Type III-C rotaxane dendrimers: Synthesis, dual size modulation and: In vivo evaluation

Article abstract of DOI:10.1039/c9cc06200a

Type III-C rotaxane dendrimers were synthesized by a divergent approach. Dual shuttling behavior and size modulation were observed from non-methylated/methylated rotaxane dendrimers under the same external stimuli. The biological distribution of dendrimers in C57BL/6J mice determined by MALDI-TOF-MS shows predominant accumulation in the spleen and liver. Drug encapsulations with chlorambucil and lithocholic acid were demonstrated.

Full text of DOI:10.1039/c9cc06200a

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Type III-C rotaxane dendrimers: synthesis, dual
size modulation and in vivo evaluation†
Cite this: Chem. Commun., 2019,
55, 13426
Chak-Shing Kwan,‡a Tao Wang,‡a Min Li,b Albert S. C. Chan,c Zongwei Cai
*
a
a
Received 13th August 2019,
Accepted 14th October 2019
and Ken Cham-Fai Leung
*
DOI: 10.1039/c9cc06200a
rsc.li/chemcomm
Type III-C rotaxane dendrimers were synthesized by a divergent with three-dimensional switching and demonstrated their applic-
approach. Dual shuttling behavior and size modulation were observed ability in drug encapsulation.9 Due to the complexity of the
from non-methylated/methylated rotaxane dendrimers under the structures and the difficulties in chemical synthesis, only a few
same external stimuli. The biological distribution of dendrimers in examples of type III RDs have been successfully reported9–13 and
C57BL/6J mice determined by MALDI-TOF-MS shows predominant their switching properties have been demonstrated.9,13
accumulation in the spleen and liver. Drug encapsulations with
chlorambucil and lithocholic acid were demonstrated.
Type III-C rotaxane dendrimers were first categorized by
Stoddart et al. in 2017,2 as the branches extend from the rings
rather than the rods. In particular, type III-C RDs have mechanical
Rotaxane dendrimers (RDs) are a class of mechanically inter- bonds in between and constituted the branching points. Only one
locked molecules (MIMs) combining the unique linear molecular example that matches the description of type III-C RDs was
shuttling properties and motif of mechanically interlocking reported by Liu et al. in 2013,14 as the first-generation (G1)
systems into spherical dendrimers to generate hyperbranched hetero[4]rotaxane. However, their strategy failed to produce higher
mechanically interlocked macromolecules. The concept of rotax- generation RDs because of the non-functionalized macrocycle.
ane dendrimers was first defined by Lee and Kim1 in 2003 and
We envisage a divergent approach toward the chemical synthesis
further elaborated by Stoddart et al. in 2017.2 It is classified into of type III-C rotaxane dendrimers (Scheme 1). Similar to the tradi-
three main RD types (I, II and III) and each type can be further tional dendrimer synthesis,15 the branching unit grows outward from
divided into sub-categories (A, B, and C). Among those, the the core [2]rotaxane to become a hyperbranched rotaxane dendrimer.
synthesis of type III rotaxane dendrimers is the most challenging
The stimuli-responsive molecular shuttling properties and
task, as they have a dendritic polyrotaxane architecture with globular structures of type III-C RDs could be potentially applied
interlocking moieties from the core to every branching unit. in drug encapsulation and a pH responsive delivery system.9 The
Equipped with three-dimensional, near-spherical macromolecular biological distribution of these potential cargo carriers is crucial
structures as well as the collective molecular shuttling properties to achieve localized and targeted delivery. Conventionally, the
of type III rotaxane dendrimers,3 it is believed that they could in vivo study of dendrimers usually relies on a radiolabel or
potentially be utilized in various applications such as catalysis,4 fluorescence label covalently attached to dendrimers. Matrix
drug delivery, molecular electronics, etc., controlled by different assisted laser desorption-time of flight mass spectrometry
external stimuli, such as pH,5 light radiation,6 redox,7,8 etc. Our (MALDI-TOF MS) could be a versatile tool for us to analyze
group has recently reported the first successful synthesis of high-
generation type III-B G3 rotaxane dendrimers and a G4 dendron
a Department of Chemistry and State Key Laboratory of Environmental and
Biological Analysis, Hong Kong Baptist University, Kowloon Tong, Kowloon,
Hong Kong SAR, P. R. China. E-mail: cfleung@hkbu.edu.hk, zwcai@hkbu.edu.hk
b School of Chinese Medicine, Hong Kong Baptist University, Kowloon Tong,
Kowloon, Hong Kong SAR, P. R. China
c School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou,
P. R. China
† Electronic supplementary information (ESI) available: Experimental, NMR
Scheme 1 Proposed divergent (A) and convergent (B) approaches towards
the synthesis of type III-C RDs.
spectra, shuttling process, in vivo study, etc. See DOI: 10.1039/c9cc06200a
‡ These authors contributed equally.
