Polyglycerol-based amphiphilic dendrons as potential siRNA carriers for in vivo applications

Article abstract of DOI:10.1039/c3tb21364a

The development of nonviral synthetic vectors for clinical application of gene therapy using siRNA transfection technology is of particular importance for treatment of human diseases, which is yet an unsolved challenge. By employing a rational design approach, we have synthesized a set of well-defined, low-molecular-weight dendritic polyglycerol-based amphiphiles, which are decorated peripherally with the DAPMA (N,N-di-(3-aminopropyl)-N-(methyl)amine) moiety. The main differences that were introduced in the structural motif relate to dendron generation and the type of linker between the hydrophilic and hydrophobic segment. The synthesized amphiphiles were then characterized for their aggregation behaviour and further evaluated with respect to their siRNA transfection potential by comparing their physico-chemical and biological features. Our findings demonstrated that all four synthesized amphiphiles yielded high gene binding affinities. Furthermore, the ester-linked compounds (G1-Ester-DAPMA, G2-Ester-DAPMA) revealed noticeable gene silencing in vitro without affecting the cell viability in the tumor cell line 786-O. Remarkably, neither G1-Ester-DAPMA nor G2-Ester-DAPMA induced inflammatory side effects after systemic administration in vivo, which is noteworthy because such highly positively charged compounds are typically associated with toxicity concerns which in turn supports their prospective application for in vivo purposes. Therefore, we believe that these structures may serve as new promising alternatives for nonviral siRNA delivery systems and have great potential for further synthetic modifications.

Full text of DOI:10.1039/c3tb21364a

Journal of
Materials Chemistry B
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PAPER
Polyglycerol-based amphiphilic dendrons as
potential siRNA carriers for in vivo applications†
Cite this: J. Mater. Chem. B, 2014, 2,
2153
Ariane Tschiche,‡a Anna M. Staedtler,‡b Shashwat Malhotra,‡a Hannah Bauer,c
Christoph Bottcher,d Soroush Sharbati,c Marcelo Calderon,a Markus Koch,b
¨
´
Thomas M. Zollner,b Anna Barnard,e David K. Smith,e Ralf Einspanier,c
b
a
*
*
Nicole Schmidt and Rainer Haag
The development of nonviral synthetic vectors for clinical application of gene therapy using siRNA
transfection technology is of particular importance for treatment of human diseases, which is yet an
unsolved challenge. By employing a rational design approach, we have synthesized a set of well-defined,
low-molecular-weight dendritic polyglycerol-based amphiphiles, which are decorated peripherally with
the DAPMA (N,N-di-(3-aminopropyl)-N-(methyl)amine) moiety. The main differences that were
introduced in the structural motif relate to dendron generation and the type of linker between the
hydrophilic and hydrophobic segment. The synthesized amphiphiles were then characterized for their
aggregation behaviour and further evaluated with respect to their siRNA transfection potential by
comparing their physico-chemical and biological features. Our findings demonstrated that all four
synthesized amphiphiles yielded high gene binding affinities. Furthermore, the ester-linked compounds
(G1-Ester-DAPMA, G2-Ester-DAPMA) revealed noticeable gene silencing in vitro without affecting the
cell viability in the tumor cell line 786-O. Remarkably, neither G1-Ester-DAPMA nor G2-Ester-DAPMA
induced inflammatory side effects after systemic administration in vivo, which is noteworthy because
such highly positively charged compounds are typically associated with toxicity concerns which in turn
supports their prospective application for in vivo purposes. Therefore, we believe that these structures
may serve as new promising alternatives for nonviral siRNA delivery systems and have great potential for
further synthetic modifications.
Received 1st October 2013
Accepted 31st January 2014
DOI: 10.1039/c3tb21364a
www.rsc.org/MaterialsB
unwanted gene expression, or introduce new cellular func-
tions.4,5 In recent years, the antisense approach has been
Introduction
regarded as particularly promising, which involves the intra-
cellular delivery of antisense constructs that usually lead to a
reduction of target activity, termed gene silencing.6 Nonviral
gene carriers including the main classes of cationic polymers
and lipids have been extensively studied for two decades now as
alternatives to viral vectors due to their improved safety, greater
synthetic tunability and versatility, larger capacity for thera-
peutic genes, and more facile manufacturing.7–15 However, low
transfection efficiencies and toxicity issues still remain major
barriers for clinical application of nonviral gene therapy.16–18
Among the nonviral nanocarriers, dendrimers have been
utilized and examined in biomedical elds for drug and gene
delivery systems because of their dened molecular structures,
versatility with regard to molecular size and surface chemistry,
intrinsic multivalent features, and high cargo payload at
nanosized dimensions.19–24 Developing dendrimers as nano-
vectors to deliver RNA therapeutics, especially synthetic small
interfering RNAs (siRNAs), and to employ them for the specic
inhibition and knockdown of disease genes by RNA interference
(RNAi) is one of the most promising pathways within the eld of
Gene therapy holds substantial promise for the treatment of
inherited and acquired diseases such as cystic brosis, HIV,
arthritis, cancer, and other medical conditions.1–3 It is based on
correcting the origin of diseases by delivery and subsequent
expression of exogenous genetic materials, either RNA or DNA,
to replace defective genes, substitute missing ones, silence
a
¨
Institute of Chemistry and Biochemistry, Freie Universitat Berlin, Takustrasse 3,
Berlin 14195, Germany. E-mail: haag@chemie.fu-berlin.de; Web: http://www.
polytree.de; Fax: +49-30-838-53357; Tel: +49-30-838-52633
bBayer Pharma AG, Global Drug Discovery, Therapeutic Research Group Oncology/
Gynecological Therapy, Muellerstrasse 178, Berlin 13353, Germany. E-mail: nicole.
schmidt1@bayer.com; Tel: +49-30-468-194360
c
¨
Institute of Veterinary Biochemistry, Freie Universitat Berlin, Oertzenweg 19b, Berlin
14163, Germany
dResearch Center of Electron Microscopy, Institute of Chemistry and Biochemistry,
¨
Freie Universitat Berlin, Fabeckstrasse 36a, Berlin 14195, Germany
eDepartment of Chemistry, University of York, Heslington, York, YO10 5DD, UK
† Electronic supplementary information (ESI) available: Experimental details and
transfection results of A3 and A4, as well as CMC, DLS, and EthBr assay graphics.
See DOI: 10.1039/c3tb21364a
‡ These authors contributed equally to this study.
This journal is © The Royal Society of Chemistry 2014
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gene therapy.25–30 The main challenge lies in the safe and effi- glycine-functionalized amphiphiles based on polyglycerol den-
cient delivery of siRNA. An optimal gene vector has to fulll drons that had successfully delivered luciferase and GAPDH
specic requirements in order to successfully transfect cells siRNA into HeLa cells.27 Interestingly, one candidate (G2-octa-
both in vitro and in vivo. Foremost, the transporter needs to amine) with eight amine groups on its surface and a hydro-
complex and condense the genetic material, protect it from phobic C-18 alkyl chain at the core acted as an efficient siRNA
nucleolytic degradation, enable cellular uptake, and nally vector and still remained non-toxic, even at high N/P ratios (e.g.,
release it from the endosomal pathway into the cytosol, where N/P of 100).
the RNAi machinery is located.31
In an endeavor to create a gene vector which is efficient and
Although high molecular weight dendrimers like PAMAM safe for both in vitro and in vivo applications, we have now
(poly(amidoamine)),7,32,33 PPI (poly(propylene imine)),34–36 and adopted the same structural motif but incorporated distinctive
PEI (poly(ethylenimine))37–39 can lead to high transfection effi- structural variations with respect to the employed amine moiety
ciencies, they also exhibit problematic toxicity proles,40 which as well as the type of linker between the hydrophilic and
can be partially attributed to their cellular accumulation aer hydrophobic domains (Fig. 2).
gene delivery has taken place.41 An alternative approach, mainly
On the surface of the dendritic head group of the amphi-
established and advanced by Florence,42 Sanya,43 Diederich,44 philes, we attached DAPMA, an amine functionality that has
and Smith,45 was to modify low-molecular-weight, amine-func- already proven to be an effective entity for gene delivery appli-
tionalized dendritic arrays with hydrophobic portions to cations.41,49 With reference to the linkage, we decided to inte-
promote their self-assembly into supramolecular dendrimers grate exible, biodegradable ester functionalities (A1, A2) in
(“pseudodendrimers”). In this way, positively charged large contrast to rigid, non-degradable triazole groups (A3, A4), to
multivalent arrays are generated that are suitable for gene assist the effective release of the genetic cargo. It is known that
delivery applications (Fig. 1).
micelles with biodegradable features can enhance polyplex
Our research group has previously proven that such a dissociation and thus improve transfection efficiency.50,51 The
strategy can enhance gene delivery and lead to synergistic integration of rigid elements has also been shown to have a
effects if aspects of both classes of nonviral gene delivery, i.e., benecial effect with respect to biocompatibility and trans-
polymers and lipids, are combined,27 which also has been fection properties.44,52
demonstrated with related architectures.17,45–48 In fact, along
Systematically, we investigated these amphiphilic dendrons
with a recent report by Peng and co-workers,28 this was one of for their aggregation behaviour and further evaluated their
the rst successful examples of in vitro siRNA transfection using siRNA transfection potential by employing different kinds of
dendritic amphiphiles.27 More specically, we developed physico-chemical and biological techniques. The obtained
Fig. 1 Concept of self-assembling amphiphiles used for gene delivery applications.
