Ornithine-derived oligomers and dendrimers forin vitrodelivery of DNA andex vivotransfection of skin cellsviasaRNA

Article abstract of DOI:10.1039/d0tb00942c

Gene therapies are undergoing a renaissance, primarily due to their potential for applications in vaccination for infectious diseases and cancers. Although the biology of these technologies is rapidly evolving, delivery strategies need to be improved to overcome the poor pharmacokinetics and cellular transport of nucleic acids whilst maintaining patient safety. In this work, we describe the divergent synthesis of biodegradable cationic dendrimers based on the amino acid ornithine as non-viral gene delivery vectors and evaluate their potential as delivery vectors for DNA and RNA. The dendrimers effectively complexed model nucleic acids at lower N/P ratios than polyethyleneimine and outperformed it in DNA transfection experiments with ratios above 5. Remarkably, all dendrimer polyplexes at N/P = 2 achieved up to 7-fold higher protein content over an optimized PEI formulation when used for transfections with self-amplifying RNA (saRNA). Finally, transfection studies utilizing human skin explants revealed an increase of cells producing protein from 2% with RNA alone to 12% with dendrimer polyplexes, attributed to expression enrichment predominantly in epithelial cells, fibroblasts and leukocytes, with minor enrichment in NK cells, T cells, monocytes, and B cells. Overall, this study indicates the clear potential of ornithine dendrimers as safe and effective delivery vectors for both DNA and RNA therapeutics.

Full text of DOI:10.1039/d0tb00942c

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Journal of
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Lovato, A. Russo, G. Russo, R. J. Shattock, C. Alexander, F. Quaglia, A. K. Blakney, P. Gurnani and C.
Conte, J. Mater. Chem. B, 2020, DOI: 10.1039/D0TB00942C.
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Wei Wei, Guanghui Ma et al.
Macrophage responses to the physical burden of cell-sized
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DOI: 10.1039/D0TB00942C
ARTICLE
Ornithine-derived oligomers and dendrimers for in vitro delivery
of DNA and ex vivo transfection of skin cells via saRNA
Francesca Saviano,a Tatiana Lovato,b Annapina Russo,a Giulia Russo,a Robin J Shattock,c Cameron
Received 00th January 20xx,
Accepted 00th January 20xx
Alexander,b Fabiana Quaglia,a Anna K. Blakney,c* Pratik Gurnani, b* Claudia Conte a,*
DOI: 10.1039/x0xx00000x
Gene therapies are undergoing a renaisannce, primarily due to their potential for applications in vaccination for infectious
disease and cancers. Although the biology of these technologies is rapidly evolving, delivery strategies need to be improved
to overcome the poor pharmacokinetics and cellular transport of nucleic acids whilst maintaining patient safety. In this work,
we describe the divergent synthesis of biodegradable cationic dendrimers based on the amino acid ornithine as non-viral
gene delivery vectors and evaluate their potential as delivery vectors for DNA and RNA. The dendrimers effectively
complexed model nucleic acids at lower N/P ratios than polyethyleneimine and outperformed it in DNA transfection
experiments with ratios above 5. Remarkably, all dendrimer polyplexes at N/P=2 achieved up to 7-fold higher protein
content over an optimized PEI formulation when used for transfections with self-amplifying RNA (saRNA). Finally,
transfection studies utilizing human skin explants revealed an increase of cells producing protein from 2% with RNA alone
to 12% with dendrimer polyplexes, attributed to expression enrichment predominantly in epithelial cells, fibroblasts and
leukocytes, with minor enrichment in NK cells, T cells, monocytes, and B cells. Overall, this study indicates the clear potential
of ornithine dendrimers as safe and effective delivery vectors for both DNA and RNA therapeutics.
and that some vectors are more suitable for particular nucleic
acid lengths.14-16 This is particularly important as gene therapies
Introduction
can vary from short oligonucleotides (< 25 bases),17 to mRNAs
(~1-2 kb) and long (~10 kb) self-amplifying RNAs (saRNA).18
There are also further complications owing to the need for
formulations to navigate the challenges associated with
application dependent administration, such as intramuscular
injection for vaccination.19
Gene therapies are a powerful means to treat or cure disease,
offering the possibility to introduce artificial genetic material
into target cells, leading to the production or inhibition of
disease-related proteins.1 Although, gene therapy research has
matured into some commercially viable technologies,2
a
number of challenges still remain in improving the efficacy of
these therapies.3 Due to the susceptibility towards nucleases
and poor intracellular transport of naked nucleic acids,
successful gene therapy relies on the efficient condensation,
protection and delivery of genetic material using vectors.
