Mannosylated polyion complexes for in vivo gene delivery into CD11c+ dendritic cells

Article abstract of DOI:10.1021/mp5005492

Dendritic cells (DCs) possess unique abilities in initiating primary immune responses and thus represent prime targets for DNA-based vaccinations. Here, we describe the design and synthesis of mannosylated polyion complexes (PICs) composed of cationic pol

Full text of DOI:10.1021/mp5005492

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Article
Mannosylated Polyion Complexes for In Vivo
Gene Delivery into CD11c+ Dendritic Cells
Lior Raviv, Michal Jaron-Mendelson, and Ayelet David
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/mp5005492 • Publication Date (Web): 22 Dec 2014
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Mannosylated Polyion Complexes for In Vivo Gene Delivery into
CD11c+ Dendritic Cells
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Lior Raviva, Michal JaronꢀMendelsonb, and Ayelet Davida,*
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a Department of Clinical Biochemistry and Pharmacology, Faculty of Health Sciences,
and Ilse Katz Institute for Nanoscale Science and Technology, BenꢀGurion University
of the Negev, BeerꢀSheva, 84105, Israel
b
Shraga Segal Department of Microbiology and Immunology, Faculty of Health
Sciences, BenꢀGurion University of the Negev, BeerꢀSheva 84105, Israel
* Corresponding Author
Department of Clinical Biochemistry and Pharmacology
Faculty of Health Sciences, BenꢀGurion University of the Negev
BeerꢀSheva 84105, Israel
Tel: +972ꢀ8ꢀ6477364; Fax: +972ꢀ8ꢀ6479303
Eꢀmail: ayeletda@bgu.ac.il
Running title: Mannosylated polyion complexes for targeting dendritic cells
Keywords: Dendritic cells, gene delivery system, mannose receptor, mannoseꢀreceptor
ligands, multivalency
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ABSTRACT
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Dendritic cells (DCs) possess unique abilities in initiating primary immune responses
and thus represent prime targets for DNAꢀbased vaccinations. Here, we describe the
design and synthesis of mannosylated polyion complexes (PICs) composed of cationic
polyethylenimine (PEI) and hydrophilic polyethylene glycol (PEG) segments, and
bearing monoꢀ and triꢀvalent mannose as ligand for targeting mannose receptor
(MR/CD206)ꢀpositive DCs. Aminoꢀterminated mannose (Man)ꢀcontaining ligands in
monoꢀ and triꢀvalent presentations (Manꢀ and Man3ꢀ, respectively) were prepared and
conjugated to PEG via an Nꢀhydroxysuccinimide (NHS)ꢀactivated terminal. Thiolated
PEI was conjugated to the mannosylated PEG via the maleimide (MAL)ꢀactivated
terminal. The resulting positively charged diꢀblock copolymers bearing mannoses (Manꢀ
PEGꢀbꢀPEI and Man3ꢀPEGꢀbꢀPEI) were selfꢀassembled with DNA to form PICs with
lower surface charge than did their PEI building block and mean hydrodynamic
diameters in the range of 100ꢀ450 nm, depending on the N/P ratio. Man3ꢀPEGꢀbꢀPEI
demonstrated a 3ꢀ4ꢀfold greater transfection efficiency in MRꢀpositive dendritic cell
lines (THPꢀ1, DC2.4), relative to ManꢀPEGꢀbꢀPEI, exhibited low cytotoxicity when
compared with PEI and showed low transfection efficiency in nonꢀdendritic HeLa cells.
In preliminary in vivo experiments, ManꢀPEGꢀbꢀPEI/DNA and Man3ꢀPEGꢀbꢀPEI/DNA
demonstrated 2ꢀ3ꢀfold higher gene delivery efficiency into CD11c+ DCs collected from
inguinal lymph nodes of C57/BL6 mice, when compared to PEI/DNA complexes, as
shown by GFP expression measurements, 24 h post subcutaneous injection. The results
indicate that the mannosylated PICs are safe and effective gene delivery system,
showing in vivo specificity towards CD11c+ DCs that overꢀexpress the MR.
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ABBREVIATIONS
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APCs
CRDs
DMSO
EtBr
MAL
Man
antigenꢀpresenting cells
carbohydrate recognition domains
dimethyl sulfoxide
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ethidium bromide
maleimide
mannose
MR
mannose receptor
MHC
MTT
NHS
PBS
major histocompatibility complex
3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolinium bromide
Nꢀhydroxysuccinimide
phosphateꢀbuffered saline
polyethylene glycol
PEG
PEI
polyethylenimine
SATA
TEA
NꢀsuccinimidylꢀSꢀacetylthioacetate
triethylamine
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INTRODUCTION
Dendritic cells (DCs) are the most potent professional antigenꢀpresenting cells known 1.
