Set of Highly Stable Amine- and Carboxylate-Terminated Dendronized Au Nanoparticles with Dense Coating and Nontoxic Mixed-Dendronized Form

Article abstract of DOI:10.1021/acs.langmuir.8b03196

The synthesis of a novel poly(propyleneimine) (PPI) dendron in gram scale as well as its use in the formation of a highly stable, dendronized gold nanoparticle (AuNP)-based drug delivery platform is described herein. The AuNP-based platform is composed of three complementary parts: (i) a 15 nm AuNP core, (ii) a heterofunctional thioctic acid-terminated tetraethylene glycol spacer, and (iii) a third-generation PPI dendron with a unique protonation profile and diverse end-group functionalization that allows for further derivatization. The prepared dendronized AuNPs are able to withstand several rounds of lyophilization cycles with no sign of aggregation, are stable in phosphate-buffered saline and Hanks' buffer as well as in serum, and are resistant to degradation by glutathione exchange reactions. This nanocarrier platform displays a dense coating, with >1400 dendrons/AuNPs, which will enable very high payload. Furthermore, while amine-terminated AuNPs expectedly showed cytotoxicity against the MCF-7 breast cancer cell line from a NP concentration of 1 nM, the mixed monolayer AuNPs (coated with 40/60 amine/carboxylate dendrons) interestingly did not exhibit any sign of toxicity at concentrations as high as 15 nM, similar to the carboxylate-terminated AuNPs. The described dendronized AuNPs address the current practical need for a stable NP-based drug delivery platform which is scalable and easily conjugable, has long-term stability in solution, and can be conveniently formulated as a powder and redispersed in desired buffer or serum.

Full text of DOI:10.1021/acs.langmuir.8b03196

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Interface Components: Nanoparticles, Colloids, Emulsions, Surfactants, Proteins, Polymers
Set of Highly Stable Amine- and Carboxylate-terminated Dendronized Au
Nanoparticles with Dense Coating and Nontoxic Mixed-dendronized Form
Arunendra Saha Ray, William Emmanuel Ghann, Phoebe Sisoen Tsoi, Brian Szychowski, Lance T.
Dockery, Yewon Joanna Pak, Wenjing Li, Maureen A Kane, Peter W Swaan, and Marie Christine Daniel
Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.8b03196 • Publication Date (Web): 04 Feb 2019
Downloaded from http://pubs.acs.org on February 4, 2019
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Set of Highly Stable Amine- and Carboxylate-
terminated Dendronized Au Nanoparticles with
Dense Coating and Nontoxic Mixed-dendronized
Form
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†
†♯
†₸
†
Arunendra Saha Ray , William E. Ghann , Phoebe S. Tsoi , Brian Szychowski , Lance T.
†
⁋⸸
‡
‡
⁋
Dockery , Yewon J. Pak , Wenjing Li , Maureen A. Kane , Peter Swaan , Marie-Christine
Daniel†*
†Department of Chemistry and Biochemistry, University of Maryland Baltimore County
(UMBC), Baltimore, Maryland 21250, USA.
⁋
Department of Pharmaceutical Sciences, Center for Nanobiotechnology, University of Maryland,
Baltimore, 20 Penn Street, Baltimore, Maryland 21201, USA.
‡
Mass Spectrometry Center, University of Maryland School of Pharmacy, Baltimore, Maryland
2
1201, USA.
ABSTRACT: The synthesis of a novel poly(propyleneimine) (PPI) dendron in gram scale as well
as its use in the formation of a highly stable, dendronized gold nanoparticle (AuNP)-based drug
delivery platform is described herein. The AuNP-based platform is comprised of three
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complementary parts: (i) A 15 nm AuNP core (ii) A hetero-functional thioctic acid (TA)-
terminated tetraethylene glycol spacer (TEG) (iii) A third generation PPI (G3-PPI) dendron with
unique protonation profile and diverse end-group functionalization that allows for further
derivatization. The prepared dendronized AuNPs are able to withstand several rounds of
lyophilization cycles with no sign of aggregation, are stable in PBS and Hanks buffers as well as
in serum, and is resistant to degradation by glutathione exchange reactions. This nanocarrier
platform displays a dense coating, with >1400 dendrons/AuNP, which will enable very high
payload. Furthermore, while amine-terminated AuNPs expectedly showed cytotoxicity against
MCF-7 breast cancer cell line from a nanoparticle concentration of 1 nM, the mixed monolayer
AuNPs (coated with 40/60 amine/carboxylate dendrons) interestingly did not exhibit any sign of
toxicity at concentrations as high as 15 nM, similarly to the carboxylate-terminated AuNPs. The
described dendronized AuNPs address the current practical need for a stable nanoparticle-based
drug delivery platform which is scalable and easily conjugable, has long-term stability in solution
and can be conveniently formulated as a powder and redispersed in desired buffer or serum.
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INTRODUCTION
The current limitations of conventional drug therapy include narrow therapeutic window, systemic
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toxicity, lack of tissue/organ specificity and real-time- diagnosis. The search for multiplexed
molecules that can provide the desired triad of functions (biorecognition, specific cytotoxicity and
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bio-barrier evasion) is still ongoing and is an area of active research. However, it is extremely
difficult for a single molecule to fulfill all these requirements. One way to simultaneously address
these needs is through the judicious selection of nanocarriers (liposomes4, 5, dendrimers6, 7
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,
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nanoparticles8, 9,10,11,12). Indeed, due to its size and particular structure, the use of a nanocarrier for
the delivery of an existing drug can confer the drug better target specificity (thereby limiting its
systemic toxicity), longer circulation time, the ability to overcome some bio-barriers and potential
for real-time monitoring.10-12
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Currently, multiple pharmaceutical companies have embarked on developing nano-based
drug delivery strategies through a careful consideration of the intrinsic challenges and
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opportunities presented by this nascent technology. The FDA has in turn approved some of the
promising drug nanocarriers such as Doxil (PEG-liposome encapsulation of Doxorubicin), Onco
TCS (liposomal formulation of vincristine), Abraxane (albumin bound Paclitaxel nanoparticles),
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Zevalin(Radio-immunoconjugate), and Zinostatin (Polymer-protein conjugate) . The success of
these drug nanoformulations evidences the potential of this methodology for improving cancer
drug therapy.
