Combinatorial synthesis and high-throughput screening of alkyl amines for nonviral gene delivery

Article abstract of DOI:10.1021/bc400158w

Efficient delivery of plasmid DNA and siRNA into cells is essential for biological and biomedical research. Although significant efforts have been made to develop efficient nonviral vectors, such as cationic lipids and polymers, most of the vectors require multistep synthesis, which complicates both fast structural optimizations and combinatorial synthesis of such vectors. Here, we present a facile, single-step method based on an alkylation of amines, allowing for the fast parallel synthesis of libraries of cationic lipid-like molecules (lipidoids). We exploited the method to synthesize 200 lipidoids, which were screened for their transfection efficiency in HEK293T cells. The screen resulted in about 2% of new lipidoids capable of efficient cell transfection similar or higher than the efficiency of Lipofectamine 2000. In addition, we observed an enhancement of cellular transfection by combining single- with double-chain lipidoids, which was attributed to the different roles of the single- and double-tailed lipids in the mixed liposomes.

Full text of DOI:10.1021/bc400158w

Article
pubs.acs.org/bc
Combinatorial Synthesis and High-Throughput Screening of Alkyl
Amines for Nonviral Gene Delivery
Linxian Li,†,‡ Fengjian Wang,§ Yihang Wu,† Gary Davidson,*,† and Pavel A. Levkin*,†,§
†Institute of Toxicology and Genetics, Karlsruhe Institute of Technology, 76344 Karlsruhe, Germany
§
‡Department of Organic Chemistry and Department of Applied Physical Chemistry, University of Heidelberg, 69120 Heidelberg,
Germany
S
* Supporting Information
ABSTRACT: Efficient delivery of plasmid DNA and siRNA into cells is
essential for biological and biomedical research. Although significant
efforts have been made to develop efficient nonviral vectors, such as
cationic lipids and polymers, most of the vectors require multistep
synthesis, which complicates both fast structural optimizations and
combinatorial synthesis of such vectors. Here, we present a facile,
single-step method based on an alkylation of amines, allowing for the fast
parallel synthesis of libraries of cationic lipid-like molecules (lipidoids).
We exploited the method to synthesize 200 lipidoids, which were screened
for their transfection efficiency in HEK293T cells. The screen resulted in
about 2% of new lipidoids capable of efficient cell transfection similar or
higher than the efficiency of Lipofectamine 2000. In addition, we observed
an enhancement of cellular transfection by combining single- with double-
chain lipidoids, which was attributed to the different roles of the single- and double-tailed lipids in the mixed liposomes.
excessive purifications,38 thus limiting fast structural optimiza-
INTRODUCTION
■
tions. Simple one- or two-step synthetic methods leading to
Chemical reagents that efficiently deliver nucleic acids into cells
lipid-like molecules could significantly increase the rate of
have revolutionized biological research since the calcium
discovery of novel efficient gene-delivery systems. So far only
phosphate transfection process was first described nearly 40
few such methods permitting combinatorial synthesis of lipids
years ago.1 Chemically based transfection reagents include
have been reported. For example, Anderson et al. described two
cationic lipids,2−6 cationic linear and branched polymers,7−13
methods based on aza-Michael addition and amine addition to
dendrimers,14−18 cationic proteins/peptides,19−22 and inorganic
epoxides to synthesize lipid-like molecules for siRNA
nanoparticles.23−28 Another commonly used nucleic acid
delivery.39−43 Recently, we described a two-step method
delivery system is based on viral vectors.29−34 The types of
based on thiol-yne chemistry for the combinatorial synthesis
nucleic acids most commonly used for cell transfections are
of cationic lipids for gene delivery applications.44
plasmid DNA molecules harboring a gene of interest or small
Here we demonstrate that simple alkylation of amines with
double-stranded RNA molecules, termed small interfering
long alkyl halides can be used to create lipid-like molecules
RNAs (siRNAs), that specifically interact with and degrade
the complementary cellular mRNA. Cellular transfection is
capable of cell transfection. We show an application of this
method for a fast combinatorial synthesis of 200 lipid-like
therefore used for both gain-of-function as well as loss-of-
molecules. The produced library was used in a high-throughput
function genetic experimentation, allowing valuable insights to
gene function. In vivo cellular gene delivery via specially
designed vectors also shows high potential for therapeutic
applications.
cell transfection screen, which resulted in identification of about
2% of the synthesized lipidoids possessing transfection
efficiencies similar to or higher than that of Lipofectamine
2000 (L2K).