Typically, viral vectors outcompete non-viral delivery systems
on transfection efficiency, however immunogenicity and
manufacture issues have marred their clinical use.4, 5
Although polymeric gene delivery vectors can be
synthesized at scale and with relative ease, their broad
molecular weight distribution may result in variability which is
carried through to biological activity. Pioneered by Tomalia and
co-workers,
macromolecules with monomer units repeated over each
dendritic generation. Compared with traditional
dendrimers
are
perfectly
branched
There have now been a multitude of studies for the
development of non-viral gene delivery vectors reported,
typically comprising cationic lipids or cationic macromolecules,
as formulation aids for nucleic acids.6-12 They operate through
the electrostatic condensation of negatively charged nucleic
acids into small nanoparticles (lipoplexes for lipids, polyplexes
for polymers), protecting the genetic material from endogenous
nucleases.13 It is now well established that the delivery vector
must be explicitly tuned depending on the nucleic acid cargo,
macromolecules, their synthesis is non-trivial, often achieved
through iterative divergent or convergent strategies.20
However, their perfect molecular weight distribution (Ð = 1.0),
coupled with their high surface valency for ligand display, have
drawn significant attention for healthcare applications due to
their reproducible interactions with biological systems. The
success of dendrimers within a biomedical context also extends
to gene delivery, with numerous cationic derivatives, such as
poly(amidoamine) (pAMAM), and polypropyleneimine (pPI)
displaying reproducible and strong nucleic acid complexation
coupled with high transfection efficiencies.21-29
a-Drug Delivery Laboratory, Department of Pharmacy, University of Napoli Federico
II, Via Domenico Montesano 49, 80131 Napoli, Italy; b-Division of Molecular
Therapeutics and Formulation, School of Pharmacy, University of Nottingham, NG7
2RD, UK; c - Department of Infectious Disease, Imperial College London, School of
Medicine, St Mary's Hospital, Praed Street, London W2 1NY
The ABB’ (A = COOH, B = α-NH2, B’ = side chain NH2) nature
of amino acids with protonatable amine-functional side chains
(lysine, ornithine etc.) represent excellent building blocks for
dendrimer synthesis. Moreover, the polypeptide structure, and
amino acid degradation products impart good compatibility
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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with cells and tissues and allow for in vivo degradation of the Dynamic light scattering and zeta-potential
ARTICLE
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vector through peptide bond cleavage by proteases. Given the The hydrodynamic diameter (dH), polydisDpOerI:s1it0y.10in3d9/eDx0(TPBD0I0)9a4n2Cd
popularity of linear poly(lysine) (pLys) in gene delivery zeta potential of polyplexes were determined on a Zetasizer
studies,30, 31 the analogous cationic pLys dendrimers have also Nano ZS (Malvern Instruments Ltd.). The polyplex dispersion
been extensively explored. 32-36 However, to date, no dendritic was diluted in Milli-Q water at intensity in the range 104-106
macromolecules based on other similar amino acids have been counts/s and measurements were performed at 25°C.
reported for gene delivery applications. Intriguingly, there are
BOC protection of L-ornithine
now a number of studies indicating that linear poly(ornithines)
display more effective nucleic acid condensation, and thus a 10- L-ornithine monohydrochloride (4.25 g, 25.22 mmol) was
fold increase in transfection efficiency in an array of cell lines dissolved in deionized water (40 mL) under vigorous stirring and
compared to linear poly(lysine), due to the removal of one cooled to 0°C through an ice bath. The initial pH of the solution
carbon between the poly(peptide) backbone and pendant (around 6) was adjusted to 9 through the addition of NaOH.
amine.37, 38
Then BOC anhydride (12.35 g, 56.63 mmol) was dissolved in THF
We therefore decided to investigate ornithine-derived (10 mL) and added dropwise to the cooled solution. The
materials and describe here the synthesis of cationic reaction was allowed to warm up to room temperature and
dendrimers based on an ornithine repeat unit and evaluate proceed for 60 hours. The progress of the reaction was
their potential as pDNA and saRNA delivery vectors. Dendrimers monitored through a ninhydrin assay. At the end of the
with increasing generation were synthesized through iterative reaction, THF was evaporated under reduced pressure, and the
additions
via
a
divergent
strategy
from
a
remaining aqueous solution acidified with a 1 M solution of HCl
hexamethylenediamine core, and then formulated with model until the solution reached pH 2. The water phase was then
calf thymus DNA and plasmid DNA to form polyelectrolyte extracted with ethyl acetate (3 x 15 mL), and the organic
complexes (polyplexes). Utilising formulations initially fraction dried over MgSO4, filtered and the product was
optimized by a rapid screening methodology,39 we examined recovered after evaporation as a white solid.
transfection efficiencies of pDNA and saRNA in 2D cell 1H NMR (400 MHz, DMSO-d6): δ 12.42 (s, 1H), 7.02 (d, J = 8.0 Hz,
monolayers mediated by the ornithine dendrimers. Finally, we 1H), 6.76 (t, J = 5.7 Hz, 1H), 1.37 (d, J = 3.1 Hz, 18H).
probed their capability to enhance saRNA transfection in human
General procedure for L-ornithine coupling
skin explants and investigated which cell types contributed to
protein expression relative to those cells which internalized the
A
general procedure for coupling BOC-Orn to
RNA. The data combined show the potential for these materials hexamethylenediamine (synthesis of G1), or the previous
to be used in gene delivery and especially in vaccine generation dendrimers (synthesis of G2 and G3) is described as
formulations.
follows. Multifunctional amine (G0, G1, G2) and BOC-Orn were
dissolved in DMF under vigorous stirring in a 50 mL round
bottomed
flask.
2-(1H-benzotriazol-1-yl)-1,1,3,3-
tetramethyluronium hexafluorophosphate (HBTU) and
triethylamine was added to the flask and the reaction was
allowed to proceed for 24 h at ambient temperature. The
protected dendrimer was precipitated from a 10% sodium
citrate solution yielding a white precipitate, which was filtered
and washed with diethyl ether.
Materials and Methods
Materials
All solvents were of analytic or HPLC grade and purchased from
Sigma Aldrich or Fisher Scientific unless otherwise stated. All
deuterated solvents were purchased from Sigma Aldrich. L-
ornithine monohydrochloride, BOC anhydride, NaOH,
hexamethylendiamine, HBTU, triethylamine, sodium citrate,
tris-borate-EDTA, agarose, sodium chloride (NaCl), magnesium
chloride (MgCl2), branched polyethylenimine (PEI) (2.5 kDa)
Tris-base, DNase I from bovine pancreas were used as received
from Sigma Aldrich. Calf thymus DNA was provided by
Invitrogen. Blue/orange loading dye and ethidium bromide
solution were provided by Promega. The plasmid pEGFP N1 was
synthesized as previously described40
General procedure for BOC deprotection
The protected dendrimer was dissolved in trifluoroacetic acid
and stirred at ambient temperature for 2 h. The solution was
concentrated under reduced pressure, and the deprotected
dendrimer isolated via precipitation in diethyl ether.