The use of dendrimers for various drug delivery applications is also an area of intense
research.6, 7, 14, 15 Dendrimers are highly branched, globular macromolecules with multiple arms
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emanating from the core. Their well-defined dendritic architecture allows for several advantages
over the use of conventional polymers such as: (i) controlled multivalence for linkage of multiple
chemical moieties,17 (ii) very low polydispersity which in turn results in reproducible
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pharmacokinetic behaviors. Additionally, the drug loading capacity of the dendrimers can be
tuned by varying the generation number (the number of surface groups available for drug
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interactions doubles or triples with each increasing generation). Likewise, the conjugation of the
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drug(s) to the dendrimer can be modulated through the nature of chemical linkage (pH sensitive ,
light sensitive , biodegradable19, 20) and many of these conjugates have shown increased solubility
and/or decreased systemic toxicity. Covalent dendrimer-drug conjugates that have been
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synthesized for ongoing studies have included platinum complexes (cisplatin, oxaliplatin,
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carboplatin), 5-fluorouracil, Ara-C, Doxorubicin, and Paclitaxel. However, organic core-bearing
dendrimers are limited in their sizes due to steric hindrance at higher generations and in turn in the
surface area to which molecules of interest might be conjugated.
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The creation of non-classical dendronized nanoparticles (NPs), also called nanoparticle-cored
dendrimers (NCDs)22, 23 can circumvent the challenges of increased steric hindrance with higher
dendrimer generation by combining a relatively large inorganic gold core with the defined
branched structure of organic dendrons (branched organic macromolecules with tree-like shape).24-
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This convergent strategy synthesis also allows for increasing the number of dendritic branches
from <10 (in the dendron) to hundreds or thousands (on the dendronized NP, depending on the NP
core size) in a single step.27, 28
We have previously reported the synthesis of an amine-terminated poly(propyleneimine)
PPI dendron and its use to prepare stable water-soluble dendronized AuNPs.29 This PPI dendron
displayed a protected thiol group at its focal point, the thiol function providing strong anchoring
to AuNP surface and the protective group avoiding thiol interference during further dendron
derivatization. While this dendron led to very stable gold nanoparticles, the use of the protective
group added two steps to the preparation of the overall dendron (protection/deprotection), with the
protection step presenting the poorest yield of the whole synthesis (<50%).
Herein, we report the use of thioctic acid at the focal point of the PPI dendron, as well as
the synthesis of the carboxylate version of the PPI dendron, in addition to the aminated version.
This brings significant advantages to both the synthesis and potential derivatization of PPI
dendrons as well as the preparation of extremely stable gold nanoparticles, capped with either the
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carboxylate-PPI dendron, the amine-PPI dendron, or a mixture of both. Indeed, since thioctic acid
displays a cyclic disulfide, it is not expected to interfere in most common organic reactions so
there is no need of protective group during the dendron preparation or conjugation: this permits to
increase the overall yield of dendron synthesis and to work at gram scale. This also extends the
choice for further derivatization of the PPI dendrons since there is no more restriction on
derivatization conditions due to potential protective group removal, and no more limitations on the
chemistry of derivatization due to potential dendritic labels cleavage during deprotection. The use
of thioctic acid also adds further stability to the dendronized gold nanoparticles, since it is a
bidentate ligand, as compared to a monodentate thiol group, and endows these reported
dendronized AuNPs with high stability both in serum as well as after multiple freeze-drying cycles.
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Dendron
Dendron
TA
TEG
TEG
TA
TA-TEG-G3NH2
TA-TEG-G3CO H
2
Figure 1. Set of PPI dendrons prepared: TA-TEG-G3NH
2
and TA-TEG-G3CO H. The sulfur
2
atoms (red) of Thioctic Acid (TA, purple) are the anchoring points for TA-TEG-Dendron through
Au-S bonds; The role of the TEG spacer (blue) is to reduce steric hindrance between arms of
Generation 3 PPI dendrons (black); The terminal functional groups (brown/green) on PPI dendrons
serve as points of conjugation and/or functional group interchange.
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EXPERIMENTAL SECTION
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Synthesis of G2-CN and G3-CN PPI Dendron: Synthesis was performed following protocols
established in our lab and published in prior work.32
Synthesis of TA-TEG Spacer:
TA-PFP (1) - Thioctic acid (10.0 g, 48.5 mmol) was dissolved in 10 mL of dichloromethane in a
round-bottom flask equipped with
dicyclohexylcarbodiimide (10.0 g, 48.5 mmol) was slowly added, and the mixture was stirred for
5 min. Pentafluorophenol (8.92 g, 48.5 mmol) dissolved in 10 mL of dichloromethane was then
a
magnetic stir bar. While stirring, N,N′-
1
slowly added to the mixture and allowed to stir at room temperature (rt) overnight. The mixture
was filtered and quenched with 80 mL of water, and the product was extracted with
dichloromethane. The combined organic layers were further washed with brine, dried over sodium
sulfate, filtered, and concentrated under reduced pressure. Purification by flash column
chromatography (SiO
2
, 9:1 hexane: ethyl acetate) yielded a yellow oil (16.2 g, 90% yield). R =
f
1
0
.85 (9:1 hexane:ethyl acetate); H NMR (400 MHz, CDCl
3
) δ (ppm) 3.62-3.54 (m, 1H), 3.08-3.21
(m, 2H), 2.64-2.73 (t, 2H), 2.42-2.52 (m, 1H), 1.87-1.96 (m, 1 H), 1.64-1.86 (m, 4H), 1.46-1.64
1
9
13
(m, 2H). F NMR (376 MHz, CDCl
100 MHz, CDCl ) δ (ppm): 169.37, 142.50, 140.00, 139.90, 138.70, 56.24, 40.29, 38.61, 34.59,
3.21, 28.56, and 24.57.
TA-TEG-OH (5) - TA-PFP (1) (4.50 g, 12.1 mmol), HO-TEG-NH
3
) -152.65 (d, 2F), -157.92 (t, 1F), -162.19 (m, 2F). C NMR
(
3
3
2
(4) (2.55 g, 13.2 mmol) and
triethylamine (1.68 mL, 11.63 mmol) were dissolved in 30 mL THF and stirred overnight at room
temperature. The solvent was evaporated under reduced pressure and the crude product purified
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via column chromatography (10:1 EtOAc/MeOH) to obtain 3.85 g of the product as yellow oil (83
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%
yield); R
f
= 0.58 (SiO
2
, 10:1 EtOAc/MeOH). H NMR (400 MHz, CDCl
3
) δ (ppm) 3.76-3.38
(m, 17H), 3.20-3.06 (m, 2H), 2.44 (m, 1H), 2.18 (t, 2H), 1.89 (m, 1H), 1.74-1.57 (m, 4H), 1.51-
+
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.35 (2H, m). HRMS (ESI) m/z calcd. for C16
H32NO
5
S
2
(M+H ) 382.1716, observed 382.1714.