Although viral vectors are the most common gene delivery
reagents in clinical trials,35 lipid-based vectors are most often
EXPERIMENTAL PROCEDURES
■
used for in vitro delivery of nucleic acids into cells. Several
Materials. Ethanol (99%), 1-(2-aminoethyl)pyrrolidine
(98%), 1-(2-aminoethyl)pyrrolidine (99%), N,N-diethylethyle-
nediamine (99%), 3-(diethylamino)propylamine (99%), 3,3′-
commercialized cationic lipids are widely used as transfection
reagents, for example, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-
trimethylammonium chloride (DOTMA) (in Lipofectin)36,37
and N,N-dimethyl-N-[2-(sperminecarboxamido)ethyl]-2,3-bis-
(dioleyloxy)-1-propaniminium pentahydrochloride (DOSPA)
(in Lipofectamine).2 Such cationic lipids are usually synthesized
via multistep synthesis routes, requiring protecting groups and
Received: March 28, 2013
Revised: July 23, 2013
Published: August 5, 2013
© 2013 American Chemical Society
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diamino-N-methyldipropylamine (96%), 1-bromtetradecane
(98%), 1-ioddodecane (98%), 1-iodhexadecane (98%), 1-
bromoctadecane (98%), 1-bromononane (99%), and 1-
iodooctane (98%) were purchased from Sigma-Aldrich
(Germany). 3-(Dimethylamino)-1-propylamine (98%) was
purchased from Fluka (Germany). Tris(2-aminoethyl)amine
(97%), 1,4-bis(3-aminopropyl)piperazine (98%), and phene-
thylamine (99%) were purchased from Alfa Aesar (Germany).
1,3-Diaminopropane (99%), diethylenetriamine (98%), ethyl-
enediamine (99%), 3-(methylamino)propylamine (99%), 1,8-
diamino-3,6-dioxaoctane (99%), ethanolamine (99%), 1-amino-
2-methoxyethane (98%), N-(2-hydroxyethyl)-1,3-diaminopro-
pane (99%), 2-(2-aminoethylamino)ethanol (98%), 2-piper-
azin-1-ylethylamine (98%), 1,2-diaminocyclohexane (99%), 1-
iododecane (99%), 1-bromoundecane (97%), 1-iodohexane
(99%), and 1-iodoheptane (97%) were purchased from Merck
(Germany). Plasmid DNA mixture consisting of 67.5 ng of
pCS2+β-galactosidase and 7.5 ng of pEGFP-1 (Clontech,
Germany) was used per well of a 96-well plate. 1,2-Di(9Z-
octadecenoyl)-sn-glycero-3-phosphoethanolamine (DOPE) was
bought from Gorden Pharma Switzerland LLC (Liestal,
Switzerland). Lipofectamine (L2K) was purchased from Life
Technologies (Germany).
Library Synthesis. Cs2CO3 (1.5n equiv, where n is the
number of NH bonds present in the corresponding amine) in
THF (2 mL) was placed in a 20-mL vial. An amine (1 equiv,
0.25 mmol) and alkyl halide (n equiv) were added to the vial.
The resulting mixture was shaken (180 rpm) at 40 °C for 20 h.
After cooling, the solvent was removed under reduced pressure,
followed by addition of 1 mL of n-hexane to the residue. The
resulting mixture was transferred to a 1-mL Eppendorf tube and
centrifuged at 13,000 rpm. Supernatant was transferred to a
new 20-mL vial. The solvent was again removed under reduced
pressure to give crude product as a mixture of mono- and
multialkylated amines that were further used without additional
purification.
quartet, quin = quintet, dd = doublet of doublets, dt = doublet
of triplets, m = multiplet. Coupling constants (J) are given in
1
hertz (Hz).13C NMR spectra were recorded as H-decoupled
spectra at room temperature on the Bruker DRX-500 (125
MHz). Spectra were calibrated to the signal of CDCl3 (central
line of triplet δC = 77.00).