Fig. 2 Dendritic amphiphiles investigated in this study (A1 ¼ G1-Ester-DAPMA, A2 ¼ G2-Ester-DAPMA, A3 ¼ G1-Trz-DAPMA, A4 ¼ G2-Trz-
DAPMA).
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results demonstrate that all constructs lead to a comparably Fluorescence emission spectra were taken using a Jasco FP-6500
strong gene binding. However, only the ester-bridged amphi- spectrouorimeter equipped with a thermostated cell holder, a
philes (A1, A2) revealed no cytotoxic effects and caused DC-powered 150 W xenon lamp, a Hamamatsu R928 photo-
reasonable gene silencing in 786-O tumor cells in vitro. Notably, multiplier, and a variable slit system. Both excitation and
systemic administration of A1 and A2 in mice demonstrated emission slits were set at 3 nm. The uorescence of pyrene was
that these constructs do not induce inammatory side effects, recorded from 350 to 600 nm aer excitation at 330 nm. Prior to
which renders these novel architectures highly interesting for measurements, a pyrene stock solution of 1 mM (in HEPES
siRNA delivery in future applications of therapeutic RNAs.
saline buffer, 9.4 mM NaCl, pH 7.4) was freshly prepared by rst
dissolving pyrene in a small amount of methanol, evaporating
the solvent until dryness through slow passage of N2, followed
by the addition of buffer. Aer complete pyrene solubilization
and dilution to 0.5 mM, the amphiphiles were added as solid in
varying amounts (1 mM–10 mM). The solutions were kept for 2 h
at room temperature to promote the self-assembly pꢀrocess.
Fluorescence measurements were carried out at 22 ꢁ 2 C and
taken in triplicate and averaged.
Cryo-transmission electron microscopy (cryo-TEM). For all
sample preparations, aqueous amphiphile solutions were used
at a concentration of 4.5 ꢂ 10ꢃ3 M. Droplets of the corre-
sponding sample solution (5 ml) were applied to perforated
(1 mm hole diameter) carbon lm covered 200 mesh copper
grids (R1/4batch of Quantifoil Micro Tools GmbH, Jena, Ger-
many), which had been hydrophilized prior to use by 60 s
plasma treatment at 8 W in a BALTEC MED 020 device. The
Experimental section
Materials
Air and moisture sensitive reactions were carried out in dried
glassware under an argon atmosphere. Anhydrous solvents were
either commercially purchased from Acros Organics™ in
septum-sealed bottles or chemically dried using a MBRAUN SPS
800 solvent purication system. Compounds 2, 3, and 10 were
synthesized according to reported procedures.49,53 All other
chemicals were of reagent grade quality and used without
further purication from the suppliers Acros Organics™,
Fluka®, Sigma-Aldrich®, Roth®, Invitrogen™, and Merck™.
Measurements
Chromatography, spectroscopy, and spectrometry. Thin supernatant uid was removed with a lter paper until an ultra-
layer chromatography (TLC) analysis was carried out on silica- thin layer spanning the holes of the carbon lm was obtained.
coated aluminium plates from Merck™ either using silica gel The samples were immediately vitried by propelling the grids
60, F254, or silica gel 60 RP-18 F254s. Preparative column chro- into liquid ethane at its freezing point (90 K) and by operating a
matography was conducted on silica gel 60 (0.040–0.063 mm, guillotine-like plunging device. The vitried samples were
230–400 mesh ASTM). Detection was accomplished by UV irra- transferred under liquid nitrogen cooling into a Tecnai F20 FEG
diation (254 nm; 366 nm) and different staining solutions such transmission electron microscope (FEI Company, Oregon, USA)
as potassium permanganate, cerium molybdate, ninhydrin, using the Gatan (Gatan Inc., California, USA) cryoholder and
bromocresol green and Dragendorff reagent. Size-exclusion -stage (model 626). Microscopy was carried out at 94 K sample
chromatography (SEC) was employed by using Sephadex® LH- temperature using the microscope's low dose protocol at a
20 (from GE Healthcare).
calibrated primary magnication of 50 000 and an accelerating
NMR spectra were recorded on a Bruker™ ECX 400 (1H: 400 voltage of 160 kV (FEG-illumination). Images were recorded
MHz, 13C: 100.5 MHz), a Jeol™ Eclipse (1H: 500 MHz, 13C: 125.8 using an EAGLE 4k-CCD camera (FEI Company, Oregon, USA)
MHz), or on a Bruker™ Biospin (1H: 700 MHz, 13C: 176.1 MHz) operated with binning factor 2 (2048 by 2048 pixel). The defocus
spectrometer at 25 ꢀC in deuterated chloroform (CDCl3), was chosen in all cases to be 2 m. It is important to note that
methanol (CD3OD), or water (D2O). Chemical shis (d) are given although the determined diameter measurements were prone
in parts per million (ppm) according to calibration to the cor- to error due to the very small size of the assemblies, more
responding solvents CDCl3 (1H: 7.26 ppm, 13C: 77.00 ppm), reliable diameter values could be derived from sample areas
CD3OD (1H: 4.87 ppm, 13C: 49.05 ppm), and D2O (1H: 4.79 ppm). where micelles were densely packed. Fourier transforms of
13C NMR spectra were recorded with 1H broadband decoupling. corresponding images revealed a diffraction pattern which
Multiplicities are indicated by s (singlet), d (doublet), t (triplet), indicates repetitive distances which can be said to correlate with
q (quartet), quin (quintet), and m (multiplet). Coupling the diameter of the micelles (cf. Fig. 3B).
constants (J) are given in Hz.
DLS and zeta potential. DLS and zeta potential measure-
Electrospray-ionization time-of-ight mass spectrometry ments were conducted at 25 ꢀC using a Zetasizer Nano ZS
(ESI-TOF-MS) experiments were carried out using an Agilent analyzer™ with an integrated 4 mW He–Ne laser, l ¼ 633 nm
6210 ESI-TOF, Agilent Technologies™. The calculated masses (Malvern Instruments™ Ltd, U.K.). The amphiphiles were
refer to the respective isotopes with the highest intensity. The measured in HEPES saline buffer (2 mM, EDTA 10 mM, NaCl
measured masses typically represent the main MS peak, while 9.4 mM) or in phosphate buffer (10 mM) at pH 7.4. Polyplex
the found isotope pattern intensities matched those calculated. solutions were obtained as follows: rst a 200 mM DNA (21-mer
Critical micelle concentration (CMC). The critical micelle oligonucleotides) solution was freshly prepared in HEPES buffer
concentration (CMC) was determined in buffered aqueous solution. With respect to a certain N/P ratio, the appropriate
solution (HEPES saline buffer, 9.4 mM NaCl, pH 7.4) by using amount of amphiphile was added. It should be noted that the
the methodology of pyrene uorescence probing.54 concentrations of the respective amphiphiles during polyplex
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cultured at 37 ꢀC and 5% CO2 in RPMI 1640 medium (Bio-
chrom), supplemented with 10% fetal bovine serum
(Biochrom). In addition, the human renal carcinoma cell line
786-O-Luc, constitutively expressing the rey luciferase, was
used for transfection and simultaneous cell viability studies.