o
TA-TEG-OTs (6) - To TA-TEG-OH (5) (8.0 g, 21 mmol) in 40 mL THF cooled to 0 C was added
a solution of sodium hydroxide (2.7 g, 67.5 mmol) dissolved in10 mL deionized water, and the
solution was stirred for a few minutes. Toluene sulfonyl chloride (12.0 g, 63.0 mmol) in 30 mL
THF was then added slowly using a dropping funnel. The reaction was allowed to stir at room
temperature overnight. The following day, THF was removed under pressure with no additional
heat on the rotary evaporator. The clear oil was then taken up in 100 mL DCM and extraction
against water and brine was performed. The combined organic layers were dried over sodium
sulfate, filtered and concentrated under reduced pressure. The crude product was purified via
column chromatography (SiO
2
, 95:5 EtOAc/MeOH) to yield 8.7 g of product as a bright yellow
1
oil (78 % yield) R = 0.60 (SiO
f
2
, 95:5 EtOAc/MeOH); H NMR (400 MHz, CDCl
3
) δ (ppm) 7.75
(d, 2H), 7.31 (d, 2H), 6.07 (s, 1H) 4.11-4.07 (m, 2H), 3.67-3.35 (m, 16H), 3.08 (m, 2H), 2.38-2.52
1
3
(
m, 3H), 2.15 (t, 2H), 1.90-1.85 (m, 1H), 1.71-1.51(m, 4H) 1.51-1.33(m, 2H). C NMR (100 MHz,
) δ (ppm) 173.17, 172.91, 145.00, 132.95, 130.00, 129.96, 128.04, 70.50, 60.47, 56.52,
0.30, 39.23, 38.54, 36.39, 34.71, 28.98, 25.45. HRMS (ESI) m/z calcd. for C23
CDCl
3
4
7 3
H37NNaO S
+
(
M+Na) 558.1630, observed 558.1621.
Synthesis of Spacer-Dendron Conjugates:
TA-TEG-G2CN (7): G2CN was prepared according to previously published work . To a solution
of G2CN (1.3 g, 2.8 mmol) in 10 mL anhydrous CH CN, KOH (0.63 g, 11.21 mmol), K CO (1.55
2
9
3
2
3
g, 11.21 mmol) and TBAB (87 mg, 0.27 mmol) was added. The reaction mixture was allowed to
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stir at 45 C for 2 h in an oil bath after which TA-TEG-OTs (6) (3.16 g, 5.88 mmol) was added to
the reaction mixture. The final reaction mixture was set to reflux for 2 days. The reaction mixture
was then dissolved in 25 mL dichloromethane and filtered to remove the solids. The organic layer
was then poured in deionized water and extracted against water and brine. The combined bright
orange organic layer was dried over sodium sulfate, filtered and concentrated to a clear orange oil.
The orange oil was subsequently dissolved in minimum amount of dichloromethane and washed
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three times with ethyl acetate, Et
2
O and hexane, resulting in the separation of a clear yellow oil
1
(
1.8 g, 77% yield). The supernatants collected from the washes were discarded. H NMR (400
MHz, CDCl ) δ (ppm) 7.08 (d, 2H), 6.82 (d, 2H), 4.09 (s, 2H), 3.83-3.42 (m, 16H), 3.11 (m, 2H),
3
1
3
2.80-2.48 (m, 31H), 2.15-1.40 (m, 11H). C NMR (400 MHz, CDCl
3
) 173.30, 156.98, 133.04,
1
2
29.73, 118.93, 114.47, 77.67, 77.35, 69.79, 56.48, 51.33, 49.61, 45.21, 40.26, 39.18, 36.28, 34.68,
+
5.43. HRMS (ESI) m/z calcd. for C42
H
66
N
8
O
5
S
2
[M+H] 827.4670, observed 827.4699.
TA-TEG-G2NH
2
(8): To a solution of 7 (1.56 g, 1.89 mmol) in anhydrous THF (40 mL) under
nitrogen in a 2-neck round bottom flack was added borane dimethyl sulfide complex (14 mL, 5
equiv. per nitrile group, 20 equiv. total) using a glass syringe. The reaction mixture was stirred at
room temperature (gel formation was evident on the sides of the flask after few hours). The borane
dimethyl sulfide complex was added in two more additions at 4 h intervals. The resulting reaction
mixture was allowed to stir overnight at room temperature under nitrogen. The next day, cold
0
methanol was added slowly at 0 C until no further bubbling or reaction was observed, and was
then removed under reduced pressure. Fresh methanol (30 mL) was added to the residue and the
solution was heated under reflux overnight. Upon cooling to room temperature, the solvent was
removed under reduced pressure to yield a light yellow oil. The oily residue was taken up in 20
mL water, washed with ethyl acetate (3x10 mL) and diethyl ether (3x10 mL) and lyophilized
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overnight to yield the product as a viscous yellow oil. The oil was dissolved in deionized water
and further purified through a size exclusion column (LH-20) to yield a yellow oil (1.27 g, 79 %
yield). Reduction of the nitriles via borane dimethyl sulfide also concurrently reduced the amide
1
1
1
1
1
1
1
1
1
1
2
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2
2
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0
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8
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bond of the thioctic acid to an amine and the completion of this reduction was observed in the
1
reflected mass spectra. H NMR (400 MHz, CD
3
OD) δ (ppm) 7.11 (d, 2H), 6.85 (d, 2H), 4.08 (s,
1
3
2
H), 3.85-3.36 (m, 16H), 3.12 (m, 2H), 2.95-2.28 (m, 40H), 1.95-1.40 (m, 21H). C NMR (400
MHz, CDCl
3
) 157.30, 132.71, 129.49, 78.25, 70.44, 48.58, 47.94, 47.73, 47.52, 47.30, 39.72,
+
3
8
4.70, 29.52, 26.80. HRMS (ESI) m/z calcd for C42
29.6176.
H
84
N
8
4
O S
2
[M+H] 829.6057, observed
TA-TEG-G3CO
2
Me (9): To a solution of 8 (0.65g, 0.78 mmol) in 20 ml of MeOH, LiBr (110 mg,
0
1
.25 mmol) was added and the reaction mixture was stirred for 30 min at 0 C. Methyl acrylate (3
mL, 5 equiv. per amine, 35 mmol) was added in a dropwise manner to the cooled reaction mixture
which was allowed to warm up to room temperature and further stirred for an additional 48 h.
After 48 h., the excess methyl acrylate was removed from the reaction mixture via repeated
stripping of the resulting oil with MeOH (4 times) followed by CH
2
Cl (4 times). The resulting
2
yellow oil was taken in 20 mL CH Cl and extracted against water, and brine. The combined
2
2
organic layers were then dried over sodium sulfate and filtered to yield an clear light yellow oil
1
(
1.1 g, 88%) H NMR (400 MHz, CDCl
3
) δ (ppm) (CDCl ) 7.03 (d, 2H) , 6.77 (d, 2H), 4.04 (s,
3
1
3
2
H), 3.78-3.38 (m,43 H) , 3.12 (m, 2H), 2.85-2.28 (m, 62 H), 1.85-1.24 (m, 25 H), C NMR (400
MHz, CDCl
3
) δ (ppm) 173.27, 157.37 , 129.54, 114.36, 70.36 , 69.61,67.34, 48.13, 48.92, 31.89,
+
2
1
9.51, 27.13, 23.39. HRMS (ESI) m/z calcd for C78
603.9447.
