. #Present address:
Department of Pathology, Johns Hopkins University School of Medicine, 1550 Orleans Street, Baltimore, MD 21231. ¥Present address: Drug
Discovery Program, Lombardi Comprehensive Cancer Center, Washington, D.C. 20057. ꢀPresent address: Department of Pathology, University
of California, San Diego, School of Medicine and Moores UCSD Cancer Center, 3855 Health Sciences Drive No. 0803, La Jolla, CA 92093-0803.
Received August 29, 2009
Mannosylated cationic vectors have been previously used for delivering DNA vaccines to antigen
presenting cells (APCs) via mannose receptors expressed on the cell surface of APCs. Here we show that
cationic amphiphiles containing mannose-mimicking quinic acid and shikimic acid headgroups deliver
genes to APCs via mannose receptor. Cationic amphiphile with shikimic acid headgroup was more
efficacious than its mannosyl counterpart in combating mouse tumor growth by dendritic cell (the most
professional APC) based genetic immunization.
Introduction
immunization,6b and mannosylated cationic liposomes has
been reported for delivering DNA vaccine to APCs.6c-e
Grandjean and co-workers have demonstrated that lysine
based clusters of mannose mimicking carbocyclic acids such
as quinic and shikimic acid are also effective ligands for the
mannose receptor of dendritic cells.7a,b Using a model lipopep-
tide vaccine, they have demonstrated that such mannose
mimicking ligands hold potential for solubilizing lipopeptide
vaccines (which are otherwise prone to aggregation), and the
resulting mixed micelles are taken up mainly via endocytosis by
DCs in vitro.7c However, systemic studies aimed at exploring
the use of cationic amphiphiles containing such mannose-
mimicking shikimic and quinic acid headgroups have not yet
been undertaken. To this end, herein we show that cationic
mannose-mimicking amphiphiles with quinic and shikimic acid
headgroups (1 and 2, respectively, Figure 1) can target DNA to
antigen presenting cells via mannose receptors. Importantly,
we show that subcutaneous administration of DCs pretrans-
fected with electrostatic complex of pCMV-MART1 plasmid
DNA (encoding MART1 antigen of human melanoma tumor)
and cationic liposomes of mannose mimicking amphiphile with
shikimic acid headgroup (2) provides more tumor protective
effect in C57BL/6 mice challenged with aggressive B16F1
melanoma tumor than subcutaneous administration of the
corresponding lipoplex of mannosylated cationic glycolipid
(3, Figure 1). Taken together, the present findings demonstrate
for the first time that use of a cationic amphiphile with
mannose-mimicking shikimic acid headgroup provides en-
hanced therapeutic benefits compared to its mannosyl analo-
gue in the emerging field of DC-based DNA vaccination.
Development of vaccines is considered as one of the most
remarkable triumphs of medical science. Traditionally, vac-
cines comprise proteins, live attenuated viruses, or killed
bacteria. More recently, DNA vaccination, the administration
of antigen encoded DNA, is gaining increasing attention as an
emerging therapeutic approach for the treatment of many
complex disorders including cancer, infectious disease, and
allergies.1a,b DNA vaccines are capable of inducing humoral
and cellular immune responses and are regarded as potentially
safer than their attenuated virus counterparts.2 To elicit an
immune response, the antigen encoded DNA first needs to be
captured by body’s antigen presenting cells (APC) including
dendritic cells (DC), macrophages, and B-lymphocytes. Trans-
fected APCs can then process the expressed antigenic proteins
through its proteosome complexes into small peptide frag-
ments and can present these small peptide fragments to
immune systems (CD8þ and CD4þ T-lymphocytes) in a
recognizable fashion in complexation with major histocom-
patibility complexes (MHC) class I and class II molecules.3a-d
However, antigen presenting cells are hard to transfect. Use of
cationic microparticles,4a,b cationic liposomes,4c and cationic
peptide,4d etc. have previously been reported for direct trans-
fection of APCs in DNA vaccination.