786-O-Luc cells were cultured in growth medium composed of
RPMI 1640 (Biochrom), supplemented with 10% FCS, 1%
200 mM glutamine, 1% 100 mM pyruvate, 1% 1 M HEPES, 4.5 g
Lꢃ1 glucose and 50 mg mLꢃ1 hygromycin B at 37 ꢀC, 5% CO2. As
a general note, it should be pointed out that the concentrations
of the respective amphiphiles during polyplex formation were
above each CMC. Aer mixing and incubation with siRNA for
30 min, the sample solutions were diluted and directly
subjected to the relevant cells.
Fig. 3 Representative cryo-TEM images of G1-Ester-DAPMA (A1). (A)
Small spherical particles with diameters of 3–5 nm coexist with (B)
extended two-dimensional arrays possessing a hexagonal ultrastruc-
ture. Fourier transform of these arrays reveals a repetitive distance of
4.44 nm indicating a highly ordered packing of A1.
Cell viability and cytotoxicity assays. Cytotoxicity and cell
viability were measured using the end-point viability WST-1
assay (Roche) as well as the xCELLigence system (Roche) for
formation were above each CMC. Aer mixing and incubation continuous monitoring of cell viability and growth in real-time,
for 30 min, the sample solutions were diluted and directly as described previously.27 The assays were conducted 24 hours
measured. All measurements were carried out using folded aer seeding 1 ꢂ 104 HeLa cells per well in 96-well plates using
capillary cells (DTS 1060) in three replicate measurements.
the amphiphiles A1–A4 and Lipofectamine 2000 transfection
Ethidium bromide displacement assay. The experimental reagent (Invitrogen) as the positive control. Untreated cells
protocol for the ethidium bromide (EthBr) displacement assay served as the negative control (termed “Control”). Each plate
is based on reported procedures.55–58 Fluorescence spectroscopy contained blanks, controls, and serial dilutions of the
measurements were performed using a JASCO FP-6500 spec- substances in four replicates. The WST-1 assay was performed
trouorometer using an excitation wavelength of 520 nm. The according to the manufacturer's instructions and carried out
uorescence intensity of samples at different N/P ratios was with non-targeting siRNA (ON-TARGETplus Non-targeting
recorded from 560 to 700 nm. The DNA binding properties were siRNA, Dharmacon). The colorimetric WST-1 assay is based on
studied at low salt concentration in HEPES saline buffer (pH the enzymatic cleavage of the tetrazolium salt WST-1 to for-
7.4, 2 mM HEPES, 9.4 mM NaCl), supplemented with EthBr mazan by cellular mitochondrial dehydrogenases present in
(1.26 mM). Solutions containing DNA (21-mer oligonucleotides) metabolically active cells. It directly reects the number of
and EthBr in buffer were rst incubated at room temperature viable cells in culture. The xCELLigence system allows contin-
for 5 min to ensure interactions. Consequently, an appropriate uous cell monitoring by measuring the impedance via inte-
quantity of the corresponding amphiphile was added in order to grated microelectrodes on the bottom of each well. The
reach the desired N/P ratio. The uorescence of the DNA solu- electrode impedance, displayed as Cell Index (CI) values,
tion with EthBr was set at 100%. The uorescence values were increases with the amount of attached cells on the electrodes. In
normalized against the set 100% value and expressed as relative addition, cell interactions with the electrodes due to
reduction of the uorescence intensity. Control experiments morphology affect the impedance. Therefore, the CI values
were conducted by measuring solutions which only contained reect the biological state of monitored cells, including the cell
EthBr and amphiphiles. Experiments were performed in number, viability, adhesion, and morphology. The xCELLigence
triplicate.
experiment was divided into three parts: background
Agarose gel electrophoresis. The siRNA–amphiphile inter- measurement, cell monitoring and compound activity moni-
actions were measured by means of agarose gel electrophoresis toring. For each treatment quadruplicate wells were used for
retardation assay employing FAM-labeled siRNA (GUCAACG- statistical analysis. 50 mL of medium was added to each well and
GAUUUGGUCGUA, synthesized by Eurogentec).27 One day prior the plate was adjusted for 15 min at room temperature. Aer
to electrophoresis the amphiphiles A1–A4 were dissolved in transferring the E-plate (Roche) to the Real-Time Cell Analyzer
double distilled water. The uorescently labeled siRNA was (RTCA) instrument, a second equilibration step for another
complexed with each amphiphile solution by incubation for 15 min in the incubator (37 ꢀC, 5% CO2) was performed.
30 min at room temperature. Here, it was ensured that the Thereaer the experiment started by measuring the back-
concentrations were above the respective CMCs of the amphi- ground of the medium. Then, the E-plate was taken out and 100
philes. The polyplexes were then loaded on 4% high-resolution mL cell suspension (1 ꢂ 104 cells per well) was added to the
agarose gels (MetaPhor Agarose, Lonza) and subjected to elec- wells. The E-plate was le for 15 min on a clean bench allowing
trophoresis at 70 V. The labeled siRNA complexes were visual- even distribution of cells. The E-plate was put in the RTCA
ized aer excitation at 495 nm using the Fusion SL imager instrument and aer 15 min for pH adjustment the cell moni-
(Vilber Lourmat).
toring step (measurement every 15 min for 24 h) was started.
Cell culture. Cell viability and cytotoxicity studies were per- Aer 24 h, the medium was changed (removal of 100 mL old
formed with the cell line HeLa (ATCC no. CCL-2), which was medium, addition of 50 mL fresh medium) and 50 mL of each
2156 | J. Mater. Chem. B, 2014, 2, 2153–2167
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polyplex solution were added. The E-plate was put in the incu- temperature, overnight. The solution was concentrated to
bator for 20 min to allow equilibration. Then, the compound dryness under vacuum, and the residue was taken up in chlo-
activity monitoring step was started (measurement every 30 s for roform (25 mL) and water (5 mL). The organic layer was sepa-
6 h, aerwards every 15 min for 40 h).
rated, washed with saturated sodium chloride (2 ꢂ 15 mL) and
The cell viability was additionally investigated in 786-O-Luc water (2 ꢂ 15 mL), and dried over MgSO4. The solvent was
tumor cells following amphiphile treatment. Cells were removed under vacuum, and the crude esteried product was
cultured in 96-well plates at a density of 1 ꢂ 104 cells per well. directly used for the next step. The crude product was treated
Aer 24 hours the growth medium was discarded and replaced overnight with a mixture of triuoroacetic acid–methanol (1 : 3)
by 100 mL Opti-MEM (Gibco). The amphiphiles G1-Ester-DAPMA to remove the acetal groups. The reaction mixture was then
(A1) and G2-Ester-DAPMA (A2) were dissolved in water and evaporated under reduced pressure and puried via column
complexed with luciferase specic (GCAAGAUCGCCGU- chromatography (eluent CHCl3–MeOH, 90 : 20) to afford the
GUAAUAUU, Dharmacon) or non-targeting siRNA (ON-TAR- desired amphiphiles 6 and 7 as viscous oils in 75% and 65%
GETplus Non-targeting siRNA, Dharmacon) by incubating yields, respectively.
5 pmol siRNA with corresponding amounts of amphiphiles
Compound 6. Obtained as a viscous oil (11.8 g, 75% over two
1
depending on N/P ratios 10, 20, and 30. Lipofectamine RNAi- steps). H NMR (MeOD-d4, 400 MHz) d 0.88 (3H, t, J ¼ 6.9 Hz,
MAX transfection reagent (Invitrogen) was used as the positive alkyl CH3), 1.27 (28H, s, alkyl CH2), 1.56–1.64 (2H, m, CH2–CH2–
control following the manufacturer's protocol. Untreated cells O), 2.29–2.37 (2H, m, CH2–CH2–CO–O), 3.43–3.67 (13H, m,
were used as the negative control. In a total volume of 50 mL the dendron), 3.68–3.77 (2H, m, dendron); 13C NMR (MeOD-d4, 101
polyplexes were added to the cells and incubated for 24 h at MHz) d 13.18, 22.44, 24.73, 24.73, 28.88, 28.92, 29.14, 29.14,
ꢀ
37 C, which was then replaced by RPMI growth medium and 29.19, 29.33, 29.50, 31.78, 33.67, 33.89, 48.55, 63.08, 69.79,
incubated for another 24 h. To determine the number of viable 70.84, 70.98, 71.16, 71.46, 72.54, 72.66, 77.28, 173.69. HRMS: m/
cells within this study, the CellTiter-Glo Luminescent Cell z calcd for C27H54O8Na [M + Na]+: 529.3711. Found: 529.3696.