Synthesis of G1
BOC-orn (BOC)-OH (3.1020 g, 9.34 mmol) was coupled to
hexamethylendiamine (505.3 mg, 4.35 mmol) using the
reaction procedure stated above, utilizing (2-(1H-benzotriazol-
1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU)
(3.3808 g, 8.91 mmol in 20 mL of DMF) and trimethylamine (3.6
mL) as coupling agent and organic base respectively in 20 mL of
DMF. The product was collected as a white powder. The
protected G1 dendrimer (2.80 g, 3,76 mmol) was deprotected
Instrumentation
1H NMR spectroscopy
1H-, 13C-NMR, 2D-NMR (COSY, HSQC, HMBC) spectra were
recorded at 25 °C on a Bruker Advance III 400 MHz
spectrometer. All chemical shifts are reported in ppm (δ)
referenced to the chemical shifts of residual solvent resonances
of d6-DMSO (2.50 ppm). MestReNova 6.0.2 copyright 2009
(Mestrelab Research S. L.) was used for analysing the spectra.
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as stated above in trifluoroacetic acid (13 mL) and recovered as 0.25; 0.50; 1; 2; 5; 10; 20. In order to allow polyplex formation,
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a yellow/brown sticky solid (yield 87%).
the solutions were stirred for 45 minutesDaOtI:ro10o.1m03t9e/mD0pTeBr0a0t9u4r2eC.
1H NMR (500 MHz, DMSO-d6) δ 8.52 (t, J = 5.6 Hz, 1H), 8.27 (d, J Size and zeta-potential analysis
= 5.3 Hz, 3H), 7.96 (s, 3H), 3.75 (h, J = 6.0, 5.6 Hz, 1H), 3.15 – Polyplexes for size and zeta-potential analysis were prepared at
3.06 (m, J = 6.6 Hz, 2H), 2.79 (dq, J = 13.9, 4.9, 4.1 Hz, 2H), 1.79 the above N/P ratios, with dendrimer/PEI concentrations fixed
– 1.67 (m, 2H), 1.57 (p, J = 7.8 Hz, 2H), 1.43 (p, J = 6.4 Hz, 2H), at 0.1 mg mL-1 with the DNA concentration varied for each N/P
1.32 – 1.21 (m, 2H). 13C NMR (126 MHz, DMSO-d6) δ 168.46, ratio. Size measurements were performed directly with the
52.13, 39.74, 38.68, 29.23, 28.65, 26.51, 23.05.
prepared polyplex solutions. For zeta-potential measurements,
polyplex solutions were diluted 1:1 with 10 mM NaCl (aq).
Gel retardation assay
Synthesis of G2
Polyplexes for gel retardation assays were prepared with a fixed
BOC-orn (BOC)-OH (279.7 mg, 0.81 mmol) was coupled to G1 DNA concentration of 200 ng mL-1, with the dendrimer/PEI
(279.7 mg, 0.81 mmol) using the reaction procedure stated concentrations varied for each N/P ratio. 20 µL of polyplex and
above, utilizing HBTU (2.3601 g, 6.22 mmol in 5 mL of DMF) and 4 µL loading dye (6x) were loaded on to a 1% agarose gel +
triethylamine (1.05 mL) as coupling agent and organic base GelRed in Tris-borate-EDTA 0.5x (TBE) buffer and subjected to
respectively in 10 mL of DMF. The product was collected as a electrophoresis for 45 minutes at 70V and visualized with an UV
white powder. The protected G2 dendrimer (300 mg, 0.19 illuminator.
mmol) was deprotected as stated above in trifluoroacetic acid
(5 mL) and recovered as a yellow/brown sticky solid (yield 82%). Plasmid DNA purification
1H NMR (400 MHz, DMSO-d6) δ 8.65 – 8.54 (m, 2H), 8.25 (s, 5H), Plasmid pEGFP-C1 was transformed into the Escherichia coli
8.07 (t, J = 5.5 Hz, 1H), 7.92 (s, 5H), 4.24 (q, J = 7.9, 7.4 Hz, 1H), DH5 and propagated in LB medium overnight at 37°C.41
3.89 – 3.82 (m, 1H), 3.75 (t, J = 6.6 Hz, 1H), 3.19 – 3.06 (m, 1H), MTT assay
3.09 – 3.02 (m, 1H), 3.01 (d, J = 6.6 Hz, 1H), 2.80 (t, J = 7.4 Hz, HCT 116p53−/− cells were obtained from the American Type
4H), 1.73 (dt, J = 14.0, 6.6 Hz, 3H), 1.58 (tq, J = 23.0, 12.7, 10.1 Culture Collection, cultured at 37°C in a humidified atmosphere
Hz, 3H), 1.37 (t, J = 6.9 Hz, 2H), 1.23 (s, 2H). 13C NMR (126 MHz, containing 5% CO2 and grown continuously in DMEM
DMSO) δ 168.46, 52.13, 39.74, 38.68, 29.23, 28.65, 26.51, supplemented with 10% FBS, 100 unit/mL penicillin and 100
23.05.
