Enhanced Spacer Length between Mannose Mimicking Shikimoyl and Quinoyl Headgroups and Hydrophobic Region of Cationic Amphiphile Increases Efficiency of Dendritic Cell Based DNA Vaccination: A Structure-Activity Investigation

Article abstract of DOI:10.1021/acs.jmedchem.6b01556

In the field of dendritic cell based genetic immunization, previously we showed that liposomes of cationic amphiphiles containing mannose-mimicking shikimoyl headgroup are promising DNA vaccine carriers for dendritic cell (DC) transfection. The present structure-activity study reports on the influence of spacer length (between mannose-mimicking headgroups and quaternary nitrogen centers) in modulating the DC-transfection efficiencies. Further, we report on the anti-melanoma immune response inducing properties of the promising cationic amphiphiles in syngeneic C57BL/6J mice under prophylactic settings.

Full text of DOI:10.1021/acs.jmedchem.6b01556

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Brief Article
Enhanced Spacer Length between Mannose Mimicking
Shikimoyl- and Quinoyl- Head Groups and Hydrophobic Region
of Cationic Amphiphile Increases Efficiency of Dendritic Cell
based DNA Vac-cination: A Structure-Activity Investigation
Chandrashekhar Voshavar, Rakeshchandra Reddy Meka, Sanjoy
Samanta, Srujan Kumar Marepally, and Arabinda Chaudhuri
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01556 • Publication Date (Web): 26 Jan 2017
Downloaded from http://pubs.acs.org on January 28, 2017
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Enhanced Spacer Length between Mannose Mimicking Shikimoyl-
and Quinoyl- Head Groups and Hydrophobic Region of Cationic
Amphiphile Increases Efficiency of Dendritic Cell based DNA Vac-
cination: A Structure-Activity Investigation
Chandrashekhar Voshavar† *, Rakesh C. R. Meka† , Sanjoy Samanta† , Srujan Marepally† , and
Arabinda Chaudhuri † § *
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† Biomaterials group, CSIR-Indian Institute of Chemical Technology, Hyderabad 500 007, India
§ Academy of Scientific and Innovative Research (AcSIR), Taramani, Chennai 600 113, India
KEYWORDS. Liposomal DNA vaccine carriers; cationic transfection lipids; dendritic cells; DNA vaccination; man-
nose-mimicking ligands.
ABSTRACT: In the field of dendritic cell based genetic immunization, previously we showed that liposomes of cationic
amphiphiles containing mannose-mimicking shikimoyl head-group are promising DNA vaccine carriers for DC transfec-
tion. The present structure-activity study reports on the influence of spacer length (between mannose-mimicking head
groups and quaternary nitrogen centers) in modulating the DC-transfection efficiencies. Further, we report on the anti-
melanoma immune response inducing properties of the promising cationic amphiphiles in syngeneic C57/BL6J mice un-
der prophylactic settings.
Introduction
lysine moiety between the shikimoyl- & quinoyl- head
groups and hydrophobic region of cationic amphiphiles
DNA immunizations in animal models have been used to
elicit protective immunity against various infectious
possess significantly enhanced DC-transfection effica-
17
cies.16,
However, systematic structure-activity studies
pathogens and malignancies.1, Generation of cellular
2
aimed at probing the role of varying methylene spacers
between quaternary nitrogen center and glycomimicking
head groups in modulating DC transfection efficacies
have not yet been undertaken. To this end, herein we
report on the design, synthesis and bio-activity evaluation
of a novel series of cationic amphiphiles (1-10, Fig. 1A) in
which the spacer lengths between the mannose-
mimicking shikimoyl- or quinoyl- head groups and the
hydrophobic tails have been varied using amino acids
containing 1-5 methylene units. We show that ex vivo
DC-transfection efficiencies can be modulated by increas-
ing the number of methylene groups. Further, we show
that ex vivo immunization with melanoma antigen
(MART1) encoded DNA vaccine with liposomes of the
most efficient DC-transfecting lipids containing five
methylene units in the spacer arm induces long lasting
anti-melanoma immune responses in C57BL/6J mice.