An emerging approach for enhancing the efficacy of genetic
immunization is based on targeting DNA vaccines to APCs
via mannose receptor, a 180 kDa multidomains unique trans-
membrane receptors expressed on their cell surfaces.5 For
instance, use of mannan (a ligand for the mannose receptor)
coated liposomes for intranasal delivery of HIV-1 DNA
vaccine,6a mannan-coated cationic nanoparticles for topical
Results and Discussion
We synthesized two cationic amphiphiles containing
mannose-mimicking quinic acid (1) and shikimic acid (2)
*To whom correspondence should be addressed. Phone: þ91-40
-27193201. Fax: þ91-40-27193370. E-mail: arabinda@iict.res.in.
r
2010 American Chemical Society
Published on Web 01/05/2010
pubs.acs.org/jmc

1388 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Srinivas et al.
Figure 1. Syntheses of mannose-mimicking cationic ampiphiles 1 and 2 and their mannosyl analogue 3.
headgroups as shown schematically in Figure 1A. Briefly,
the quinic and shikimic acids were first converted to their
tetra-O-acetyl and tri-O-acetyl derivatives, respectively. The
acetyl derivatives of quinic and shikimic acids (intermediates
I and II, respectively, Figure 1A) upon peptide coupling
with N-2-aminoethyl-N,N-di-n-hexadecylamine followed by
sequential quaternization with methyl iodide, acetyl deprotec-
tion with sodium methoxide/methanol, and chloride ion
exchange over amberlyst resin afforded the target cationic
mannose mimicking amphiphiles (Figure 1A).
The control mannosylated cationic glycolipid (3, Figure 1B)
was synthesized by selective deprotection of the anomeric
O-acetyl group of penta-O-acetyl-D-mannose followed by
conversion of the anomeric OH group to its trichloroacetami-
dine derivative. The resulting trichloroacetamidine derivative
upon reaction with N-2-hydroxyethyl-N,N-di-n-hexadecyla-
mine afforded intermediate III (Figure 1B). The intermediate
III, upon quaternization with methyl iodide followed by acetyl
deprotection with K2CO3/methanol and chloride ion exchange
over amberlyst resin afforded the control mannosylated catio-
nic glycolipid (3, Figure 1B).
ished when RAW 264.7 cells were pretreated with mannan, a
natural ligand of mannose receptor (Figure 2B). No such
decrease of transfection efficiencies in presence of mannan
was observed in NIH 3T3 cells (mouse fibroblast cells used as
a control non-APCs). Rather the transfection efficacies in
NIH3T3 cells were found to be somewhat enhanced presum-
ably because of some favorable nonspecific interactions be-
tween the lipoplexes and thecellsurface receptors (Figure2C).
We also carried out cellular uptake experiments using lipo-
plexes of lipids 1-3 containing fluorescently (FITC) labeled
plasmid DNA and using lipoplexes prepared with fluores-
cently labeled (Rho-PE) cationic liposomes in RAW 264.7
cells. In both these cellular uptake experiments, the number of
fluorescently labeled cells was found to be remarkably less
when cells were preincubated with mannan than for untreated
cells (Figures S12-S17, Supporting Information). Such find-
ings in cellular uptake experiments and the results summar-
ized in Figure 2 are consistent with the supposition that
the mannose mimicking amphiphiles 1 and 2 and the control
mannosylated cationic glycolipid 3 deliver DNA to APCs via
mannose receptor.
To establish that the mannose mimicking cationic amphi-
philes 1 and 2 and the control mannosylated cationic amphi-
phile 3 target DNA to APCs via mannose receptor, first we
evaluated their transfection efficiencies in RAW 264.7 cells
(murine macrophage cells as a model antigen presenting cells)
using luciferase reporter gene expression assay. All three
amphiphiles in combination with equimolar cholesterol as
colipid were transfection efficient across the entire lipid/DNA
charge ratios 1:1 to 8:1 (Figure 2A), and the transfection
efficacies of all the amphiphiles 1-3 were significantly dimin-
Among the various APCs, DCs are the most professional
antigen presenting cells (since they express the costimulatory
molecules, e.g., CD86, necessary for efficient antigen presen-
sation to naive T cells) and play a very critical role in antigen
presentation under systemic settings.8 The therapeutic ap-
proach based on expressing tumor associated antigen
(TAA) or viral antigen in DCs is finding increasing exploita-
tion ingenetic immunization for treating cancerandinfectious
diseases.4d,9a-c Since such approach of using DCs pretrans-
fected with expression constructs for TAA genes is effective

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1389
Figure 2. (A) Transfection efficiencies of 1-3 in RAW 264.7 cells with equimolar cholesterol as colipid across the lipid/DNA charge ratios
1:1 to 8:1. The RLU (% control) values (filled bars) refer to the average values of the RLU/mg of protein (done in triplicate) in the presence of
mannan compared to the average values of the RLU/mg of protein (done in triplicate) in the absence of mannan (taken as 100, open bars) in
RAW 264.7 cells (B) and in NIH3T3 cells (C) using lipid/DNA charge ratios of 8:1.