g/mL streptomycin. HCT 116p53−/− cells were seeded onto 96-
well plates (2ꢀ×ꢀ104 cells/well) and cultured in 200 μL of cell
medium with FBS at 10%. After 24 h, cells were treated and
Synthesis of G3
BOC-orn (BOC)-OH (1.23 g, 3.69 mmol) was coupled to G2 incubated with PEI and G3 polyplexes for 24 h. In particular,
(325.5 mg, 0.41 mmol) using the reaction procedure stated polyplexes at 2, 5 and 10 N/P ratios were prepared and tested
above, utilizing HBTU (1.63 g, 4.29 mmol in 10 mL of DMF) and at DNA concentrations of 0.1 – 0.8 µg/ mL. Then, cell viability
triethylamine (1.0 mL) as coupling agent and organic base was evaluated as a function of mitochondrial activity using the
respectively in 10 mL of DMF. The product was collected as a MTT assay. The absorbance was measured at 570 nm using a
white powder. The protected G2 dendrimer (234 mg, 0.19 microplate reader (Biotek Synergy H1 Hybrid multiplate reader).
mmol) was deprotected as stated above in trifluoroacetic acid
(5 mL) and recovered as an off-white sticky solid (yield 78%). 1H In vitro DNA transfection
NMR (400 MHz, DMSO-d6) δ 8.75 – 8.55 (m, 1H), 8.24 (s, 2H), HCT 116p53−/− cells were grown in 12-well plates until 80-90%
8.11 (d, J = 15.2 Hz, 2H), 8.02 (d, J = 8.0 Hz, 2H), 4.38 – 4.09 (m, confluent and were then treated with polyplexes complexed to
1H), 3.83 (dq, J = 42.0, 6.5, 6.0 Hz, 1H), 3.11 (t, J = 7.1 Hz, 1H), pEGFP-C1 DNA plasmid (0.2 g per well) which encodes for the
3.02 (s, 1H), 2.81 (q, J = 9.3, 8.2 Hz, 2H), 1.77 (dq, J = 12.2, 5.8 green fluorescence protein (GFP). After 24 h of incubation, the
Hz, 2H), 1.68 – 1.55 (m, 3H), 1.54 – 1.44 (m, 1H), 1.37 (s, 1H), medium was aspirated, cells were washed with cold PBS,
1.23 (s, 1H). 13C NMR (101 MHz, DMSO) δ 171.75, 52.12, 39.54, harvested and lysed as previously described 42. Total protein
39.33, 28.68, 23.09.
extracts (200 g) from samples were subsequently analyzed for
the expression of transfected DNA by measuring the
fluorescence of the GFP. The fluorescence was measured with a
Buffering capacity
Polymer (1 mg of dendrimer or PEI) was dissolved in 10 mL of Cary Eclipse fluorescence spectrophotometer (Varian).
NaCl (0.1 M) and the pH was adjusted to 10 through addition of Excitation and emission wavelengths were 488 and 507 nm,
NaOH (0.1 M). The polymer solution was titrated with HCl (0.1 respectively. The amount of total protein in the cell extracts was
43
M) and the pH value of solution was measured until pH 5.
quantified using the Bradford-assay as previously described
.
Results were plotted as the GFP fluorescence intensity in cells
normalized against the protein amount in the cell extracts.
Formulation and characterization of DNA polyplexes
General procedure
Stocks solutions of dendrimers G1, G2, G3, PEI and calf thymus In vitro transcription of saRNA
DNA were prepared at double the concentration required in the Self-amplifying RNA encoding the replicase derived from the
polyplexes in HEPES buffer (20 mM, pH 7.4). Complexes were Venezuelan Equine Encephalitis Virus (VEEV) and either firefly
prepared by mixing the solutions 1:1 to produce N/P ratio of luciferase (fLuc) or enhanced green fluorescent protein (eGFP)
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was produced using in vitro transcription. pDNA was consent from patients, under protocols approved by the Local
View Article Online
DOI: 10.1039/D0TB00942C
transformed into E. coli, cultured in 100 mL of LB with 100 Research Ethics Committee (MED_RS_11_014). The tissue was
µg/mL carbenicillin (Sigma Aldrich, UK) an isolated using a obtained from patients undergoing elective abdominoplasty or
Plasmid Plus MaxiPrep kit (QIAGEN, UK). The concentration and mastectomy surgeries and processed as previously described.19
purity of pDNA was measured on a NanoDrop One Upon arrival in the laboratory the subcutaneous layer of fat was
(ThermoFisher, UK) and subsequently linearized using MluI for removed and the tissue was excised into 1 cm2 pieces. Explants
3 h at 37 ˚C. For in vitro transfections, capped RNA was were incubated at 37˚C with 5% CO2 in 12-well plates with
synthesized using 1 µg of linearized DNA template in a cDMEM, which was replaced daily. Explants were injected
mMessage mMachine™ (Ambion, UK) and purified using a intradermally (ID) using a Micro-Fine Demi 0.3 mL syringe
MEGAClear™ column (Ambion, UK) according to the (Becton Dickinson, UK) with 5 µg of eGFP saRNA polyplexes.
manufacturer’s protocol. RNA for ex vivo experiments was After 72 h, explants were digested into single cell suspensions
prepared as previously described.19 Uncapped RNA transcripts by incubating in 2 mL of DMEM supplemented with 1 mg/mL
were synthesized using 1 µg of linearized DNA in a MEGAScript™ collagenase P (Sigma, UK) and 5 mg/mL dispase II (Sigma, UK)
reaction (Ambion, UK) according to the manufacturer’s for 4 h at 37 ˚C on a rotational shaker. Digests were then filtered
protocol. Transcripts were then purified by overnight LiCl through a 70 µm cell strainer and centrifuged at 1750 RPM for
precipitation at -20˚C, pelleted by centrifugation at 14,000 RPM 5 min. Cells were then resuspended in FACS buffer (PBS + 2.5%
for 20 min at 4 ˚C, washed 1X with 70% EtOH, centrfigued for FCS) and stained with Fixable Aqua Live-Dead Cell stain
14,000 RPM for 5 min at 4˚C, and then resuspended in UltraPure (ThermoFisher, UK) diluted at 1:400 in FACS buffer for 20 min.