immune response against complex diseases in humans
indicated the possibility for clinical application of this
immunization technique.3 The stability, ease of produc-
tion, strong induction of immunity and long lasting cyto-
toxic T lymphocyte (CTL) responses make DNA vaccines
as an attractive strategy compared to traditional vac-
cines.4 Dendritic cells (DCs) are known to be professional
antigen presenting cells (APCs) that play a critical role in
6
the induction and regulation of immune responses.5,
Dendritic cell based genetic immunization (DNA vaccina-
tion) involves administration of tumor antigen encoded
4,
7
plasmid DNA which primes the immune system.3,
Such DC-based genetic immunizations are being increas-
ingly used in cancer immunotherapy.8, 9 Several different
DNA vaccine carriers, both viral and non-viral, have been
developed for ex vivo loading of DCs.10, 11 Over expression
of endocytic mannose receptors (MRs) on DC cell surfac-
es has been elegantly exploited in the past for developing
efficient DNA vaccine carriers in DC-based immunother-
apy.12-14 Previously, we demonstrated that liposomes of
cationic amphiphiles containing mannose-mimicking
shikimoyl- and quinoyl- head-groups are promising DNA
vaccine carriers for DC-transfection.15 Subsequently we
showed that introduction of a lysine or a guanidinylated-
Results and Discussion
Chemistry. Synthetic routes adopted for preparing the
mannose receptor specific glycomimicking cationic am-
phiphiles 1-10 and control mannosyl analog 11 are shown
schematically in Fig. 1A & Fig. S2, respectively. Lipids 1-
10 were synthesized by coupling O-acetylated
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B.
Figure 1A. Synthesis of mannose receptor targeting cationic
amphiphiles 1-10.
Figure 2. Flow cytometric GFP expression profiles in
Figure 1B. Structures of control mannosyl analog (Lipid 11)
and co-lipid (Lipid 12).
mbmDCs transfected with lipoplexes of lipids 1-12 & pα5GFP
(A). Reduced GFP expression in mbmDCs pre-incubated
with mannan for lipoplexes of lipids 5, 10 & 11 & pα5GFP (B).
Filled traces are for untreated control mbmDCs.
shikimic acid and quinic acids to the 1o amine group of
the hydrophobic derivatives of glycine, β-alanine, γ-amino
butyric acid, 5-amino valeric acid and 6-amino caproic
acid (Intermediates IIA, Fig. 1A) followed by deacetyla-
tion of the resulting product (Fig. 1A). Briefly, N-BOC
protected amino acids (IA, Fig. 1A) upon peptide coupling
with N-2-aminoethyl-N,N-di-n-hexadecylamine followed
by acid mediated N-BOC deprotection afforded primary
amine intermediates IIA (Fig. 1A). The intermediates IIA
upon coupling with O-acetyl protected shikimic and
quinic acids15 provided intermediates IIIA (Fig. 1A).
Quaternization of intermediate IIIA with excess methyl
iodide, O-acetyl deprotection of the resulting quaternized
iodides with potassium carbonate & methanol, and finally
chloride ion exchange chromatography of the deacetylat-
ed products over amberlite IRA 400 Cl- resin afforded the
title/target mannose mimicking cationic amphiphiles 1-10
(Fig. 1A). The control mannosyl analog (Lipid 11, Fig. 1B)
was synthesized from D-Mannose using the synthetic
scheme shown in Fig. S2, supporting information. The
details of the synthetic procedures, spectral characteriza-
tions and reverse phase HPLC profiles of all the target
compounds (Lipids 1-11) are provided in the supporting
information.