Their ability to capture, process, and present foreign antigens, migrate to lymphoidꢀrich
tissues, and stimulate cellular antigenꢀspecific immune responses makes them an ideal
tool for cancer immunotherapy. Genetic approaches to antigen loading, based on
physical, viralꢀ and nonꢀviralꢀbased transfer of vaccine DNA into DCs, offers the
advantage of a continuous supply of antigen for presentation of vaccine antigens on
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MHC class I and II products . To date, adenovirus vectors have been found to be the
most efficient gene delivery system for DCs. However, adenoviruses are highly
immunogenic and can elicit potent antiꢀviral immune responses that restrict their
application in medical therapy. Accordingly, different types of nonꢀviral delivery
vehicles have been developed for gene delivery, with lipidꢀ or polymerꢀbased
complexes (lipoplexes or polyplexes, respectively) currently being the most commonly
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employed . Polyplexes consist a cationic segment as a core component that can selfꢀ
assemble through electrostatic interactions with oligonucleotides to produce polyion
complexes (PICs), within which DNA is encapsulated 4ꢀ6. Polyethylenimine (PEI) is one
of the most useful polycations for DNA binding and condensation as it demonstrates
high transfection efficiency and pH buffering capacity, which is believed to protect
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DNA from degradation and to enable its escape from the endosomal compartment
.
However, the positive surface charge of PEI/DNA polyplexes results in aggregation,
adsorption to plasma proteins, nonꢀspecific interaction with cells, recognition by
immune system components, and cytotoxicity 9ꢀ11. Modification of PEI by polyethylene
glycol (PEG) can increase polymer solubility, protect PICs from macrophage uptake,
prolong circulation time in vivo, and decrease nonꢀspecific interactions with serum
12ꢀ15.
protein
Yet, PEGꢀPEI/DNA complexes can show reduced cellular uptake as
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compared to positively charged PICs due to reduced interactions with the negatively
charged cellular membrane. Together with the fact that DCs compose only 1ꢀ3% of cells
in peripheral tissues 16ꢀ19, the system may have a very limited efficiency unless targeted.
The attachment of cellꢀspecific ligand to the PEGꢀPEI/DNA complex surface is
expected to improve transfection efficiency and enhance cellular uptake via receptorꢀ
mediated endocytosis.
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Mannose receptor (MR/CD206) is a 175 kDa transmembrane protein highly expressed
on DCs and macrophages and upꢀregulated in tumorꢀassociated macrophages 20. It
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contains eight Cꢀtype carbohydrate recognition domains (CRDs) per polypeptide
and mediates the endocytosis and processing of antigens that expose mannose and
fucose residues for presentation by major histocompatibility complex (MHC) class II
molecules 23. It has been previously demonstrated that mannosylated lipoplexes and
polyplexes can be used for gene delivery into macrophages and DCs 24ꢀ32. The eight
adjacent CRDs in MR may further help to increase the binding affinity and specificity
of lipoplexes and polyplexes containing, Dꢀmannoseꢀ and Lꢀfucoseꢀcontaining glycans
in a multivalent display. Several research groups have synthesized multivalent
mannosylated ligands and demonstrated higher in vitro binding affinity for the MR than
the monoꢀmannosylated analogs 33, 34. However, the influence of mannose valency on
the targeting of DCs in vivo has not been tested yet.
In this study we synthesized mannosylated polyion complexes (PICs) composed of
cationic linear PEI (25 kDa) and hydrophilic PEG (3.5 kDa) segment, and bearing
monoꢀ and triꢀvalent mannoses for actively targeting MRꢀpositive DCs through
multivalent interactions. We, moreover, evaluate their targeting ability and activity both
in vitro and in vivo. We found that the PICs bearing manosylated ligands in triꢀvalent
presentation (Man3) demonstrated higher gene delivery efficiency into CD11c+ cells
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collected from inguinal lymph nodes, when compared to monoꢀmannosylated PIC or
nonꢀmannosylated PICs. To the best of our knowledge, this is the first study to evaluate
Man3ꢀPEGꢀbꢀPEI copolymer for gene delivery applications and it is the first attempt to
evaluate the effect of the Man valency on PEGꢀbꢀPEI/DNA complexes on gene
expression efficiency in vivo.
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EXPERIMENTAL SECTION
Materials
All chemicals were of reagent grade and obtained from Sigma–Aldrich (Rehovot,
Israel), unless otherwise noted. Linear PEI "Max" Mw 40,000 (Equivalent to Mw
25,000 Da in Free Base Form) was purchased from Polysciences Inc. (Eppenheim,
Germany). NꢀsuccinimideꢀPEGꢀmaleimide ester (NHSꢀPEGꢀMAL; MW 3,500 Da) was
purchased from JenKem Technology USA Inc. (Allen, TX). The plasmid DNA
encoding firefly luciferase and GFP (pCMVLuc and pGFP) were a gift from Prof.
Moshe Oren (Weizmann Institute of Science, Rehovot, Israel). Dialysis tubing with
12,000ꢀ14,000 g/mol Mw cutꢀoff was purchased from Cellu Sep T4, Membrane
Filtration Products, Inc. (Segiun, TX, USA).