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label-free type III-C RDs in vivo. To broaden the scope of rotaxane
dendrimers, herein we present a facile divergent synthesis of
type III-C rotaxane dendrimers equipped with dual shuttling
functions for size modulation. The binding of two drug molecules
(chlorambucil and lithocholic acid) has been studied. In vivo
biological distributions of the label-free MIMs by MALDI-TOF
MS were also investigated. To the best of our knowledge, it is the
first rotaxane dendrimer that possesses dual (expansion and
contraction) collective size modulation for drug encapsulation.
The key-precursor in type III-C RD synthesis is the
di-functionalized crown ether in the first step of [2]rotaxane for-
mation. Di-succinimide (NHS) functionalized [2]rotaxane (2-HÁPF6)
was synthesized in about 67% yield (the detailed synthetic route is
illustrated in the ESI†). The NHS moiety was transformed to an azide
moiety (3-HÁPF6) followed by the formation of two new mechanical
bonds through ubiquitous copper catalyzed azide alkyne cyclo-
addition (CuAAC), giving G1 [4]rotaxane dendrimer (G1-H3Á3PF6) in
63% yield. A tetra-functionalized-NHS G1 [4]rotaxane dendrimer
(4-H3Á3PF6) was synthesized by replacing DB24C8 with di-
functionalized DB24C8 in the first step. The four NHS units on
4-H3Á3PF6 were transformed to azide ended tetra-functionalized
G1 [4]rotaxane dendrimer (5-H3Á3PF6) and used in the formation
of G2 [8]rotaxane dendrimer (G2-H7Á7PF6). The synthesis of
G2-H7Á7PF6 involved the formation of four new mechanical
bonds in one step CuAAC. Encouragingly, we were able to isolate
the final pure G2-H7Á7PF6 in 56% yield. Triazole methylation
of G1-H3Á3PF6 and G2-H7Á7PF6 was performed for the further
investigation on switching (Fig. 1).
Fig. 2 Stacked 1H NMR spectra (400 MHz, CD2Cl2, 298 K) of (A) G1-H3Á
3PF6, (B) neutral G1, (C) methylated Me6G2-H3Á9PF6 and (D) methylated
and deprotonated Me6G1-6PF6.
All type III-C RDs were well characterized thoroughly by 1H
and 13C NMR spectroscopies. The starting [2]rotaxane was char-
acterized by 1H NMR with two NHS moieties in di-NHS [2]rotaxane
(2-HÁPF6). When it was transformed into di-azido [2]rotaxane
(3-HÁPF6), the original succinimide proton peak at d = 2.76 ppm
was transformed into two sets of new peaks at d = 4.27 ppm, and
d = 4.45 ppm corresponding to the azide moiety (Fig. S5, ESI†).
The 1H NMR spectra of G1-H3Á3PF6 were clearly identified (Fig. 2A),
dibenzylammonium (DBA) He and Hk shared an identical chemical
shift at d = 4.54 ppm, and the chemical shift at d = 4.42 ppm was
Fig. 1 Graphical representation and partial structural formula of G2 rotaxane dendrimers.
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attributed to the protons Hi near the amide. Three triazole peaks MTA, a well-known secondary station for DB24C8.17 Deprotonation
were located in the downfield region, corresponding to the three of Me6G1-H3Á9PF6 (Fig. 2C and D) led to DBA protons He and Hk
1
sets of triazole. From the H NMR spectrum of G2-H7Á7PF6, the shifting upfield (DdHe,k = À0.86 ppm), and all the MTA protons Hc,
peak broadening phenomenon was observed because of more Hm, and Hg shifted as two sets of resonances, revealing that the
repeating units (ESI,† Fig. S7). Nonetheless, G1 and G2 type III-C macrocycles were oscillating between two equal MTA and slow on the
1
RDs shared a very similar pattern in the H NMR spectrum with NMR time-scale.18 The methyl protons of MTA shifted dramatically,
reasonable peak integration, indicating the successful synthesis of due to the shielding effect by the cavity of aromatic macrocycles. The
G1-H3Á3PF6 and G2-H7Á7PF6.