H
138
N
8
O
22
S
2
[M+H] 1603.9445, observed
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TA-TEG-G3CO
LiOH.H
was stirred for 24 h. After 24 h., an additional 15 mL of water was added to the reaction along with
0 mL of CH Cl . The extraction against CH Cl (10 mL) was performed 3 times and the combined
2
H (10): To the solution of 9 (0.5 g, 0.31 mmol) in 10 mL MeOH:H 0 (3:1),
2
2
O (42 mg, 6 equiv. per ester branch, 16.75 mmol) was added and the reaction mixture
0
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1
2
3
4
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8
9
0
1
2
2
2
2
aqueous layers were further purified via dialysis (MWCO 1000) against pH7 water. The water was
changed 4 times at an interval of 4-6 h. Post dialysis, the solution was lyophilized, redissolved in
DI water, further purified through a size exclusion column (LH-20) in water and again lyophilized
to yield an off white solid (0.38 g, 83%). Prior to MS acquisition, the product solution was desalted
via a PD-10 column. MS of the sample in 0.1% acetic acid strongly showed the presence of triply
charged and doubly charged species at m/z 493.3 [M+3H] and 739.5 [M+2H]. A low intensity
+
1
signal for the [M+H] was also observed at 1477.9 via MS. H-NMR (400 MHz, CD
3
OD) δ (ppm)
7
.15 (s, 2H), 6.87 (d, 2H), 4.08 (s, 2H), 3.81-3.54 (m, 16 H), 3.11 (m, 2H), 3.12-2.43 (b, 62 H),
1
3
1.85-1.24 (b, 33 H). C NMR (400 MHz, CDCl
0.27, 67.69, 52.67, 51.58, 40.04, 38.04, 34.70, 33.83, 29.45, 28.95. ζ potential at pH 7 was - 40.6
mV. ATR-FTIR (powder); ν= 3395 O-H stretch, ν = 1725 C=O stretch, ν =1450 OH bend, ν =1085
3
) 179.36, 178.55, 157.36, 129.49, 114.35, 70.42,
7
C-O bending; HRMS (ESI) m/z calcd. for C69
H N
120 8
O
22
2
S : 1476.8 Da; observed mass: 1476.9 Da
(both are monoisotopic masses).
TA-TEG-G3CN (11): G3CN was prepared as previously reported in the literature. G3CN (0.96 g,
.6 mmol) was dissolved in 10 mL of anhydrous acetonitrile. To the G3CN dendron, 4 equiv. of
KOH (0.36 g, 6.4 mmol) and K CO (0.88 g, 6.4 mmol) was added along with 63 mg (0.19 mmol).
1
2
3
Tetrabutyl ammonium bromide (TBAB) was added and allowed to stir for 1 h. in an oil bath at
00C. To this mixture, TA-TEG-OTs (6) (1.8 g, 4.2 mmol) dissolved in 15 mL CH
CN was added
5
3
and set to reflux for 2 days. After 2 days, the reaction mixture was filtered to remove any solids
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and the organic mixture was removed under pressure to yield a viscous orange oil. The orange oil
was dissolved in 20 mL of CH
combined organic layers were dried over sodium sulfate, filtered and concentrated to a clear orange
oil which was dissolved in a minimum of CH Cl and washed 4 times with hexane and diethyl
2
Cl
2
and extracted twice against water and once against brine. The
1
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9
0
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1
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9
0
1
2
3
4
5
6
7
8
9
0
2
2
ether to produce a clear orange oil. The supernatant from the washes were discarded. (1.48 g, 74
1
%
yield). H NMR (400 MHz, CDCl
3
) δ (ppm) 7.05 (d, 2H), 6.78 (d, 2H), 4.05 (s, 2H), 3.78-3.35
1
3
(
m, 16H), 3.07 (m, 2H), 2.78-2.43 (m, 62H), 2-1.1 (m, 20). C NMR (400 MHz, CDCl
3
) 129.70,
1
14.60, 70.61, 69.87, 67.50, 49.67, 37.69, 34.24, 29.12. HRMS (ESI) m/z calcd. for
+
C
66
H
106
N
16
O
5
S
2
[M+H] 1267.7924, observed 1267.8062.
(12): To a solution of 11 (1.5 g, 1.18 mmol) in anhydrous THF in a 2-neck round
bottom flask under nitrogen, borane dimethyl sulfide complex (4.5 mL, 5 equiv. per nitrile branch,
7 mmol) was slowly added using a syringe. The reaction mixture was allowed to stir (gel
TA-TEG-G3NH
2
4
formation was evident after few hours). Two further additions of borane dimethyl sulfide complex
were performed at 3-4 h. intervals. The reaction mixture was allowed to stir overnight at room
0
temperature under inert conditions. The next day, methanol was added slowly at 0 C until no
further bubbling or reaction was observed. The solvent was then removed under reduced pressure.
Fresh methanol (30 mL) was added to the reaction and was heated under reflux overnight. Upon
cooling to room temperature, the solvent was removed under reduced pressure to yield a light
yellow oil. The oily residue was taken up in 20 mL water and extracted against ethyl acetate (2x10
mL) and diethyl ether (2x10 mL). The combined aqueous layers were lyophilized overnight to
yield the product as a viscous clear off white oil. The oil was dissolved in deionized water and
further purified through size exclusion column (LH-20) followed by dialysis (MWCO 1000). The
dialysis water was changed 3 times at 4-6 h. intervals. Post dialysis, the aqueous solution was
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lyophilized to yield a very light yellow powder (1.08 g, 71 % yield). H NMR (400 MHz, CDCl
3
)
δ (ppm) 7.25 (d, 2H), 6.92 (d, 2H), 4.09 (s, 2H), 3.78-3.35 (m, 16H), 3.07 (m, 2H), 3.28-2.43 (m,
1
3
78H), 2.26-1.16 (m, 38H). C NMR (400 MHz, H
0.67, 40.62, 39.78, 39.78, 39.37, 31.31, 26.7; ATR-FTIR (powder): ν = 3357, 3262 primary amine
N-H stretch, ν =1654 N-H bending. ζ potential at pH 5 was +31.2 mV. HRMS (ESI) m/z calcd. for
2
O) 162.88, 130.45, 114.99, 72.82, 70.42, 70.10,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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9
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1
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8
9
0
6
+
+
C
66
H
140
N
16
O
4
S [M+H] 1286.0685, observed: [M+H] 1286.0869.