Cell lines and culture media
THPꢀ1 monocytic leukemia cells were cultured in RPMIꢀ1640 medium supplemented
with 10% fetal bovine serum, 2 mM Lꢀglutamine, 100 units/ml penicillin and 100 µg/ml
of streptomycin. For differentiation to immature DCs, the following cytokine cocktail
was added to the THPꢀ1 cells medium: rhILꢀ4 (100 ng/ml = 1500 units/ml), rhGMꢀCSF
(100 ng/ml = 1500 units/ml) 35. DC2.4 cells were cultured in RPMIꢀ1640 medium
supplemented with 10% fetal bovine serum, 2 mM Lꢀglutamine, 20 units/ml penicillin
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and 20 µg/ml of streptomycin supplemented with 50 µM 2ꢀmercaptoethanol. HeLa cells
were cultured in DMEM supplemented with 10% fetal bovine serum, 2 mM Lꢀ
glutamine, 100 units/ml penicillin and 100 µg/ml of streptomycin. Cells were grown at
37°C in a humidified atmosphere of 5% (v/v) CO2 in air.
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Synthesis of a monovalent Man targeting moiety, 4ꢀaminophenyl αꢀDꢀ
mannopyranoside (ManꢀPAP)
4ꢀnitrophenyl αꢀDꢀmannopyranoside (ManꢀPNP) was reduced to 4ꢀaminophenyl αꢀDꢀ
mannopyranoside (ManꢀPAP) in methanol (MeOH) with ammonium formate
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(NH4HCO2) and in the presence of 10% palladium on carbon (Pd/C), as described .
Briefly, PNPꢀMan (151 mg, 0.2 mmol), NH4HCO2 (250 mg, 4 mmol) and Pd/C (25 mg)
were dissolved in 30 ml MeOH. The mixture was gently warmed to 50°C while stirring
to afford PAPꢀMan. The reaction mixture was then filtered, the solvent was evaporated,
the product was dissolved in double distilled water and purified on PDꢀ10 desalting
column. PAPꢀMan was identified by thin layer chromatography (TLC).
Synthesis of trivalentꢀMan targeting moiety, Man3ꢀ(amidohexanamido)ꢀTris
(Man3ꢀAHT)
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Man3ꢀAHT was synthesized based on a previously described procedure . Briefly, Tris
(0.97 g, 8 mmol) was first coupled with Cbzꢀaminohexanoic acid (2.4 g, 9 mmol) in
absolute ethanol (500 ml) in the presence of 2ꢀethoxyꢀNꢀ(ethoxycarbonyl)ꢀ1,2ꢀ
dihydroquinoline
(EEDQ)
(2.66
g,
10.8
mmol)
to
afford
(6ꢀ
(benzyloxycarbonamido)hexanamido)ꢀtris(hydroxymethyl)methane (CbzꢀAHT) (Fig.
1A). αꢀDꢀMan (180 mg, 1 mmol) was transformed into 2,3,4,6ꢀtetraꢀOꢀacetylꢀαꢀDꢀ
mannopyranosyl bromide using 45% (w/v) HBr in glacial acetic acid (1 ml) (Fig. 1B),
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and
then
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with
[6ꢀ(benzyloxycarbonamido)hexanamido]ꢀ
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tris(hydroxymethyl)methane (CbzꢀAHT) (0.5 mmol) in 10 ml mixture of
nitromethane:toluene (1:1 (v/v)) in the presence of mercuric cyanide (0.5 mmol) to give
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the
[6ꢀ(benzyloxycarbonamido)hexanamido]ꢀtris(2,3,4,6ꢀtetraꢀOꢀacetylꢀαꢀDꢀ
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mannopyranosyloxymethyl)methane (CbzꢀAHTꢀ[Man(OAc)4]3) conjugate (Fig. 1C).
CbzꢀAHTꢀ[Man(OAc)4]3 (0.2 mmol)was purified on Sephadex LHꢀ20 resin and deꢀ
acetylated in dry methanol containing 0.5 M sodium methoxide (NaOCH3, 7.5 mmol) to
give CbzꢀAHTꢀ[Man]3. The Cbz protecting group was removed by hydrogenation in
methanol using 10% Pd/C in the presence of ammonium formate (NH4HCO2) to give
the desired Man3ꢀAHT. H1ꢀNMR and matrixꢀassisted laser desorption/ionization time of
flight (MALDIꢀTOF) analysis were performed after each synthesis step to confirm the
structure of Man3ꢀAHT and intermediate products (Fig. 1s, in Supplemental data),
Synthesis of mannosylated PEGꢀMAL
Man3ꢀAHT or ManꢀPAP was conjugated to NHSꢀPEGꢀMAL via the NHSꢀactivated
terminal in dimethyl sulfoxide (DMSO) to give Man3ꢀPEGꢀMAL or ManꢀPEGꢀMAL
(Fig. 2A). Briefly, NHSꢀPEGꢀMAL (0.013 mmol) was dissolved in DMSO (400 µl).
AHTꢀMan3 or PAPꢀMan (0.13 mmol) were dissolved in DMSO (500 µl) in the
presence of TEA (0.13 mmol) and then added to PEG solution. The reaction was
carried out for 3 h at room temperature with shaking. ManꢀPEGꢀMAL or Man3ꢀPEGꢀ
MAL were precipitated in ether, isolated and characterized by 1HꢀNMR.