Viability Assay (Promega) was used according to the manu-
facturer's protocol.
Compound 7. Obtained as a viscous oil (2.2 g, 65% over two
steps). H NMR (MeOD-d4, 400 MHz) d 0.88 (3H, t, J ¼ 6.9 Hz,
1
Transfection study. The transfection efficacy of the amphi- alkyl CH3), 1.27 (28H, s, alkyl CH2), 1.56–1.64 (2H, m, CH2–CH2–
philes was examined via quantitation of rey luciferase O), 2.33 (2H, t, J ¼ 7.4 Hz, CH2–CH2–CO–O), 3.43–3.55 (25H, m,
reporter gene activity from treated 786-O-Luc cells. Therefore, dendron), 3.61–3.67 (5H, m, dendron), 3.73–3.77 (5H, m, den-
the same treatment procedure as for the cell viability assay with dron); 13C NMR (MeOD-d4, 101 MHz) d 13.30, 22.49, 24.80,
786-O-Luc cells was used. Luciferase activity was measured 28.96, 29.24, 29.43, 29.57, 31.82, 33.94, 63.17, 68.66, 69.84,
using the Luciferase Assay System (Promega) according to the 70.87, 71.02, 71.12, 71.63, 71.71, 72.12, 72.64, 72.67, 78.46,
manufacturer's protocol. The luminescence signal was detected 78.55, 78.60, 173.72. HRMS: m/z calcd for C39H78O16Na [M +
via a Tecan Innite 200Pro.
Na]+: 825.5188. Found: 825.5192.
Determination of pro-inammatory cytokines. To determine
General procedure for the synthesis of compounds 8 and 9.
the effect of G1-Ester-DAPMA (A1) and G2-Ester-DAPMA (A2) on The reaction was performed under an inert gas atmosphere and
the cytokine secretion in vivo, three BALB/c mice (Charles River) exclusion of water. A solution of p-nitrophenyl chloroformate
per group were treated intravenously with A1 and A2 at doses of (4.76 g, 23.6 mmol, 12 eq.) in 20 mL dry DCM was added
8 mg kgꢃ1 and 20 mg kgꢃ1, respectively, complexed with non- dropwise to dry DCM (20 mL) and dry pyridine (0.30 mL, 23.6
targeting siRNA (ON-TARGETplus Non-targeting siRNA, Dhar- mmol, 12 eq.), while stirring at 0 ꢀC in an ice bath. On addition,
macon) at N/P 25. Invivofectamine 2.0 Reagent (Invitrogen) was a white precipitate was formed. Subsequently, a solution of
complexed with non-targeting siRNA according to the manu- compound 6 (1.0 g, 1.97 mmol, 1 eq.) or 7 (300 mg, 0.37 mmol, 1
facturer's manual and used as the positive control. HyPure eq.) dissolved in dry DCM (60 mL) and dry pyridine (0.37 mL,
water was administered as the negative control. Retrobulbar 23.6 mmol, 1.2 eq.) was added at 0 ꢀC via a dropping funnel over
blood was taken 1 h aer administration and serum was har- a period of 2 h. The mixture was stirred in a thawing ice bath for
vested. Cytokine levels in the serum were determined using the 14 h. The reaction mixture was then diluted with DCM (50 mL)
Meso Scale Discovery Multi-Spot Assay System, Mouse ProIn- and washed with NaHSO4 (2 ꢂ 50 mL, 1.33 M) and sat. brine
ammatory 7-Plex Assay Ultra-Sensitive Kit corresponding to (50 mL). The organic phase was dried over MgSO4, ltered, and
the manufacturer's protocol.
the ltrate evaporated in vacuo. The reaction mixture was
roughly puried by HPLC (silica column, DCM–MeOH 98 : 2,
64 mL minꢃ1). Due to the relative instability and since thorough
purication was not required at this stage, the intermediates 8
Synthesis
General procedure for the synthesis of compounds 6 and 7. and 9 were used in their crude form for further synthesis.
Stearoyl chloride 1 (18.8 g, 62.4 mmol for 2/2.6 g, 8.61 mmol for
General procedure for the synthesis of compounds 11 and
3), and acetal protected dendrons 2 (10.0 g, 31.2 mmol) or 3 12. Each solution of the crude compounds 8 (500 mg,
(3.0 g, 4.3 mmol) were dissolved in 25 mL of THF and cooled to 0.43 mmol) or 9 (175 mg, 0.08 mmol), which were dissolved in
ꢀ
ꢀC in an ice bath. Triethylamine (8.6 mL, 62.4 mmol for dry DCM (120 mL), was added dropwise over 2 h at 0 C into a
0
2/1.2 mL, 8.61 mmol for 3) was then added and the reaction solution of mono-Boc-DAPMA (1.24 g, 5.08 mmol, 12 eq., dis-
mixture was stirred rst at 0 ꢀC for 2 h and then at room solved in 50 mL dry DCM) employing dry reaction conditions.
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Immediately, the solution turned yellow due to the displace- HRMS: m/z calcd for C59H123N12O12 [M + H]+: 1191.9378. Found:
ment of p-nitrophenol. A solution of DMAP (0.20 g, 1.69 mmol, 1191.9376.
ꢄ0.5 eq. per p-nitrophenyl branch) and DIPEA (0.15 mL,
Compound A2. Obtained as a white foam (131 mg, quant.).
1.69 mmol, 1.0 eq. per p-nitrophenyl branch) in dry DCM 1H NMR (MeOD-d4, 400 MHz) d 0.89 (3H, t, J ¼ 6.7 Hz, alkyl
(30 mL) was added and the reaction mixtures were stirred at CH3), 1.28 (28H, s, alkyl CH2), 1.58–1.60 (2H, m, CH2–CH2–CO–
room temperature for 72 h. The solvent was then removed O), 1.94 (16H, brs, NH–CH2–CH2) 2.12–2.14 (16H, m, NH–CH2–
under reduced pressure. Purication was performed both by CH2), 2.31–2.34 (2H, t, J ¼ 7.0 Hz, CH2–CH2–CO–O) 2.89, 2.90
column chromatography (CHCl3–MeOH–NH4OH 90 : 9 : 1) and (24H, s, N–CH3), 3.03–3.06 (16H, m, CH2–N–CH3), 3.19–3.26
size exclusion chromatography (SEC) using a Sephadex™ LH-20 (48H, m, 16 ꢂ CH2–N–CH3 & 32 ꢂ CH2–NH), 3.51–3.73 (29H, m,
(CHCl3–MeOH 1 : 1). Drying under high vacuum yielded the dendron), 4.03–4.07 (3H, m, dendron), 4.31–4.33 (3H, m, den-
products 11 and 12 as yellowish oils.
dron); 13C NMR (MeOD-d4, 176 MHz) d 173.71, 161.70, 161.51,
Compound 11. Obtained as a yellowish viscous oil (1.57 g, 157.34 & 156.97 (TFA), 115.92 & 113.84 (TFA), 71.29, 70.85,
1
50%). H NMR (MeOD-d4, 500 MHz) d 0.89 (3H, t, J ¼ 6.7 Hz, 69.94, 69.71, 68.62, 63.29, 53.99, 52.94, 52.81, 39.00, 37.37,
alkyl CH3), 1.28 (28H, s, alkyl CH2), 1.43 (36H, s, Boc CH3), 1.59– 36.40, 33.92, 31.65, 29.35, 29.11, 29.05, 28.86, 24.76, 24.37,
1.63 (2H, m, CH2–CH2–CO–O), 1.64–1.74 (16H, m, NH–CH2– 22.31, 22.09, 13.03. HRMS: m/z calcd for C103H216N24O24 [M +
CH2), 2.30 (12H, s, N–CH3), 2.32–2.37 (2H, m, CH2–CH2–CO–O), 2H]2+: 1086.8204. Found: 1086.8152.