2
Synthesis and studies of gold nanoparticles:
Synthesis of Citrate AuNPs: All glassware and stir bars were cleaned with aqua regia and rinsed
a minimum of 10 times with ultrapure water. A gold salt stock solution was prepared by dissolving
HAuCl (0.1 g in 10 mL) in ultrapure water to yield a 28.31 mM stock solution. Sodium citrate
4
stock solution was prepared by dissolving 1 g sodium citrate in 20 mL water (0.17 M). 2.65 mL of
gold salt stock solution was added to 247.5 mL ultrapure water in a two-neck round bottom flask
equipped with a condenser and a stir bar, and heated to reflux in an oil bath. Once the solution was
refluxing, 4.41 mL of sodium citrate stock solution was added to the reaction mixture and stirred
vigorously under reflux. The gold salt to citrate molar ratio used was 1:10. After 20 minutes, the
heat was stopped and the solution was kept stirring while cooling down to room temperature. UV-
Vis and DLS spectra were recorded to characterize the AuNPs. The DLS size by number was 21.4
nm.
AuNP Ligand Exchange Reactions: 200 mL of citrate AuNPs (2.46 nM) was centrifuged at
0
10,000 g, for 60 min at 6 C to remove some of the excess citrate. The clear supernatant was then
removed very carefully and discarded to yield a dark red pellet which was subsequently dissolved
in 30 mL ultrapure water. The absorbance of the citrate AuNP solution at 450 nm was used to
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calculate the AuNP concentration. After the calculation of citrate-stabilized AuNP concentration,
the number of ligand required for complete coverage of the AuNP surface was calculated and a
minimum of 40X the molar amount was used for ligand exchange using both synthesized dendrons
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8
9
0
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2
3
4
5
6
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9
0
(
Figure S24).
Singly dendronized AuNPs - The amine-terminated dendron (TA-TEG-G3NH
in 2 mL ultrapure water (pH 5.5) and the carboxylate-terminated dendron (TA-TEG-G3CO
was dissolved in ultrapure water adjusted to pH 9 using NaHCO to ensure complete solubility of
2
) was dissolved
H)
2
3
respective dendrons. Prior to the addition of the dendron solution to a rapidly stirring solution of
AuNPs, the samples were filtered through a 0.22 m syringe filter. Upon addition of the dendron
solution, the bright reddish AuNP solution turned to a slightly darker red. The solutions were
allowed to stir under ambient condition for 20-22 h. prior to purification and removal of excess
0
ligands via centrifugation at 10,000 g, 45 min at 6 C (2-3 rounds).
Mixed-monolayer dendronized AuNPs – First, two solutions of amine-terminated dendron (TA-
TEG-G3NH
2
) and carboxylate-terminated dendron (TA-TEG-G3CO H), respectively, were
2
prepared separately by dissolving each in 1 mL ultrapure water adjusted to pH 7 using NaHCO3.
Then the appropriate volumes from each solution were combined to obtain a mixture composed of
6
0% TA-TEG-G3CO
2
H and 40% TA-TEG-G3NH (molar ratio). This mixture was filtered
2
through a 0.22 m syringe filter and added to a solution of AuNPs that had been previously
centrifuged and redispersed in ultrapure water, as explained for the singly dendronized AuNPs.
The AuNP solution turned to a very slightly darker red (if the solution turns purplish, then add a
drop or two of 1M HCl). The AuNP solution was allowed to stir under ambient condition for 20-
22 h. prior to purification and removal of excess ligands via centrifugation at 10,000g, 45 min at
60
C (2-3 rounds).
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TGA: The dendronized AuNPs were purified extensively prior to the dendron quantification
studies via thermogravimetric analysis. The crude dendronized AuNPs were centrifuged 2 times
0
for 75 min at 12,000g at 5 C. After each centrifuge cycle, the colorless supernatant was carefully
0
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removed and the remaining dark red pellet was re-suspended in ultrapure water. Post
centrifugation, the AuNP solution was dialyzed (MWCO: 12,000 Da) three times against ultrapure
0
water. The resulting AuNP solution was centrifuged a third time at 10,000g, 5 C for 60 min. The
supernatant was discarded and the reddish black pellet was suspended in 15 mL ultrapure water
and transferred to a lyophilization chamber. The AuNP samples were lyophilized for 3 days to
ensure complete removal of trace amounts of water. The resulting black colored AuNP powders
were used for TGA analysis. TGA analysis was run in duplicates for both samples with sample
weights ranging from 1.3 mg to 1.6 mg.
Lyophilization Studies: The dendronized AuNPs from AuNP ligand exchange reaction were
purified according to the procedure outlined in the TGA sample preparation prior to the dialysis
stage. Post centrifugation of AuNP, the supernatant was discarded and the reddish black pellet was
suspended in 20 mL ultrapure water. From the 20 mL batch, a 5 mL aliquot was taken for running
UV-Vis and DLS data collection. After the acquisition of UV-Vis and DLS spectra, the sample
was lyophilized for 2 days. The resulting powder was dissolved in exactly the same volume of 5
mL water and was lyophilized. The prior steps of dissolution of AuNPs, data acquisition and
lyophilization was performed for two additional rounds on the same sample, yielding 3 data points
per dendronized AuNP. Care was taken to maintain the same AuNP concentration (2.6 nM)
through the three lyophilization rounds.
Stability Studies: The lyophilized AuNP powders derived as per the procedure in prior steps were
dissolved in 2 mL neat solution of buffers and salts with the exception of the serum solution. The
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concentration for AuNP used for the stability studies were 1.8 nM. The sample preparation for the
study of stability in serum was performed by first dissolving the AuNPs in 250 L of ultrapure
water to which 1.75 mL human serum was added. All DLS data for stability studies were acquired
1
1
1
1
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0
0
at 37 C with an incubation time of 3 minutes. UV-Vis data of samples were acquired immediately
following DLS data acquisition. The samples were maintained through the entire 7 day period in
0
a water bath maintained at 37 C.
Evaluation of Cytotoxic Profile of Dendronized AuNPs: MCF-7 cells were grown in a 250 mL
0
cell culture flask using high-glucose DMEM serum at 37 C in a humidified atmosphere of 5%
CO
2
. Cell line was maintained by passaging when cells exhibited 80% confluence. In order to
, AuNP-G3CO H and AuNP-G3CO H/NH (60/40),
assess the cytotoxic profile of AuNP-G3NH
2
2
2
2
the respective purified nanoparticle formulations were lyophilized overnight to yield a black
powder. The AuNP powders were suspended in DMEM media via mixing with a sterile 1 mL
pipette to generate 15.6 nM initial concentrations. All cytotoxic tests and re-dispersions were
carried out within a BSL-2 laminar flow hood. MCF-7 cells were seeded and dosed according to
the protocol outlined above. Cell viability was determined through the equation: Cell viability (%)
=
(Sample absorbance at 460 nm - background absorbance at 600 nm)/ (Absorbance of negative
control at 460 nm - background absorbance at 600 nm).