Figure 3. (A) Humoral immune responses in C57BL/6J mice upon subcutaneous administration of mbmDCs pretransfected with lipoplexes
of 1-3 and p-CMV-β-gal as a model genetic vaccine. The 6-8 week old female C57BL/6 mice (each weighing 20-22 g, n = 3) were immunized
subcutaneously with 5 ꢀ 105 cells of pretransfected mbmDCs (twice with a 7-day interval). Two weeks after second immunization, serum
samples were collected from mice and assayed for β-gal antibodies by ELISA. The Y-axis represents absorbance obtained with a 1:300 dilution
of serum: (/) P < 0.005 for lipids 1 and 2 compared with values for untreated mbmDCs. (B, C) Humoral (Th2) and cellular (Th1) immune
responses in C57BL/6J mice upon subcutaneous administration of mbmDCs pretransfected with lipoplexes of 1-3 and the therapeutic DNA
vaccine pCMV-MART1. Subcutaneous administration of mbmDCs pretransfected with pCMV-MART1 lipoplexes of 1-3 elicit Th1 and Th2
immune responses. The 6-8 week old female C57BL/6 mice (each weighing 20-22 g, n = 5) were immunized subcutaneously with 5 ꢀ 105
mbmDCs (twice with a 7-day interval). Two weeks later, mice were sacrificed to isolate splenocytes and used immediately (without invitro
restimulation) in ELISA assays for IFN-γ (B, (/) P < 0.01, (//) P < 0.05, (///) P < 0.0005) and for IL-4 (C, (/) P < 0.05, (//) P < 0.01, (///)
P < 0.01). /, //, /// refer to comparison between mannose mimicking amphiphile 2 and 1, 2 and 3, and 2 and untreated mbmDCs, respectively.
and safe and because DCs, the most professional antigen
presenting cells, express high levels of mannose receptors on
their cell surface,5 we next measured the abilities of 1-3 to
transfect DCs using pR5GFP (plasmid DNA encoding green
fluorescence protein). First, we confirmed the expression of
major histocompatibility complex II (MHC-II, surface and
intracellular) and the costimulatory molecules CD86, CD11c,
CD40 and the mannose receptors (the common immature DC
markers) in mouse bone marrow derived DCs (mbmDCs)
isolated from the bone marrow of male C57BL/6 mice by flow
cytometry (Figure S10, Supporting Information). Although
low in efficiency (∼3%), flow cytometric analysis confirmed
expression of GFP in mbmDCs transfected by lipids 1-3
(Figure S11, Supporting Information). It is worth mentioning
that mbmDCs are hard to transfect and successful genetic
immunization has previously been reported using mbm DCs
transfected with cationic peptide, CL22, with DC transfec-
tion efficiency as low as 1%.4d Before probing the relative
therapeutic potentials of 1-3 in genetic immunization, next
we examined whether subcutaneous administration of the
mbmDCs transfected with the lipoplexes of 1-3 and pCMV-
SPORT-β-gal plasmid (as a model DNA vaccine) was com-
petent in inducing antigen-specific humoral response in vivo.
Importantly, mbmDCstransfected withthe mannose mimick-
ing amphiphiles 1 and 2 were found to be more efficient than
the mbmDCs transfected with mannosylated lipid 3 and
untreatedmbmDCsinelicitinganti-β-Galactosidaseantibody
responses (Figure 3A).