2.49 (16H, brs, CH2–N–CH3), 3.07 (8H, t, J ¼ 6.7 Hz, CH2–NH),
3.10–3.16 (8H, m, CH2–NH), 3.51–3.81 (9H, m, dendron), 4.04–
Results and discussion
Synthesis
4.30 (6H, m, dendron); 13C NMR (MeOD-d4, 126 MHz) d 13.17,
22.43, 24.76, 26.53, 26.63, 27.54, 28.90, 28.94, 29.15, 29.35,
29.49, 31.77, 33.68, 33.89, 38.22, 38.71, 40.78, 53.50, 54.75, Four distinct low-molecular-weight amphiphilic constructs (cf.
54.82, 63.24, 69.76, 71.20, 78.65, 156.84, 157.21, 173.89. HRMS: Fig. 2) have been synthesized differing in linker chemistry and
m/z calcd for C79H155N12O20 [M + H]+: 1592.1481. Found: dendron generation. Thus, specic properties with reference to
1592.1414.
biodegradability, size, and the number of multivalent interac-
Compound 12. Obtained as a yellowish viscous oil (0.45 g, tions were introduced in order to create an efficient gene carrier
1
40%). H NMR (MeOD-d4, 400 MHz) d 0.88 (3H, t, J ¼ 6.7 Hz, with a particular attributed structural motif. The potential gene
alkyl CH3), 1.28 (28H, s, alkyl CH2), 1.41 (72H, s, Boc CH3), 1.62– vectors were designed to utilize hydrophilic polyglycerol den-
1.69 (34H, m, CH2–CH2–CO–O & NH–CH2–CH2), 2.15, 2.20 & drons of two different generations [G1, G2] combined with a
2.23 (24H, s, N–CH3), 2.31–32 (2H, m, CH2–CH2–CO–O), 2.36– simple C-18 alkyl chain representing the hydrophobic domain,
2.43 (32H, m, CH2–N–CH3), 2.99–3.06 (32H, m, CH2–NH), 3.09– in order to unite the advantageous features of both lipid and
3.12 (8H, m, dendron), 3.51–3.75 (21H, m, dendron), 4.06–4.25 dendritic gene delivery vehicles. As mentioned above, it is
(6H, m, dendron); 13C NMR (MeOD-d4, 176 MHz) d 174.88, necessary to modify the dendritic polyglycerol scaffold with a
158.47, 149.32, 79.97, 76.40, 72.52, 71.28, 64.66, 56.37, 55.91, hydrophobic unit to allow the formation of multivalent supra-
54.57, 53.89, 47.48, 42.58, 41.08, 39.90, 39.77, 39.25, 35.06, molecular architectures and thus facilitate efficient complexa-
32.99, 30.55, 30.49, 30.27, 30.11, 29.00, 27.88, 26.12, 25.23, tion and condensation of the genetic material, which is an
23.69, 14.35. HRMS: m/z calcd for C143H280N24O40 [M + 2H]2+
:
important prerequisite for transfection ability.27
1487.0228. Found: 1487.0299.
Due to their water solubility and physiological safety, which
General procedure for the synthesis of compounds A1 and is based on a very biocompatible glycerol building block,59
A2. TFA (6.0 mL, excess) was slowly added to a solution of polyglycerol dendrons were selected to serve as the hydrophilic
compounds 11 (0.10 g, 0.06 mmol) or 12 (0.10 g, 0.03 mmol) in part within the amphiphilic structure. As a dendritic scaffold,
DCM (6.0 mL) and stirred overnight at room temperature. The they provide multiple hydroxyl groups which can be syntheti-
solvent was removed in vacuo and the residue washed alter- cally modied, thus offering the opportunity for multivalent
nately with hexane and diethyl ether. Purication was accom- interactions with biological substrates,60 resulting in consider-
plished via SEC (Sephadex™ LH20, MeOH) to remove any trace ably stronger binding affinities compared to monovalent
amounts of impurities. Freeze drying yielded the compounds A1 interactions.61 In addition, the degree of functionality and thus
and A2 as white foams.
the size can be varied by choosing a certain dendron generation.
Here, the amphiphiles were functionalized at their periph-
Compound A1. Obtained as a white foam (98 mg, quant.). 1H
NMR (MeOD-d4, 500 MHz) d 0.88 (3H, t, J ¼ 6.7 Hz, alkyl CH3), eral positions with a particular triamine: N,N-di-(3-amino-
1.28 (28H, s, alkyl CH2), 1.57–1.60 (2H, m CH2–CH2–CO–O), 1.93 propyl)-N-(methyl)amine (DAPMA) that already proved to be an
(8H, brs, NH–CH2–CH2), 2.11–2.14 (8H, m, NH–CH2–CH2), 2.31– effective entity for gene delivery purposes.49 It is known for a few
2.34 (2H, m, CH2–CH2–CO–O), 2.88 & 2.89 (12H, s, N–CH3), decades now that in the presence of multivalent cations, such as
3.03–3.06 (8H, m, CH2–N–CH3), 3.19–3.28 (24H, m, 8 ꢂ CH2–N– the naturally occurring oligoamines spermine and spermidine,
CH3 & 16 ꢂ CH2–NH), 3.59–3.73 (9H, m, dendron), 4.02–4.33 DNA undergoes a dramatic condensation to a compact, usually
(6H, m, dendron), 13C NMR (MeOD-d4, 126 MHz) d 13.10, 22.17, highly ordered structure,62 which is essential for the gene
22.39, 24.49, 24.75, 24.77, 28.88, 28.92, 29.12, 29.32, 29.45, delivery process, since as one of the rst steps the genetic
31.73, 33.67, 33.90, 36.49, 37.40, 39.10, 52.91, 54.07, 63.26, material needs to be transported efficiently through the cellular
69.70, 69.84, 71.35, 157.10, 157.46, 162.08 (CF3COOH) 174.00. membrane. In the specic case of DAPMA, it has been suggested
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that the sterically hindered tertiary amine deforms DNA in dendrons.49 In addition to that, it is believed that primary amine
order to achieve the interactions, resulting in strong local groups generally participate in nucleic acid binding, condense
binding and thus in efficient DNA compaction.49 Moreover, this it into nanoscale particles and promote its cellular uptake,
specic triamine ligand has been shown to lower the toxicity while tertiary amines may act as “proton sponge” entities in
when compared with diamine- and tetraamine modied endosomes and enhance the release of the genetic material into
Scheme 1 Synthesis of G1-Ester-DAPMA (A1) and G2-Ester-DAPMA (A2).
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the cytoplasm.63 Therefore, we reason that this specic amine enables electrostatic interactions with negatively charged
unit (DAPMA) is an ideal candidate for gene delivery nucleic acids.
applications.
To examine the physico-chemical characteristics of the
Besides the dissimilarity in the size of the hydrophilic head synthesized dendritic amphiphiles A1–A4 and to evaluate their
group by using different generations of polyglycerol dendrons applicability as potential gene delivery systems, the aggregation
(G1, G2), variations were also introduced regarding their behavior as well as size and surface charge were studied using
degradation prole that may assist the efficient release of the different physical characterization methods such as critical
nucleic acids from the polyplex. Therefore, two amphiphiles micelle concentration (CMC) determination, cryogenic trans-
were equipped with a biodegradable ester bond at the focal mission electron microscopy (cryo-TEM), dynamic light scat-
point [G1-Ester-DAPMA (A1), G2-Ester-DAPMA (A2)] in contrast tering (DLS), and zeta potential measurements (Table 1).
to a non-degradable rigid triazole linker within the other two
Initially, the aggregation of the amphiphiles was studied via
scaffolds [G1-Trz-DAPMA (A3), G2-Trz-DAPMA (A4)] (cf. Fig. 2).
determination of the CMC values, since self-assembly of
For the synthesis of G1-Ester-DAPMA (A1) and G2-Ester- amphiphilic entities and thus formation of thermodynamically
DAPMA (A2) (Scheme 1), we rst reacted each of the acetal group stable micellar aggregates only occurs, when the concentration
protected polyglycerol dendrons [G1.0]-OH (2) and [G2.0]-OH (3) of the amphiphile exceeds a critical concentration, known as
with commercially available stearoyl chloride (1), thereby the CMC. In fact, the CMCs were identied by means of uo-
forming an ester bond at the focal point which represents a rescence spectroscopy using pyrene as a uorescence probe in
biodegradable connection between both domains of the aqueous solution. This hydrophobic dye is a preferred uores-
amphiphile. Aer removing the 1,2-diol protecting groups cent probe due to its strong uorescence in nonpolar domains
under acidic conditions, the peripheral hydroxyl entities were and its weak radiation in polar media. Upon micellization,
converted into activated carbonate functionalities by using pyrene partitions preferentially into the hydrophobic micro-
p-nitrophenyl chloroformate. In the next step, mono-Boc-pro- domain of the micelles which is accompanied by a sharp
tected DAPMA units (10) were attached via nucleophilic increase in uorescence.