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RESULTS AND DISCUSSION
Scheme 1. Synthesis of TA-TEG-OTs spacer from commercially available thioctic acid (TA) and
tetraethylene glycol (HO-TEG-OH) precursors.
0
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Design and Synthesis of TA-TEG-PPI Dendrons
In order to ensure the production of a scalable, easily amenable to multifunctionalization, and very
stable water-soluble dendronized nanoparticle system that can carry very large payloads, we
devised a dendron platform composed of three essential components (Figure 1): (1) a PPI dendritic
system, which can display either carboxylate or amine termini for convenient further derivatization
with desired payload, and whose commercial precursors are affordable, and readily available from
commercial sources (2) a tetraethylene glycol (TEG) spacer, that allows for maximum coverage
of the nanoparticle surface by reducing steric hindrance between dendritic branches, and (3) a
thioctic acid (TA) group at the end of the TEG spacer, which imparts very strong AuNP anchoring
ability to the dendron.31
The use of TA as anchoring point of the dendron to the AuNPs brings two major advantages over
the use of a thiol group: (i) TA provides a stronger attachment to gold surfaces due to the bidentate
nature of its terminal cyclic disulfide group,31-33 and (ii) it eliminates the need for a protective
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group, thus removing 2 steps (protection/deprotection) from the synthetic route. This absence of
protective group alleviates any concern in the choice of reaction conditions during further
derivatization of the dendritic branches.
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0
The PPI dendron platform was obtained by first preparing the TA-TEG spacer and the PPI dendron
and then coupling them together. This synthetic approach allowed us to prepare the TA-TEG
spacer (Scheme 1) at multigram scale (with each step affording over 85% yield) and the final
dendron platform in gram amounts (Schemes 2 and 3).
Scheme 2. Synthesis scheme for TA-TEG-G3NH
2
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2
2
2
2
2
2
2
2
2
2
3
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4
4
4
4
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4
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4
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5
5
5
5
5
5
5
5
5
6
Scheme 3. Synthesis Scheme of TA-TEG-G3CO H
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
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2
3
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8
9
0
Two different synthetic routes were used to prepare the amine-terminated PPI dendron and the
carboxylate-terminated PPI dendron due to the nature of the chemistry involved in the formation
of classical PPI dendron. Indeed, the usual synthetic scheme for the growth of PPI dendrimers or
dendrons involves a repetitive sequence of hetero Michael addition using acrylonitrile followed by
3
4
reduction of the nitrile groups to primary amines. This sequence of steps directly leads to the
amine-terminated PPI dendron.
Consequently, for the formation of TA-TEG-G3NH , we first prepared the third generation nitrile-
2
terminated dendron (G3CN) with a phenol group as focal point, then coupled it to the spacer (TA-
TEG-OTs) through nucleophilic substitution at the phenol group, and finally reduced the dendritic
nitrile groups to amines using borane dimethyl sulfide. Coupling G3CN instead of G3NH to the
2
spacer avoids any competition of the amine termini with the phenol group, which could result in
attachment of the spacer not only to the focal point but also to the dendritic branches. It is
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interesting to note that the last step, reducing TA-TEG-G3CN into TA-TEG-G3NH concurrently
2
reduces the amide bond in the spacer, between TA and TEG, which is evidenced by the mass
spectrometry data (Figure S5): indeed, the observed mass on the MS data lacks 14 Daltons (16
Daltons less due to the loss of the oxygen of the amide group and 2 Daltons more from the 2
protons of the reduced amide to secondary amine) compared to the calculated mass for TA-TEG-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
G3NH
2
2
containing the amide bond. Other characterization data were collected for TA-TEG-
1
G3NH
, and are displayed in Figure 2. The H NMR spectrum in D
2
O(Figure 2A) shows the peaks
from the protons of the dendritic branches spanning from 2 ppm to about 3.3 ppm. The protons
from the TEG part give peaks between 3.4 and 4.1 ppm and the thioctic acid portion gives rise to
peaks mostly between 1 and 2 ppm, but also hidden within the dendritic peaks, between 2 and 3.4
ppm. The aromatic focal point of the dendron is represented by the two peaks at 6.9 and 7.2 ppm.
The presence of the terminal primary amine groups is clearly illustrated by the FTIR bands at
-1
-1
3357-3262 cm (primary amine N-H stretch) and 1654 cm (N-H bending). Finally, the positive
zeta potential (30.1 mV) of TA-TEG-G3NH at pH 7 also reflects the presence of the primary
2
amine groups, which are expected to be protonated at this pH.
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1
10
A)
B)
100
9
8
7
6
0
0
0
0
0
1
2
3
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9
0
1
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0
1
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3
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5
6
7
8
9
0
3
500
2500
1500
500
cm-1
C)
-
100
0
100
Zeta Potential (mV)
1
Figure 2. Characterization of TA-TEG-G3NH
in D O with water suppression. B) ATR-FTIR of TA-TEG-G3NH
amine N-H stretch, ν = 2928 C-H unsaturated carbon, ν =1654 N-H bending, ν = 1467 C-H bend.
2
dendron. A) H NMR of TA-TEG-G3NH
2
dendron
2
2
powder ν = 3357, 3262 primary
C) Zeta Potential (ζ) of TA-TEG-G3NH
2
at pH 7.
For the preparation of TA-TEG-G3CO
2
H, the last Michael addition needs to involve methyl
acrylate in order to lead to carboxylate termini after hydrolysis of the esters. Since on one hand
coupling G3CO H to the spacer would not be efficient due to solubility issues as well as potential
side reactions, and on the other hand the dendron-spacer coupling reaction requires the use of bases
that would hydrolyze G3CO Me during its coupling to the spacer, we decided to couple the spacer
2
2
to the second generation nitrile-terminated dendron (G2CN). After obtaining TA-TEG-G2CN, we
then continued the growth of the dendron through Michael addition using methyl acrylate and
3
5
obtained TA-TEG-G3CO
2
H by hydrolysis using lithium hydroxide. Interestingly, following this
synthetic path results in the addition of a ninth branch to the final TA-TEG-G3CO
2
H. Indeed, in
order to keep growing the dendritic branches after coupling to the spacer, TA-TEG-G2CN is
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reduced to TA-TEG-G2NH
2
:during thisreduction, the amide bond between TA and TEG also gets
3
6
reduced into a secondary amine, which results in TA-TEG-G2NH displaying nine amines (8
2
dendritic primary amines and one secondary amine from the spacer). Consequently, the use of an
excess (required for the dendritic branches) of methyl acrylate during the following reaction leads
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
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0
1
2
3
4
5
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9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
1
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1
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9
0
to nine –CH
2
CH
2
CO
2
Me branches in TA-TEG-G3CO
CH CO
2
Me. The final hydrolysis results in TA-
1
TEG-G3CO
2
H, which contains nine CH
2
2
2
H branches, as evidenced by H NMR
integration as well as mass spectrometry data. The MS data displays two prominent peaks: one
corresponding to the doubly-protonated species (M+2H), and one for the triply-protonated species
(M+3H) (Figure S7). Other characterization data were collected for TA-TEG-G3CO H, and are
2
1
displayed inFigure 3. The H-NMR spectrum in D
2
O(Figure3A) shows the peaks from the protons
of the dendritic branches spanning from 1.6 ppm to about 2.95 ppm. The protons from the TEG
part gives peaks between 3.5 and 4.1 ppm and the thioctic acid portion gives rise to a peak at
around 1.35 ppm, but has also other peaks hidden within the dendritic peaks, between 1.7 and 3.4
ppm. The aromatic focal point of the dendron is represented by the two peaks at 6.8 and 7.1 ppm.