-nally used co-lipids namely, cholesterol and DOPE (1,2-
dioleoyl-sn-glycero-3-phosphoethanola-mine). However,
we did not observe significant lipid:DNA binding proper-
ties (representative DNA binding data with Cholesterol as
co-lipid are shown in Fig. S3). Subsequently, instead of
cholesterol or DOPE, we used lipid 12 as co-lipid which
previously showed high DNA binding, membrane fuso-
genicity and stability profiles.18 Lipids 1-11 showed high
DNA binding properties when lipid 12 was used as co-
lipid (Fig. S4A). DNAse I sensitivities were evaluated for
the lipoplexes of transfection efficient lipids 5, 10 & 11
along with lipid 12 which further confirmed the strong
DNA binding properties of these lipids across the li-
pid:DNA charge ratios 8:1-1:1 (Fig. S4B). Interestingly, the
sizes of the liposomes of lipids 1-12 were found to be large
in general (240-980 nm) and surface potentials varied
across the range 3-40 mV (Table S1). Similarly, the sizes
of lipoplexes were found to be large (300-900 nm) across
the lipid:DNA charge ratio 8:1-2:1 (Table S2A) and the
global surface potentials of all the lipoplexes within the
charge ratios 8:1-2:1 were found to be positive within the
range of 4-16 mV (Table S2B). It is important to note
that the dynamic light scattering experiments provide
hydrodynamic radii of the liposomal aggregates.
Physico-chemical characterizations. First, we exam-
ined the DNA binding properties of the liposomes of li-
pids 1-10 (100-350 nm size) prepared with two conventio-
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ILꢀ4
IFNꢀγ
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Figure 3. Cellular (A) and Humoral (B) immune responses in C57BL/6J mice (n = 4) s.c. immunized with mbmDCs pre-
transfected with lipoplexes of lipids 5, 10-12 and pCMV-MART1. Splenocytes were isolated and used directly without any in vitro
re-stimulation for measuring secreted IFN- γ (A, *P< 0.001) and IL-4 (B, *P< 0.005). (C) Target cell lysis by CD8+ T cells from
immunized mice. Untreated mbmDCs were used as controls. Experimental details are as described in supporting information.
Since the quinoyl head-groups of lipids 6-10 possess four
hydroxyl groups compared to three hydroxyl group in
lipids 1-5 with shikimoyl- head groups, the liposomes of
lipids 6-10 could be more hydrated than liposomes of
lipids 1-5. Thus, the observed higher size range for the
liposomes of lipid 6-10 than those for lipids 1-5 may
partly originate from more water of hydration in the
head-group region. Similarly, the co-lipid 12 used in the
present study, in addition to having one hydroxyl head-
group, contains two amide linkers near its polar head-
group region which may bring additional water of hy-
dration compared to liposomes prepared using choles-
terol as co-lipid. The observed higher size range of lipo-
somes prepared with lipid 12 as co-lipid than those for
liposomes prepared with cholesterol as co-lipid (con-
tains only one hydroxyl head-group) may also owe its
origin to higher degree of head-group hydration.
In vitro DC Transfection Biology. Isolated dendritic
cells from mouse bone marrow (femur & tibia) were first
subjected to phenotypic analysis using cell surface
markers unique to DCs by flow cytometry. After con-
firming the expressions of the distinguishing DC-surface
markers (including Mannose receptor, MHC-II, F4/80,
H2kB, CD86, CD11c, CD45R and CD40) in the isolated
mbmDCs (Fig. S5), we measured the DC-transfection
efficiencies of the lipids 1-12. The cultured mbmDCs
were transfected with lipoplexes of lipids 1-12 and
pα5GFP plasmid (plasmid DNA encoding green fluores-
cent protein) and the transfection efficiencies were
measured by flow cytometry. Lipids 5, 10 & 11 were
found to be the most efficient (up to 6-8% DC-
transfection efficiencies) (Fig. 2A). Importantly, the
liposomes of only lipid 12 (used as co-lipid in preparing
liposomes of lipids 1-11) were found to be essentially
transfection incompetent (Fig. 2A). Notably, the DC-
transfection efficiencies of lipids 5, 10 & 11 were found to
be significantly reduced (by >50%) in DCs pre-treated
with mannan, a high affinity natural ligand of mannose
receptor (Fig. 2B). This finding is consistent with man-
nose receptor selective uptake of the lipoplexes of lipids
5, 10 & 11 in DCs. Thus the findings summarized in Fig.
