Branched-chain and dendritic lipids for nanoparticles

Article abstract of DOI:10.1139/cjc-2016-0462

Lipid nanoparticles (LNPs) for drug-delivery applications are largely derived from natural lipids. Synthetic lipids, particularly those incorporating branched hydrocarbons and hyper-branched hydrocarbon architectures, may afford enhanced lipophilicity with enhanced fluidity and thereby lead to LNP stabilization. Hydrocarbon anchors based on serinol diesters were prepared from linear Cn (n = 14, 16, 18) and branched (n = 16) acids with Boc-protected serinol. These diesters were further dimerized on an iminodiacetamide backbone to provide eight branched-chain and dendritic lipid anchors. Derivatization of these core structures provided eight PEG-lipids and seven thiopurine linked lipid-drug conjugates. LNPs were prepared by microfluidic mixing from mixed lipids in ethanol diluted into aqueous media. The lipid-drug conjugates incorporated 5 mol% of a phosphocholine and 5 mol% of a commercial PEG-lipid to form LNPs with a thiopurine drug loading of 15 wt%. The PEG-lipids prepared were formulated at 1.5 mol% as a surface stabilizer to LNPs containing dsDNA lipoplexes. The stability of the LNPs was assessed under different storage conditions through monitoring of particle size. For both LNPs from lipid-Thiopurine conjugates and the PEG-lipid systems, there is strong preliminary evidence that hydrocarbon branching results in LNP stabilization. Four of the lipid-drug conjugate formulations were stable to cell culture conditions (10% serum, 37 °C) and the toxicity of these LNPs was assessed in two cell lines relative to the free thiopurines in the medium. The observed toxicity is consistent with cellular uptake of the LNPs and reductive release of the cargo thiopurine within the cell.

Full text of DOI:10.1139/cjc-2016-0462

120
ARTICLE
Branched-chain and dendritic lipids for nanoparticles
Michael W. Meanwell, Connor O’Sullivan, Perry Howard, and Thomas M. Fyles
Abstract: Lipid nanoparticles (LNPs) for drug-delivery applications are largely derived from natural lipids. Synthetic lipids,
particularly those incorporating branched hydrocarbons and hyper-branched hydrocarbon architectures, may afford enhanced
lipophilicity with enhanced fluidity and thereby lead to LNP stabilization. Hydrocarbon anchors based on serinol diesters were
prepared from linear Cn (n = 14, 16, 18) and branched (n = 16) acids with Boc-protected serinol. These diesters were further
dimerized on an iminodiacetamide backbone to provide eight branched-chain and dendritic lipid anchors. Derivatization of
these core structures provided eight PEG-lipids and seven thiopurine linked lipid–drug conjugates. LNPs were prepared by
microfluidic mixing from mixed lipids in ethanol diluted into aqueous media. The lipid–drug conjugates incorporated 5 mol%
of a phosphocholine and 5 mol% of a commercial PEG-lipid to form LNPs with a thiopurine drug loading of 15 wt%. The PEG–lipids
prepared were formulated at 1.5 mol% as a surface stabilizer to LNPs containing dsDNA lipoplexes. The stability of the LNPs was
assessed under different storage conditions through monitoring of particle size. For both LNPs from lipid–thiopurine conjugates
and the PEG-lipid systems, there is strong preliminary evidence that hydrocarbon branching results in LNP stabilization. Four of
the lipid–drug conjugate formulations were stable to cell culture conditions (10% serum, 37 °C) and the toxicity of these LNPs was
assessed in two cell lines relative to the free thiopurines in the medium. The observed toxicity is consistent with cellular uptake
of the LNPs and reductive release of the cargo thiopurine within the cell.
Key words: lipid synthesis, PEG-lipid, lipid–drug conjugate, electrospray mass spectrometry, lipid nanoparticle stability.
Résumé : Les nanoparticules de lipides (NPL) dans les applications d’administration des médicaments sont largement dérivées
de lipides naturels. Or, des lipides synthétiques, en particulier ceux qui comporteraient des chaînes d’hydrocarbures ramifiées
ou des architectures d’hydrocarbures hyper-ramifiées, pourraient présenter une lipophilie et une fluidité accrues, et ainsi
permettre la stabilisation des NPL. Nous avons synthétisé des diesters de sérinol comme points d’ancrage pour les hydrocarbures
a` partir d’acides linéaires Cn (n = 14, 16, 18) et ramifiés (n = 16) et de sérinol protégé par Boc. Nous avons par la suite formé un
dimère de ces diesters sur un squelette d’iminodiacétamide pour produire huit composés a` ancrages lipidiques de structure
ramifiée ou dendritique. La dérivation de ces structures de base a produit huit lipides liés au PEG et sept conjugués lipide–
médicament liés a` la thiopurine. Nous avons synthétisé les NPL par mélange microfluide a` partir de lipides mélangés dans
l’éthanol puis dilués en milieu aqueux. Les conjugués lipide–médicament ont incorporé 5 mol % de phosphocholine et 5 mol %
d’un lipide lié au PEG vendu sur le marché pour former les NPL dont la charge du médicament lié a` la thiopurine était de 15 % p/p.
Les lipides liés au PEG que nous avons synthétisés ont été mis en formule a` 1,5 mol % comme stabilisant de surface des NPL
contenant des lipoplexes d’ADN bicaténaire. Nous avons évalué la stabilité des NPL dans différentes conditions d’entreposage en
mesurant la taille des particules. Dans le cas des NPL a` base de conjugués lipides-thiopurine comme dans celui des systèmes de
lipides liés au PEG, les résultats préliminaires appuient fortement l’hypothèse selon laquelle la ramification des chaînes
d’hydrocarbures permet de stabiliser les NPL. En effet, quatre des préparations de conjugué lipide–médicament étaient stables en
conditions de culture cellulaire (sérum a` 10 %, 37 °C). En outre, nous avons évalué la toxicité de ces NPL dans deux lignées cellulaires
comparativement a` celle de thiopurines libres dans le milieu. La toxicité observée est cohérente avec l’absorption des NPL par la cellule
et la libération réductrice de la charge de thiopurine dans la cellule. [Traduit par la Rédaction]
Mots-clés : synthèse des lipides, lipide lié au PEG, conjugué lipide–médicament, spectrométrie de masse a` ionisation par
électronébulisateur, stabilité des nanoparticules de lipides.
in the target cell.3,8,9 The ultimate utility of a particular LNP is
based on an array of formulation variables — lipid composition
and proportion, drug loading, surface modifying agents, and mix-
Introduction
Lipid nanoparticles (LNPs) are an emerging drug delivery strat-
egy in biomedical research and therapy.1–5 LNPs consist of a con-
densed lipidic core — solid, nanostructured, and (or) fluid — with
ing parameters — that control particle size, lifetime in biological
media, and cellular uptake.3 The formulation space appears to be
a hydrophilic outer layer that may bear additional functionality.
vast and, yet, is predominantly based on a relatively restricted set
The drug loading in the particle can be noncovalent, as in the
of naturally derived phospholipids possessing two fatty acid
electrostatic complexes (lipoplexes) of plasmid DNA or siRNA
with cationic lipids,6,7 or covalent, as in the so-called lipid–drug
conjugates (LDCs) in which a hydrophilic drug is linked as a pro-
drug to a lipid via a functionality that can be metabolically cleaved
chains of C16–C18 as glycerol diesters. Although this may limit the
potential toxicity of the LNP, it also imposes geometrical re-
straints10 on the packing of lipids and the overall stability of the
LNP.
Received 30 August 2016. Accepted 28 October 2016.
M.W. Meanwell and T.M. Fyles. Department of Chemistry, University of Victoria, Victoria, BC V8W 3V6, Canada.
C. O’Sullivan and P. Howard. Department of Biochemistry and Microbiology, University of Victoria, Victoria, BC V8P 5C2, Canada.
Corresponding author: Thomas M. Fyles (email: tmf@uvic.ca).
Copyright remains with the author(s) or their institution(s). Permission for reuse (free in most cases) can be obtained from RightsLink.
Can. J. Chem. 95: 120–129 (2017) dx.doi.org/10.1139/cjc-2016-0462
Published at www.nrcresearchpress.com/cjc on 1 November 2016.

Meanwell et al.
121
Scheme 1. Branched-chain and dendritic lipid designs.