Preparation of Cationic Liposomes in the Initial
Screen. A 200 μL solution of DOPE (0.0067 mol/L) and
100 μL of a solution of lipidoid (0.0175 mol/L) in ethanol were
mixed and vortexed for 10 s. Then, 700 μL of a 200 mM
sodium acetate buffer (pH = 5) was added, and the solution
was vortexed for 30 s.
Preparation of Cationic Liposomes for the Optimiza-
tion of Hits. Stock solutions of DOPE (7.44 mg/mL, 0.01
mol/L) and lipidoid (0.01 mol/L) were made in absolute
ethanol. Stock solutions of DOPE and lipidoids were mixed to
produce 300 μL solutions with three different lipid/DOPE
mole ratios (2:1, 1:1, and 1:2) in Eppendorf tubes, followed by
vortexing for 10 s and mixing with 700 μL of 200 mM sodium
acetate buffer (pH = 5). The solution was then vortexed again
for 10 s.
Preparation of Cationic Liposomes To Study Syner-
gistic Effects. Stock solutions of DOPE (7.44 mg/mL, 0.01
mol/L) and lipidoids 1C16-D (0.01 mol/L) and 1C16-S (0.01
mol/L) were made in absolute ethanol. 1C16-D and 1C16-S
were first mixed at four different mole ratios (3:1, 2:1, 1:1, 1:2)
to produce solutions of combined lipidoids. The final molar
concentration of the lipidoids and DOPE were the same as the
concentration of cationic liposomes for the optimization of hits.
Stock solutions of DOPE and combined lipidoids were further
mixed in Eppendorf tubes to produce 300 μL of solutions
containing lipidoids/DOPE mixtures of 1:1 mole ratios,
followed by vortexing for 10 s and mixing with 700 μL of
200 mM sodium acetate buffer (pH = 5). The solution was
then vortexed again for 10 s before using as a transfection
reagent.
ESI-MS and HR-MS Characterization. Electrospray
ionization mass spectrometry (ESI-MS) was performed using
an API 4000 Quadrupol mass spectrometer (Applied
Biosystems, Foster City, CA, USA) equipped with a Turbo V
electrospray ion source (TurboIonSpray probe, Applied
Biosystems) operating in positive ion mode at a source
temperature of 400 °C. Nitrogen was used as nebulizer, curtain,
collision, and auxiliary gas. Instrument controlling and data
acquisition were carried out using the Analyst Software V 1.4
(Applied Biosystems, Foster City, CA, USA). All parameter
settings were optimized by flow injection experiments with
standard solutions infused into the mass spectrometer using a
syringe pump (Harvard Apparatus Inc., South Natick, USA) at
an infusion flow rate of 10 μL/min. Lipidoids were dissolved in
methanol and tested in ESI-MS at 5 μg/mL concentration.
High-resolution mass spectra (HR-MS) were recorded using
the Bruker ICR APEX-QE, Vacuum Generators ZAB-2F,
Finnigan MAT TSQ 700 and JEOL JMS-700.
Dynamic Light Scattering. Dynamic light scattering
(DLS) measurement was performed using the Malvern
Zetasizer Nano ZS (Malvern, Germany). Cationic liposome
samples were prepared by diluting 32 μL of liposomes with 768
μL of 50 mM NaOAc/HOAc buffer (pH = 5). Lipoplex
samples with different DNA amounts (75 ng, 150 ng, and 200
ng DNA per well of a 96-well plate) were prepared as follows:
(a) 16 μL of liposome solutions was diluted with 384 μL of 50
mM NaOAc/HOAc buffer (pH = 5), and 30 μL of DNA
(GFP/LacZ = 1:10; 0.1 μg/μL) was diluted with 370 μL of 50
mM NaOAc/HOAc pH = 5 buffer; (b) 16 μL of liposomes was
diluted with 384 μL of 50 mM NaOAc/HOAc pH = 5 buffer,
and 60 μL of DNA (GFP/LacZ = 1:10; 0.1 μg/μL) was diluted
with 340 μL of 50 mM NaOAc/HOAc pH = 5 buffer; (c) 16
μL of liposomes was diluted with 384 μL of 50 mM NaOAc/
HOAc pH = 5 buffer, and 80 μL of DNA (GFP/LacZ = 1:10;
0.1 μg/μL) was diluted with 320 μL of 50 mM NaOAc/HOAc
pH = 5 buffer. Diluted liposomes and DNA solutions were
mixed to form 800 μL of lipoplex solutions. Particle size
(hydrodynamic diameter) and surface charge (ζ-potential)
were measured sequentially by using a standard operating
procedure. The particle size was presented as the z-average
diameter, which is the intensity-weighted mean hydrodynamic
size of particles measured by DLS.