2A-B clearly show that the DC-transfection efficiencies
of cationic amphiphiles with mannose mimicking shi-
kimoyl- & quinoyl- head-groups increase with increasing
spacer unit length between the quaternary nitrogen at-
om and DC-targeting ligand. Toward gaining some in-
sights into whether the varying transfection efficiencies
of the lipoplexes of lipids 1-12 with different spacer
lengths (in between the polar head-groups and hydro-
phobic tails) owe their mechanistic origin to varying
cellular uptake efficiencies, we initially performed both
transfection and cellular uptake experiments (using Rh-
PE labeled liposomes) in mannose-receptor positive
mouse macrophage cells (RAW 264.7 cells, a model an-
tigen presenting cells). The degrees of cellular uptake of
the lipoplexes of Rh-PE labeled liposomes of lipids 1-12
were obtained from the average fluorescence intensities
of the transfected RAW264.7 cells using ImageJ soft-
ware.
Representative epifluorescence images from
which quantitative cellular uptake data were computed
using ImageJ software are shown in Fig. S6 for the lipo-
plexes of lipids 5, 10-12. Interestingly, for the lipid se-
ries 1-5 containing shikimoyl- head-groups, the most
efficient liposomes of lipid 5 also showed best cellular
uptake properties (now included in Fig. S7A-B). How-
ever, the degree of cellular uptake for the most efficient
liposomes of lipid 10 in the quinoyl- head-group con-
taining lipid series 6-10 was not found to be the highest
(Fig. S7A-B). Thus, cellular uptake is unlikely to play
the most important role in modulating the in vitro APC
transfection efficiencies of the presently described lipids
1-10. Presumably, subsequent intracellular events (such
as cytosolic release of plasmid DNA from endosomes,
endosomal/lysomal degradation of plasmid DNA, etc.)
also play some role in influencing the transfection prop-
erties of these lipids. Clearly, further studies need to be
carried out in future toward demonstrating the mecha-
nistic origin of the varying efficacies of lipids 1-10 in
transfecting antigen presenting cells under in vitro set-
tings.
Immune response assays. After confirming the DC-
transfection efficiencies of lipids 5, 10 & 11, we evaluated
their efficacies in mounting cellular immune response in
mouse model using lipid 12 as control. Initially, cellular
immune responses (anti β-gal response) in mice were
measured after subcutaneous administration of DCs
pre-transfected with lipoplexes of lipids 5 & 10-12 with
pCMV-SPORT-β-gal as a model DNA vaccine. ELISA
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assay with serum samples of immunized mice revealed in inducing both humoral and cellular immune respons-
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significant anti-β-gal antibody humoral immune re-
sponse for lipoplexes of lipids 5, 10 & 11 compared to
that in mice immunized with lipoplexes of lipid 12 (Fig.
S8). Next, IL-4 and IFN-γ (two well known signature
cytokines for humoral and cellular immune responses,
respectively)19 were evaluated with splenocytes collected
from mice subcutaneously immunized with DCs pre-
transfected with lipoplexes of lipids 5 & 10-12 using
pCMV-MART-1 DNA (encodes Human MelanA/MART1
antigen of human melanoma tumor where 68.6% of
amino acid sequence matches with its murine equiva-
lent).20, 21 The results showed that lipids 5 & 10 are capa-
ble of inducing significant cellular immune response
against MART-1 antigen as indicated by the elevated IL-
4 and IFN- γ secretion (Fig. 3A & B) than control lipids
11 & 12. Furthermore, cytotoxic T lymphocytes (CTL)
assay was carried using splenocytes isolated from the
mice immunized with pre-transfected DCs (using sple-
nocytes as effector cells and B16F10 cells as target cells).
Data from CTL assay revealed significant MART-1 anti-
gen specific T-cell responses for lipids 5 & 10 than lipids
11 & 12 (Fig. 3C).
es. Finally, the efficiencies of the cationic liposomes of
the most promising lipids 5, 10 along with that of con-
trol mannosyl analog 11 and co-lipid 12 in mounting long
lasting immune response against B16 melanoma by DC-
based genetic immunization were evaluated in C57BL/6J
mice under prophylactic settings. MART1 antigen is
known to induce immune response in mouse against the
aggressive murine B16 melanoma.15, 16, 22, 23 C57BL/6J mice
were immunized (twice with a seven day interval) by
subcutaneous administration of mbmDCs pre-
transfected with the lipoplexes of lipids 5, 10-12 and
plasmid DNA encoding MART-1 antigen using untrans-
fected DCs and naked plasmid as negative controls.