Specific lipids within LNP formulations are designed to per-
form specialized functions. For example, poly (ethylene glycol)-
derivatized lipids (PEG-lipids) are used to reduce nonspecific
protein binding by the LNP surface and thereby increase the life-
time of LNPs in serum.2,11,12 Current commercially available PEG-
lipids (usually diC18) insufficiently anchor the polymer to the LNP,
limiting both storage lifetime and circulation times and leading
to immunogenic activity; these effects may be linked to hydrolysis
of esters within the PEG-lipid,13 but PEG-lipid loss to the medium
is also implicated.14 PEG anchoring can be compared with the
anchoring of oligosaccharides to cell surfaces achieved in nature
by lipid A — a disaccharide bearing up to six branched fatty acid
chains.15 Lipid A anchors invest nearly twice the hydrocarbon,
incorporated as shorter branched chains, to anchor the hydrophilic
surface oligosaccharide relative to a comparable PEG-lipid.
Naturally derived lipids may also be similarly poor as the core
structures for the formation of LDC nanoparticles. Typically, a
hydrophilic drug is conjugated as the head group of a diacylglyc-
erol. Hydrophobic self-assembly is expected to immobilize the
LDC into a LNP. To achieve high drug or particle loading and
thereby reduce side effects, the hydrophobic component must be
as small as possible relative to the drug head group.3 The inherent
shape of a diacylglycerol tends towards a lamellar lipid arrange-
ment10 that will inhibit the formation of closed LNPs structures.
Even if formed, the specific chain packing of a natural diacylglyc-
erol may compromise particle integrity even with sufficient hy-
drocarbon present to drive the self-assembly of the LNP.
Lipid self-assembly into LNPs and other lipid morphologies
is a dynamic balance between lipid shape, charge, and hydro-
phobicity.10 Many successful formulations, particularly for the
formation of LNPs with solid cores, are based on lipid mixtures of
glycerol diesters, neutral glycerol triesters and other harder fats,
and fluidizing glycerol monoesters along with surfactants to sta-
bilize the particle surface.16 Engineering lipid mixtures in this
context not only is equivalent to adjusting the average lipid shape
and charge to control overall LNP size and charge, but also leads to
complex phase behavior and phase separation and potentially to
compromised LNP stability and performance.16
branched chains to anchor surface oligosaccharides, we initially
focussed on designs incorporating branched hydrocarbon chains
that are available commercially as Guerbet-derived acids.17,18 We
subsequently realized that branching could also be achieved with
dendrimer-like dimerization on an imino-diacetamide backbone
(Scheme 1). The latter structures are the hydrophobic inverse of
lipids bearing dendritic head groups.19,20 Branching, shorter
chains, and chain multiplicity all are expected to reduce the crys-
tallinity of the hydrocarbon and enhance the miscibility of the
chains with other components.
Our focus here is on serinol diesters of fatty acids21 that offer
the potential to explore alternative hydrocarbon anchoring topol-
ogies and their consequences for LNP formation and stability as
either PEG-lipid anchors or as the driving components of LDC
nanoparticle formation. Serinol substructures occur in sphingo-
lipids22 and serinolipids,23–26 but it is usually the amine, not the
hydroxyl group, that is acylated. Serinol-derived diesters, with
linear C14–C18 chains bearing (poly) ammonium head groups, are
widely reported as transfection reagents.27–31
The goal of this initial study is to develop the synthesis and
characterization of the core lipids and simple derivatives for the
formulation of LNPs and to uncover if hydrocarbon chain branching
affords any advantages in LNP stabilization. As shown in Scheme 1,
we focussed on a PEG-lipid derivative and a thiopurine lipid–drug
conjugate as initial targets.
PEG-lipids play a number of roles in both the formulation and
circulation stability of LNPs. When formulating LNPs, the PEG
surface coating prevents aggregation and contributes to obtaining
stable, small, and mono-disperse nanoparticles.12,13 While provid-
ing a steric barrier to protect the LNP, the PEG also serves to
significantly decrease the surface charge. This combination of
steric barrier and reduced surface charge is generally assumed to
prevent the LNPs from associating with serum proteins and ulti-
mately leads to extended circulation times compared with their
nonpegylated counterparts.13,32 We anticipated some differential
between branched-chain and linear isomers with respect to parti-
cle size stability if there is underlying merit to this lipid core
design.
As naturally derived lipids provide only limited diversity of
component shapes and charges, we recognized an opportunity to
explore synthetic lipids as a route to access a wider range of these
key properties. Inspired by the reliance of lipid A on shorter
Thiopurines are frontline drugs in the treatment of childhood
acute lymphoblastic leukaemia33 and are widely used as immuno-
suppressants in the treatment of autoimmune disorders (e.g.,
Crohn’s disease, rheumatoid arthritis) and as anti-rejection agents
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Can. J. Chem. Vol. 95, 2017
1
in organ transplant patients.34,35 The thiopurines are activated by
a phosphoribosyl transferase to produce thiopurine nucleotide
triphosphates (TTP), which are incorporated into DNA34 causing
mitotic death due to DNA damage.36 The cytosol of most tumour
cells is more reducing than in normal cells due to elevated free
thiol/glutathione levels, and this tumour cell-specific reactivity
has been exploited for tumour targeting, imaging, and drug deliv-
ery.8,9,37–39 Accordingly, we incorporated a disulfide linkage to
release the drug in tumorigenic cell lines with elevated reducing
potential in the cytosol. In these initial studies, we were con-
cerned with two issues: LNP particle size stability in storage and
cell culture conditions and LNP uptake into suitable cells with
reductive release of the drug.
Here, we report the synthesis of straight-chain, branched,
and dendritic lipid anchors derivatized as both PEG-lipids and
thiopurine lipid–drug conjugates. Experiments to monitor the
particle-size stability of LNP formulations containing these lipids
established that chain branching enhances LNP stability. Cell cul-
ture bioassays of LDC activity are consistent with LNP uptake and
reductive thiopurine release from the LDCs.
a waxy white solid was afforded in a 73% yield (1.21 g). H NMR
(300 MHz, CDCl3) ␦: 4.06 (m, 2H), 4.05 (m, 2H), 3.28 (m, 1H), 2.32 (t,
4H, J = 7.8 Hz), 1.61 (m, 4H), 1.24 (s, 48H), 0.87 (t, 6H). 13C NMR
(75 MHz, CDCl3) ␦: 173.8, 66.0, 49.5, 34.4, 32.1, 29.9, 29.9, 29.8, 29.7,
29.6, 29.5, 29.4, 25.2, 22.9, 14.3. MS ((+) ESI): calcd for C35H70NO4
568.53 amu; found, 568.33 amu.
+
,
3c
By the general procedure: step 1, thionyl chloride (5.66 mL,
78.2 mmol) and 1c (2.30 mL, 7.82 mmol); step 2, 2 (0.488 g,
2.55 mmol) in pyridine (2.46 mL, 30.6 mmol) and the acid chloride
(2.10 g, 7.64 mmol); step 3, TFA (5 mL) and the product of step 2; a
light yellow oil was afforded in a 75% yield (1.09 g). 1H NMR
(300 MHz, CDCl3) ␦: 4.06 (m, 2H), 4.04 (m, 2H), 3.26 (m, 1 H), 2.34
(m, 2 H), 1.57, 1.44 (m, 8 H), 1.24 (s, 40 H), 0.86 (t, 12 H, J = 7.0 Hz). 13
NMR (75 MHz, CDCl3): 176.2, 65.5, 53.4, 49.4, 45.7, 32.4, 31.8, 31.6,
29.5, 29.4, 29.2, 29.2, 27.4, 27.4, 22.6, 22.6, 14.0, 14.0. MS ((+) ESI):
calcd for C35H70NO4+, 568.53 amu; obtained, 568.33 amu.