NMR Characterization. 1H NMR spectra were recorded at
room temperature on the Bruker DRX-500 (500 MHz). Unless
stated otherwise, all spectra were recorded in deuterochloro-
form purchased from Sigma-Aldrich. All chemical shifts are
given in ppm (δ) units relative to tetramethylsilane (singlet δH
= 0.00). Calibration was achieved using the residual solvent
signal of chloroform at δH = 7.27. Analyses followed first order
and the following abbreviations were used throughout the text:
s = singlet, br. s = broad singlet, d = doublet, t = triplet, q =
In Vitro Transfection. For the HEK293 cell transfection
screen, 0.4 μL of liposomal reagent, 75 ng of plasmid DNA
(67.5 ng pCS-LacZ and 7.5 ng pCMV-EGFP), and 4 × 104 cells
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Figure 1. Combinatorial synthesis of lipidoids via alkylation of amines. (A) Twenty amines and 10 alkylhalides (C6−C12, C14, C16, and C18) were
used in the synthesis. (B) Scheme of the synthesis of one typical lipidoid from the library.
were used per well in 96-well plates using a one-step protocol
that combines cell plating and transfection. Liposomal reagent
and DNA were each diluted in a final volume of 10 μL of 50
mM sodium acetate buffer (pH 5.0), combined, and mixed with
pipet action to give 20 μL of lipoplex solution. After lipoplex
incubation at room temperature for 30 min, 80 μL of a freshly
trypsinized cell suspension in DMEM supplemented with 10%
FCS was added, mixed gently with a pipet, and transferred to a
96-well. After 16 h of incubation at 37 °C in 5% CO2, Hoechst
33342 was added to a final concentration of 1 μg/mL by
pipetting 10 μL of a 10 μg/mL solution. After a further 30 min
of incubation to allow staining of DNA within nuclei, cells were
imaged on an Olympus IX81 automated fluorescent micro-
scope. Images for bright field, Hoechst, and GFP were acquired.
Cell transfection efficiencies were estimated using CellProfiler45
after counting the number of GFP positive cells and dividing by
the total number of cells (nuclei). Relative transfection
efficiency was determined by dividing the transfection
efficiencies of lipidoids with that of L2K.
approximately 500 molecules. All lipidoid mixtures were
analyzed for their ability to delivery DNA into cells. After
efficient lipidoid mixtures were identified, selected mixtures
could be purified to isolate individual lipidoids possessing high
transfection efficiencies.
Initial Screen. Lipidoids were screened for transfection
activity by delivering eGFP plasmid into HEK293T cells in
culture. A helper lipid, phosphatidylethanolamine (DOPE), was
admixed with individual lipidoids in order to aid fusion with
cellular membranes and enhance endosomal escape of DNA
within cells.46−48 Lipidoids were mixed with DOPE at molar
ratio of 1:1 in ethanol then diluted in NaOAc/HOAc pH = 5.0
buffer to yield liposomal suspensions containing 30% ethanol
and approximately 2 mg/mL total lipid. The acidic buffer was
used in order to protonate the amino head groups of the
lipidoids. We used two plasmids because there is a requirement
to have a fixed amount of total DNA for the transfection
protocol; however, this amount (75 ng per 96-well) is too
much for the EGFP expression construct alone and would
result in the overexpression of toxic levels of the GFP protein in
cells. For this reason we “make up” to the total amount of DNA
using an inert expression construct that is not toxic for cells
(the pCS-LacZ, encoding the β-galactosidase gene). Cell
transfection was performed using a one-step method, where
freshly trypsinized and resuspended cells were added directly to
the lipoplexes, followed by transfer to culture wells. The
following day the nuclei of adherent cells were stained with
Hoechst, and imaged automatically with a fluorescent micro-
scope. In this assay, Hoechst staining was used to count the
total cell number and also indicate cell toxicity. CellProfiler
software45 was used to calculate transfection efficiency by
quantifying the ratio of GFP transfected to total cell number.