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Days after tumor challenge
Days after tumor challenge
Figure 5. Protection against spontaneous growth of i.v.
administered pLuc-B16F10 cells in mice (n = 5) immunized
with mbmDCs ex vivo pre-transfected with indicated lipo-
plexes. Mice immunized with untransfected DCs was used
as control group. A. Luciferase activities in lung extract
prepared from one sacrificed mice from each group on day
30 post tumor challenge. B. Representative images of ex-
cised lungs. C. Long term survival study of the remaining
mice (n = 4) challenged with pLuc-B16F10 cells. Experi-
mental details are in supporting information.
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Days after tumor challenge
Two weeks after the last immunization, in one set of
experiments mice were challenged (s.c) with lethal dose
of B16F10 cells. In another set of experiments, following
a widely used secondary lung metastasis model,24 mice
were i.v. challenged with lethal dose of B16F10 cells sta-
bly transformed with luciferase gene (pLuc-B16F10)
which spontaneously accumulated in lung. Mice s.c.
immunized with DCs pre-transfected with lipoplexes of
lipids 5 & 10 & MART1 encoded plasmid DNA showed
significant protection against melanoma (subcutaneous
tumor challenge). No tumor was detected for 29 days
in mice s.c. immunized with lipoplexes of lipids 5 & 10
(Fig. 4A). Importantly, lipoplexes of lipid 5 was found
to be the most efficient in mounting long-lasting (80%
Figure 4. (A) Protection against melanoma challenge in
mice (n = 5) immunized with mbmDCs pre-transfected
with lipoplexes of lipids 5, 10-12 and pCMV-MART1. Mice
groups immunized with naked pCMV-MART1 and with
untransfected DCs were used as control groups. Two
weeks after, mice were challenged with B16F10 cells (1 x 105
cells in 100µL HBSS). Results represent the means +/- SD
(*P < 0.05 for lipids 5, 10-12 & naked MART-1 treated group
compared with control group. (B) Survival study of immun-
ized mice subsequently challenged (s.c.) with B16F10 cells.
Experimental details are in supporting information.
Results from immune response & CTL assays indicate
that lipids 5 & 10 hold more potential than lipids 11 & 12
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tional Centre for Cell Sciences (NCCS), Pune, India.
of s.c. immunized mice survived for 120 days post tumor
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challenge) protection against melanoma (Fig. 4B). The
survivability for other groups were significantly less
(Fig. 4B). Lipoplexes of lipid 5, 10 & 11 were found to be
highly efficient in protecting mice from i.v. adminis-
tered Luc-B16F10 cells growing on mouse lung during
the first 30 days post luc-B16F10 challenge (Fig. 5A-B).
However, long-lasting immune response against mela-
noma (150 days post luc-B16F10 challenge) was observed
in 75% of mice s.c. immunized with DCs pre-transfected
with the lipoplexes of pCMV-MART1 and lipids 5 and
while the survival rate for the other groups were found
to be significantly less (Fig. 5C).
B16F10, pLuc-B16F10 Cells were grown at 37° C in Dul-
becco’s modified Eagle’s medium (DMEM) with 10% FBS
in a humidified atmosphere containing 5%CO2/95% air.
6-8 weeks old C57BL/6J mice (male & female, each
weighing 20-22 g) were purchased from National Insti-
tute of Nutrition, Hyderabad, India. All the in vivo ex-
periments were performed in accordance with the Insti-
tutional Bio-Safety and Ethical Committee Guidelines
using an approved animal protocol.
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Statistical Analysis. Experimental data presented is the
mean ± SD of triplicate experiments. Data were com-
pared among the different groups for individual experi-
ment using the Student t test and p < 0.05 was consid-
ered as significant.