C
3d
By the general procedure: step 1, thionyl chloride (5.10 mL,
70.3 mmol) and 1d (2.000 g, 7.030 mmol); step 2, 2 (0.510 g,
2.67 mmol) in pyridine (2.15 mL, 26.7 mmol) and the acid chloride
(2.020 g, 6.668 mmol); step 3, TFA (5 mL) and the product of step 2;
Experimental
Synthetic procedures
1
a waxy white solid was afforded in a 67% yield (1.09 g). H NMR
General procedure for G1 lipid anchor synthesis
(300 MHz, CDCl3) ␦: 4.09 (m, 2H), 4.07 (m, 2H), 3.30 (m, 1H), 2.34 (t,
In a round-bottom flask, a stirred solution of thionyl chloride
(10.0 equiv.) and 1a–1d (1.0 equiv.) was refluxed for 2 h under a
CaSO4 drying tube. The reaction was monitored by 1H NMR anal-
ysis of aliquots. The reaction was allowed to cool, and subse-
quently, the thionyl chloride was removed under vacuum on a
rotary evaporator to obtain the acid chloride. In a round-bottom
flask, 2 (1.0 equiv.) was dissolved in pyridine (10.0 equiv.) and the
acid chloride (2.5 equiv.) was added dropwise. The mixture was
then heated to 70 °C and left to stir for 3 h under a CaSO4 drying
tube. The reaction was monitored by 1H NMR analysis of aliquots.
Once completed, the reaction was cooled and diluted with DCM.
The solution was then washed three times with 1 mol/L HCI and
three times with 1 mol/L NaOH. The organic layer was then dried
over anhydrous sodium sulfate and then gravity filtered. The sol-
vent was removed under vacuum to obtain the Boc-protected an-
chor. Excess TFA was added dropwise to the Boc-protected anchor
in a round-bottom flask. The reaction was left stirring for 1 h at
room temperature and was monitored by TLC (silica gel, EtOAc/
Hexanes as eluent, visualized by KMnO4). Following completion of
the reaction, TFA was removed on a rotary evaporator. NaOH
(1 mol/L) was then added and 3a–3d was extracted with DCM and
washed three times with 1 mol/L NaOH. The organic layer was
dried with anhydrous sodium carbonate and subsequently gravity
filtered. DCM was removed on a rotary evaporator yielding 3a–3d.
No chromatography was necessary.
4H, J = 7.5 Hz), 1.63 (m, 4H), 1.26 (s, 56H), 0.89 (t, 6H, J = 6.8 Hz). 13
C
NMR (75 MHz, CDCl3) ␦: 173.8, 65.8, 49.6, 34.4, 32.1, 29.9, 29.9, 29.8,
29.7, 29.6, 29.5, 29.4, 25.2, 22.9, 14.3. MS ((+) ESI): calcd for
C39H78NO4+, 624.59 amu; obtained, 624.53 amu.
General procedure — G2 lipid anchor synthesis — preparation of
5a–5d and 6a–6d
In a round-bottom flask, 4 (1 equiv.) was dissolved in dry DCM/
DMF (1:1; 10 mmol/L). HBTU (2.4 equiv.), 3a–3d (2.2 equiv.), and
triethylamine (4.0 equiv.) were subsequently added. The solution
was stirred for 20 h at room temperature and was monitored by 1H
NMR. Once completed, the reaction solution was diluted with
EtOAc and washed three times with 1 mol/L HCl and three times
with H2O. The organic layer was then dried over anhydrous so-
dium sulfate, followed by gravity filtration. EtOAc and DCM were
rotary evaporated to yield a Boc-protected product. Excess TFA
was added dropwise to the Boc-protected product in a round-
bottom flask. The reaction was left to stir for 1 h and monitored by
1H NMR. Following completion of the reaction, TFA was removed
on rotary evaporator and 1 mol/L NaOH was added to the round-
bottom flask; then, the mixture was extracted with DCM and
washed three times with 1 mol/L NaOH. The organic layer was
then dried over sodium carbonate, followed by gravity filtration.
DCM was removed on a rotary evaporator and 5a–5d was isolated.
The crude product was directly characterized; no purification
was necessary (characterization: H NMR, 13C NMR, ESI–MS). In a
1
3a
round-bottom flask, Boc-glycine (1.1 equiv.) was dissolved in dry
DCM/DMF (1:1; 10 mmol/L). To this, HOBt (1.2 equiv.), HBTU
(1.2 equiv.), 5a–5d (1.0 equiv.), and triethylamine (3.0 equiv.) were
then added, and the resulting solution was left stirring at room
temperature for 20 h. When the reaction was complete, moni-
By the general procedure: step 1, thionyl chloride (6.35 mL,
87.6 mmol) and 1a (2.000 g, 8.757 mmol); step 2, 2 (0.596 g,
3.13 mmol) in pyridine (2.53 mL, 31.3 mmol) and the acid chloride
(1.931 g, 7.835 mmol); step 3, TFA (5 mL) and the product of step 2;
1
a waxy yellow solid was afforded in a 66% yield (1.06 g). H NMR
1
tored by H NMR, the reaction solution was diluted with EtOAc
(300 MHz, CDCl3) ␦: 4.07 (m, 2H), 4.05 (m, 2H), 3.28 (m, 1H), 2.33 (t,
4H, 7.7 Hz), 1.62 (m, 4H), 1.48 (s(br), 2H), 1.26 (s, 40H), 0.88 (t, 6H,
7.0 Hz). 13C NMR (75 MHz, CDCl3): 173.6, 65.8, 49.3, 34.2, 31.9, 29.6,
29.6, 20.4, 29.3, 29.2, 29.1, 24.9, 22.7, 14.1. MS ((+) ESI): calcd for
C31H62NO4+, 512.47 amu; found, 512.33 amu.
and washed once with 1 mol/L HCl and three times with H2O. The
organic layer was then dried over anhydrous sodium sulfate and
then gravity filtered. DCM and EtOAc were removed by rotary
evaporation to yield a crude Boc-protected. Excess TFA was added
in a round-bottom flask, and the reaction was left for 1 h. Follow-
ing completion, as monitored by 1H NMR, TFA was removed on a
rotary evaporator. NaOH (1 mol/L) was added to convert to the
amine and the glycine amide (6a–6d) was extracted with diethyl
ether and subsequently washed three times with 1 mol/L NaOH.
The organic layer was dried with anhydrous sodium carbonate
3b
By the general procedure: step 1, thionyl chloride (5.61 mL,
78.0 mmol) and 1b (2.000 g, 7.800 mmol); step 2, 2 (0.558 g,
2.92 mmol) in pyridine (2.31 mL, 29.2 mmol) and the acid chloride
(2.000 g, 7.299 mmol); step 3, TFA (5 mL) and the product of step 2;
Published by NRC Research Press

Meanwell et al.
123
and then gravity filtered. Diethyl ether was removed to obtain the
crude product, which was utilized without further purification.
and the Boc-protected product of step 1; as a white waxy solid in a
51% yield (0.248 g). Data for 5d: 1H NMR (300 MHz, CDCl3) ␦: 7.07 (d,
2H, J = 8.6 Hz), 4.46 (m, 2H), 4.25 (m, 4H), 4.13 (m, 4H), 3.26 (s, 4H),
2.32 (t, 8H, J = 7.8 Hz), 1.60 (m, 8H) 1.25 (s, 112H), 0.87 (t, 12H, J =
6.8 Hz). 13C NMR (75 MHz, CDCl3) ␦: 174.0, 170.8, 62.9, 52.8, 48.1,
34.3, 32.1, 29.9, 29.7, 29.6, 29.5, 29.4, 25.1, 22.9, 14.3. MS ((+) ESI):
calcd for C82H158N3O10+, 1345.19 amu; found, 1345.07 amu. Con-
tinuing the general procedure: step 3, Boc glycine (0.013 g,
0.074 mmol) in dried DCM/DMF (3.7 mL/3.7 mL; 10 mmol/L), HOBt
(0.012 g, 0.085 mmol), HBTU (0.032 g, 0.085 mmol), 5d (0.0950 g,
0.0706 mmol), and triethylamine (0.030 mL, 0.21 mmol); step 4,
TFA (5 mL) and the Boc-protected product of step 3 gave a light
brown oil that was used without further purification.
5a and 6a
By the general procedure: step 1, 4 (0.104 g, 0.444 mmol) was
dissolved in dry DCM/DMF (22.2 mL/22.2 mL; 10 mmol/L). HBTU
(0.404 g, 1.066 mmol), 3a (0.500 g, 0.977 mmol), and triethylamine
(0.25 mL, 1.8 mmol) were subsequently added; step 2, TFA (5 mL)
and the product of step 1; a waxy light yellow solid was afforded in
53% yield (0.264 g). Data for 5a: 1H NMR (300 MHz, CDCl3) ␦: 7.02 (d,
2H, J = 8.6 Hz), 4.46 (m, 2H), 4.24 (m, 4H), 4.12 (m, 4H), 3.25 (s, 4H),
2.31 (t, 8 H, J = 7.8 Hz), 1.59 (m, 8H), 1.25 (s, 80H), 0.87 (t, 12H, J =
6.9 Hz). 13C NMR (75 MHz, CDCl3) ␦: 174.0, 170.8, 62.9, 52.8, 48.0,
34.3, 32.1, 29.9, 29.9, 29.7, 29.6, 29.5, 29.4, 25.1, 22.9, 14.3. MS ((+)
ESI): calcd for C66H126N3O10+, 1120.94 amu,;found, 1121.00 amu.
