In Situ Formation of Polymeric Nanoassemblies Using an Efficient Reversible Click Reaction

Article abstract of DOI:10.1002/anie.202004017

Polymer–drug conjugates are promising as strategies for drug delivery, because of their high drug loading capacity and low premature release profile. However, the preparation of these conjugates is often tedious. In this paper, we report an efficient meth

Full text of DOI:10.1002/anie.202004017

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Title: In Situ Formation of Polymeric Nanoassemblies Using an
Efficient Reversible Click Reaction
Authors: Bin Liu, Ruiling Wu, Shuai Gong, Hang Xiao, and Sankaran
Thayumanavan
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In Situ Formation of Polymeric Nanoassemblies Using an Efficient
Reversible Click Reaction
Bin Liu, Ruiling Wu, Shuai Gong, Hang Xiao, S. Thayumanavan*
Abstract: Polymer-drug conjugates are promising as strategies for
drug delivery, because of their high drug loading capacity and low
premature release profile. However, the preparation of these
conjugates is often tedious. In this paper, we report an efficient
method for polymer-drug conjugates using an ultrafast and reversible
click reaction in a post-polymerization functionalization strategy. The
reaction is based on the rapid condensation of aryl boronic acid
functionalities with salicylhydroxamates. The polymer, bearing the
latter functionality, has been designed such that the reaction with
boronic acid bearing drugs induces an in situ self-assembly of the
conjugates to form well-defined nanostructures. We show that this
method is not only applicable for molecules with an intrinsic boronic
acid group, but also for the other molecules that can be linked to aryl
boronic acids through a self-immolative linker. The linker has been
designed to cause traceless release of the attached drug molecules,
the efficiency of which has been demonstrated through intracellular
delivery.
because of the inherently low efficiency of the post-polymerization
method, the direct polymerization approach is more commonly
used for generating polymer-drug conjugates.[29-32] However,
syntheses of functional monomers, that are compatible with the
polymerization and self-assembly conditions, make the direct
polymerization method quite tedious.[29-32] Moreover, in general,
the covalently conjugated systems also require a mechanism by
which the drug is released from the nanoparticle in its active form.
Nanosized molecular carriers offer the ability to improve drug
pharmacokinetics and enhance the accumulation of therapeutics
at the tumor site, a potentially promising approach for tissue
targeting in cancer therapy.[1-18] The drug molecules can either be
non-covalently encapsulated or covalently conjugated to these
nanoparticles. The non-covalent approach is convenient in that a
broad range of drugs can be incorporated into the hydrophobic
interiors of an amphiphilic assembly.[5-8,18-22] In many of the
systems, however, the instability in encapsulation causes the
molecules to be prematurely released in biological systems, when
undergoing distribution-induced dilution.[23-28] Also, many of these
systems also exhibit low drug loading efficiency. The covalent
conjugation strategy has the inherent capability of addressing
both drug loading efficiency and encapsulation stability issues.
Covalent polymer-drug conjugates are obtained either through
direct polymerization of drug-containing monomers or with a post-
polymerization modification process.[29-32] Among the two,
Figure 1. Schematic illustration of polymer-drug reaction for conjugates with in-
situ induced nanoassembly of drug-conjugates formation and stimuli induced
pristine drug release process.
In sum, non-covalent encapsulation has the advantage of
simply mixing the drug and nanoassembly components to pre-
pare drug-containing nanoparticles, while the covalent approach
provides high thermodynamic stability of the conjugates. A useful
strategy would combine the advantages of procedural ease of the
non-covalent method with the stability and efficiency of
encapsulation offered by the covalent conjugation method.
Additionally, it is critical that the strategy also allows for the drug
to be efficiently released in its pristine active form at the target
location.