The average transfection efficiencies were calculated and are
RESULTS AND DISCUSSION
■
Synthesis of Alkylamine Lipidoids. In order to produce a
library of lipidoids, parallel synthesis was performed using 20
different amines and 10 alkyl halides (C6−C12, C14, C16,
C18) (Figure 1A). Direct alkylation was performed in the
presence of Cs2CO3 to trap the acid produced during alkylation
(Figure 1B). After 20 h of alkylation reaction, the alkylamines
were isolated by extraction with hexane. Using this one-step
method, a library of 200 lipidoid mixtures was synthesized in a
single day. Since each lipidoid mixture contained both single
and double as well as in some cases multialkylated amines, the
overall number of individual lipidoids in the library came to
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Figure 2. Results of in vitro cell transfection screening of the lipidoid library. (A) Graph showing relative transfection efficiency with respect to
Lipofectamine 2000 of selected lipidoids with transfection efficiencies above 20%. Lipidoids/DOPE (1:1) mixtures were screened by delivering
eGFP plasmid into HEK293T cells in 96-well plates. (B) Graph showing correlations between the nature of cationic head groups, length of
hydrophobic tails, and the efficiency of cell transfection.
shown in Supplementary Tables S3−S6. Lipofectamine 2000
(L2K) was used as a positive control. Relative transfection
efficiency was determined by dividing the average transfection
efficiencies of lipidoids with that of L2K.
superior performance in cell transfection. These results indicate
that both the nature of cationic head groups and the length of
hydrophobic tails are important determinants in transfection
efficiency.
In this screen, 12 lipidoid mixtures showed transfection
activity in HEK293T cells, 4 of which had an efficiency equal to
or greater than that of L2K (Figure 2A). Notably, for these top
4 hits, increasing the length of hydrophobic tails from C14 up
to C18 resulted in a corresponding increase in transfection
efficiency (Figure 2B), which is in accordance with our previous
study showing a particular importance of the length of
hydrophobic tails for transfection efficiency.44 With respect to
the amine head groups, those lipidoids containing either 1-(2-
aminoethyl)pyrrolidine (1) or 3-(dimethylamino)-1-propyl-
amine (4) (Figure 1A) showed the highest activity. It is
notable that the dimethylamine headgroup has been already
used in several nonviral gene delivery systems, including both
lipid-44 and polymer-based49 vectors, where it has shown
Cell Transfection Optimization. To further optimize and
analyze the performance of the identified lipidoids in cell
transfection, we repeated synthesis of the 12 hits identified in
the initial screen. These lipids were used to prepare liposomal
transfection reagents using three different lipidoid/DOPE mole
ratios (2:1, 1:1, 1:2). Figure 3 shows that although 1C12, 1C16,
4C16, 4C18, and 10C14 maintain high transfection efficiency,
several hits identified during the initial screen show a decreased
efficiency. A difference in the ratio of mono- and disubstituted
amines produced by the two synthesis reactions likely explains
the variation. This was confirmed by testing the efficiency of
mono- and disubstituted lipidoids mixed at different ratios (see
below). Interestingly, the hits with the longest hydrophobic
chains (C16 and C18) were more effective when combined
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Figure 6. Optimization of in vitro DNA delivery into HEK293T cells
by selected hits at three different lipidoid/DOPE molar ratios.
Figure 3. Relative transfection efficiencies in HEK293T cells (with
respect to Lipofectamine 2000) of selected hits of three different
lipidoid/DOPE molar ratios (2:1, 1:1, 1:2). Amount of DNA is 75 ng
for 0.4 μL of liposome solution (400 ng total lipid), which is
equivalent to a lipidoid/DNA mass ratio of 5.3:1.
mainly the double-chain lipid (1C12-D). Purification of the
1C16 sample led to the isolation of lipidoids bearing both
single (1C16-S) and double (1C16-D) alkyl chains (Figure 4).
HR-MS and NMR confirmed the structures (see Supporting
Information).