Continuing the general procedure: step 3, Boc-glycine (0.033 g,
0.19 mmol) in dried DCM/DMF (9.0 mL/9.0 mL; 10 mmol/L), HOBt
(0.029 g, 0.21 mmol), HBTU (0.080 g, 0.21 mmol), 5a (0.20 g,
0.179 mmol), and triethylamine (0.075 mL, 0.53 mmol); step 4, TFA
(5 mL) and the Boc-protected product gave a light brown solid that
was used without additional purification.
Asymmetric disulfide synthesis — preparation of 8
In a round-bottom flask, 6-mercaptopurine monohydrate (0.500 g,
2.94 mmol) and 11-thioundecanoic acid (0.641 g, 2.94 mmol) were
dissolved in DMSO (9.0 mL; 0.33 mol/L). While the reaction was
stirred, solid DDQ (0.669 g, 2.94 mmol) was slowly added over
10 min, and then, the mixture was left at room temperature for
1 h. The reaction was monitored by TLC (silica gel, MeOH/DCM as
eluent, visualized by iodine). Following completion, the product
was precipitated by adding 20 mL of water to the reaction solu-
tion. The resulting mixture was left for 6 h before being vacuum
filtered and a red solid was isolated. The crude product was redis-
solved in hot methanol, cooled in an ice water bath, and then,
vacuum filtered. A white powder was afforded in a 60% yield
5b and 6b
By the general procedure: step 1, 4 (0.093 g, 0.40 mmol) was
dissolved in dry DCM/DMF (20.0 mL/20.0 mL; 10 mmol/). HBTU
(0.364 g, 0.960 mmol), 3b (0.500 g, 0.880 mmol), and triethylamine
(0.22 mL, 1.6 mmol) were subsequently added; step 2, TFA (5 mL)
and the Boc-protected product of step 1; a light brown waxy solid
was afforded in 55% yield (0.272 g). Data for 5b 1H NMR (300 MHz,
CDCl3) ␦: 7.03 (d, 2H, J = 8.7 Hz), 4.47 (m, 2H), 4.26 (m, 4H), 4.14 (m,
4H), 3.27 (s, 4H), 2.33 (t, 8H, J = 7.3 Hz), 1.60 (m, 8H), 1.27 (s, 96H),
0.89 (t, 12H, J = 6.8). 13C NMR (75 MHz, CDCl3) ␦: 174.0, 170.8, 62.8,
52.7, 48.0, 34.31, 32.1, 29.9, 29.7, 29.6, 29.5, 29.4, 25.1, 22.9, 14.3. MS
((+) ESI): calcd for C74H142N3O10+, 1233.07 amu; found, 1233.00.
Continuing the general procedure: step 3, Boc-glycine (0.030 g,
0.17 mmol) in dried DCM/DMF (8.1 mL/8.1 mL; 10 mmol/L), HOBt
(0.026 g, 0.20 mmol), HBTU (0.074 g, 0.20 mmol), 5b (0.201,
0.162 mmol), and triethylamine (0.068 mL, 0.49 mmol); step 4, TFA
(5 mL) and the Boc-protected product of step 3 gave a light brown
oil that was used without further purification. MS ((+) ESI): calcd
for C76H145N4O11+, 1290.09 amu; found, 1290.00 amu.
1
(0.622 g). H NMR (300 MHz, DMSO-d6) ␦: 8.84 (s, 1H), 8.57 (s, 1H),
2.96 (t, 2H, J = 7.0 Hz), 2.21 (t, 2H, J = 7.3 Hz), 1.67 (quintet, 2H, J =
7.5 Hz), 1.50, 1.40 (m, 4H), 1.25 (s, 10H). 13C NMR (75 MHz, DMSO-d6)
␦: 174.4, 151.7, 39.5, 38.1, 33.6, 28.8, 28.7, 28.5, 28.1, 27.6, 24.5. (MS
((+) ESI): calcd for C16H25N4O2S2+, 369.14 amu; found, 369.13 amu.
Similarly, in a round-bottom flask, 6-thioguanine (0.500 g,
2.99 mmol) and 11-thioundecanoic acid (0.652 g, 2.99 mmol) were
dissolved in DMSO (0.15 mol/L). While the reaction was stirred,
solid DDQ (0.679 g, 2.99 mmol) was slowly added over 10 min,
and then, the mixture was left at room temperature for 1 h. The
reaction was monitored by TLC (silica gel, MeOH/DCM as eluent,
visualized by iodine). Following completion, the product was pre-
cipitated by adding water to the reaction solution. The resulting
mixture was left for 6 h before being vacuum filtered and a red
solid was isolated. The crude product was redissolved in hot ace-
tone, cooled in an ice water bath, and then, vacuum filtered. A
yellowish powder was afforded in 64% yield (0.707 g). 1H NMR
(300 MHz, DMSO-d6) ␦: 7.98 (s, 1H), 6.45 (s, 2H), 2.90 (t, 2H, J =
7.1 Hz), 2.17 (t, 2H, J = 8.4 Hz), 1.62 (quintet, 2H, J = 7.0), 1.46, 1.35 (m,
4H), 1.21 (s, 10H). 13C NMR (75 MHz, CDCl3) ␦: 174.5, 159.9 156.9,
140.1, 39.5, 38.10, 33.6, 28.8, 28.7, 28.5, 28.01, 27.7, 24.5. MS ((+) ESI):
calcd for C16H26N5O2S2+, 384.15 amu; found, 384.27 amu.
5c and 6c
By the general procedure: step 1, 4 (1 equiv.) was dissolved in dry
DCM/DMF (1:1; 10 mmol/L). HBTU (0.363 g, 0.960 mmol), 3c
(0.500 g, 0.880 mmol), and triethylamine (0.22 mL, 1.6 mmol) were
subsequently added; step 2, TFA (5 mL) and the Boc-protected
product of step 1; a colorless oil was afforded in 60% yield (0.296 g).
Data for 5c 1H NMR (300 MHz, CDCl3) ␦: 7.07 (d, 2H, J = 8.6 Hz), 4.46
(m, 2H), 4.25 (m, 4H), 4.11 (m, 4H), 3.23 (s, 4H), 2.34 (m, 4H), 1.56,
1.45 (m, 16H), 1.24 (s, 80H), 0.86 (t, 24H, J = 7.4 Hz). 13C NMR
(75 MHz, CDCl3) ␦: 176.4, 170.4, 62.3, 52.6, 48.0, 45.6, 34.6, 34.4,
32.9, 32.7, 32.5, 32.3, 32.2, 31.8, 31.6, 31.5, 29.6, 29.5, 29.4, 29.2,
29.2, 29.0, 27.4, 27.4, 25.2, 22.6, 22.6, 20.6, 14.0, 14.0, 11.3. MS ((+)
ESI): calcd for C74H142N3O10+, 1233.07 amu; found, 1232.87 amu.
Continuing the general procedure: Boc-glycine (0.030 g, 0.17 mmol)
in dried DCM (1.62 mL), HOBt (0.026 g, 0.20 mmol), HBTU (0.074 g,
0.20 mmol), 5c (0.200 g, 0.162 mmol), and triethylamine (0.068 mL,
0.49 mmol); step 4, TFA (5 mL) and the Boc-protected product of
step 3 gave a light brown oil that was used without further puri-
fication. MS ((+) ESI): calcd for C76H145N4O11+, 1290.09 amu; found,
1289.93 amu.