In conclusion, our present structure-activity findings
demonstrate that mannose-receptor selective cationic
amphiphile containing 5 methylene units in the spacer
arm between the hydrophobic tail and mannose-
mimicking shikimoyl- & quinoyl- head groups (lipids 5
& 10) hold promise for use in ex vivo DC-transfection
based DNA vaccination under prophylactic settings.
Importantly, use of lipoplexes of pCMV-MART1 and li-
pid 5 in ex vivo DC-transfection were found to be the
most efficient in mounting long-lasting anti-melanoma
immune response in mice challenged with B16F10 cells
using two different challenge modes.
ASSOCIATED CONTENT
Supporting Information
Detailed information on the syntheses and purifications, 1H
NMR & ESI-Mass spectra and the reverse phase analytical
HPLC profiles of the purified lipids 1-11; details for DC-
transfection, isolation and culture of mbmDCs, DC-based
immunization protocols, ELISA assays, and FACS protocol.
This material is available free of charge via the internet at
http://pubs.acs.org.
Experimental Section
AUTHOR INFORMATION
Corresponding Authors
General procedures and reagents. ESI-Mass spectra
for all the lipids and their intermediates were acquired
by micromass Quatro LC triple quadrapole mass spec-
trometer or LCQ ion trap mass spectrometer (Thermo
Finnigan, San Jose, CA, USA). 1H NMR spectra were rec-
orded on a Varian AV 300 MHz NMR Spectrometer. Cell
culture media, fetal bovine serum, Amberlite Cl- ion
exchange resin, TMS-triflate, EDCI, HOBt and choles-
terol were purchased from Sigma-Aldrich (St. Louis,
USA). Antibiotics and agarose were procured from Hi-
media, India. TFA, trichloro acetonitrile and methyl
iodide were purchased from Spectrochem, India. D-
Mannose was procured from SD Fine, India. Column
chromatography was performed with silica gel (Acme
Synthetic Chemicals, India, 60-120 mesh). Unless oth-
erwise stated all reagents were purchased from local
commercial suppliers and were used without further
purification. Mouse GM-CSF, IL-4, IFN-γ, ELISA and
CTL assay kits were purchased from Thermo Scientific
and Promega USA. Mouse FITC conjugated CD11c,
CD206, CD40, CD86, CD45R, H2kB, F4/80, anti-MHC II
antibodies were purchased from Chemicon, USA. FACS
data was analyzed and processed using FCS Express 4
Research Edition (USA) software. Structures of all the
synthetic intermediates were confirmed by 1H NMR and
ESI-MS. The purity of all the target compounds was con-
firmed by reverse phase analytical HPLC and found to
be >95%.
* E-mail: arabinda@iict.res.in, Tel: +91-9542816932
chanduadb@gmail.com, Tel: +91-9542816932
Present Addresses
CV is at BioSatva Technologies, Golnaka, Hyderabad
500013, India; RCRM is at Department of Microbiology &
Immunology, School of Medicine, University of Maryland,
Baltimore, MD 21201 USA; SS is at Department of Cancer
Biology, UMASS Medical School, Worcester, MA 01605,
USA; SM is at Centre for Stem Cell Research, CMC Campus,
Bagayam, Vellore 632002, India
Funding Sources
This work was supported by the Department of
Biotechnology and Council of Scientific and Industrial
Research, Government of India, New Delhi (NWP-0036,
CSC0123 & CSC0302).
Notes
Conflict of Interest: The authors declare no competing
financial interest.
ACKNOWLEDGMENT
We thank Dr. Van den Eynde from Ludwig Institute for
Cancer Research, Brussels, Belgium, Dr. N. M. Rao & Dr. G.
Pande from Centre for Cellular and Molecular Biology, Hy-
derabad, India, for providing us with pCMV-MART1, p-
CMV-SPORT-β-Gal, pCMV-Luc and pCMV-α5-GFP plas-
mids, respectively.
Cell lines and Animal models. B16F10 & pLuc-B16F10
(murine melanoma cells) were procured from the Na-
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logue as DNA Vaccine Carrier in Dendritic Cell Based Genetic
Immunization. J. Med. Chem. 2010, 53, 1387-1391.