Synthesis of mannosylated PEGꢀbꢀPEI, Man3ꢀPEGꢀbꢀPEI or ManꢀPEGꢀbꢀPEI
A thioacetate group was introduced into PEI (25000 Da) (0.004 mmol) by activation
with NꢀsuccinimidylꢀSꢀacetyl thioacetate (SATA) (0.024 mmol) in 5 ml DMSO in the
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presence of triethylamine (TEA) and converted into a free sulphydryl group by
subsequent treatment with 0.5 M hydroxylamine (NH2OH) in PBS containing EDTA
disodium (25mM), according to the manufacturer’s protocol (Fig. 2B). Thiolated PEI
(PEIꢀNHꢀCOꢀCH2ꢀSH) (0.001 mmol) was then conjugated to the mannosylated PEG
(0.01 mmol) via the MALꢀactivated terminal in 3 ml PBS (pH 6.5 ) (Fig. 2C), based on
a previously described procedure 37. Man3ꢀPEGꢀbꢀPEI or ManꢀPEGꢀbꢀPEI were
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dialyzed (12,000ꢀ14,000 Da cutꢀoff), lyophilized and characterized by HꢀNMR and
phenol/sulfuric acid assay 38. PEGꢀbꢀPEI blockꢀcopolymers were synthesized as a
control in a similar manner.
Complexation of mannosylated blocks copolymer with DNA and characterization
Blockꢀcopolymers and DNA (pCMVLuc or pGFP, 50 µg)) were separately dissolved
in50 µl of 150mM NaCl. Following the addition of blockꢀcopolymer solution into the
DNA solution, the mixture was vortexed for 15 seconds, spun down and incubated for
20 min at room temperature for complexation. Mean hydrodynamic size of the
complexes was characterized by dynamic light scattering (DLS, CGSꢀ3 LSEꢀ5004,
ALVꢀLaser Vertriebsgesellschaft mꢀb.H., Langen, Germany) at a scattering angle of 90°
through a 400 ꢁm pinhole at room temperature, and Surface charge was determined by
ζꢀpotential measurements (Zetasizer Nano ZS, Malvern Instruments Ltd,
Worcestershire, UK) in 150mM NaCl. Average values of the ζꢀpotential were calculated
with the data from 9 measurements.
Ideal N/P ratio determination
Ideal N/P ratio for complexation (the ratio of amine groups on the blockꢀcopolymer to
phosphate groups on the DNA) was determined Ethidium bromide (EtBr) exclusion
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assay 39. Briefly, triplicates of pGFP (4.3 µg) were complexed with increasing amounts
of PEI, PEG-bꢀPEI, ManꢀPEG-bꢀPEI or Man3ꢀPEG-bꢀPEI (N/P: 0, 4, 8, 12) in 96ꢀwell
dark, as described above. After 20 min incubation, 20 µl EtBr solution (0.1 mg/ml) were
added. Fluorescence was measured on a Plate Luminol Luminescence Analyzer at
λex=518 nm and λem=605 nm. Results are given as relative fluorescence, with the
fluorescence of DNA alone (N/P=0) with ethidium bromide considered as 100%.
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Transfection efficacy in vitro
THPꢀ1 or DC2.4 cells were plated in 12ꢀwell plates (1.5×105 cells/well) and grown 24 h
before transfection. 2.8 µg of pCMVLuc or pGFP (8.5 nmol) were used to prepare
PEI/DNA, PEGꢀbꢀPEI/DNA, ManꢀPEGꢀbꢀPEI/DNA and Man3ꢀPEGꢀbꢀPEI/DNA
complexes at N/P=8, as described above, and the resulting Man3ꢀPEGꢀbꢀPEI/pCMVLuc
and ManꢀPEGꢀbꢀPEI/pCMVLuc complexes were used to transfect the cells in 10%
serum containing medium. PEI/pCMVLuc and PEGꢀbꢀPEI/pCMVLuc controls were
tested likewise. Following 24h incubation with complexes, cells were washed twice
with PBS and lysed by 15 minutes incubation with Cell Culture Lysis Buffer
(Promega), with shaking. Luciferase gene expression was then monitored using the
Luciferase 1000 Assay System (Promega), and Relative luminescent Units (RLU) were
measured by a microplate reader (Infinite M200, TECAN, Switzerland). Protein content
in the cells was determined by Bradford assay using the Protein Assay Dye Reagent
(BioꢀRad) to normalize gene expression measurements. Normalized transfection
efficiency values are presented as RLU/mg protein. GFP expression was monitored to
quantify pGFP transfection efficiency. Transfected cells were visualized by
fluorescence microscopy, and analyzed by flowꢀcytometry (Guava Easy Cyte mini,
Guava Technologies). Results are expressed as % GFP transfected cells.