substitution to the dendritic backbone of compounds 8 and 9 in
The determined CMC values given in Table 1 show that all
50% and 40% yield over two steps, respectively, involving the four amphiphiles (A1–A4) self-assemble at a millimolar level
liberation of p-nitrophenol. The Boc-protected species 11 and 12 ranging from 1.3–2.9 mM. These rather high CMC values can be
were then treated overnight with TFA to afford G1-Ester-DAPMA ascribed to the high cationic charge density, which is the result
(A1) and G2-Ester-DAPMA (A2) in quantitative yields, as their of the conjugated oligoamine surface ligand DAPMA. This is
triuoroacetate salts (Scheme 1).
especially true, when comparing the CMCs of G1-Trz-DAPMA
Regarding the synthesis of the amphiphiles G1-Trz-DAPMA (A3) and G2-Trz-DAPMA (A4) to similar constructs27 that only
(A3) and G2-Trz-DAPMA (A4), the hydroxylated, non-degradable differ in the attached amine unit and exhibit CMC values in the
precursors (13, 14; cf. ESI, Scheme S1†) were initially generated micromolar range (10–60 mM). Interestingly, the G2 amphi-
according to our previously published procedure.27 The DAPMA philes (A2, A4) possess slightly lower CMC values than their G1
functionalization of the dendron's deprotected surface occurred counterparts (A1, A3). A similar observation has been made by
in a similar fashion to those for the compounds 6 and 7. Trappmann et al., who showed that in their case a larger degree
Correspondingly, the free hydroxyl groups of the triazole- of hydrophilicity can lead to lower CMC values.66 As expected,
bridged amphiphiles (13, 14; cf. ESI, Scheme S1†) were con- the different spacer units only had a small effect on the CMC,
verted into the respective carbonate species (15, 16; cf. ESI, since G1-Trz-DAPMA (A3) and G2-Trz-DAPMA (A4), which
Scheme S1†), followed by the addition of mono-Boc-protected
DAPMA (10) in the presence of DMAP and DIPEA, to give the
aminated triazole amphiphiles (17, 18; cf. ESI, Scheme S1†) in
39% and 27% yields over two steps, respectively. Aer the nal
Table 1 CMC, size (dH), and zeta potential of A1–A4
dH (DLS)c
[nm]
d (TEM)a,d
[nm]
Zeta potentiala
[mV]
treatment with triuoroacetic acid, the two triazole-based target
molecules G1-Trz-DAPMA (A3) and G2-Trz-DAPMA (A4) were Sample
CMCa,b [M]
obtained in quantitative yields (cf. ESI, Scheme S1†).
A1
A2
A3
A4
2.9 ꢂ 10ꢃ3
1.6 ꢂ 10ꢃ3
2.7 ꢂ 10ꢃ3
1.3 ꢂ 10ꢃ3
n.a.e
4.0 (ꢁ 0.2)
3.0 (ꢁ 1.0)
5.0 (ꢁ 0.2)
3.0 (ꢁ 1.0)
51.7 (ꢁ 5.2)
41.5 (ꢁ 4.2)
52.9 (ꢁ 5.3)
42.7 (ꢁ 4.3)
3.4 ꢁ 0.2
n.a.e
Physico-chemical characterization of amphiphiles A1–A4
3.8 ꢁ 0.5
Amphiphiles generally tend to aggregate towards micellar
a
b
HEPES saline buffer (pH 7.4, 9.4 mM NaCl). T ¼ 25 ꢀC. Pyrene
assemblies in aqueous media due to the hydrophobic effect.64
By suitable synthetic modication of the head group and
balancing the hydrophobic tail, even structurally persistent
supramolecular architectures can be generated,65,66 which stand
in contrast to conventional micelles that form loose, short-lived
dynamic aggregates. This particular behavior can, for instance,
be exploited for transporting therapeutic material like genes,
particularly if the head group exposes a positive net charge that
c
concentration: 0.5 mM.
Phosphate buffer (pH 7.4, 10 mM);
c ¼ 4.5 mM. T ¼ 25 ꢀC. Size distribution by volume, PDI ¼ 0.2–0.4.
d
c ¼ 4.5 mM. TEM diameter values of micelles are estimated from
individual micelles in the case of G2 derivatives; in the case of the G1
species, areas of two-dimensionally ordered spherical micelles were
analyzed in reciprocal space (Fourier analysis), where diffraction rings
e
indicate the mean micelle diameter. G1 derivatives turned out to
assemble towards extended two-dimensionally ordered structures
besides spherical micelles. Because of this structural bimodality DLS
measurements are not applicable.66
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comprise a triazole ether linkage, exhibited slightly lower CMC
values than the ester-bridged, less hydrophobic analogs, G1-
Ester-DAPMA (A1) and G2-Ester-DAPMA (A2).
Oligonucleotide binding and condensation
The process of efficient binding and condensation of oligonu-
cleotides represents an elemental step within the gene delivery
route, since it enables the cellular internalization of the genetic
material. Due to the fact that cell membranes are typically
negatively charged owing to their content of glycoproteins,
proteoglycans, and glycerolphosphates,67,68 the complex
between the gene carrier and the nucleic acid, termed polyplex,
should preferentially exhibit a positive net charge for effectively
penetrating the cell membrane. Moreover, the induced
complexation of the genetic material causes its collapse and
condensation due to the decrease of the repulsive forces
between the anionic charges. As a result, much smaller oligo-
nucleotide particles are formed which can be endocytosed by
the cell.
The aggregation behavior was further studied by DLS, which
allows determination of hydrodynamic diameters (dH). As
shown in Table 1, the G2 amphiphiles A2 and A4 formed very
small micelles with diameters between 3 and 4 nm. Surpris-
ingly, these values are in the same range as the theoretical size
of the amphiphilic monomer units G2-Ester-DAPMA (A2) and
G2-Trz-DAPMA (A4) (ꢄ3.3–3.6 nm), which indicates that the
formed spherical particles are only composed of a small
number of self-assembled amphiphiles, presumably dimers. In
the case of generation one amphiphiles G1-Ester-DAPMA (A1)
and G1-Trz-DAPMA (A3), the obtained DLS values could not be
used because cryo-TEM measurements signied a bimodal
aggregation behavior (see below).
Notably, increasing the net positive surface charge of
polyplexes will promote the uptake but also raise cytotoxicity
due to membrane destabilization. Accordingly, the complexes
have to strike a balance between cellular uptake and cytotoxicity
to achieve optimal delivery efficiencies. Hence, only the actually
required amount of nitrogen atoms or rather cationic charges
should be employed. This can be realized by nding the
appropriate N/P ratio which describes the molar ratio of amine
(N) to phosphate (P) groups. Relevant N/P ratios have to be
determined individually since they are dependent on a variety of
factors such as size, shape, charge, and ligand modication of
the used gene carrier.
In order to examine the ability of the synthesized
compounds A1–A4 to efficiently complex and condense the
genetic material, the binding affinity to DNA was studied rst by
using the ethidium bromide (EthBr) displacement assay (cf. ESI,
S11–S13 for additional information†). Although the resulting
data rather reect a competition assay than an absolute binding
strength, it is a powerful comparative method, because it gives
valuable information about the gene carriers' general tendency
to bind oligonucleotides.55 According to the obtained, very low
CE50 values (ꢄ0.3) (cf. ESI, Table S1†) – that generally reect the
relative ability to bind anionic DNA per cationic charge – all
scaffolds (A1–A4) proved to be overall strong DNA binders. This
is supported by CE50 values of dendritic amphiphiles that were
decorated with primary amine groups.27 There, the DNA
binding was signicantly weaker as CE50 values of 0.8–1.3 were
obtained. In contrast to the currently employed triamine unit
(DAPMA), a simple glycine unit (monoamine) was connected to
each dendron branching in that case, thereby indicating the
benecial effect of using an inherently multivalent amine
group, such as DAPMA.