protons adjacent to amide Hi were not shifted implying that
Both G1-H3Á3PF6 and G2-H7Á7PF6 were subjected to methyla- the macrocycles did not move to the amide unit. In Me14G2-14PF6
tion to methylate all the triazole units into the N-methyltriazolium (ESI,† Fig. S9), very similar chemical shifts can be detected, except for
(MTA) ion. The total number of triazole units was 6 in G1 and 14 the MTA protons that shifted as one set of peaks. G1, G2 and Me6G1-
in G2. After methylation, methylated G1 [4]rotaxane dendrimers 6PF6, Me14G2-14PF6 exhibited distinct switching behaviours com-
(Me6G1-H3Á9PF6) carrying 9+ charges and methylated G2 [8]rotaxane pared to their initial compounds. In addition, all RDs can be restored
dendrimers (Me14G2-H7Á21PF6) became the second most densely to their initial structures with at least 5 acid–base switching cycles,
charged MIMs16 on record carrying 21+ charges.
showing that the RDs were capable of multiple pH switching cycles
The successful synthesis of all the type III-C RDs was further without degradation (ESI,† Fig. S18–S25).
confirmed by high resolution electrospray-ionization mass spectro-
Unlike our reported type III-B RDs, dual (expansion and
metry (HR ESI-MS). [M-3PF6]3+ species was observed in G1-H3Á3PF6. contraction) size modulation of type III-C RDs was achieved.
In G2-H7Á7PF6 (ESI,† Fig. S93), two ion species representing Based on the NMR result, supposedly, the initial RDs will be
[M-7PF6]7+ and [M-6PF6]7+ with m/z 1439.1611 and m/z 1703.0207 contracted after deprotonation, whereas the size of the methylated
were found and are consistent with the theoretical value, implying RDs will be slightly expanded. We then analyzed the morphology
that all the targeted type III-C RDs were successfully synthesized. and size changes of the RDs with atomic force microscopy (AFM).
The mass spectrum of Me6G1-H3Á9PF6 (ESI,† Fig. S94) showed The AFM analysis revealed that all the RDs were nearly spherical
peaks at m/z 570.0549, 693.0532 and 832.7225 corresponding to in morphology and monodispersed when they were deposited on
the species of [M-9PF6-H]7+, [M-7PF6]7+ and [M–6PF6]6+, respec- the mica/silicon wafer surface. G1-H3Á3PF6 (B1.50 nm) and G1
tively. In the mass spectrum of Me14G2-H7Á21PF6 (ESI,† Fig. S95), (B1.51 nm) showed a similar height (Fig. 3A and B), while the
peaks at m/z 1520.9983, 1335.8846, 1187.7024, 1066.5527, and height of Me6G1-6PF6 (B1.96 nm) was slightly greater than that of
À
965.6718 refer to the species of losing eight to twelve PF6
Me6G1-H3Á9PF6 (B2.11 nm) (Fig. 3C and D). In G2 RDs, obvious
counterions. The ESI-MS evidence further confirmed that all height differences were observed. After the deprotonation of
triazoles inside the RDs were exclusively methylated.
After we confirmed the chemical synthesis, we explored the The height decreased by about 21% from 5.06 Æ 0.07 nm to
switching properties of type III-C RDs. The most interesting properties 4.10 Æ 0.06 nm because the mechanical bonds squeeze into the
of type III-C RDs are the acid–base responsive globular switching core after the deprotonation (Fig. 1C), whereas in Me14G2-H7Á21PF6,
(Fig. 1). The deprotonation of RDs was performed with BEMP resin.
G2-H7Á7PF6 to G2, contraction was observed (Fig. 3E and F).
After deprotonation, significant NMR peak shifting was observed in
all RD spectra. The methylated RDs (Me6G1-H3Á9PF6 and Me14G2-H7Á
21PF6) exhibited a different switching process in comparison to the
ones without methylation. Interestingly, in the non-methylated RDs
(G1-H3Á3PF6 and G2-H7Á7PF6), the macrocycles at the periphery
moved toward the amide17 instead of the triazole unit, and only
the core macrocycle oscillated between triazoles. By taking the neutral
G1 [4]rotaxane dendrimers (G1) as an example (Fig. 2A and B), after
deprotonation, He and Hk shifted upfield significantly (DdHe,k
=
À0.84 ppm), proving that the macrocycles were not located at the
amine groups. In contrast, Hi experienced a downfield shift (DdHi =
0.32 ppm), attributed to the oxygen of DB24C8 interacting with the
amide protons through hydrogen bonding. In the downfield region,
only the triazole protons at the core Hm shifted as two sets of
resonances, by shuttling between two triazoles, and the shuttling
was slow on the NMR timescale. The chemical shift of the other two
triazole protons Hc and Hg was similar to that in the initial state. Also,
similar chemical shifts were observed in deprotonated neutral G2
[8]rotaxane dendrimers (G2) (ESI,† Fig. S8), revealing that the mole-
cular shuttling process occurred after the deprotonation.