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Cytotoxicity studies
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The cytotoxicity of PEI, PEGꢀbꢀPEI, Man3ꢀPEGꢀbꢀPEI and ManꢀPEGꢀbꢀPEI in
complexes (N/P=8) against DC2.4 cells was assessed using a modified 3ꢀ(4,5ꢀ
dimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀdiphenyltetrazolinium bromide (MTT) assay. Briefly, cells
were seeded into 96ꢀwell plates (1.5×105 cells/well), allowed to grow for 24 h (37°C,
5% CO2), and then transfected with Man3ꢀPEGꢀbꢀPEI/pGFP and ManꢀPEGꢀbꢀ
PEI/pGFP complexes, prepared as described above. PEI/pGFP and PEGꢀbꢀPEI/pGFP
were used as controls. Following 48 h of incubation,, the medium was discarded, and
MTT solution (5 mg/ml MTT in PBS) was added into each well to a final
concentration of 0.5mg/ml. Following 3 h incubation, the he medium was discarded
and 200 µl of DMSO were added to dissolve formazan crystals. The absorbance of
each sample was measured at 570 nm using a microplate reader (Infinite M200,
TECAN, Switzerland). Cell viability is expressed as the percentage of nonꢀtransfected
(control) cells.
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Free mannose competition assay
THPꢀ1, DC2.4 and HeLa cells were seeded in 96ꢀwell plates (1.5×105 cells/well), for 24
h at 37°C. Thirty minutes before transfection, the medium in the mannose+ group was
replaced with medium containing 10 mM mannose. Cells were transfected with Man3ꢀ
PEGꢀbꢀPEI/pGFP, ManꢀPEGꢀbꢀPEI/pGFP, PEI/pGFP and PEGꢀbꢀPEI/pGFP at a N/P
ratio of 8. Three hours after transfection, wells were washed twice with PBS and 1 ml of
fresh medium (without mannose) was added to the wells. Twentyꢀfour hours later, the
cells were collected, washed with PBS and analyzed by flow cytometry to quantify GFP
positive cells, (excitation achieved at 488 nm, with emission measured at 515ꢀ535 nm).
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Uptake by DCs in vivo
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PEI/DNA, PEGꢀbꢀPEI/DNA, ManꢀPEGꢀbꢀPEI/DNA and Man3ꢀPEGꢀbꢀPEI/DNA at
N/P=8 were prepared by dissolving the copolymers in water for injection. Further
dilution was achieved by adding an equal volume of a sterile isotonic 10% glucose
(w/v) solution to obtain a final concentration of 5% glucose. Plasmid GFP solution (15
ꢁg) was added to an equal volume of copolymer solution, the mixtures were vortexed
for 15 seconds and left undisturbed at room temperature for 20 min. C57/BL6 mice
were injected subcutaneously at the tail base with a total volume of 80 ꢁL. Twentyꢀfour
hours later, the mice were sacrificed the subiliac lymph nodes were removed and
digested in complete RPMI media containing 500 U/mL of collagenase at 37°C for 30
min. The samples were then homogenized for 5 sec. The resulting singleꢀcell suspension
was washed with PBS containing 1% BSA and reꢀsuspended in FACS buffer. CD11c+
cells were stained with APCꢀlabeled antiꢀmouseꢀCD11c antibody (BD Pharmingen,
Franklin Lakes, NJ). Fluorescence intensity from GFP produced by the CD11c+ cells
was quantified by flow cytometry.
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Statistical analysis
Statistical calculations were carried out using GraphPad Prism v.5 software (GraphPad
Software, San Diego, CA). To identify statistically significant differences, oneꢀway
ANOVA with Bonferroni’s postꢀtest analysis was performed. Differences were
considered significant if P<0.05(*), P<0.01(**) or P<0.001(***) and are marked
accordingly in the figures.
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MR recognizes mannoseꢀ and fucoseꢀterminated glycans on the surface of pathogens
and plays a crucial role in antigen crossꢀpresentation 40, 41. Several ManꢀPEI conjugates
formulations have been synthesized and used for MRꢀtargeted gene delivery into
antigenꢀpresenting DCs and macrophages. While some of these studies demonstrated
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that ManꢀPEI conjugates are very effective in gene delivery via mannose receptor
their transfection potential was limited due to their relative toxicity at the optimal
polycation/DNA ratio of 1:1 (w/w) 42. In the present study, we designed and synthesized
PEGylated PICs displaying mannosylated ligands in monoꢀ and triꢀvalentꢀorientation to
target MRꢀexpressing DCs. The PEG shield was added to reduce the positive charge of
PEI and decrease its potential toxicity. Multivalent mannosylated ligand was employed
to improve the binding affinity and selectivity to DCs, which is especially important in
DNA vaccination that demands highly efficient delivery of antigenꢀencoded DNA to
specific cells.
Monovalentꢀ and trivalentꢀmannosylated ligands (ManꢀPAP and Man3ꢀAHT,
respectively) were synthesized as described in the Experimental section (Fig. 1) and
their identity and purity was confirmed by TLC and MALTIꢀTOF, respectively. Manꢀ
PAP migrated with Rf = 0.57 on the TLC plate developed in ethyl
acetate:isopropanol:acetone (5:3:2). MALDIꢀTOF analysis identified Man3ꢀAHT
(calculated molecular weight, 720 g/mol) as molecular weight species of 743 and 759
g/mol, corresponding to M + Na and M + K adducts, respectively. ManꢀPAP and Man3ꢀ
AHT were then coupled to MALꢀPEGꢀNHS, and the resulting ManꢀPEGꢀMAL or
Man3ꢀPEGꢀMAL was conjugated to thiolated linear PEI to yield the final ManꢀPEGꢀbꢀ
PEI or Man3ꢀPEGꢀbꢀPEI blockꢀcopolymers (Fig. 2). PEGꢀbꢀPEI was synthesized and
used as a control. 1HꢀNMR spectra of PEGꢀbꢀPEI, ManꢀPEGꢀbꢀPEI and Man3ꢀPEGꢀbꢀ
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PEI collected in deuterated water (Fig. 3) show the characteristic shifts of PEI (δ2.8)
and PEG (δ3.6) in all three blockꢀcopolymers. The estimated Man content of Man3ꢀ
PEGꢀbꢀPEI was there times higher than in ManꢀPEGꢀbꢀPEI, as determined by a
phenol/sulfuric acid assay (Fig. 3, inset). This confirms the success of the conjugation of
the Manꢀ and Man3ꢀbased targeting moieties to the PEGꢀbꢀPEI copolymer.