In order to supplement the described aggregation phenomena
in the case of A2 and A4 by a direct structural analysis and to
elucidate the self-assembly behavior of G1-Ester-DAPMA (A1) and
G1-Trz-DAPMA (A3), cryo-TEM measurements were performed on
aqueous sample solutions at a general concentration of 4.5 mM,
which is well above the amphiphiles' corresponding CMC values.
All amphiphiles (A1–A4) showed a tendency to form small
spherical particles, though the respective G1 derivatives A1 and
A3 revealed a second supramolecular species by showing a more
complex two-dimensionally ordered hexagonal aggregation
pattern (cf. Fig. 3). This nding renders DLS measurements non-
applicable in the particular case of A1 and A3. The relevance of
the second assembly phenomenon is not yet fully understood
and remains to be claried in future investigations.
The spherical particles formed by all four amphiphiles (A1–
A4) showed diameters in the range of 3–5 nm and did not
denote internal structural differentiations. Interestingly, the
diameters are far below the values obtained from the dendritic
non-ionic analogs,66 where dimensions and density proles
indicate the formation of molecular bilayer structures. Due to
the presence of the strongly hydrated amine sphere and hence
the corresponding large head group volume, we suppose that
small assemblies consisting of only very few molecules are
obtained. For geometrical reasons this might even lead to the
formation of dimeric assemblies in the case of the G2 deriva-
tives A2 and A4. It is further assumed that in such a dimeric
assembly the diameter of the head groups (2–4 nm) determines
the whole size of the assemblies, whereas hydrophobic alkyl
chains can be easily accommodated within the core volume
without a signicant impact on the overall assembly size.
In accordance with the large number of peripheral amine
groups, the conducted zeta measurements (Table 1) showed
that all four compounds displayed high positive surface charges
between 41 and 53 mV, which supports the applicability of these
systems as prospective gene carriers. Interestingly, the G1
amphiphiles A1 and A3 exhibited slightly higher surface
charges, which presumably relates to the different aggregation
patterns observed with these compounds. Moreover, this can be
explained by less repulsion of the positive charges within the
hydrophilic head groups of the generation one amphiphiles, as
already observed for related structures.27,47
The binding capacity of all four amphiphiles (A1–A4) was
further investigated using the agarose gel electrophoresis
retardation assay that directly measures interactions between
oligonucleotides and potential gene carriers. As a sign of
successful DNA or RNA neutralization and condensation, the
constructs should either reduce or completely retard the elec-
trophoretic mobility of the genetic material. The obtained
results (Fig. 4) show that all four amphiphiles (A1–A4) are able
to efficiently complex and condense siRNA at N/P ratios between
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Fig. 4 Binding capacity and migration of amphiphile–siRNA complexes using agarose gel electrophoresis. Complexes of FAM-labeled siRNA and
the amphiphiles G1-Ester-DAPMA (A1), G2-Ester-DAPMA (A2), G1-Trz-DAPMA (A3), and G2-Trz-DAPMA (A4) with increasing N/P ratios (lane
1–7: 5, 10, 20, 30, 50, 70, and 100, each) are shown. Complex-free FAM-siRNA (siRNA) and Lipofectamine 2000 complexed FAM-siRNA (Lipo) are
displayed in the last two slots on the right. Images were acquired by detecting the green fluorescence signal.
10 and 20. Complex-free siRNA and a complex of siRNA and condensed to a more pronounced extent which leads to
Lipofectamine 2000 were loaded as controls. In accordance with smaller polyplexes. However, relatively large polyplex particles
the results obtained by the EthBr assay, the G2 derivatives (A2, have been observed with lower N/P ratios (N/P 2–5), which
A4) are slightly more efficient than their lower generation underlines the necessity of applying a minimum N/P ratio of
counterparts (A1 and A3), i.e., amphiphile G2-Ester-DAPMA (A2) 10 for all four amphiphiles (A1–A4) to yield sufficiently
reduces the siRNA mobility at N/P 10 to a more pronounced condensed polyplexes that are able to be internalized by cells
extent than G1-Ester-DAPMA (A1).
(ca. 200 nm).
As mentioned above, in order to facilitate efficient gene
The conducted zeta potential measurements give overall
expression, on the one hand, slightly positively charged poly- positive net charges ranging from 12–28 mV. Interestingly, even
plexes are believed to promote interactions with predominantly at the lowest N/P ratio of 2, positive net charges can still be
negatively charged cell membranes.67,68 On the other hand, the detected with every amphiphilic transporter (A1–A4) which
process of DNA/siRNA condensation plays a vital role in the reects the strong interaction and as a result leads to a
transfection pathway. Therefore, the hydrodynamic diameters reasonable neutralization of the DNA. In accordance with the
plus surface charges of the amphiphile–DNA polyplexes were DLS results, the identied zeta potentials of the polyplexes
then determined by DLS and zeta potential measurements.
illustrate the general tendency that the zeta potential rises with
The obtained data (Fig. 5) indicate that all four compounds increasing N/P ratios, which is in agreement with theoretical
(A1–A4) which already exhibited high DNA binding affinities, expectations.27 Although the concentrations of the sample
as revealed by the EthBr assay as well as by gel electrophoresis solutions were convenient for zeta potential and DLS
studies, in turn condense DNA effectively into polyplexes of measurements, they turned out to be too low for cryo-TEM
ꢄ200 nm in diameter, at N/P ratios 10–20. Predictably, on investigations, so that direct structural data of polyplexes could
raising the N/P ratios, the complexed oligonucleotides are not be obtained under the experimental conditions used.
Fig. 5 Hydrodynamic diameter (size) and zeta potential of DNA polyplexes obtained with amphiphiles A1–A4 using varying N/P ratios (2, 5, 10,
and 20). Amphiphile–DNA polyplexes were measured in HEPES saline buffer (pH 7.4, 9.4 mM NaCl). DLS size distribution by volume, PDI ¼ ꢄ0.1–
0.4. Zeta potential of DNA ¼ ꢃ29.5 ꢁ 3.2 mV (size not determinable, due to high polydispersity).
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and release of the siRNA together with low cytotoxicity and high
transfection efficiency. In order to cover a broad range of
possible N/P ratios and in accordance with the ndings
extracted from the gel retardation assay, amphiphile–siRNA
polyplexes with N/P 10, 30, and 50 were evaluated.
The results of the WST-1 assay (Fig. 6A–D) relative to nT
siRNA treated cells (control cells which were treated with nT
siRNA but without transfection reagent; henceforth referred to
In vitro cytotoxicity
Cytotoxicity is an important aspect to be considered when
evaluating nanocarriers for their specic transfection potency.
Therefore, the cytotoxic potential of all four amphiphiles (A1–
A4) was determined by the WST-1 assay as well as by the
xCELLigence system (Roche) using non-targeting (nT) siRNA
and HeLa cells. As mentioned earlier, the most appropriate N/P
ratio has to be a compromise between sufficient complexation
Fig. 6 Cytotoxicity determined by the two different methods: WST-1 assay (left) and xCELLigence (right). HeLa cells were transfected with non-
targeting (nT) siRNA. The results of the WST-1 assay (A–D) and of the xCELLigence system (E–H) are shown relative to nT siRNA control cells that are
treated with nT siRNA, but without transfection reagent (WST-1 assay ¼ 1 (dashed line); xCELLigence ¼ 0 (baseline)). The control group “Control”
(integrated within the xCELLigence graphics) characterizes untreated cells. N/P ratios 10, 30, and 50 were analyzed for all four amphiphiles. G1-Ester-
DAPMA (A1) is shown in panels A and E, G2-Ester-DAPMA (A2) in B and F, G1-Trz-DAPMA (A3) in C and G, and G2-Trz-DAPMA (A4) in D and H.
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as “nT siRNA cells”) illustrate that all examined amphiphiles
(A1–A4) showed no toxicity while using an N/P ratio of 10.