Fig. 3 AFM images of type III-C rotaxane dendrimers on the mica surface
(A–F) (1 mm  1 mm) or the silicon surface (G and H) (1 mm  1 mm) and the
height profiles.
As expected, if RDs were methylated, a distinct shuttling process
was exhibited. After the methylation (Fig. 1), all triazoles turned to
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the removal of all protons leads to the oscillation of all mechanical in a basic environment, while methylated rotaxane dendrimers
bonds between MTA and causes extension (Fig. 1D). The height of tend to expand under basic conditions and contract under acidic
Me14G2-H7Á21PF6 was increased by about 8% from 5.11 Æ 0.11 nm conditions. These results of molecular ‘‘breathing’’ reveal that
to 5.58 Æ 0.16 nm (Fig. 3G and H). The dynamic light scattering both non-methylated and methylated rotaxane dendrimers could
(DLS) measurement was in agreement with AFM, supporting that be potentially applicable in binding acidic or basic drugs for
the size modulation was achieved after acid/base treatment (ESI,† actively pH-controlled drug release. Chlorambucil and lithocholic
Fig. S27 and S28).
acid were capable of binding with the deprotonated G1 and G2.
The neutral G1 and G2 were able to bind with guest molecules For the first time, MALDI-TOF MS has been used to investigate the
through electrostatic interaction and hydrophobic interaction.9 Two in vivo distribution of label-free, monodispersed type III-C rotax-
small molecular weight drug molecules, chlorambucil and lithocholic ane dendrimers as a potential cargo carrier. The in vivo experi-
acid, were separately used for the binding study. The 1H NMR titration ment indicated that G1/G2 tend to accumulate and remain in the
results (ESI,† Fig. S26–S29) showed that G1 was able to bind with two reticuloendothelial system enriched spleen and liver. This study
chlorambucil or three lithocholic acid molecules out of its three DBA has provided a new analytical method for evaluating label-free
sites, while G2 was capable of binding with six chlorambucil or seven dendrimers, dendritic materials and MIMs before biomedical
lithocholic acid molecules out of its seven DBA sites.
Since type III-C RDs are not fluorescent in nature but thanks to experiments are currently underway in our laboratory.
its monodispersity, we finally investigated the inter-organ distribu- We acknowledge the financial support from The State Key
applications and clinical trials. Drug delivery and more biological
tion of RDs in C57BL/6J mice with MALDI-TOF MS (Fig. 4 and ESI,† Laboratory of Environmental and Biological Analysis, The Inter-
Fig. S34–S47). In both G1 and G2, the accumulation in the spleen disciplinary Research Clusters (IRCS/17-18/03) and The President’s
and liver was higher than in other organs, implying that the highly Award for Outstanding Performance in Research Supervision to
lipophilic G1 and G2 were mainly retained in the reticuloendothelial K. C.-F. L. from The Hong Kong Baptist University. We also
system enriched organs (Fig. 4). The amount of G1 and G2 in each acknowledge Mr David T.-W. Chik for the helpful discussion on
organ after administration was increased gradually from 12 h to the MALDI experiment. We acknowledge the discussions with
24 h, and started to decrease from 36 h to 48 h due to excretion from Professor Sir Fraser Stoddart.
organs. Only a trace amount of RDs was found in the spleen and
none was detectable in other organs after 48 h, suggesting that the
retention-time of RDs in the mice was about 48 h and they were
Conflicts of interest
excreted from the organs. Moreover, a fragment or its metabolite ion
cannot be observed in the spectra (12 h to 48 h) indicating that both
G1 and G2 were stable in a physiological environment and did not
undergo degradation in vivo.
There are no conflicts to declare.
Notes and references
In conclusion, new type III-C RDs were successfully synthe-
sized and characterized by various spectroscopic and microscopic
techniques. Dual switching processes of G1/G2 and methylated
G1/G2 rotaxane dendrimers were demonstrated by NMR spectro-
scopy, and AFM and DLS analyses. The morphology of type III-C
rotaxane dendrimers tends to expand in an acidic and contract
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