We were inspired by reaction induced self-assembly (RISA)
approaches via tuning the hydrophilic-lipophilic balance (HLB),
such as the polymerization induced self-assembly (PISA), for
preparing nanoparticles. [33-35] While the fundamentals of the self-
assembly process have been quite nicely explored, we became
interested in developing systems with clear biomaterials
implications. In this paper, we use a simple and efficient
[*]
B. Liu, R. Wu, S. Gong, Prof. S. Thayumanavan
Department of Chemistry, University of Massachusetts, Amherst,
Massachusetts 01003, USA
Email: thai@chem.umass.edu
Prof. H. Xiao
Department of Food Science, University of Massachusetts, Amherst,
Massachusetts 01003, USA
Prof. H. Xiao, Prof. S. Thayumanavan
Center for Bioactive Delivery, Institute for Applied Life Sciences,
University of Massachusetts, Amherst, Massachusetts 01003, USA
Prof. H. Xiao, Prof. S. Thayumanavan
Molecular and Cellular Biology Program, University of
Massachusetts, Amherst, Massachusetts 01003, USA
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.202004017
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reversible click reaction as the post-polymerization modification
strategy to prepare polymer-drug conjugates (Figure 1).
used for in situ conjugation of small molecule drugs onto polymers
with high efficiency. Furthermore, the pH-induced reversibility of
this reaction should also offer controlled drug release under mild
acidic conditions.
Specifically, we utilize
a fast and efficient boronic acid-
salicylhydroxamate reversible click reaction for the drug
conjugation, which then also induces the in situ self-assembly of
the conjugates. In addition to the highly efficient conjugation
reaction, the versatility of this approach is further demonstrated
with two more features in the drug release process. First, since
the click reaction is reversible, the process naturally lends itself to
stimulus-induced release of drug molecules from the conjugate.
In addition to utilizing boronic acid itself as the handle for causing
the stimulus-induced release, we also show that the strategy
lends itself to introducing other cue-induced release of drug
molecules, using linker engineering. The versatility of the method
to accommodate a variety of functional groups demonstrates its
broad applicability to drug molecules (beyond boronate-based
ones). Similarly, the ability to tune molecular release in response
to different stimuli offers to engineer drug release profiles for a
variety of biological microenvironments. We disclose these
findings in this manuscript.
To test the utility of such a process, we used a potent boronic
acid-based drug. As boronic acid moiety is considered to be a
biostere of carboxylic acid, boronic acid-based drugs have been
developed, especially as proteasome inhibitors. For instance,
bortezomib (BTZ) is an FDA-approved anticancer drug with a
boronic acid functionality. (Figure 2a).[41-43] In the present work,
BTZ was first chosen as the candidate drug for the study here. To
conjugate this molecule to the polymer, the latter must be
functionalized with the salicylhydroxamate (SaH) group. Thus,
polymer P1 was prepared by an initial RAFT co-polymerization of
trityl-protected
salicylhyroxymate
monomer
and
PEG-
methacrylate monomer (MW=500 Da), followed by the
deprotection of the trityl group. The purpose of the PEG-based
co-monomer is to make the polymer sufficiently hydrophilic and
potentially biocompatible, while the SaH functionality in the other
co-monomer offers the handle for drug conjugation. The ratio of
the two monomers in P1 was designed to be 1:1, which was
confirmed by NMR (See Supplemental Experimental Procedures,
Figure S19). For fluorescence microscopy studies,
a
corresponding rhodamine-labelled polymer P2 was synthesized
through copolymerization of a small percentage (0.5% mole ratio)
of a third dye-functionalized monomer. Synthetic protocols and
characterizations of the polymers P1 (Mw=10.1 kDa, Ɖ=1.37) and
P2 (Mw=10.3 kDa, Ɖ=1.46) are detailed in supporting information.
Figure 2. Characterization of the in-situ reaction induced polymer-drug
conjugates. a) Chemical illustration of the formation of polymer P1-BTZ
conjugate and stimulus induced drug release, b) DLS for size of nanoassembly
of polymer P1 and P1-BTZ conjugate, c) correlation function of P1 and P1-BTZ
conjugate, d) Structure of rhodamine b labelled polymer P2-BTZ conjugate, e)
Normalized UV absorbance for rhodamine b labelled polymer P2 and P2-BTZ
conjugate.
Figure 3. Cell uptake and toxicity of the polymer-drug conjugates. (a) confocal
images for cell uptake of polymer-BTZ conjugate toward different cell lines (BF:
bright field). Cells were incubated with rhodamine b labelled polymer-BTZ
conjugate for 4 h. (b) Cytotoxicity (MTT assay) of polymer-BTZ conjugate toward
different cell lines at different times. The concentration is based on the drug
(BTZ).