Finally, we evaluated the therapeutic potentials of the
mannose mimicking amphiphiles 1 and 2 and their mannosy-
lated counterpart 3 under systemic settings in DC-based
genetic immunization. Human MelanA/Mart1 antigen shares
68.6% amino acid sequence identity with its murine counter-
part10 and has been used in inducing immune response
in mouse against the murine B16 melanoma.11a,b Subcuta-
neous administration of the pCMV-MART1 plasmid DNA
encoding the human melanoma MART1 antigen in com-
plexation with the mannose mimicking amphiphile 2 with
shikimic acid headgroup was found to be effective in inhibit-
ing the growth of the B16 melanoma in C57BL/6 mice when
the latter was challenged with aggressive B16F1 melanoma
tumor after genetic immunization (Figure 4). However, the
corresponding lipoplexes of the mannose mimicking amphi-
phile with quinic acid headgroup (1) and its mannosyl analo-
gue (3) were found to be significantly less efficient in inhibiting
the B16 melanoma tumor (Figure 4). Since cytokine produc-
tion and tumor regression in mice model have recently been
reported to be mediated by activation of toll-like receptor 9
(TLR9) by lipoplexes,14a,b we also carried out the tumor
challenge experiment using a control lipoplex prepared with
lipid 2 and a nonspecific plasmid DNA (pCMV-β-gal). The
tumor growth inhibition observed with such control lipoplex

1390 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3
Srinivas et al.
further mechanistic insights into the observed tumor rejection
property of lipoplex 2.
In conclusion, the present findings demonstrate that ca-
tionic amphiphiles containing mannose-mimicking quinic
acid and shikimic acid headgroups target DNA vaccines to
APCs via mannose receptor. Cationic amphiphile with
mannose-mimicking shikimic acid headgroup provides en-
hanced tumor protective therapeutic benefits compared to
its quinic acid and its mannosyl analogue in DC-based DNA
vaccination. Measurements of the amounts of the secreted
INF-γ and IL-4 from the spleenocytes of the immunized
mice indicated a mixed Th1/Th2 immune response to be
responsible for the observed tumor protective properties of
the mbmDCs transfected with pCMV-MART1/2 lipoplex.
Future structure-activity studies are expected to further
enhance the therapeutic benefits of cationic amphiphiles
containing mannose-mimicking shikimic/quinic acid head-
groups in DC-based genetic immunization.
Figure 4. Tumor growth inhibition efficiencies of lipoplexes of 1-3
and pCMV MART1 in DC based genetic immunization. The 6-8
week old female C57BL/6 mice (each weighing 20-22 g, n = 5) were
immunized by subcutaneous administration of mbmDCs pretrans-
fected with lipoplexes of 1-3 and p-CMV-MART1, twice with a
7-day interval. Two weeks after second dose mice were challenged by
subcutaneous injection of B16F1 cells. Tumor volumes (V = 1/2ab2
where a is maximum length of the tumor and b is minimum length of
the tumor measured perpendicular to each other) were measured
with a slide calipers for up to 30 days. Results represent the mean (
SD for n = 5 tumors: (/) P < 0.005 compared to the control
untreated mbmDCs.
Experimental Section
General Methods and Reagents. Mass spectral data were
acquired by using a commercial LCQ ion trap mass spectro-
meter (ThermoFinnigan, SanJose, CA) equipped with an ESI
source or micromass Quattro LC triple quadrapole mass spec-
trometer for ESI analysis. The FABMS analyses were per-
formed on a Micromass AUTOSPEC-M mass spectrometer,
Manchester, U.K. 1H NMR spectra were recorded on a Varian
FT 200 MHz or AV 300 MHz NMR spectrometer. Quinic
and shikimic acids were purchased from Fluka (Switzerland).
D-Mannose was procured from SD Fine Chemicals, Hyderabad,
India. EDCIa and HOBt were purchased from Sigma-Aldrich.
Column chromatography was performed with silica gel (Acme
Synthetic Chemicals, India, 60-120 mesh). The purities of all
the target lipids 1-3 were confirmed to be g95% by reversed
phase analytical HPLC analysis using two different mobile
phases (A, pure methanol; B, 95:5, v/v, methanol/water).
Preparation of Liposomes. The liposomes of 1-3 were pre-
pared by the conventional method. Briefly, lipid and cholesterol
(at a mole ratio of 1:1) were dissolved in chloroform. The solvent
was then evaporated under a thin stream of nitrogen gas,
vacuum-dried for 8 h, and hydrated in deionized water over-
night to give a final lipid concentration of 1 or 5 mM. Initial
optimization experiments revealed that liposomes of lipids 1-3
containing mixed colipids, namely, equimolar mixture of
DOPE, cholesterol, and DOPC, were more efficient than lipo-
somes prepared with only equimolar cholesterol in transfecting
dendritic cells (data not shown). The hydrated lipid film was first
vortexed for 30 s and then sonicated until clarity using a Branson
450 sonifier at 100% duty cycle and 25 W output power. The
resulting clear aqueous liposomes were used in preparing lipo-
plexes.
was remarkably less than that observed for pCMV-MART
1/lipid 2 lipoplex (Figure S18, Supporting Information).