General procedure for LDC monomer synthesis
In a round-bottom flask, 8 (1.1 equiv.) was dissolved in dried
DCM/DMF (1:1; 10 mmol/L). To this, HBTU (1.2 equiv.), 3a–3d
(1.0 equiv.), and triethylamine (2.0 equiv.) were then added, and
the resulting solution was left stirring at room temperature for
20 h. When the reaction was complete, visualized by 1H NMR, the
reaction solution was diluted with EtOAc and washed once with
1 mol/L HCl and three times with H2O. The organic layer was dried
over anhydrous sodium sulfate and then gravity filtered. DCM and
EtOAc were removed by rotary evaporation. The crude product
was purified by flash column chromatography on silica, using
EtOAc/hexanes as the eluent.
5d and 6d
MP-C14
By the general procedure: step 1, 4 (0.088 g, 0.36 mmol) was
dissolved in dried DCM/DMF (18.2 mL/18.2 mL; 10 mmol/L). HBTU
(0.329 g, 0.867 mmol), 5d (0.500 g, 0.801 mmol), and triethylamine
(0.20 mL, 1.5 mmol) were subsequently added; step 2, TFA (5 mL)
By the general procedure: 6-MP (0.113 g, 0.308 mmol) was dis-
solved in dried DCM/DMF (14.6 mL/14.6 mL; 10 mmol/L). HBTU
(0.133 g, 0.352 mmol, 1.20 equiv.), 3a (0.150 g, 0.293 mmol), and
triethylamine (0.082 mL, 0.59 mmol) were subsequently added; a
Published by NRC Research Press

124
Can. J. Chem. Vol. 95, 2017
1
light yellow solid was afforded in a 28% yield (0.072 g). H NMR
(300 MHz, CDCl3) ␦: 11.75 (s, 1H), 8.90 (s, 1H), 8.22 (s, 1H), 6.02 (d, 1H,
J = 8.3 Hz) 4.52 (m, 1H), 4.30 (m, 2H), 4.11 (m, 2H), 2.91(t, 2H, J =
7.0 Hz), 2.32 (t, 4H, J = 7.9 Hz), 2.02 (t, 2H, J = 7.9 Hz), 1.66 (m, 6H),
1.26, 1.12 (m, 54H), 0.88 (t, 6H, J = 7.0). MS ((+) ESI): calcd for
C47H84N5O5S2+, 862.59 amu; found, 862.53 amu.
CDCl3) ␦: 11.23 (s, 1H), 7.87 (s, 1H), 6.08 (d, 1H, J = 8.4 Hz), 5.24 (s, 2H),
4.51 (m, 1H), 4.29 (m, 2H), 4.11 (m, 2H), 2.87 (t, 2H, J = 7.0 Hz), 2.31 (t,
2H, J = 7.5 Hz), 2.17 (t, 2H, J = 7.6 Hz), 1.68, 1.60 (m, 6H), 1.24, 1.17 (m,
62H), 0.87 (t, 6H, J = 7.0 Hz). 13C NMR (75 MHz, CDCl3): 173.9, 173.5,
159.4, 62.8, 47.7, 39.2, 36.6, 34.1, 31.9, 29.7, 29.6, 29.4, 29.3, 29.2,
29.1, 29.0, 29.0, 28.8, 28.4, 28.1, 25.6, 24.9, 22.7, 14.1. MS ((+) ESI):
calcd for C51H93N6O5S2+, 933.66 amu; found, 933.73 amu.
MP-C16
By the general procedure: 6-MP (0.0648 g, 0.176 mmol) was dis-
solved in dried DCM/DMF (9.0 mL/9.0 mL; 10 mmol/L). HBTU
(0.112 g, 0.211 mmol), 3b (0.0648 g, 0.176 mmol), and triethylamine
(0.076 mL, 0.53 mmol) were subsequently added; a white solid was
afforded in a 63% yield (0.102 g). 1H NMR (300 MHz, CDCl3) ␦: 12.88
(s, 1H), 8.91 (s, 1H), 8.29 (s, 1H), 6.05 (d, 1H, J = 8.2 Hz), 4.50 (m, 1H),
4.26 (m, 2H), 4.09 (m, 2H), 2.91 (t, 2H, 7.1 Hz), 2.30 (t, 4H, J = 7.6 Hz),
2.20 (t, 2H, J = 7.1 Hz), 1.65 (m, 6H), 1.24, 1.17 (m, 62H), 0.87 (t, 6H, J =
6.9 Hz). 13C NMR (75 MHz, CDCl3) ␦: 174.1, 173.8, 160.5, 152.6, 149.9,
142.0, 131.4, 62.9, 48.0, 39.3, 36.9, 34.3, 32.1, 29.9, 29.9, 29.8, 29.7,
29.6, 29.5, 29.4, 29.1, 28.7, 28.3, 25.9, 25.1, 22.9, 14.3. MS ((+) ESI):
calcd for C51H92N5O5S2+, 918.65 amu; found, 918.60 amu.
TG-bC16
By the general procedure: 6-TG (0.071 g, 0.19 mmol) was dis-
solved in dried DCM/DMF (8.8 mL/8.8 mL; 10 mmol/L). HBTU
(0.080 g, 0.21), 3c (0.100 g, 0.176 mmol), and triethylamine
(0.049 mL, 0.35 mmol) were subsequently added; a light yellow
1
liquid was afforded in a 37% yield (0.061 g). H NMR (300 MHz,
CDCl3) ␦: 7.88 (s, 1H), 6.05 (d, 1H, J = 8.2 Hz), 5.20 (s, 2H), 4.50 (m, 1H),
4.30 (m, 2H), 4.12 (m, 2H), 2.87 (t, 2H, J = 7.1 Hz), 2.35 (m, 2H), 2.15
(t, 2H, J = 7.4 Hz), 1.68, 1.57, 1.45 (m, 14H), 1.24, 1.16 (m, 50H) 0.86 (t,
12H, J = 6.5 Hz). 13C NMR (75 MHz, CDCl3) ␦: 176.7, 173.3, 159.4, 62.5,
48.0, 45.7, 39.1, 36.6, 32.3, 31.8, 31.6, 29.7, 29.6, 29.4, 29.3, 29.2,
29.1,29.0, 29.0, 28.8, 28.4, 28.0, 27.4, 27.4, 25.5, 22.6, 22.6, 14.1,
14.00. MS ((+) ESI): calcd for C51H93N6O5S2+, 933.66 amu; found,
933.60 amu.
MP-bC16
By the general procedure: 6-MP (0.136 g, 0.370 mmol) was dis-
solved in dried DCM/DMF (17.5 mL/17.5 mL; 10 mmol/L). HBTU
(0.160 g, 0.422 mmol), 3c (0.200 g, 0.352 mmol), and triethylamine
(0.126 mL, 0.704 mmol) were subsequently added; a light yellow oil
was afforded in a 42% yield (0.137 g). 1H NMR (300 MHz, CDCl3) ␦:
12.8 (s, 1H), 8.90 (s, 1H), 8.29 (s, 1H), 5.97 (d, 1H, J = 8.8 Hz), 4.48 (m,
1H), 4.26 (m, 2H), 4.09 (m, 2H), 2.90 (t, 2H, J = 7.3 Hz), 2.32 (m, 2H),
2.2 (m, 2H), 1.68, 1.55, 1.42 (m, 14H), 1.22, 1.16 (m, 50H), 0.85 (t, 12H,
J = 6.9 Hz). 13C NMR (75 MHz, CDCl3) ␦: 176.5, 173.3, 152.2, 142.0,
62.4, 48.0, 45.6, 39.1, 36.7, 32.3, 32.0, 31.8, 31.6, 29.7, 29.5, 29.4,
29.2, 29.2, 28.9, 28.5, 28.2, 27.4, 27.4, 25.6, 22.6, 22.6, 14.0, 14.0. MS
((+) ESI): calcd for C51H92N5O5S2+, 918.65 amu; found, 918.53 amu.
General procedure for PEG-lipids
An equimolar mixture of 7 and the amino lipid was made to a
concentration of 0.12 mol/L in pyridine in a vial. The vial was then
sealed, and the solution was heated to 55 °C and stirred for 48 h.
Following completion, as monitored by TLC (silica gel, MeOH/
DCM as eluent, visualised by iodine), the pyridine was removed on
a rotary evaporator and the resulting mixture was purified by
flash column chromatography on silica gel, using MeOH/DCM as
the eluent. The amounts used and the characteristic ESI–MS are
given in full in the Supplementary material.
