.
Angewandte
Communications
2
+
Figure 2. HisGFP association to pGi-Ni and to pPEI carrier. A) Elec-
trophoretic mobility of hisGFP alone (first lane) and with increasing
2
+
Scheme 1. Synthesis of pGi. a) Diglycolic anhydride, 4-dimethylamino-
pyridine (DMAP), CH Cl . b) N,N’-dicyclohexylcarbodiimide, N-hydrox-
ysuccinimide. c) Polyornithine, DMAP, dimethylformamide/water.
d) Trifluoroacetic acid.
amounts of pGi-Ni . *In the last lane, the complex was mixed with
+
2
2
2
EDTA. B) Mobility of [pGi-Ni :hisGFP] following association with
pPEI. C) AFM image of hisGFP-containing delivery complex and size
distribution.
can be administrated in vivo at doses that do not induce
observable adverse effects in rodent.
amounts. A complete association was achieved at a pPEI/
hisGFP ratio of 2.5. Atomic force microscopy (AFM) of [pGi-
2
+
The pGi was prepared according to Scheme 1. The
Ni :hisGFP/pPEI] at a ratio 1:4/4.8 showed a distribution of
round-shaped particles with a mean height of 35 nm (Fig-
ure 2C).
[11]
carboxylate-protected NTA 1 was reacted with diglycolic
anhydride. The dangling carboxylic acid was activated to
a succinimidyl ester 2, which was coupled to polyornithine.
Unreacted cationic amines were converted to carboxylic acid
by reaction with diglycolic anhydride and protecting groups
were removed with trifluoroacetic acid. Extensive dialysis
yielded pGi, a polymer grafted with approximately 150 NTA
Insight into how His-tagged molecules associate onto the
2
+
linear pGi-Ni at maximum docking capacity was next
explored by AFM and cryo-electron microscopy (cryo-EM).
2
+
The hisGFP alone or bound to pGi-Ni were seen only using
cryo-EM as individual dots or serpentine filaments, respec-
tively (Figure S1). For finer resolution and confirmation, we
aimed at substituting the soft and low contrasting protein with
a harder and higher contrasting material. The 2 nm-diameter
mercaptobenzoic acid-coated gold nanocluster was selected
because it has a molecular weight of 28 kDa similar to hisGFP,
is easily prepared in large quantities and its surface may be
functionalized through exchange of mercaptobenzoic acid
2
+
groups as estimated by NMR spectroscopy. Ni was then
2
+
immobilized on pGi and provided pGi-Ni to an estimated
Ni immobilization on 50% of the NTA groups.
2
+
The docking capacity of pGi-Ni was evaluated by mixing
the polymer with 75 equivalents of a fluorescently labeled
1
.6 kDa hexahistidine-containing peptide (one peptide per
immobilized nickel ion). The free peptides were then
separated from the polymer-bound ones in an ultrafiltration
device, allowing determination of a maximum docking of 20
peptides per polymer. Repetition of this experiment with His-
tagged green fluorescent protein (hisGFP, 29 kDa) yielded
a lower docking capacity of 10 proteins per polymer. The
bulkiness of the protein, which presumably covers two times
more NTAs than the peptide, explains these different
capacities. The effect of stoichiometry on the association
was analyzed by monitoring the variation in hisGFP electro-
phoretic mobility within an agarose gel (Figure 2A).
[12]
with thiolated biomolecules. The gold clusters were pre-
pared and reacted with a thiolated hexahistidine-containing
peptide to afford the His-tagged gold nanoclusters
(hisAuNC). We verified that they formed a fully dispersed
2
+
colloidal solution and associated with pGi-Ni similarly to
His-tagged proteins (Figures S2 and S3). The hisAuNC were
2
+
docked onto pGi-Ni at the maximum docking capacity of
2
+
the polymer (measured as 12 hisAuNC per pGi-Ni ) and
unbound hisAuNC were removed by dialysis for AFM and
cryo-EM analyses (Figure 3A).
2
+
The hisGFP alone (lane 1) migrated in the gel as a single
The purified [pGi-Ni :(hisAuNC) ] were seen as fila-
12
2
+
band. Addition of pGi-Ni modified the protein mobility
towards a faster moving band in a dose-dependent manner. A
band with an electrophoretic mobility similar to that of
hisGFP alone was observed when the nickel ion bridging the
hisGFP and the polymer were displaced with EDTA (last
mentous assemblies composed of distinct 2 nm particles. In
the diluted EM condition, about 10 to 20 distinct dots were
seen to cluster and roughly align in oblong superstructures,
corresponding presumably to single or dimeric [pGi-
2
+
Ni :hisAuNC]. These observations confirmed the gel mobi-
lity assays and demonstrated the ability of the linear polymer
to bind and congregate His-tagged materials. The purified
2
+
lane). Association between [pGi-Ni :hisGFP] and pPEI was
similarly assayed (Figure 2B). Four hisGFPs were docked per
2
+
2+
pGi-Ni and the cationic pPEI was added in increasing
[pGi-Ni :(hisAuNC) ] was then mixed with pPEI (final ratio
12
1
ꢀ 2015 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2015, 54, 10583 –10586