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To test the DNA condensation ability of Man3ꢀPEGꢀbꢀPEI/DNA, ManꢀPEGꢀbꢀ
PEI/DNA, PEGꢀbꢀPEI/DNA and PEI/DNA, the quenching of ethidium bromide
fluorescence was analyzed. According to the results depicted in Fig. 4, an N/P>4 was
sufficient for complexation using PEI, PEGꢀbꢀPEI and mannosylatedꢀPEGꢀbꢀPEI.
Further experiments were, therefore, performed with complexes at N/P=8. In order to
determine the size and surface charge of the formed PICs at N/P=8, their corresponding
mean hydrodynamic size and ζꢀpotentials were measured. The ζꢀpotential of PEGꢀbꢀ
PEI/DNA, ManꢀPEGꢀbꢀPEI/DNA and Man3ꢀPEGꢀbꢀPEI/siRNA were significantly
lower than that measured for PEI polyplexes (Table 1). All PICs displayed positive ζꢀ
potentials owing to an excess of PEI over DNA. The ζꢀpotential of the mannosylated
PEGꢀbꢀPEI/DNA complexes rose upon increasing the N/P ratio from 5 to 10, however,
the surface charge of these complexes was significantly lower than that of PEI/DNA
polyplexes (Table 1). DLS measurements were then used to determine the mean
hydrodynamic size of the polyplexes (Table 1). Although PEI exhibit excellent DNA
condensing properties at the N/P ratio used, the attained particles were larger than
expected, being over 1000 nm in size, most probably due to aggregate formation 43.The
aggregation process of PEI/DNA polyplexes in high ionic strength medium (150 mM
NaCl) was reported in many previous works 39, 44. PEGylated PEI/DNA complexes
demonstrated a smaller hydrodynamic size, with a mean diameter of about 100 nm for
N/P ratios of 5 and 10. The size of ManꢀPEGꢀbꢀPEI/DNA complexes grew upon
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increasing the N/P ratio from 5 to 10 and was about 250 nm in size at the highest N/P
ratio measured. Man3ꢀPEGꢀbꢀPEI/DNA complexes generally increased in size with
augmentation of the N/P ratio (from 129 to 460 nm). The increase in the mean diameter
of the mannosylated polyplexes relative to PEGꢀbꢀPEI/DNA could be attributed to the
projection of hydrophilic mannose molecules on the exterior of the polyplexes that
could cause steric interruptions and lead to a less packed particle with a larger
hydrodynamic diameter. It was previously shown that the PEG chains in PEGꢀb–PEI
copolymers must attain a minimum molecular weight of about 5 kDa to prevent
complex aggregation 45. In our case, using a 3.5 kDa PEG chain was sufficient to
prevent aggregation. We assume that mannosylation contributes to surface shielding of
the PICs to provide enhanced stability and reduced cytotoxicity.
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We employed the murine dendritic cell line DC2.4 as an APC cell line model to
evaluate transfection efficiency of our newly designed gene delivery system. DC2.4
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cells are known to express MR . Cells were transfected with Man3ꢀPEGꢀbꢀPEI, Manꢀ
PEGꢀbꢀPEI, PEGꢀbꢀPEI or PEIꢀencapsulated pCMVLuc or pGFP at N/P=8. Twentyꢀ
four hours after transfection, luciferase gene expression and GFP activity were tested.
PEI/DNA polyplexes showed the highest gene activity in vitro, whereas addition of
PEG significantly reduced gene activity (Fig. 5 and 6). This reduction could be
attributed to the lower surface charge of the PEGꢀbꢀPEI/DNA complexes, as confirmed
by the ζꢀpotential measurements, and the low adsorption to the negatively charged cell
membrane. Man3ꢀPEGꢀbꢀPEI demonstrated a 3ꢀ4ꢀfold greater transfection efficiency
relative to ManꢀPEGꢀbꢀPEI in MR positive DC2.4 cells, and showed almost equal
efficiency when compared to PEI/DNA. The transfection efficiency of the
mannosylated PICs was significantly inhibited by free mannose in DC2.4 and THPꢀ1
cells (Fig. 7A and B, respectively), indicating the contribution of mannoseꢀbased
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targeting moieties to the increase in transfection efficiency. Of the different PICs tested,
only PEI/DNA complexes were effective in transfecting HeLa cells that do not express
the MR on the cell surface (Fig. 7C). Indeed, the activity of PEI/DNA complexes was
not affected by the presence of free mannose, confirming the cell specificity of the
mannosylated PICs. The results further demonstrate that DCs are more difficult to
transfect than are HeLa cells. While ~50% of HeLa cells were transfected with
PEI/DNA complexes, only ~15% of the DCs were affected by the same formulation.