H2O (Ambion, UK). Purified transcripts were then capped using Cells were then washed and stained with a panel of antibodies
the ScriptCap™ m7G Capping System (CEllSCript, Madison, WI, to identify each cell type for 30 min. The antibody panel
USA) and ScriptCapt™ 2’-O-Methyltransferase Kit (CellScript, included CD3-V450 (BioLegend, UK), CD14-Qdot605
Madison, WI, USA) simultaneously according to the (BioLegend, UK), CD19-BV650 (BioLegend, UK), CD56-BV711
manufacturer’s protocol. Capped transcripts were then purified (BioLegend, UK), CD1a-PerCP-eFluor710 (BioLegend, UK),
by LiCl precipitation as detailed above, resuspended in CD11c-PE (BioLegend, UK), CD90-PE-Cy7 (BioLegend, UK) and
UltraPure H2O and stored at -80˚C until further use.
CD45-AF700 (BioLegend, UK). Cells were then fixed in 1.5%
paraformaldehyde and refrigerated until flow cytometry
analysis. Samples were analyzed on a LSRFortessa™ (BD
Preparation of saRNA polyplexes
Stock solutions were prepared by dissolving the polymers and Biosciences, UK) with FACSDiva software (BD Biosciences, UK)
RNA in molecular grade H2O at a concentration of 1 mg/mL. with 100,000 acquired cell events. Gating and analysis was
Polyplexes were then prepared in a buffer of 20 mM HEPES and performed in FlowJo Version 10 (FlowJo LLC, Oregon, USA).
5 wt.% glucose at N/P ratios of 2, 5 and 10.
t-SNE representation of flow cytometry data
In vitro transfection of saRNA polyplexes
t-Distributed Stochastic Neighbor Embedding (tSNE) analysis of
Transfections were performed in HEK293T.17 cells (ATCC, USA) unsupervised clusters of live cells was performed in FlowJo
that were maintained in culture in complete Dulbecco’s using 1000 iterations, a perplexity of 30, a learning rate of
Modified Eagle’s Medium (cDMEM) (Gibco, ThermoFisher, UK) 15196, the Exact (vantage point tree) KNN algorithm and the
containing 10% (v/v) fetal calf serum (FCS), 5 mg/mL L- Barnes-Hut gradient algorithm. Unsupervised clusters are
glutamine and
5
mg/mL penicillin and streptomycin shown in grey, whereas the gated cells are superimposed on top
(ThermoFisher, UK). Cells were plated at a density of 50,000 with green (eGFP+), blue (Cy5=) and red (cell phenotype).
cells/well in a 96 well plate 24 h prior to transfection. At the
time of transfection, the media was completely removed and Statistical analysis
replaced by 50 µL of transfection media (DMEM with 5 mg/mL Graphical and statistical analysis were performed in GraphPad
L-glutamine). 100 µL of the polyplex solution was added to each Prism, version 8. Statistical differences were analyzed using
well and incubated for 4 h. Then, the media was replaced with either multiple t tests adjusted for multiple comparisons or one-
cDMEM and the cells were allowed to culture for 24 hours, at way ANOVA with multiple comparisons, with p<0.05 used to
which time 50 µL of media was removed and 50 µL of ONE-Glo™ indicate significance.
D-luciferin substrate (Promega, UK) was added and mixed well
by pipetting. The total volume was transferred to a white 96-
well plate (Costar, UK) and analyzed on a FLUOstar Omega plate
Results and discussion
reader (BMG LABTECH, UK). Cy5-labelled saRNA was purchased
from TriLink (USA).
Dendrimer synthesis and complexation with DNA
Dendrimers of three generations (G1-G3) were synthesized via
a divergent iterative addition-deprotection strategy utilizing
BOC-protected ornithine (BOC-Orn, Figure 1, Figure S2).
Initially, two equivalents of BOC-Orn were coupled to a
hexamethylenediamine scaffold (G0) via conventional HBTU
chemistry and subsequently deprotected using TFA to produce
Flow cytometry analysis of eGFP expression in human skin
explants
Surgically resected specimens of human skin tissue were
collected at Charing Cross Hospital, Imperial NHS Trust, London,
UK. All tissues were collected after receiving signed informed
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G1. This process was repeated twice using 4 then 8 equivalents
of Boc-Orn to prepare G2 and G3 respectively.
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DOI: 10.1039/D0TB00942C
4 x
2 x
TFA
HBTU
TEA
DMF
HBTU
TEA
DMF
G0
G1
TFA
8 x
TFA
HBTU
TEA
DMF
G2
G3
O
NH2
NHBoc
COOH
BocHN
OH
NHBoc
=
Figure 1. Representative iterative addition-deprotection divergent synthetic approach to produce ornithine dendrimers G1, G2 and G3 from Boc-protect ornithine and
hexamethylenediamine (G0).
46
Deprotection reactions were confirmed by the disappearance polyplexes transport intracellularly,45,
and thus we first
of the singlet at ~1.5 ppm in 1H NMR spectra for each required to evaluate nucleic acid binding and release properties
dendrimer, whilst each iterative coupling was validated by the outside a cellular environment.
appearance of a signal at ~4.3 ppm attributed to the CH2 To investigate the DNA binding capabilities of the cationic
protons next to the new amide bond (Figure 1, Figure S3, Figure dendrimers, a range of dendrimer/DNA complexes at different
S4).