Preparation of LNPs
MP-C18
Stock solutions of lipids were made up in ethanol with a lipid
concentration of 10 mmol/L. In an Eppendorf tube, the lipid mix-
ture of PEG-lipid (5.00 mol %), DMPC (5.00 mol %), and LDC
(90.00 mol %) was made up to a total volume of 0.25 mL with
additional ethanol. The mixture was then heated to 43 °C for
5 min in the oven.
By the general procedure: 6-MP (0.062 g, 0.168 mmol) was dis-
solved in dried DCM/DMF (8.0 mL/8.0 mL; 10 mmol/L). HBTU
(0.073 g, 0.19 mmol), 3d (0.100 g, 0.160 mmol), and triethylamine
(0.045 mL, 0.32 mmol) were subsequently added; a white solid was
afforded in a 32% yield (0.050 g). 1H NMR (300 MHz, CDCl3) ␦: 8.91
(s, 1H), 8.27 (s, 1H), 6.01 (d, 1H, J = 8.3 Hz), 4.50 (m, 1H), 4.26 (m, 2H),
4.09 (m, 2H), 2.90 (t, 2H, J = 7.2 Hz), 2.30 (t, 4H, J = 7.8 Hz), 2.20 (t,
2H, J = 7.8 Hz), 1.68, 1.59 (m, 6H), 1.24, 1.15 (m, 70H), 0.87 (t, 6H, J =
6.9 Hz). 13C NMR (75 MHz, CDCl3) ␦: 173.8, 173.5, 152.3, 62.7, 47.7,
39.1, 36.7, 34.1, 31.9, 29.7, 29.6, 29.5, 29.3, 29.2, 29.1, 29.1, 29.0, 28.9,
28.8, 28.4, 28.0, 25.6, 24.9, 22.7, 14.1. MS ((+) ESI): calcd for
C55H100N5O5S2+, 974.72 amu; found, 974.67 amu.
Prior to using, the NanoAssemblr™ cartridge was washed with
PBS buffer in a 3 mL syringe (left port) and ethanol in a 3 mL
syringe (right port) with a 12 mL/min flow rate and a 1:1 (aqueous:
ethanol) flow ratio. A total of 4 mL of wash was collected and
discarded. The NanoAssemblr™ microfluidic mixer was then used
to make the LNPs. A 3 mL syringe (left port) was loaded with 2 mL
PBS buffer and another 3 mL syringe (right port) was loaded with
the 0.25 mL lipid mixture. The flow rate was set to 4 mL/min, the
flow ratio was set to 3:1 (aqueous:EtOH), and the total volume
collected was 1 mL, with the initial 300 uL at the beginning and
50 uL at the end being discarded. An aliquot (0.50 mL) of the
collected formulation was then diluted to 0.40 mmol/L (thiopurine)
with PBS buffer, transferred to a 3 mL Slide-A-Lyzer Dialysis
TG-C14
By the general procedure: 6-TG (0.118 g, 0.308 mmol) was dis-
solved in dried DCM/DMF (14.6 mL/14.6 mL; 10 mmol/L). HBTU
(0.133 g, 0.352 mmol), 3a (0.150 g, 0.293 mmol), and triethylamine
(0.082 mL, 0.586 mmol) were subsequently added; a light yellow
solid was afforded in 18% yield (0.047 g). 1H NMR (300 MHz, CDCl3)
␦: 7.92 (s, 1H), 6.10 (d, 1H, J = 8.10 Hz), 5.31 (s, 2H), 4.52 (m, 1H), 4.29
(m, 2H), 4.12 (m, 2H), 2.88 (t, 2H, J = 7.3 Hz), 2.32 (t, 4H, J = 7.3 Hz),
2.18 (t, 2H, J = 7.3 Hz), 1.68, 1.59 (m, 6H), 1.25, 1.18 (m, 54H), 0.88 (t,
6H, J = 6.9 Hz). 13C NMR (75 MHz, CDCl3) ␦: 174.1, 173.7, 159.7, 139.6,
63.0, 48.0, 39.4, 36.9, 34.3, 32.1, 29.9, 29.9, 29.8, 29.7, 29.6, 29.5,
29.4, 29.4, 29.3, 29.1, 28.7, 28.3, 25.8, 14.3. MS ((+) ESI): calcd for
C47H85N6O5S2+, 877.60 amu; found, 877.67 amu.
®
Cassette G2 (10 000 molecular weight cutoff), and dialyzed against
PBS buffer for 5 h. The PBS buffer was refreshed after 3 h, and the
removal of ethanol from the formulation was monitored using
potassium dichromate. Diameters of the SNPs were determined
by dynamic light scattering (DLS) experiments (Brookhaven In-
strument, ZetaPALS particle sizing software). The SNPs were then
stored at 4 °C.
Stability assay methods
TG-C16
The stability of the formulated LNPs was assessed under six
conditions: LNPs were stored at three different temperatures, i.e.,
4 °C, ambient, and 37 °C, and incubated in two different media,
namely PBS buffer and PBS buffer with 10% bovine growth serum
(BGS; by volume). To a polystyrene cuvette, 200 uL of LNPs solution
By the general procedure: 6-TG (0.068 g, 0.18 mmol) was dis-
solved in dried DCM/DMF (8.8 mL/8.8 mL; 10 mmol/L). HBTU
(0.112 g, 0.211 mmol), 3b (0.120 g, 0.18 mmol), and triethylamine
(0.076 mL, 0.53 mmol) were subsequently added; a light yellow
1
solid was afforded in a 49% yield (0.081 g). H NMR (300 MHz,
Published by NRC Research Press

Meanwell et al.
125
Scheme 2. Synthesis of serinol-derived branched and dendritic lipids. Boc, tert-butoxycarbonyl; DCM, dichloromethane; DMF,
N,N,-dimethylformamide; Gly, glycine; HBTU, N,N,N=N=-tetramethyl-O(1H-benzotriazol-1yl) uronium hexfluorophosphate; HOBT,
hydroxybenzotriazole; NHS, N-hydroxysuccimidyl; pyr, pyridine; TFA, trifluoroacetic acid.
and 2.30 mL of PBS buffer, or 2.05 mL of PBS buffer and 0.25 mL of
serum, were added and the diameters measured in a DLS experi-
ment. Cuvettes were then sealed and incubated under the indi-
cated conditions. Diameter measurements were taken regularly.
Full data are given in the Supplementary material.
6MP-C16, F[5,12] = 18.16 and P < 0.001, followed by Tukey’s multiple-
comparison posthoc test to determine whether there were signif-
icant differences between groups.
A one-way ANOVA was also performed for the experiments on
HeLa cells: 6-TG, F[7,16] = 24.69, P < 0.001; 6-MP, F[7,16] = 13.37 and
P < 0.001; 6TG-C16, F[6,14] = 26.51 and P < 0.001; 6TG-bC16, F[6,14]
2.06 and P > 0.05; 6MP-C16, F[6,17] = 2.24 and P > 0.05; 6MP-bC16,
[6,14] = 0.57 and P > 0.05, followed by Tukey’s multiple-comparison
posthoc test to determine whether there were significant differ-
ences between groups.
=
Bioassay methods
MCF7 cells were seeded on 24 well plates at 5.0 × 104 cells/well,
18 h later were treated with the indicated compounds in growth
medium (Dulbecco’s Modified Eagle’s Medium (DMEM) supple-
mented with 10% BGS). DMSO was used at a concentration of
400 ␮mol/L in growth medium as a control for 6-thioguanine
(6-TG) and 6-mecaptopurine (6-MP), and Na3PO4 buffer (pH 7,
[Na3PO4] = 10 mmol/L [NaCl] = 0.1 M) was used at a concentration of
40 ␮mol/L in growth medium as a control for 6TG-C16, 6TG-bC16,
6MP-C16, and 6MP-bC16. After 72 h of treatment, cells were
washed with phosphate-buffered saline (PBS), fixed for 10 mins
with 4% paraformaldehyde in PBS, stained for 30 mins with 0.1%
crystal violet in H2O, washed twice with H2O, and let dry. Then,
10% acetic acid was added to wells and incubated on a shaker for
10 mins. Absorbance was measured at 590 nm using a PerkinEl-
mer Victor3V 1420 multilabel plate counter. The absorbance from
stained wells without cells was subtracted from experimental val-
ues to eliminate background absorbance from crystal violet ad-
hering to the plate. The percent viability was calculated as follows:
(absorbance compounds/absorbance control buffer) × 100. A sub-
set of the data is given in the figures; the full data are given in the
Supplementary material.