Still, Man3ꢀbearing PICs were capable of transfecting 12ꢀ15% of antigenꢀpresenting
DCs. Since only few DCs are required to initiate an immune response 2, 47, Man3ꢀPEGꢀ
bꢀPEI emerges as a promising gene carrier for immunotherapeutic manipulations.
During the design of the targeting ligand we hypothesized that our trivalent ligand
comprises three Man molecules would allow multivalent interactions with multiple
CRDs on the MR, and thus increase transfection efficiency. The transfection
experiments showed that Man3ꢀPEGꢀPEI/DNA exhibited 3ꢀfold greater transfection
efficiency than did ManꢀPEGꢀPEI in MRꢀpositive cells. Nonetheless, the maximum
enhancement level was 3.8ꢀfold for trivalent over monovalent Man, which amounts to
an only 1.3ꢀfold enhancement when corrected to a perꢀsugar basis. This is less
remarkable than other instances of multivalent interactions that are triggered by
clustering effects. Moderate enhancement in binding affinity mediated by multivalent
ligand has been previously documented 34, 36. The most acceptable explanation for this
phenomenon is that the orientation of the mannose molecules in Man3ꢀAHT does not
perfectly match the distance between the CRDs on MR. This situation would only cause
an increase in the statistical chance for a mannose molecule to encounter a CRD, thus
causing an additive rather than a multivalency effect.
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The cytotoxicity of the different PICs was assessed by determining the growth
inhibitory activity of each complex against DC2.4 cells 24 h after transfection with the
various PICs together with or without pGFP (Fig. 8). The results indicated the relatively
high cytotoxicity of PEI/DNA polyplexes, with only 42% of the cells being viable after
treatment. This value is in agreement with previous reports in the literature 48ꢀ50. This
cytotoxicity can be attributed to the positive charge of PEI that causes nonꢀspecific
adsorption to cells via electrostatic interactions with the negatively charged cell
membrane. PEGylated and mannosylated PICs demonstrated reduced in vitro
cytotoxicity when compared to the PEI building block, with 83ꢀ96% cell viability postꢀ
transfection being seen with the former pair.
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In preliminary experiment, we investigated the transfection efficiency of our gene
delivery systems in vivo. Man3ꢀPEGꢀbꢀPEI/DNA, ManꢀPEGꢀbꢀPEI/DNA, PEGꢀbꢀ
PEI/DNA or PEI/DNA polyplexes containing pGFP at N/P=8 were injected into the tail
base of C57/BL6 mice. This particular site was chosen for injection because of
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enhanced lymphatic drainage to the inguinal lymph node in mice . Twentyꢀfour hours
postꢀinjection, the percentages of GFP positive cells in the entire cell population and in
the CD11c + cells taken from the inguinal lymph nodes were measured. When
examining the entire cells population extracted from the lymph node, no significant
change was observed in GFP activity between the treatments. Of the different PICs,
only PEI showed detectable (0.333%) GFP activity (Fig. 9A). However, Man3ꢀPEGꢀbꢀ
PEI/DNA and ManꢀPEGꢀbꢀPEI/DNA were 3.2ꢀ and 1.9ꢀfold more efficient,
respectively, in transfecting DCs hosted at the injection site draining lymph nodes, as
compared to PEI/DNA complexes (Fig. 9B, Fig. 2s). The decrease in transfection
efficiency of the PEI/DNA complexes in vivo as opposed to in vitro can be explained by
the lack of cell specificity of the positively charged PEI/DNA complexes. It is possible
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that the PEI/DNA complexes adhered to the cell membranes at the site of injection, thus
lowering their concentration at the draining lymph nodes. This assumption needs to be
further validated in future by testing tissue samples taken from the site of injection. In
future experiments, we will further test whether the mannosylated PICs are taken up by
APCs at the site of injection or instead migrate to the draining lymph nodes and are
taken up there by APCs.
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CONCLUSIONS
In this work, we have successfully synthesized an antigenꢀpresenting DCꢀtargeted gene
delivery system composed of mannosylatedꢀPEG conjugated to linear PEI. ManꢀPEGꢀbꢀ
PEI and Man3ꢀPEGꢀbꢀPEI showed good in vitro complexation with DNA at N/P ratios
>4, had diminished surface charge, reduced hydrodynamic size and lower in vitro
cytotoxicity than did their PEI building block. All mannosylated PICs demonstrated in
vitro selectivity towards MRꢀexpressing cells and superior in vivo CD11c+ transfection
efficiency, as compared with the PEI building block. The mannosylatedꢀPEGꢀbꢀPEI
vehicle was shown to be a safe and efficient gene carrier to DCs, suitable for in vivo
gene delivery. The route of injection evaluated in this work mimics an infection in the
periphery, when peripheral DCs become activated and migrate to the draining lymph
nodes to initiate adaptive immune responses. In future studies, we shall evaluate
whether loading this system with a tumorꢀspecific antigen encoding DNA could result
in DCꢀtargeting in vivo to induce an effective immune response and enhance vaccine
efficiency, especially when combined with agents that elicit complementary activation
signals.