N/P ratios (0.25 – 20) were formulated with G1, G2 and G3 with
The buffering capacities of dendrimers G1-G3 were determined model calf thymus DNA (Figure S5). These polyplexes were
through a pH titration and compared to PEI. All dendrimers (G1- compared to analogues prepared with PEI. All polyplexes
G3) and PEI displayed some buffering compared to a NaCl prepared with PEI, G2 and G3 displayed particle sizes below 300
solution. Within the family of dendrimers, it was observed that nm, however, dendrimer polyplexes had significantly lower PDI
the buffering capacities increased with each generation of values than PEI, particularly at low N/P ratios. In contrast, G1
dendrimer, requiring 20, 50 and 90 µL of 0.1 M HCl to acidify the did not form stable polyplexes at any N/P ratio evidenced by
solution from pH 9 to pH 5 for G1, G2 and G3 respectively. This their large sizes and negative zeta-potentials, probably due to
is likely due to the expanding number of ionisable nitrogens per the low polycation density. This was further supported by the
generation, coupled with the increasing density of these groups lack of DNA retardation in the electrophoresis assay, in line with
which modifies the relative pKa per each protonation event. previous studies evaluating dendritic polylysine. Comparing
None of the dendrimers displayed buffering comparable to PEI, between G2 and G3 polyplexes, the higher generation displayed
however PEI has a very high density and greater number of smaller particle sizes, below 200 nm, potentially due to its
protonatable amines than the relatively low molar mass greater charge density enabling greater electrostatic attraction,
dendrimers synthesised here (Figure 2A). The increasing thus more efficient DNA condensation, in line with the most
buffering capacity with each successive generation of recent cationic materials investigated. G2 & G3 exhibited
dendrimer might be expected to enhance endosomal escape, stronger binding capabilities of model calf thymus DNA than PEI,
according to the ‘proton sponge’ hypothesis,44 however, there on the contrary of different PLL based polyplexes,47 thus fully
have been some questions relating to the mechanisms by which complexing the nucleic acid above N/P ratios 2 (compared to
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N/P 5 for PEI), as evidenced by the electrophoresis retardation
assay. This is supported by the zeta-potential data which
indicates the G2 and G3 polyplexes had positive potentials with
lower polymer content (lower N/P ratio), compared to PEI
which was negative until N/P 5. We postulate that this is due to
the greater charge density on the dendrimer surface allowing
for more efficient condensation than the conventional gaussian
polymer chains of PEI.
View Article Online
10
9
DOI: 10.1039/D0TB00942C
A
G1
G2
8
G3
PEI
7
0.1 M NaCl
6
5
4
0
20
40
60
80 100 120 140
Cytotoxicity and transfection of dendrimer-DNA polyplexes
Volume 0.1 M HCl (µL)
The preliminary complexation studies suggested that the G3
poly(ornithine) materials were likely to be the most effective in
transporting DNA into cells in 2D culture, owing to their smaller
size relative to the other polymers in complexes with DNA. We
selected a representative colon cancer cell line, HCT 116, as a
target for these initial feasibility studies for nucleic acid
transport. Accordingly, HCT 116p53-/- cells were incubated with
G3 and PEI polyplexes formulated at N/P 2, 5, 10, across four
concentrations of pEGFP-C1 DNA plasmid (0.1 – 0.8 g/mL).
Cells treated with G3/pEGFP-C1 polyplexes exhibited excellent
cell viability, above 90%. In contrast we observed some
cytotoxic behaviour in cells treated with PEI polyplexes above
0.4 µg DNA at all N/P ratios (Figure 2B). In general, this suggests
that G3 dendrimer polyplexes are significantly more
biocompatible than those with PEI. This is likely due to the
greater hydrophilicity of G3 than PEI, which is known to exhibit
membrane damaging properties.
B
C
0.1 g/mL
0.2 g/mL
100
80
60
40
20
0
0.4 g/mL
0.8 g/mL
PEI
G3
PEI
G3
PEI
G3
N/P 2
N/P 5
N/P 10
15
10
5
Untreated
PEI
G3
0
2
5
0
1
d
e
t
P
P
/
/
a
P
/
N
N
e
r
t
N
n
U
Figure 2. A) Potentiometric pH titration curves for dendrimers (G1, G2 and G3) and PEI,
measured from pH 10 to 3 adjusting the pH with HCl (0.1 M). B) Cell viability of HCT 116
p53-/- cells after treatment with PEI and G3 pEGFP polyplexes at DNA concentrations of
0.1 – 0.8 µg/ mL. The cell viability from untreated cells was normalised to 100%. Results
are presented as percentage (meanꢀ±ꢀstandard deviation) (nꢀ=ꢀ3) of the control cells. C)
Transfection efficiency of PEI /DNA and G3 polyplexes/DNA at different N/P ratios using
pEGFP-C1 plasmid in HCT 116p53−/− cells. Transfection efficiency of different delivery
reagents was quantified by measuring the transgene expression level (GFP fluorescence
intensity) 24 h after transfection by fluorescence spectroscopy. Results from three
independent experiments (meanꢀ±ꢀstandard deviation) (nꢀ=ꢀ3) are displayed.
To evaluate transfection efficiency, HCT 116p53-/- cells were
incubated with G3 or PEI polyplexes complexed to pEGFP-C1
(0.2 g), at N/P ratios 2, 5 and 10. After 24 hours, the
transfection efficiencies were quantified by measuring the
relative GFP expression by fluorescence spectroscopy in cell
lysates (200 g). Although at N/P=2 G3 displayed lower GFP
expression than PEI, the transfection efficiency was 2-3 fold
higher in cells treated with G3 polyplexes at N/P ratio of 5 and
10 compared to PEI polyplexes. Interestingly, there was no
correlation between GFP expression and N/P ratio for PEI
polyplexes, whereas G3 polyplexes showed N/P ratio
dependent expression, with higher ratios exhibiting more
protein production (Figure 2C). This finding may be due to the
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n=3; * indicates significance of p<0.05 as determined by multiple t tests adjusted for
observed cytotoxicity of PEI, which would limit total
transfection efficiency, thus highlighting again a greater
potential compared to the more employed PLL as gene delivery
vector.48
View Article Online
multiple comparisons.