F
Results and discussion
Synthesis
The serinol-based lipids 3a–3d were readily accessed (Scheme 2)
in good yields over three steps, requiring only extractive workup
to isolate the pure lipid anchors. The diaseterotopic serinol-
derived methylene protons appear as complex but symmetric
multiplets in the 1H NMR spectra (see Supplementary material).
Dendritic analogues 5a–5d were synthesized in moderate yields
over two steps utilizing an iminodiacetic acid backbone and 3a–
3d. Once again, extractive workups were all that were necessary
to access the pure lipids. The methylene protons present in the
serinol fragment were observed here as two distinct multiplets in
the 1H NMR (see Supplementary material).
Conjugates of 3a–3d with thiopurine drugs requires a suitable
disulfide that we elected to incorporate via a C11 spacer. Oxidation
with DDQ of an equimolar mixture of 11-thioundecanoic acid and
either 6-mercaptopurine or 6-thioguanine provides the asymmet-
ric disulfides 8 with excellent selectivity following simple precip-
itation and recrystallization. Only the asymmetric disulfide 8 was
A one-way ANOVA was performed for the experiments on
MCF-7 cells: 6-TG, F[7,16] = 243.8 and P < 0.001; 6-MP, F[7,16] = 18.77
and P < 0.001; 6TG-C16, F[5,12] = 23.58 and P < 0.001; 6TG-bC16,
1
F[5,12] = 24.97 and P < 0.001; 6MP-C16, F[5,12] = 18.21 and P < 0.001;
observed by both H NMR and ESI–MS. This type of heteroselec-
Published by NRC Research Press

126
Can. J. Chem. Vol. 95, 2017
Fig. 1. Electrospray mass spectrum of PEG-G2-C14 (0.1% TFA and 0.1% NaCl in methanol). The compound shows four distinct ion series for
PEG-oligomers bearing 4Na+ (yellow inverted triangle), 3Na+ (red squares), 2Na+ and H+ (grey squares), and 2Na+ (blue circles). A total of 64 of
the 71 isotopic ion clusters are assigned to a specific value of the number of ethyleneoxy units in the PEG (N). The inset shows the sum of ion
intensities for a given value of N (where observed and assigned). The curve is a Gaussian fit to the data (center, 43.1 0.13; width, 11 1.7; r2 =
0.969). [Colour online.]
tively has been demonstrated for aromatic thiols forming disul-
fides with alkyl thiols.40 Amide coupling of 8 with lipid anchors
3a–3d afforded the seven LDC monomers in poor to moderate
yields — providing sufficient material to explore the composition
space for the LDC nanoparticle formulations.
based in integration range from 60 to 85 mol% for the PEG-Gn-C#
samples. This level of impurity can be tolerated in the proof-of-
principle experiments described below; more detailed analyses
will require starting materials with higher purity.
ESI–MS was used to characterize the PEG-lipids as the distribu-
tion of PEG oligomers. All PEG-lipids gave ESI spectra of ions
bearing multiple cations grouped in clusters corresponding to
different oligomers in the PEG chain (Fig. 1). No ions associated
with the starting amines were observed. Nor were any ions asso-
ciated with the known impurity MeO–PEG–OMe, presumably due
to the high surfactant character of the PEG-lipids that would sup-
press ionization of weaker surfactants. The observed ions were
assigned based on isotopic mass predictions to a given value of the
number of ethyleneoxy units in the oligomer (N). The intensities
for all observed ions of a given N were summed, and a Gaussian
distribution was fit to determine the average N and distribution
width of the sample. The starting NHS ester 7 gave a value of N =
43.5 0.2 and a width of 14 1.5; with the exception of PEG-G2-
bC16, the isolated products gave N = 43.3 0.3 and a width of 14
5.3. This suggests that for the majority of samples there has been
little fractionation during synthesis and handling and that the
original distribution is relatively unaltered. The PEG-G2-bC16
sample has much broader distributions of ions within each oli-
gomer series observed, and the summed data do not fit well to a
Gaussian distribution (r2 < 0.7). The identity of the compound is
not in doubt, but the spectra suggest that some fractionation has
occurred or that the ionization of this compound differs from the
rest of the series or both. Spectra similarly analyzed are given for
all products in the Supplementary material.
Linkage of the PEG to the lipid anchors required some optimi-
zation. Initially, we expected to use an isocyanate-terminated
PEG2000 derivative; two different commercial samples failed to
give any evidence of the expected carbamate products (NMR, IR,
ESI–MS). N-hydrosuccinimide PEG2000 7 is reported to react read-
ily at hindered sites in proteins at ambient temperature;41 7 did
react with 3a–3d in pyridine at a slow rate; moderate heating over
48 h in pyridine was required to produce the first generation
PEG-lipids (PEG-G1-C#) in yields of 66%–70% following chromato-
graphic purification. The NHS–PEG derivative 7 did not react with
the secondary amine of 5a–5d at an appreciable rate, so we
elected to install a glycine spacer to produce a primary amine
(6a–6d) for coupling. The final step of the dendritic PEG-lipid
synthesis proceeded from coupling of 7 with the unpurified
6a–6d under the same conditions as used previously with 3a–3d
to give PEG-G2-C# in 33%–54% chromatographed yield for the
three steps from 5a–5d.
Structural confirmation of the PEG lipids based on 1H NMR
proved to be complex. Amide formation for 3a–3d was confirmed
by the downfield shift of the methine proton on the serinol back-
bone from 3.28 ppm (amine) to 4.41 ppm (amide); a similar shift of
the glycine-derived methylene signals in PEG-G2-C# samples sim-
ilarly confirms amide coupling (see Supplementary material). Fur-
thermore, due to lack of free bond rotation at the amide, the
diastereomeric methylene protons on the serinol backbone expe-
rience a greater difference in chemical environment and, corre-
spondingly, better resolved symmetrical multiplets in the amides
relative to the starting amines. However, the commercial sample
of 7 was contaminated to the extent of about 40 mol% with a
bis-methoxy terminated oligomer MeO–PEG–OMe (see Supple-
mentary Fig. S3). This impurity was not (fully) removed from the
final samples by chromatography, and the integrations of the
methoxy signals relative to the serinol-derived signals all show
excess intensity in the methoxy singlet. The estimated purities
LNP formulation and particle size stability
Recent advances in microfluidic mixing technologies allow the
rapid, reproducible, and scalable production of LNPs.12,42–45 The
Precision Nanosystems microfluidic mixer (NanoAssemblr™) is
based on a staggered herringbone design,43 which leads to rapid
mixing via chaotic advection and efficiently gives reproducible
LNP formulations.12,42,44 The mixer takes lipidic components in a
water-miscible organic solvent such as ethanol and rapidly dilutes
them into aqueous buffer to produce LNPs by hydrophobic
Published by NRC Research Press

Meanwell et al.
127
Fig. 2. Particle size during storage in PBS at 37 °C for LNP
formulations containing 90 mol% MP-C# or TG-C#, 5 mol% DMPC,
and 5 mol% PEG-DSPE. [Colour online.]
Fig. 3. Cell viability as a function of thiopurine dose level as
assessed by a Crystal Violet assay. Top panel: for MCF-7 cells; bottom
panel: for HeLa cells. TG (6-thioguanine) and MP (6-mercaptopurine)
were added to the media at the indicated concentrations. The LNP
formulations (90 mol% MP-C# or TG-C#, 5 mol% DMPC, and 5 mol%
PEG-DSPE) were diluted to give the indicated total thiopurine
concentration. Significant differences from buffer are as follows:
**, P < 0.01; ***, P < 0.001. Error bars represent standard deviation.
[Colour online.]
association of the components. Organic solvents and small mole-
cules are readily removed by dialysis. Given the unusual struc-
tures of the branched and dendritic lipids prepared, we opted to
use a standardized procedure as opposed to individual operating
conditions and formulations requiring optimization. The reliabil-
ity of LNP formation was assessed by particle size and polydisper-
sity index (PDI), as determined by dynamic light scattering.