Furthermore, siRNA polyplexes at N/P 30 formulated with G1-
Ester-DAPMA (A1) and G2-Ester-DAPMA (A2) did not show
considerable toxic effects. However, G2-Trz-DAPMA (A4)
induced a decrease of cell viability at N/P 30 (61% reduction) as
well as to some extent G1-Trz-DAPMA (A3) (31% reduction),
indicating a higher cytotoxic potential for the triazole deriva-
tives when compared to the ester-linked analogs (A1: 7% and
A2: 28% reduction). At N/P 50 all amphiphiles led to a reduction
of cell viability by at least 54% (i.e., A1) (A2: 70%), which was
again found to be more pronounced in the case of the triazole-
bridged amphiphiles (A3: 79%, A4: 99%). Therefore, an N/P
ratio well below 50 was used for subsequent transfection studies
with all four nanotransporters (A1–A4).
In addition, the cytotoxicity of the nanocarriers was investi-
gated by the xCELLigence system (Fig. 6E–H) for continuous
monitoring of cell viability and growth in real-time. The data
from the xCELLigence system were normalized to the time
before transfection (24 h aer seeding) and were related to nT
siRNA cells, demonstrated as a baseline (delta cell index ¼ 0).
Proles from untreated cells (negative control, termed
“Control”) and cells treated with Lipofectamine (positive
control) served as references. Short-term rises (5 h period) of the
cell index aer amphiphile–siRNA polyplex treatment were
observed for all four nanocarriers A1–A4 (E–H), while A2 (F) and
A4 (H) led to stronger increases than the G1 analogs (A1, A3),
potentially caused by nontoxic changes in cell adhesion and cell
structure. Addition of Lipofectamine and the replacement of
culture medium in the control group (Control) showed a minor
effect on the cell index. Generally, it can be stated that all
amphiphiles (A1–A4) induced no toxic long-term effects as
observed by the xCELLigence system. Slight but non-signicant
Fig. 7 786-O Luc transgenic cells were transfected with luciferase
specific and non-targeting siRNA (ON-TARGETplus Non-targeting
siRNA, Dharmacon) complexed with nanocarriers A1 and A2 at N/P
ratios of 10, 20, and 30 for 48 h. Lipofectamine was used as a positive
control and untreated cells as the negative control. (A) Cell viability was
measured by using the CellTiter-Glo Luminescent Cell Viability Assay
(Promega). (B) Transfection efficacy was determined by using the
commercial Luciferase Assay System (Promega). Results are shown as
mean ꢁ SD of triplicates.
decreases of the cell index compared to the controls (Control, However, the triazole-bridged compounds A3 and A4 revealed
Lipofectamine) (i.e., A3 N/P 10, 24–50 h post-treatment) may enhanced cytotoxicity vs. the ester-linked constructs (cf. ESI,
result from alteration of viability, adhesion, or cell structure Fig. S4A†), thereby conrming the effect seen with HeLa cells.
during transfection. The differences between the outcomes of Therefore, we focused on A1 and A2 for further studies.
the two assay systems result from the varying methods, since
The transfection efficacy of the ester-linked amphiphiles A1
the WST-1 assay generally gives information about the mito- and A2 was largely inuenced by the given N/P ratio of the
chondrial metabolism activity which signies the endogenous amphiphiles (A1, A2)–anti-Luc-siRNA polyplexes, as shown in
redox potential of cells, whereas the xCELLigence system relates Fig. 7B. Both A1 and A2 showed the highest knockdown effect
to changes of the cell impedance. In conclusion, although in using N/P 30 (38% and 27%, respectively). N/P 20 caused a
several cases there is a drop of detectable mitochondrial activity decrease of luciferase activity of 34% (A1) and 19% (A2) vs.
at 48 h post-transfections (see above; WST assay), generally cell control. However, knockdown was less than that obtained with
morphology and numbers do not differ signicantly within the Lipofectamine (positive control). Polyplexes with N/P 10
tested period of two days (xCELLigence system).
induced no reduction of luciferase expression. When using non-
targeting siRNA, the luciferase activity of A1 at N/P 30 was also
partially reduced, while the same applies to the A2 complexes
(with all three tested N/P ratios). Due to the fact that these
In vitro siRNA transfection
The in vitro transfection efficacy of amphiphile–siRNA complexes amphiphile–non-targeting siRNA complexes showed no cyto-
was evaluated using luciferase as a reporter gene in the human toxicity, an unspecic effect can be assumed in this case. In
renal carcinoma cell line, 786-O-Luc. Due to the partial in vitro addition, polyplexes with an N/P ratio above 30 caused cyto-
cytotoxicity of certain amphiphiles seen in HeLa cells, the effect toxicity (data not shown). An N/P ratio of 25 was therefore used
on the cell viability of 786-O-Luc by all four constructs (A1–A4) for the following investigation (determination of pro-inam-
was evaluated rst. No reduction of cell viability was determined matory cytokines). The triazole amphiphiles A3 and A4 showed,
in A1 and A2 treated cells, as well as by the positive control Lip- in addition to enhanced cytotoxicity, no specic transfection
ofectamine compared to the untreated control cells (Fig. 7A). efficacy (cf. ESI, Fig. S4B†).
2164 | J. Mater. Chem. B, 2014, 2, 2153–2167
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This divergence in transfection efficiency can be attributed
Journal of Materials Chemistry B
Determination of pro-inammatory cytokines
to the different incorporated linker groups at the core of the
amphiphiles. The biodegradable and exible nature of the
ester linker unit (in A1 and A2) compared to a stiff, non-
degradable triazole group present in A3 and A4 could be the
reason for the low cytotoxicity and enhanced transfection
activity for A1 and A2. It can be assumed that due to their high
positive surface charges and consequential strong nucleic
acid binding (as revealed by the conducted EthBr assay)
which is clearly the result of the incorporated amine moiety
DAPMA, the complexed siRNA presumably could not be
liberated effectively in order to yield superior gene transfer
activities. Similar observations are known from other gene
carriers, as it can be difficult to nd the delicate balance
between initial protection and subsequent release of the
genetic cargo.69,70
It is known that nanoparticles can induce pro-inammatory
responses in vivo as an adverse reaction which can therefore
constrict their administration.71 To evaluate if there are limi-
tations regarding the biological application of the relevant
amphiphiles A1 and A2 due to possible inammatory side
effects, the impact of their systemic injection in mice on pro-
inammatory cytokine levels in blood was determined (Fig. 8).
BALB/c mice were treated intravenously with A1– and A2–non-
targeting siRNA complexes, respectively. Administration of Invi-
vofectamine (Invitrogen) was used as a positive control, HyPure
water as the negative control (vehicle). Blood was taken 1 h aer
administration and pro-inammatory cytokines were determined
in the serum. It was found that the secretion of the cytokines IL-6,
IL-1b, and TNF-a was not affected by the treatment with A1 and
A2. Furthermore, A1 treatment did not lead to a change in serum
Fig. 8 Three BALB/c mice per group were treated intravenously with 8 mg kgꢃ1 and 20 mg kgꢃ1 A1 (A) vs. A2 (B) complexed with non-targeting
siRNA (ON-TARGETplus Non-targeting siRNA, Dharmacon) at an N/P ratio of 25, or Invivofectamine 2.0 Reagent, Invitrogen. HyPure water was
used as the negative control (vehicle). Retrobulbar blood was taken 1 h after injection and serum was examined via a Meso Scale Discovery Multi-
Spot Assay System, Mouse ProInflammatory 7-Plex Assay Ultra-Sensitive Kit. Results are shown as mean ꢁ SD of triplicates.
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cytokine levels of IL-10 and KC. The treatment with 20 mg kgꢃ1 A2
N/P 25 caused a moderate increase of KC and IL-10 in the serum
in comparison to the vehicle control. Followed by the adminis-
tration of 20 mg kgꢃ1, both amphiphiles showed an increased
secretion of IFN-g compared to the negative control. However,
these measured average concentrations (A1: 5 pg mLꢃ1, A2: 16 pg
mLꢃ1) do not reect an acute inammatory response.72,73
Interestingly, the positive control Invivofectamine induced
higher levels of KC, IL-6, IL-10, and TNF-a compared to the
tested amphiphiles (A1, A2). In conclusion, the administration
of G1-Ester-DAPMA (A1) and G2-Ester-DAPMA (A2) caused no
induction of a pro-inammatory response compared to the
controls Invivofectamine and Hypure water. This lack of
inammatory side effects is promising for the application of
this kind of delivery vehicle in vivo.
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