ABBREVIATIONS
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8
9
APC: Antigen presenting cells; DC: Dendritic cell; FBS: Fe-
tal bovine serum; FITC: Fluorescein isothiocyanate; GM-
CSF: Granulocyte Macrophage Colony Stimulating Factor;
GFP: Green fluorescent protein. HBSS: Hank’s Balanced
Salt Solution; IFN-γ: Interferon gamma; IL-4: Interleukin-4;
mbmDC: Mouse bone marrow derived dendritic cells;
MHC: Major Histocompatibility Complex.
16. Srinivas, R.; Garu, A.; Moku, G.; Agawane, S. B.; Chaudhuri,
A. A Long-Lasting Dendritic Cell DNA Vaccination System
Using Lysinylated Amphiphiles with Mannose-Mimicking
Head-Groups. Biomaterials 2012, 33, 6220-6229.
17. Garu, A.; Moku, G.; Gulla, S. K.; Chaudhuri, A. Genetic Im-
munization With In Vivo Dendritic Cell-Targeting Liposomal
DNA Vaccine Carrier Induces Long-Lasting Antitumor Im-
mune Response. Mol. Ther. 2015, 24(2), 385-97.
18. Srujan, M.; Chandrashekhar, V.; Reddy, R. C.; Prabhakar, R.;
Sreedhar, B.; Chaudhuri, A. The Influence of the Structural
Orientation of Amide Linkers on the Serum Compatibility and
Lung Transfection Properties of Cationic Amphiphiles. Bio-
materials 2011, 32, 5231-5240.
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REFERENCES
1. Ferraro, B.; Morrow, M. P.; Hutnick, N. A.; Shin, T. H.; Lucke,
C. E.; Weiner, D. B. Clinical Applications of DNA Vaccines:
Current Progress. Clin. Infect. Dis. 2011, 53, 296-302.
19. Rengarajan, J.; Szabo, S. J.; Glimcher, L. H. Transcriptional
Regulation of Th1/Th2 Polarization. Immunol. Today 2000, 21,
479-483.
2. Cui, L.; Osada, K.; Imaizumi, A.; Kataoka, K.; Nakano, K.
Feasibility of a Subcutaneously Administered Block/Homo-
Mixed Polyplex Micelle as a Carrier for DNA Vaccination in a
Mouse Tumor Model. J. Control. Release 2015, 206, 220-231.
20. Zhai, Y.; Yang, J. C.; Spiess, P.; Nishimura, M. I.; Overwijk,
W. W.; Roberts, B.; Restifo, N. P.; Rosenberg, S. A. Cloning and
Characterization of the Genes Encoding the Murine Homo-
logues of the Human Melanoma Antigens MART1 and gp100. J.
Immunother. 1997, 20, 15-25.
3. Rice, J.; Ottensmeier, C. H.; Stevenson, F. K. DNA Vaccines:
Precision Tools for Activating Effective Immunity against Can-
cer. Nat. Rev. Cancer 2008, 8, 108-120.
21. Mockey, M.; Bourseau, E.; Chandrashekhar, V.; Chaudhuri,
A.; Lafosse, S.; Le Cam, E.; Quesniaux, V. F.; Ryffel, B.; Pichon,
C.; Midoux, P. mRNA-Based Cancer Vaccine: Prevention of B16
Melanoma Progression and Metastasis by Systemic Injection of
MART1 mRNA Histidylated Lipopolyplexes. Cancer Gene Ther.
2007, 14, 802-814.
4. Lu, S.; Wang, S.; Grimes-Serrano, J. M. Current Progress of
DNA Vaccine Studies in Humans. Expert Rev. Vaccines 2008,
7(2), 175-191.
5. Merad, M.; Sathe, P.; Helft, J.; Miller, J.; Mortha, A. The Den-
dritic Cell Lineage: Ontogeny and Function of Dendritic Cells
and Their Subsets in the Steady State and the Inflamed Setting.
Annu. Rev. Immunol. 2013, 31, 563-604.