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ACKNOWLEDGMENTS
This work was supported by the Israeli National Nanotechnology Initiative for a Focal
Technology Area (FTA) on Nanomedicines for Personalized Theranostics. We thank
Mazal Rubin for her valuable assistance during this project.
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Polyplex
ζꢀpotential (mV)
Particle size (nm)
N/P=5
N/P=10
31.0 ± 4.5
18.7 ± 2.3
20.7 ± 3.5
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N/P=5
N/P=10
>1000
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27.8 ± 1.2
NA
>1000
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PEI/DNA
PEGꢀbꢀPEI/DNA
16.5 ± 0.5
12.5 ± 1
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ManꢀPEGꢀbꢀPEI/DNA
Man3ꢀPEGꢀbꢀPEI/DNA
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Figure 1: Reaction scheme for the synthesis of Man3ꢀ(amidohexanamido)ꢀTris, Man3ꢀ
AHT. (A). CbzꢀAHT was synthesized by coupling Tris base with Cbzꢀaminohexanoic
acid in absolute ethanol in the presence of EEDQ. (B). αꢀDꢀMan was transformed 1ꢀBrꢀ
Man(OAc)4 using 45% (w/v) HBr in glacial acetic acid. (C). CbzꢀAHT was coupled
with 1ꢀBrꢀ Man(OAc)4 in a mixture of nitromethane:toluene (1:1) in the presence of
mercuric cyanide to give CbzꢀAHTꢀ[Man(OAc)4]3 conjugate. Man3ꢀAHT was obtained
after the removal of the Cbz protecting group by hydrogenation in methanol using 10%
palladium on carbon (Pd/C) in the presence of ammonium formate (NH4HCO2).
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Figure 2: Threeꢀstep synthesis of ManꢀPEGꢀbꢀPEI and Man3ꢀPEGꢀbꢀPEI. (A). Man3ꢀ
AHT or ManꢀPAP was conjugated to NHSꢀPEGꢀMAL via the NHSꢀactivated terminal
in DMSO in the presence of TEA to give Man3ꢀPEGꢀMAL or ManꢀPEGꢀMAL MALꢀ
PEGꢀNHS, respectively. (B). PEI was conjugated with SATA in DMSO in the
presence of TEA and subsequently treated with NH2OH to give PEI with free
sulphydryl group. (C). Thiolated PEI was then conjugated to the mannosylatedꢀPEG
via the MALꢀactivated terminal PEI to yield the final products, ManꢀPEGꢀbꢀPEI or
Man3ꢀPEGꢀbꢀPEI.
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Figure 3: H1ꢀNMR spectra of (A) PEGꢀbꢀPEI, (B) ManꢀPEGꢀbꢀPEI and (C) Man3ꢀ
PEGꢀbꢀPEI in D2O indicating the chemical shifts of PEG δ3.6 (s, CH2ꢀCH2) and PEI
δ2.8 (s, CH2ꢀCH2). Inset: % weight of mannose in PEGꢀbꢀPEI, ManꢀPEGꢀbꢀPEI and
Man3ꢀPEGꢀbꢀPEI conjugates calculated by a phenol/sulfuric acid assay. The results are
given as means ± SD of three independent experiments.
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Figure 4: DNA condensation ability of PEI, PEGꢀbꢀPEI, ManꢀPEGꢀbꢀPEI and Man3ꢀ
PEGꢀbꢀPEI complexes formed with pCMVLuc at different N/P ratios in a 150 mM
NaCl solution. Condensation ability was analyzed by the ethidium bromide fluorescence
quenching assay. Results are given as means ± SD of three independent experiments.
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Figure 5: Transfection efficiency of the different PICs measured by luciferase activity
in DC2.4 cells after a 24 h incubation with PEI/pCMVLuc, PEGꢀbꢀPEI/pCMVLuc,
ManꢀPEGꢀbꢀPEI/pCMVLuc or Man3ꢀPEGꢀbꢀPEI/pCMVLuc at N/P=8. Relative
luminescent units (RLU) were measured using a Plate Luminol Analyzer. Results are
given as means ± SD of three independent experiments, ***p<0.001.
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Figure 6: (A) Transfection efficiency of the different PICs visualized by GFP
fluorescence in DC2.4 cells after a 24 h incubation with PEI/ pGFP, PEGꢀbꢀPEI/ pGFP,
ManꢀPEGꢀbꢀPEI/pGFP or Man3ꢀPEGꢀbꢀPEI/pGFP at N/P=8. Pictures were taken by a
fluorescence microscope. (B) Percentage of transfected DC2.4 cells after a 24 h
incubation with PEI/pGFP, PEGꢀbꢀPEI/pGFP, ManꢀPEGꢀbꢀPEI/pGFP or Man3ꢀPEGꢀbꢀ
PEI/pGFP at N/P=8 measured by flow cytometry. Results are given as mean ± SD of
four independent experiments, ***p<0.001.
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