The presence of the terminal carboxylate groups is clearly illustrated by the FTIR bands at 3395
-1
-1
-1
cm (O-H stretch), 1725 cm (C=O stretch) and 1450 cm (OH bend). Finally, the negative zeta
potential (- 40.6 mV) of TA-TEG- G3CO H at pH 7 also reflects the presence of the carboxylate
2
groups, which are expected to be deprotonated at this pH.
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1
00
A)
B)
9
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0
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2
3
4
5
6
7
8
9
0
3
500 3000 2500 2000 1500 1000 500
cm-1
C)
-
100
0
100
Zeta Potential (mV)
1
Figure 3. Characterization of TA-TEG-G3CO
dendron in D 0 with water suppression. B) ATR-FTIR of TA-TEG-G3CO
H stretch, ν = 2969 unsaturated C-H stretch, ν = 1725 C=O stretch, ν =1450 OH bend, ν =1085 C-
O bending. C) Zeta Potential (ζ) of TA-TEG-G3CO H at pH 7.
2
H dendron. A) H NMR of TA-TEG-G3CO
2
H
2
2
H powder; ν= 3395 O-
2
Dendronized AuNPs Syntheses and Characterization:
a) Synthesis
To ensure complete exchange of the citrate molecules with TA-TEG-dendrons on the AuNPs, a
minimum of 40 times excess of the TA-TEG-dendron required for total surface coverage was used
for each ligand exchange reaction (Figure S24). In addition, we have experimentally determined
that the polydispersity indexes (PDI) of dendronized nanoparticles were not consistent between
batches when less than 40 times equivalence of dendrons were used. Furthermore, removal of
excess citrate prior to ligand exchange led to lower variability in resulting AuNPs polydispersity
after ligand exchange across different dendron platforms. It is noteworthy to point out that the
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presence of excess citrate did not affect the PDI of the AuNP-G3CO
2
H as much as it did for the
, facile and quick ligand
AuNP-G3NH
2
. We hypothesize that, during formation of AuNP-G3NH
2
exchange on the Au surface was hindered due to electrostatic attraction between the negatively
charged excess citrate and positively charged amine dendrons. On the other hand, steric repulsion
between the negatively charged dendron-carboxylate and citrate prevented any interference of the
excess citrate in the ligand exchange on the AuNPs. Similar findings have been reported on the
1
1
1
1
1
1
1
1
1
1
2
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0
3
7
instability of cationic AuNPs due to the presence of citrate. Overall, the removal of excess citrate
prior to ligand exchange circumnavigated the discussed difficulties and led to uniformity in the
preparation of all batches of dendronized AuNPs.
A) (i)
(ii)
(iii)
1
0
.8
0.6
0
0
.4
.2
0
4
50
550
650
750
850
-100
0
100
Wavelength (nm)
Size (nm)
ζ potential (mV)
B)
(i)
(ii)
(iii)
1
.8
.6
.4
0
0
0
0.2
0
-100
0
1
00
4
50
550
650
750
850
Wavelength (nm)
Size (nm)
ζ potential (mV)
Figure 4. A) Characterization of AuNP-G3CO H: (i) UV-Vis spectra comparison of citrate AuNPs
2
(
blue) vs AuNP-G3CO
2
H (green), Ligand exchange causes an SPR red-shift of 4 nm; (ii) DLS
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3
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5
5
6
comparison of citrate AuNPs (blue) vs AuNP-G3CO
2
H (green) (16.1 nm, PDI 0.05 to 20.4 nm,
H at pH 5.5. B) Characterization of AuNP-
: (i) UV-Vis spectra comparison of citrate AuNPs vs AuNP-G3NH (SPR red-shift of 3
nm); (ii) DLS comparison of citrate AuNPs vs AuNP-G3NH (15.6 nm, PDI 0.06 to 19.9 nm, PDI
PDI 0.09); (iii) Zeta Potential (ζ) of AuNP-G3CO
2
G3NH
2
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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0
1
2
3
4
5
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8
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0
1
2
3
4
5
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9
0
2
0
.17); (iii) Zeta Potential (ζ) of AuNP-G3NH at pH 5.5.
2
b) Characterization
The purified dendronized AuNPs showed excellent stability in ultrapure water (pH 5.5) (stable >
months) as a consequence of high steric stability and electrostatic repulsion between highly
charged particles as evidenced by zeta potential characterization (Figure 4): in pure water (pH 5.5)
the zeta potentials of AuNP-G3CO H and AuNP-G3NH are – 43.5 mV and 36.6 mV, respectively.
The size of the AuNPs in solution increased from 16.1 nm (DLS by number) for AuNP-citrate to
0.4 nm for AuNP-G3CO H and from 15.6 nm to 19.9 nm for AuNP-G3NH , which corresponds
6
2
2
2
2
2
to an overall increase of 4.3 nm in both cases. This indicates that the hydrodynamic size of dendron
coating is 2.15 nm. This corresponds well to the hydrodynamic diameter measured by DLS for the
dendron alone (2.3 nm by number).
Dendronized AuNPs with mixed-coating (AuNP-G3CO
2
H/NH , 50/50 molar ratio of TA-TEG-
2
G3CO H and TA-TEG-G3NH ) were also prepared and characterized (Figure 5). The ligand
2
2
exchange of citrate by the mixture of dendrons led to a surface plasmon resonance (SPR) peak
shift of 13 nm (521 nm to 534 nm), which is larger than for the singly dendronized AuNPs. Also,
the hydrodynamic diameter increased from 17.9 nm (DLS by number) for AuNP-citrate to 41.6
nm for AuNP-G3CO
2
H/NH , which represents an overall size increase of 23.7 nm and is
2
equivalent to a dendritic coating thickness of 11.8 nm. Both the absorption and DLS data seem to
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indicate the formation of a dendron multilayer around the AuNPs with mixed-coating, as opposed
to the monolayers formed around singly dendronized AuNPs.