ThetumorgrowthinhibitionresultssummarizedinFigure4
demonstrate the therapeutic promise of mannose mimicking
amphiphile with shikimic acid headgroup in DC-based DNA
vaccination. CD4þ Th cells exhibit their helper functions
through secreted cytokines. Differences in cytokine secretion
patterns among the two Th cell subsets (Th1 and Th2)
determine the type of immune response mounted against a
particular antigenic challenge. Th1 subset is responsible for
mounting cell-mediated immune response, e.g., activation of
TC cells, while Th2 subset stimulates humoral responses, e.g.,
activation of antibody producing B cells. Two defining cyto-
kines secreted by Th1 and Th2 cells are interferon γ (INF-γ)
and interleukin-4 (IL-4), respectively.12 To gain insights into
the contribution of each of these two subsets of Th cells in
providing melanoma tumor protection, we measured the
amounts of these two signature cytokines in mice spleenocytes
after genetic immunization with DCs pretransfected with
lipoplexes of pCMV-MART1 plasmid. While the amount of
secreted INF-γ was higher for lipoplex of lipid 1 than for the
lipoplexes of lipids 2 and 3 (Figure 3B), lipoplex of the
mannose mimicking amphiphile 2 provided higher amount
of IL-4 than lipoplexes of 1 and 3 (Figure 3C). Thus, a mixed
Th1/Th2 immune response is likely to be responsible for the
observed tumor protective properties of the mbmDCs trans-
fected with pCMV-MART1/2 lipoplex. Although IL-4 might
be required for tumor rejection in some animal models, in
general, INF-γ is more important than IL-4 in mediating
tumor rejection. It is not obvious at this stage of investigation
why IL-4 rather than INF-γ mediates tumor rejection in the
present model. Clearly, further mechanistic studies need to be
undertaken in the future toward gaining more insight to this
end. In addition, future studies aimed at probing possible role
of cytotoxic T cells and natural killer (NK) are likely to throw
Statistical Analysis. Data are represented as the mean ( SD
and were compared among different groups using the Student
t test. p < 0.05 was considered as significant.
Acknowledgment. We thank Dr. Van den Eynde, Ludwig
Institute for Cancer Research, Brussels, Belgium, Dr. N. M.
Rao and Dr. G. Pande, Centre for Cellular and Molecular
Biology, Hyderabad, India, for providing pCMV-MART1,
p-CMV-SPORT-β-Gal, and pCMV-Luc plasmids. We thank
aAbbreviations: EDCI, 1-ethyl-3-(3-dimethylaminopropyl)carbodii-
mide hydrochloride; HOBt, 1-hydroxybenztriazole; Chol, cholesterol;
DCM, dichloromethane; DMEM, Dulbecco’s modifid Eagles medium;
DMF, N,N-dimethylformamide; FBS, fetal bovine serum; PBS, phos-
phate buffered saline; DOPE, 1,2-dioleoyl-sn-glycero-3-phasphoetha-
nolamine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; mbm DC,
mouse bone marrow derived dendritic cells.

Brief Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 3 1391
Dr. Ashok Khar, Centre for DNA Finger Printing and
Diagnostics, Hyderabad, India, for helping us with the DC
isolation procedure from mouse bone marrow. R.S., D.P.,
Y.V.M., B.K.M. and A.G. thank the Council of Scientific and
Industrial Research, Government of India, New Delhi, and
P.P.K. thanks University Grant Commission, Government
of India, New Delhi, for providing doctoral research fellow-
ships. A.C. thanks Department of Biotechnology and the
Council of Scientific and Industrial Research, Government
of India, New Delhi (Grant NWP-0036), for providing finan-
cial support.
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Supporting Information Available: Detailed descriptions for
syntheses of lipids 1-3, 1H NMR, high resolution mass spectra,
reverse phase HPLC chromatograms, and HPLC conditions in
two mobile phases for the cationic amphiphiles 1-3, details for
transfection and cellular uptake experiments, isolation of mbm
DCs, DC-based immunization of mice, ELISA assays, and
FACS protocol. This material is available free of charge via
the Internet at http://pubs.acs.org.
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