In order to optimize the transfection efficiency of 1C12-D,
1C16-D, and 1C16-S, three different lipidoid/DOPE ratios
(2:1, 1:1, 1:2), as well as three different DNA/liposome ratios
(75, 150, or 200 ng DNA per 800 ng liposome reagent,
equivalent to lipidoid/DNA mass ratios of 10.7:1, 5.3:1, and
4:1, respectively) were tested and compared. Fluorescent
micrographs of the transfection results are shown in Figure 5.
1C12-D and 1C16-D with two hydrophobic tails maintained
significant transfection efficiency. For both 1C12-D and 1C16-
D the transfection efficiency decreased with increasing the
amount of DNA added to liposomes. The optimal lipidoid/
DNA mass ratio was found to be 10.7:1, (75 ng plasmid DNA
per 0.4 μL liposome solution per 96-well). Interestingly, 1C16-
S with only one hydrophobic tail showed very low efficiency
(Figure 6), suggesting that hydrophobicity of a single alkyl tail
was not enough to facilitate cellular transfection. In addition,
although the particle size of 1C16-D lipoplexes (liposome−
DNA complexes) stabilized at 450 nm within the first 30 min
after mixing the liposomes with DNA, the size of 1C16-S
lipoplexes continued to increase above 1 μm (Supplementary
Figure S1).
Figure 4. Structures of 1C12-D, 1C16-D, and 1C16-S lipidoids.
with a lower ratio of DOPE, which itself has two long C18 tails
(blue bars, Figure 3).
To further investigate the performance of the novel
alkylamines as gene delivery vectors, two lipidoids from the
hits (1C12, 1C16) were purified. 1C12 was shown to contain
Size and Surface Charge of Lipid Particles. Liposome’s
size and surface charge (ζ-potential) are known to have a major
Figure 5. Fluorescent microscope images of in vitro DNA delivery into HEK293T cells by purified 1C12-D, 1C16-D, and 1C16-S at three different
lipidoid/DOPE molar ratios.
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Figure 7. Particle size (A) and ζ-potential (B) of liposomes and lipoplexes formed by 1C12-D, 1C16-D, and 1C16-S at three different lipidoid/
DOPE ratios (2:1, 1:1, 1:2) and different liposome/DNA ratios. The particle size was presented as the z-average diameter, which is the intensity
weighted mean hydrodynamic size of particles measured by DLS.
effect on transfection. To investigate why the transfection
efficiency decreased with the increase of DNA amount as well
as to understand better the reason for decreased activity of the
single-tailed lipid, we next analyzed the particle size and surface
charge of lipid particles before (liposomes) and after the
complexation with DNA (lipoplexes). The experiment was
done using DLS. Figure 7A shows that the size of lipoplexes
increases from 200 to 600 nm to more than 2 μm with
increasing amounts of plasmid DNA. With respect to surface
charge, the ζ-potentials of lipoplexes decrease from +60 mV to
−60 mV upon increasing the amount of plasmid DNA (Figure
7B). Both results indicate that small particle size and high
positive surface charge are two key parameters to maintain high
transfection efficiency. Liposomes composed of single chained
lipids were marginally smaller than those composed of double-
chained lipids (black bars, Figure 7A); however, overall the size
and surface charge of lipoplexes composed of the single-tailed
lipidoid did not significantly differ from that of the double-
tailed lipids. This means that the observed decrease in efficiency
Figure 8. Synergistic effect of the combination of lipidoids with single
(1C16-S) and double (1C16-D) hydrophobic tails on cell transfection
is likely not due to the size and surface charge of liposomes or
lipoplexes. A reduced stability of the lipoplexes composed of
the single-tailed lipids could be a possible explanation. Another
conclusion from this study is that the amount of DOPE in
liposomes has only minor effect on the size and surface charge
efficiency. HEK293T cells in 96-well plate were transfected with 75 ng
total DNA (eGFP/LacZ = 1:10) and 0.4 μL of liposome solution
(lipidoid/DOPE = 1:1). This is equivalent to a lipidoid/DNA mass
ratio of 5.3:1. Ratios shown below graph indicate 1C16-S/1C16-D.
L2K = lipofectamine 2000.
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of the lipoplexes, which is also reflected in the transfection
efficiencies (Figure 6).