A) 0.7
25
20
B)
1
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1
1
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1
1
2
2
2
2
2
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0
0
.6
.5
0.4
.3
.2
.1
0
15
0
10
0
5
0
0
0
4
50
550
650
750
0
.1
10
1000
Wavelength (nm)
Size (nm)
C)
pH = 7.2
pH= 7.2
+8.1 mv
400000
-
31.7 mv
3
00000
00000
+
29.6 mv
pHpH
=
=9
9
2
pH= 4
pH =5.5
100000
-100
0
100
Zeta Potential (mv)
Figure 5. Characterization of mixed-coated AuNP-G3CO
comparison of citrate AuNPs (blue) vs AuNP-G3CO H/NH
citrate AuNPs (blue) vs AuNP-G3CO H/NH (orange); C) Zeta potential analysis at pH10 (green),
2
H/NH
2
(50/50): A) UV-Vis spectra
2
2
(orange); B) DLS comparison of
2
2
pH7 (blue) and pH5 (red) - Insets correspond to NP colors at each pH value.
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We have also tested AuNP-G3CO
2
H/NH
2
(50/50) at different pHs (Figure 5C), and as expected,
the AuNPs show an overall negative charge (- 32 mV) at pH10, due to the carboxylate groups, a
positive charge (30 mV) at pH5 due to the ammonium groups, and a nearly neutral charge (8 mV)
at pH7, likely coming from equal partial deprotonation and protonation of carboxylate and amine
groups, respectively. This evolution of charge reflects well the dendrons composition on these
mixed-monolayer dendronized AuNPs.
0
1
2
3
4
5
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7
8
9
0
1
2
3
4
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1
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3
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0
1
2
3
4
5
6
7
8
9
0
TEM images (Figure 6C) obtained by staining AuNPs with 2% uranyl acetate allowed for
visualization of the dendron corona around the nanoparticles, with an average thickness of 1.6 nm.
The apparent reduction in dendron thickness calculated from TEM measurements compared to the
DLS data (2.15 nm) can be explained by the difference in sample preparation: the DLS
measurements are done in solution and at atmospheric pressure, thus the nanoparticles coating
corresponds to fully hydrated dendrons; but the TEM measurements are done on dry samples and
under vacuum, thus the nanoparticles coating corresponds to contracted dendrons. Further STEM
images (Figures S9, S10) of concentrated dendronized AuNPs also showed spherical shapes with
no apparent aggregation upon visualization.
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A) 1.55
1.53
1.51
1.49
1.47
1.45
1.43
1.41
1.39
1.37
TA-TEG-G3NH dendron
2
C)
coating: 11.9 %
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
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0
1
2
3
4
5
6
7
8
9
0
1.35
0
100 200 300 400 500 600 700
Temperature (℃)
B) 1.145
1.125
1.105
1.085
TA-TEG-G3CO H dendron
2
coating:11.1 %
1.065
1.045
1.025
1.005
0.985
0
100 200 300 400 500 600 700
Temperature (℃)
Figure 6. Surface coating of dendronized AuNPs. A) TGA analysis of dendron coating on AuNP-
G3CO
2
2
H. B) TGA analysis of dendron coating of AuNP-G3NH
2
NPs. C) TEM image of AuNP-
G3CO
H stained with 2% uranyl acetate for visualization of dendron corona: average thickness of
1
.6 nm.
In order to quantify the extent of surface coating, TGA analyses were performed. The TGA data
indicated that the surface coating of dendrons represents 11.8 and 11.2 % of the AuNPs masses,
for AuNP-G3NH
350 and 1450 dendrons/NP, respectively, calculated via a formula accounting for the core of the
nanoparticle and molecular weight of the dendron (Figure S25). The lower number of dendrons on
AuNP-G3CO H can be explained by the presence of 9 arms in TA-TEG-G3CO H compared to
the 8 arms in TA-TEG-G3NH (see discussion above). The added lateral steric hindrance from the
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and AuNP-G3CO
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H respectively (Figure 6A, 6B), which equates to around
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9th arm leads to a slightly lower dendron packing on the nanoparticle surface. However, this slight
reduction did not contribute to a lack of nanoparticle stability, as showcased in various stability
studies (vide infra). The surface density of the dendrons on the AuNPs was calculated to be around
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high ligand density but is still realistic as it corroborates well with other observed surface
densities.38-41
We also performed some pH studies, and noticed that the zeta potential of AuNP-G3NH increases
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from 36.1 mV at pH 5.5 to 54.3 mV at pH 3 (Figure S10). This increase in positive charges is most
likely coming from the protonation of the tertiary amines of the dendrons, which we have reported
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earlier. This observation showcases the potential of these dendronized nanoparticles to act as
proton sponges at the lysosomal pH of 4.5.
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Although other dendrons also using TA as anchoring point, or also presenting carboxylate or
amine termini42 have been reported in the literature for the preparation of dendronized
nanoparticles, our TA-TEG-G3PPI dendrons bring two main advantages. The first difference is
the addition of the TEG linker between the thioctic acid group and the PPI dendron itself: this
counter-balances the steric hindrance of the eight dendritic branches of each dendron around the
gold core, thus allowing very high packing and dense coating on the AuNPs. The other benefit is
our preparation of a pair of cationic and anionic PPI dendrons that differ only in the nature of
their termini (amine or carboxylate): which allowed us to combine them and prepare the first
zwitterionic dendronized AuNPs.
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Stability Studies
a) Lyophilization
In order to test their ease of handling, we investigated the stability of our dendronized nanoparticles
against repeated lyophilization cycles. As outlined in Figure 7, both AuNP-G3CO H and AuNP-
G3NH , as well as remained stable even after three rounds of freeze-dry/reconstitution cycles,
regardless of the nature of their end group functionality: their hydrodynamic diameter showed
negligible change after each round. Similar stability was observed for AuNP-G3CO H/NH
Figure S26a). We can also note that all of the lyophilized nanoparticles readily redissolved in
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(
water, without leaving any precipitate residue. This very high stability is conferred to the
nanoparticles by the bidentate nature of the thioctic acid moiety at the end of the dendritic focal
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point, but also by the high packing density of TA-TEG-dendrons and the steric repulsion between
charged dendronized nanoparticles. So far, only very few studies have reported other thiol-
protected AuNPs to withstand several rounds of freeze-drying: one featuring cationic ligands,42
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one using anionic ligands, and one with zwitterionic ligands. Our present work shows that both
TA-TEG-G3CO H and AuNP-G3NH lead to very stable anionic and cationic AuNPs,
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respectively, and also offers the possibility of creating zwitterionic AuNPs starting from non-
zwitterionic ligands. These results provide a more versatile solution to the impracticality of storage
and long term handling of nanoparticles in solution. Indeed, since these dendronized nanoparticles
are resistant to multiple freeze-dry cycles, they can be stored as powders and reformulated directly
into the desired medium (buffers, serum, etc.) right before use. But they also have the additional
advantage of presenting two alternatives for further payload conjugation, through either the
carboxylate groups or the primary amine groups.
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