DOI: 10.1039/D0TB00942C
We, and others, have observed efficient RNA transfection by
low-medium molar mass carriers.39 In addition, a second
mechanism relating to transfection may have been involved.
The initial complexation studies revealed that G1 polymers did
not form persistent and stable polyplexes with model DNA, as
measured by light scattering. However, it is possible that weak
complexation occurred at low N/P ratios and that transport of
loose polymer-RNA aggregates nevertheless took place
efficiently into the cellular interiors. Polymer G1 may also have
had a role in aiding mRNA delivery by permeabilizing cellular
membranes, enhancing either its internalization or
translocation from intracellular compartments into the cytosol,
in line with many prior studies describing the effects of
polycations on biological membranes.50, 51
In vitro transfection efficiency of self-amplifying RNA
For RNA vaccine applications, it is necessary to deliver the
encoding nucleic acid to the cytosol and engage the
translational machinery ultimately so that antigens expressed
which induce an immune response. However, it is known that
polycations alone can induce potent immune activation,49 and
thus we decided to evaluate G1-G3 polymers for delivery of
saRNA even though the G1 and G2 complexes with the nucleic
acid were likely to be less stable than those of G3. In order to
characterize the capacity of the dendrimers to deliver saRNA
encoding for luciferase, we transfected HEK 293T.17 cells at N/P
ratios ranging from 2-10 (Figure 3). PEI was used as a positive
control at N/P=5, which was determined in the initial
experiments to be the optimal ratio for this polymer with
saRNA. We observed that ratios of N/P=2 resulted in the highest
transfection efficiency (~105 RLU) for all three dendrimers (G1-
G3), which were all significantly higher than the PEI control (~2
x 104 RLU, p=0.007, 0.014 and 0.00035, respectively).
Interestingly, increasing the N/P to 5 and 10 for G1 did not
impact the transfection efficiency, although both formulations
were also significantly better at transduction of luciferase
activity than PEI (p=0.044 and 0.00734, respectively). However,
increasing the N/P for G2 and G3 inhibited the transfection
efficiency. Overall, polymers G1-3 at N/P=2 induced the highest
transfection efficiency, and further increasing the N/P ratio did
not enhance luciferase expression. The higher transfection
efficiencies at lower N/P ratios, and with the lower generation
dendrimers (G1 and G2) may have been due to weaker
electrostatic binding between these materials and RNA
compared to those of higher mass and greater numbers of
cationic residues. It thus may have been the case that for saRNA
delivery, the polyplexes at higher N/P ratios and with higher
charge densities (G3) were less efficient in release of the nucleic
acid for translation into protein, resulting in a lower transfection
efficiency.
Transfection efficiency of saRNA/dendrimer polyplexes in ex vivo
human skin explants
We then sought to characterize the ex vivo transfection
efficiency of the dendrimers in human skin explants (Figure 4)
using saRNA encoding for green fluorescent protein (GFP).
Based on the in vitro transfection results Figure 3) we elected to
use N/P ratios of 2 and 10, using RNA alone and PEI as controls.
We observed that RNA alone resulted in GFP expression in ~2%
of cells, and that complexation with PEI did not increase the
number of GFP+ cells. However, complexation of saRNA with
G1-3 at N/P=2 resulted in ~7% of cells being positive for GFP
expression. When G1 and G2 were increased to N/P=10 the
number of GFP+ increased to ~12%, and was significantly higher
than RNA alone or PEI, with p=0.011 and 0.003, respectively.
There was no increase in the percentage of GFP+ cells when the
ratio for G3 was increased to N/P=10. These results are not in
direct alignment with the in vitro transfection results; however,
this is often the case for different transfection systems. The skin
explants are a three-dimensional co-culture of many different
cell types in a native tissue architecture as opposed to a
monoculture of cells on a two-dimensional polystyrene surface.
We subsequently analyzed which cells were expressing GFP in
the skin explants (Figure 4B-C). We observed that the cell
phenotype in the skin was predominantly epithelial (~50%),
with leukocytes (~10%) and fibroblasts (~12%), and a much
smaller proportion of Langerhans cells (~5%), NK cells (~5%),
dendritic cells (<1%), monocytes ~2%), B cells (~2%) and T cells
(~1%) (Figure 4B). We observed that RNA and PEI formulations
were expressed in similar populations, the majority of which
were still epithelial cells, fibroblasts and leukocytes, but
showing some enrichment in NK cells, T cells, monocytes, and B
cells compared to the composition of the resident skin cell
population (Figure 4C). G1 showed expression enrichment in
fibroblasts and Langerhans cells, which was augmented by
increasing the N/P ratio from 2 to 10. Interestingly G2 and G3
both showed an enrichment in epithelial cells, and to a lesser
extent NK cells and Langerhans cells. Based on these
observations we can conclude that G1 and G2 at N/P=10
enhance the percentage of cell expressing GFP in human skin
*
*
*
1000000
100000
10000
1000
*
PEI
G1
G2
*
G3
2
5 10
2
5 10
2
5 10
2 5 10
N:P
Figure 3. Transfection of polyplexes with saRNA encoding luciferase at N/P=2, 5 and 10
in HEK 293T.17 cells 24h after treatment. Bars represent mean ± standard deviation for
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explants, specifically in resident NK cells, Langerhans cells and
epithelial cells.