ASSOCIATED CONTENT
■
S
* Supporting Information
Synergistic Increase of Transfection Efficiency. In the
initial screen the lipidoid 1C16 showed higher transfection
efficiency compared with that of 1C12 (Figure 2); however,
subsequent analysis of the purified double-chained lipidoid
species showed the reverse (Figure 6). Interestingly, the
synergistic siRNA delivery was observed by combining lipid-like
materials.50 We therefore reasoned that the separation of the
single- and double-alkylated 1C16 lipidoids (Figure 4) may
account for the observed decrease in transfection efficiency. To
test this hypothesis and to investigate whether a combination of
1C16 lipidoids with different number of hydrophobic tails has a
positive synergistic effect on cell transfection, we recombined
1C16-D and 1C16-S at four different 1C16-D/1C16-S molar
ratios (3:1, 2:1, 1:1, 1:2) and, for each, prepared liposomes at
1:1 lipidoid/DOPE ratio. The final molar concentration of
lipidoids and DOPE was the same as for the samples prepared
during optimization of the cell transfection. The results of cell
transfection with these mixtures are shown in Figure 8.
Interestingly, and as hypothesized, the addition of the single-
tailed lipid 1C16-S to the double-chained 1C16-D significantly
enhances the transfection efficiency in HEK293T cells. We next
monitored the time-dependent change of the particle size
(Supporting Figure S1) and ζ-potential (Supporting Figure S2)
of lipoplexes during the first 30 min after mixing liposomes with
plasmid DNA. The ζ-potential of liposomes formed by single-
tailed lipid 1C16-S was much higher than that of double-tailed
lipid 1C16-D. After adding the single-tailed lipid 1C16-S to the
double-tailed lipid 1C16-D, the ζ-potential of liposome
increased from 46 to 63 mV. The particle size of lipoplexes
formed by 1C16-D is more stable and smaller than 1C16-S.
Increasing the proportion of 1C16-D in the 1C16-S liposomes
results in lipoplexes that are more stable and the final particle
size of lipoplexes decreases. These results indicate that the
single and double-tailed lipids play different roles in the
formation of lipoplexes. The single-tailed lipid 1C16-S increases
the surface charge of the liposomes and lipoplexes, while the
double-tailed lipid 1C16-D forms smaller and more stable
lipoplexes. The different roles of lipids also give us a hint that
we may miss some hits in the initial screen.
Experimental details of chemical synthesis, H and 13C NMR
spectra of purified compounds, and the results of dynamic light
scattering. This material is available free of charge via the
Internet at http://pubs.acs.org.
1
AUTHOR INFORMATION
Corresponding Author
*E-mail: levkin@kit.edu; gary.davidson@kit.edu.
Notes
■
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors would like to thank Dr. Peravali (ITG) for his
assistance with the Olympus IX81 automated fluorescent
imaging microscope, Dr. Brenner-Weiß (IFG) and his group,
especially Michael Nusser, for their help on API 4000
Quadruple mass spectrometer, and Sven Stahl (INT) for his
help with NMR Bruker AMX 500 spectrometer. This work was
supported by funding from the Deutsche Forschungsgemeind-
chaft (DFG) as part of the Wnt Forschergruppe, FOR 1036
(Davidson Lab) and Helmholtz Association’s Initiative and
Networking Fund (grant VH-NG-621) (Levkin Lab).
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CONCLUSIONS
■
We have presented a facile one-step method applicable for
parallel combinatorial synthesis of new vectors for gene delivery
applications. The method was used to synthesize 200
alkylamine lipidoids that were screened for their transfection
efficiency leading to the identification of 12 hits showing
efficiencies comparable or higher than that of Lipofectamine
2000. The hits were further optimized and characterized by
measuring the size and ζ-potential of liposomes and lipoplexes.
In addition, we have found that combining lipidoids with one
and two hydrophobic tails has a positive synergistic effect on
cellular transfection efficiency. This effect was attributed to the
different roles of the lipids in the mixed liposomes: the single-
tailed lipid was shown to increase the positive surface charge,
and the double-tailed lipid improved the stability of the vesicles.
We believe that the developed cost-effective method for
combinatorial synthesis of cationic lipid-like molecules and
the new insights about the effect of a liposome composition on
transfection efficiency will help in the development of more
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