Neutral hydrophobic compounds dispersed in water rarely
form stable self-assemblies on their own and usually crystallize or
precipitate; initial attempts to form MP-C# and TG-C# monomers
directly into LNPs confirmed this. Incorporation of a surfactant
into the LDC formulations was therefore necessary to improve
the solubility of the LNPs. Several different compositions were
investigated before arriving at 5 mol% 1,2-dimyristoyl-sn-glycero-3-
phosphocholine (DMPC), 5 mol% 1,2-distearoyl-sn-glycero-3-
phosphoethanolamine-N-(methoxy- (polyethylene glycol)-2000)
(ammonium salt) (DSPE-PEG), and 90 mol% MP-C# or TG-C#. The
drug loading for all of these formulations was 15 wt% calculated
from the thiopurine drug mass over the total mass of the mixture.
Particle stability was evaluated by monitoring particle size and
PDI over time in PBS or 10% serum in PBS at various temperatures
(4 °C, ambient, 37 °C). An increase in particle size and (or) PDI was
taken as an indication of particle aggregation as a result of parti-
cle instability.46 MP-C14, MP-C18, and TG-C14 showed significant
size increase (45%–100% diameter increase) after 30 days at 4 °C in
PBS buffer (Supplementary Tables S3–S9). The LNPs from MP-C16,
MP-bC16, TG-C16, and TG-bC16 maintain their apparent diame-
ters (0%–20% diameter increase) and PDI values (–10% to 10% PDI
change) for up to 60 days at 4 °C (see Supplementary material). At
37 °C in PBS, the isomeric pairs MP-C16/MP-bC16, and TG-C16/TG-
bC16 show significant differences in stability, with the branched
chain isomers giving the apparently more stable LNPs (Fig. 2).
In the MCF-7 tumour cell line, LNPs containing MP-C16, MP-
bC16, TG-C16, and TG-bC16 show activity in the same concentra-
tion range as the free drug (Fig. 3, top panel; Supplementary
Figs. S13–S18). Control experiments show the same LNPs are gen-
erally inactive in HeLa cells (Fig. 3, bottom panel; Supplementary
Figs. S19–S24), which contain normal cytosolic concentration and
distribution of thiol/glutathione.47 However, HeLa cells in this
assay are not particularly sensitive to the thiopurines as free drugs
so small differences will be missed. Taking the two bioassays in
conjunction, there is indirect evidence that the LNPs are taken up
by the MCF-7 tumour cell-line prior to reduction and thereafter
release the thiopurine in the cytosol. Although it is unclear if
there is an isomer difference in these bioassays, we are encour-
aged by the apparent stability of the LNPs under the physiological
conditions needed for cell proliferation.
Turning to assessment of the PEG-lipid compounds, these were
formulated into LNPs similar to those for siRNA transfection ex-
periments,12 using a double-stranded DNA (dsDNA) octadecamer
as the nucleic acid cargo. The PEG-G1-C# compounds (1.5 mol%)
were formulated with the cationic lipid DOTMA (1,2-di-O-
octadecenyl-3-trimethylammonium propane; 50 mol%), disteroyl-
phosphatidyl choline (DSPC, 10 mol%), and cholesterol (38.5 mol%)
at a N/P ratio of five with the dsDNA. The formulations were
compared with the commercial DSPE-PEG bearing the same PEG
oligomer length (Fig. 4; Supplementary Tables S10–S14).
Particle stability was assessed under various conditions; the
results for 37 °C in PBS are given in Fig. 4A. Initial differences in
the sizes of the LNPs indicate the formulation is not optimal for
the complete set of PEG-lipids. The branched PEG-G1-bC16 shows
a markedly different time-dependent change under these condi-
tions. A better cationic lipid for LNP formulation was available in
a proprietary mixture,12,48 as shown in Fig. 4B for PEG-G2-C16
Published by NRC Research Press

128
Can. J. Chem. Vol. 95, 2017
of their preferred formation into lamellar phases.10 Using meth-
ods based on the additive molecular parameters we have used
previously,49,50 we estimate for the protonated forms of 3b S = 1.4,
of 3c S = 2.2, of 5b S = 2.4, and of 5c S = 3.6. These values, lying well
above S = 1, indicate a propensity to form inverted lipid phases;
however, these very high values are so far outside the shape pa-
rameters associated with conventional amphiphiles that the de-
tails require considerable further experimental exploration. The
compounds are also very lipophilic with calculated clogP val-
ues49,50 of about 14 for 3bc·HCl, about 28 for 5c·HCl, and about 32
for 5b·HCl. It is perhaps relevant to the potential applications of
these unusually shaped lipids that the most extreme example —
PEG-G2-bC16 — is the compound that failed to give an acceptable
fit to the ESI–MS ions observed, suggesting that the compound
was a poorer surfactant than its isomer and congeners.
Fig. 4. Particle size during storage for LNPs incubated at 37 °C in
PBS for formulations containing 1.5 mol% of different PEG-lipids.
(A) Composition, 50:10:38.5:1.5 of DOTMA:DSPC:Cholesterol:PEG-
lipid. (B) Composition, 50:10:38.5:1.5 of ionisable lipid:DSPC:
Cholesterol:PEG-lipid. Both experiments had a dsDNA loading of
5.1 wt%. Error bars are shown only when they exceed the symbol
dimension. [Colour online.]
Formulation of the thiopurine lipid–drug conjugates into stable
LNPs required only minor amounts of added surfactant and PEG-
lipid, resulting in a drug loading of 15 wt%. The bioassays suggest
the LNPs are taken into the MCF-7 cells where elevated levels of
reductants in the cytosol liberate the thiopurines to produce the
cytotoxic effect. Relative to HeLa cells, where the cytosol level of
reductants is not elevated, the LDCs produced lower levels of tox-
icity than the free drug in the medium. Neither cell line showed
any additional toxicity due to serinol diesters in these preliminary
studies.
In storage tests evaluated by particle size change, there was a
significant difference between the C16 isomeric pairs in which the
branched-chain compounds afforded greater particle stabiliza-
tion compared with the linear isomers. In addition, PEG-G2-bC16
afforded significant stabilization relative to a commercially used
PEG-lipid for which the formulation had previously been opti-
mized to enhance storage stability. Thus, in both LNPs from
lipid-thiopurine conjugates and the PEG-lipid systems, there is
strong preliminary evidence that hydrocarbon branching results
in LNP stabilization in buffer. Whether this will translate into
enhanced effectiveness in either drug release or transfection will
require more detailed cellular studies.
and PEG-G2-bC16; the neutral glycerol PEG ether DMG-PEG
(1,2-Dimyristoyl-sn-glyceroxy-␻-methoxy poly-ethylene glycol; n ϳ45)
was the control in these experiments (Fig. 4B; Supplementary
Table S15). All three LNP formulations initially have 60–90 nm
particle diameters, and are nearly monodisperse, but it is clear
that the branched formulation containing PEG-G2-bC16 is sub-
stantially more robust than even the commercial PEG-lipid under
these conditions. Note that the lipid mixture used has been opti-
mized for use with this commercially available PEG-lipid.
Supplementary material
Supplementary material is available with the article through
the journal Web site at http://nrcresearchpress.com/doi/suppl/
10.1139/cjc-2016-0462.
Acknowledgements
The ongoing support of the Natural Sciences and Engineering
Research Council of Canada is gratefully acknowledged. We thank
Precision NanoSystems for their collaborative assistance through
the loan of the NanoAssemblr™ and for a gift of their proprietary
lipid mixture used in some experiments.
Conclusions
The synthesis of a new class of branched-chain and dendritic
lipids derived from serinol was achieved. The yields are acceptable
overall and produce materials in good quantities for formula-
tions. As in other lipid amphiphile syntheses, chromatographic
and handling losses can be significant, and many of the steps have
been developed to avoid chromatography. The lipid amphiphiles
give strong electrospray mass spectra, and in the case of the PEG-
lipids prepared, the complex spectra could be analysed to estab-
lish the characteristics of the oligomeric PEG mixture. Not all PEG
derivatives give this sort of diagnostic characterization tool. As an
example, the impurity MeO–PEG–OMe in the commercial sample
of 7 was clearly evident by 1H NMR but absent in the ESI–MS
spectra of mixtures containing it as a known impurity.
The lipids prepared have very unusual shapes compared with
naturally occurring phospholipids. The lipid shape or packing
parameter S = V/a0lc is derived from the volume of the lipid mol-
ecule (V), the projected area of the headgroup in the lipid interfa-
cial plane (a0), and the length of the hydrocarbon chains (lc).
Phospholipids based on diacylglycerols have 0.7 < S < 1 predictive
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