View Article Online
DOI: 10.1039/D0TB00942C
Figure 4. Cellular expression of saRNA after ID injection with polyplex formulations. A) Percentage of eGFP+ cells out of total live cells for each formulation after an intradermal
injection of 5 µg of saRNA in human skin explants. Explants were analyzed 72 h after initial injection. Jet PEI was formulated at ratios of N/P = 5, while dendrimer formulations were
prepared at ratios of N/P=2 or 10. Bars represent mean +/- SD for n = 3, with * indicating significance of p < 0.05 using an ordinary one-way ANOVA. B) Phenotypic identity of cell
present in human skin explants and C) GFP+ cells after intradermal (ID) injection of polyplex formulations as determined by flow cytometry. Cells identified using the following
antibodies: epithelial cells (CD45-), fibroblasts (CD90+), NK cells (CD56+), leukocytes (CD45+), Langerhans cells (CD1a+), monocytes (CD14+), dendritic cells (CD11c+), T cells (CD3+)
and B cells (CD19+).
Characterization of polyplex uptake versus expression in human
skin explants
expressing the RNA, eGFP+. All the formulations yielded clusters
of cells that had taken up RNA but were not expressing it, in
addition to eGFP+/Cy5+ populations (Figure 5A). We then
overlaid the gating for each cell phenotype in red to determine
which white cells were responsible for uptake vs. expression
(Figure 5B). We observed that T cells, monocytes, B cells,
Langerhans cells, leukocytes and NK cells internalised the RNA
and expressed eGFP for each formulation. While there was
uptake and expression in dendritic cells, there was a population
of DCs in each formulation which was only positive for Cy5,
indicating that these cells did not express the RNA to a
detectable level. The epithelial cell and fibroblast populations
for each formulation were also quite similar in that there was a
higher proportion of these cells that were positive for RNA but
which did not express GFP. Based on these observations we
suggest that the uptake and subsequent expression of RNA
varied in a phenotypic, not formulation dependent manner, and
that increased expression relied on increasing the percentage
of cells targeted by a formulation.
Based on the observations that these dendrimers enhanced
the percentage of GFP+ cells in human skin explants, we then
wanted to explore how the formulation changed which cells
internalised the polyplexes, compared to those cells which
expressed the RNA. In order to probe this interaction we utilized
Cy5 labelled saRNA that encodes eGFP, and analyzed the
phenotypes using flow cytometry (Figure 5). We utilized t-
distributed stochastic neighbor embedding (tSNE), to provide
three-dimensional principal component analysis for flow
cytometry data using unsupervised clustering. tSNE allows
resolution of high-dimentional data arising from multi-paramter
flow cytometry analysis into two dimensions, meanwhile
preserving the structure of the data and revealing inherent
clusters within the data.52 The grey lines (Figure 5 A-B) indicate
the tSNE unsupervised clustering, with Cy5+ (blue) and eGFP+
(green) gating applied on top, allowing for visualization of
clusters of cells which internalised RNA, Cy5+, and clusters
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DOI: 10.1039/D0TB00942C
ARTICLE
Figure 5. t-Distributed Stochastic Neighbour Embedding (t-SNE) analysis identifies cell populations that are positive for saRNA uptake (Cy5, blue) and saRNA expression (eGFP, green).
A) t-SNE of live cells to show unsupervised clustering after injection with 5 μg of saRNA formulated with either PEI or dendrimers (G1-3). B) Overlay of phenotypic identification (red)
as identified by cell gating overlaid with t-SNE.
compared to 16 in G3. This potentially indicates that low
Conclusions
generation dendrimers may be more effective for particular
transfection settings than others. Given the data for efficient
In conclusion, this study demonstrates the potential for
transfection in skin, we believe that some of these formulations
ornithine dendrimers as efficient non-viral delivery vectors for
may be effective vectors for RNA vaccines due to their
DNA and RNA, compared to the most popular cationic materials
substantial localisation with dermal immune cells. From a
designed for gene therapy so far. G2 and G3 dendrimers
manufacturing perspective, these materials may alleviate
outperformed the ‘gold-standard’ polymer in nucleic acid
potential
batch-batch
variability
associated
with
binding assays, complexing all available DNA at lower N/P ratios
than PEI. Furthermore, the synthesized dendrimers exhibited
significantly higher transfection efficiencies over PEI for DNA
and RNA across 2D in vitro assays and 3D ex vivo skin explant
studies. In skin, this increase was attributed to the dendrimer
mediated enrichment in protein production in NK, Langerhans
and epithelial cells, compared to RNA alone and PEI which
displayed similar phenotypic protein expression. Finally using
labelled RNA, we observed similar correlations between uptake
and expression across each formulation. Somewhat
surprisingly, in many of our transfection studies we observed
significantly greater protein expression with the G1 and G2
dendrimers, which only contained 4 and 8 ionisable nitrogens,
macromolecules with non-uniform molecular weight
distributions.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
This research is funded by the Department of Health and Social
Care using UK Aid funding and is managed by the Engineering
and Physical Sciences Research Council (EPSRC, grant number:
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19.
20.
A. K. Blakney, P. F. McKay, B. Ibarzo Yus, J. E. Hunter, E. A.
EP/R013764/1). The views expressed in this publication are
those of the author(s) and not necessarily those of the
Department of Health and Social Care. This research was also
funded through the Royal Society through a Wolfson Research
Merit Award [WM150086]. C. Conte thanks the Italian
Association for Cancer Research (AIRC) and the European Union
(iCARE/Marie Curie 2014). F.Q., A.R. and G.R. wish to thank
Regione Campania-POR Campania FESR for funding under
2014/2020 Project N. B61G18000470007. We also thank
Douglas Crackett and Paul Cooling for expert technical
assistance and Carol Turrill for outstanding administrative
support.
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Journal of Materials Chemistry B
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DOI: 10.1039/D0TB00942C
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Page 13 of 13
Journal of Materials Chemistry B
View Article Online
DOI: 10.1039/D0TB00942C
Ornithine dendrimers exhibit high activity as vectors for saRNA in cells and skin, highlighting their
potential use in RNA vaccines.