22. Ribas, A.; Butterfield, L. H.; Hu, B.; Dissette, V. B.; Chen, A.
Y.; Koh, A.; Amarnani, S. N.; Glaspy, J. A.; McBride, W. H.;
Economou, J. S. Generation of T-Cell Immunity to a Murine
Melanoma Using MART-1-Engineered Dendritic Cells. J. Im-
munother. 2000, 23, 59-66.
6. Mildner, A.; Jung, S. Development and Function of Dendritic
Cell Subsets. Immunity 2014, 40, 642-656.
7. Kutzler, M. A.; Weiner, D. B. DNA Vaccines: Ready for Prime
Time? Nat. Rev. Genet. 2008, 9, 776-788.
23. Ramirez-Montagut, T.; Turk, M. J.; Wolchok, J. D.; Guevara-
Patino, J. A.; Houghton, A. N. Immunity to Melanoma: Unrav-
eling the Relation of Tumor Immunity and Autoimmunity.
Oncogene 2003, 22, 3180-3187.
8. Palucka, K.; Banchereau, J. Cancer Immunotherapy Via
Dendritic Cells. Nat. Rev. Cancer 2012, 12, 265-277.
9. Palucka, K.; Banchereau, J. Dendritic-Cell-Based Therapeutic
Cancer Vaccines. Immunity 2013, 39, 38-48.
24. Li, S. D.; Chono, S.; Huang, L. Efficient Oncogene Silencing
and Metastasis Inhibition Via Systemic Delivery of siRNA. Mol.
Ther. 2008, 16, 942-946.
10. Humbert, J. M.; Halary, F. Viral and Non-Viral Methods to
Genetically Modify Dendritic Cells. Curr. Gene Ther. 2012, 12,
127-136.
11. Watson, D. S.; Endsley, A. N.; Huang, L. Design Considera-
tions for Liposomal Vaccines: Influence of Formulation Param-
eters on Antibody and Cell-Mediated Immune Responses to
Liposome Associated Antigens. Vaccine 2012, 30, 2256-2272.
12. Azad, A. K.; Rajaram, M. V.; Schlesinger, L. S. Exploitation
of the Macrophage Mannose Receptor (CD206) in Infectious
Disease Diagnostics and Therapeutics. J. Cytol. Mol. Biol. 2014,
1(1), 1000003.
13. Apostolopoulos, V.; Thalhammer, T.; Tzakos, A. G.;
Stojanovska, L. Targeting Antigens to Dendritic Cell Receptors
for Vaccine Development. J. Drug Deliv. 2013, 2013, 869718.
14. Yoshida, M.; Kawakami, S.; Kono, Y.; Un, K.; Higuchi, Y.;
Maruyama, K.; Yamashita, F.; Hashida, M. Enhancement of the
Anti-Tumor Effect of DNA Vaccination Using an Ultrasound-
Responsive Mannose-Modified Gene Carrier in Combination
with Doxorubicin-Encapsulated Pegylated Liposomes. Int. J.
Pharm. 2014, 475, 401-407.
15. Srinivas, R.; Karmali, P. P.; Pramanik, D.; Garu, A.; Mahi-
dhar, Y. V.; Majeti, B. K.; Ramakrishna, S.; Srinivas, G.;
Chaudhuri, A. Cationic Amphiphile with Shikimic Acid Head-
group Shows More Systemic Promise Than Its Mannosyl Ana-
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Journal of Medicinal Chemistry
1
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7
Protection against
s.c. Melanoma challenge in mice
mbmDCs ex vivo
pre-transfected with lipoplexes of
pCMV-MART1 & Lipids 5 & 10
2500
Control
Lipid 5 + MARTꢀ1
2000
Lipid 10 + MARTꢀ1
Lipid 11 + MARTꢀ1
1500
8
Lipid 12 + MARTꢀ1
9
Naked MARTꢀ1
1000
s.c.Immunization
Tumor Challenge
with mbmDCs
post immunization
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11
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500
0
0
18
20
22
25
29
Days after tumor challenge
ACS Paragon Plus Environment