The reaction between salicylhydroxamate and boronic acid
functionalities is considered to be highly efficient with the
bimolecular rate constant of ~7x106 M-2s-1.[36] With this rate, this
reaction would be considered to be the fastest click reaction
reported thus far.[36-40] We envisaged that the fast kinetics can be
The polymer-drug conjugate was prepared by simply mixing
BTZ and P1. The polymer itself did not exhibit any self-assembly
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(or formed ill-defined polymer aggregates), as discerned by the
poor correlation function observed in dynamic light scattering
(DLS) experiments (Figure 2c). However, in the presence of BTZ,
an in-situ formation of a nanoassembly with a size of ~50 nm was
observed (Figure 2b and S1). This suggests that the addition of
the more hydrophobic drug molecule causes the polymer to attain
a hydrophilic-lipophilic balance needed for self-assembly into a
nanoparticle. Drug conjugation to the polymer was demonstrated
by UV absorbance (Figure 2e). We used the absorption peak from
the rhodamine dye in P2 as the internal standard to demonstrate
the loading of BTZ onto the polymer assembly, where the P2-BTZ
exhibited similar size of 50 nm as P1-BTZ conjugates (Figure 2c
and S2). Absorbance spectra of the polymer and the polymer-
drug conjugate, normalized at 550 nm, shows a significantly
higher absorbance at ~300 nm for the latter. This increase is
attributed to the conjugation of BTZ to the polymer. We also
confirmed that the drug conjugation does not affect rhodamine
fluorescence in P2 (Figure S2c). Further, loading efficiency and
capacity of BTZ in P1-BTZ conjugates was quantified by LC-
MS/MS, which showed a loading capacity of 25% (246 μg BTZ
per mg of P1) and loading efficiency of 50% (Figure S3).
revealed a pH dependent drug release feature, which exhibited
stable encapsulation at physiological pH (7.4) with extended
release kinetics under acidic pH (5.0) (Figure S4). Further, the
polymer-drug conjugate nanoassembly was evaluated with three
different cancer cell lines (HeLa, MDA-MB-231, and MCF-7).
Rhodamine-labelled P2 was used to track the location of the
conjugate in cells with confocal microscopy, while the cell nucleus
was stained with hochest 3332. The conjugate exhibited good cell
uptake for all the cell lines as the bright red color was dispersed
throughout the cells, around the nucleus after 4 h (Figure 3a).
Moreover, the assembled drug conjugate can efficiently kill all of
three cancer cell types with clear dose- and time-dependent
profiles (Figure 3b), where the polymer itself did not exhibit any
toxicity even at a high concentration of 1 mg/mL (Figure S4). We
also noted that the conjugates cause the cell death at a slower
rate (compare with Figure S5 and Table S1), which is attributed
to the controlled release of the drug from the conjugate (Figure
S6).
The results above are promising for the intracellular delivery of
drugs that contain an intrinsic boronic acid group. However, most
small molecule drugs do not bear a boronic acid functional group.
To extend the methodology to other functional groups that are
commonly found in drug molecules, we were interested in
developing linkers that bridge the boronic acid functional group
and the drug molecules. Since chemical modifications might
significantly decrease the potency of drugs, we targeted a
stimulus-responsive degradable linker that offers to release the
drug in its traceless active form at the targeted site. To this end,
we first used self-immolative linkers that are responsive to
reactive oxygen species (ROS), as highly oxidizing environment
is found in certain pathological sites such as in tumor cells, where
the concentration is 2 to 3 orders of magnitude higher than in
normal cells and can reach up to 100 µM. [44-47] The boronic acid
functional group has two functions in that it is the key click reaction
handle and is also inherently ROS responsive.
The molecular design, which allows for the tertiary alcohol of
camptothecin (CPT) to be used as the handle for conjugation, is
shown in Figure 4a (see SI for synthetic details). When the
nanoconjugate is subjected to ROS conditions, the aryl boronic
acid moiety would be oxidized to the corresponding phenol, which
would induce a 1,6-benzyl self-immolation cascade [48-52] to
release the CPT molecule in its original form. CPT is an anti-
cancer drug with a well-established pharmacology profile.[53] The
procedure for preparation of the prodrug with a boronic acid
terminal functional group is detailed in the Supporting Information.
The formation of the nanocarrier P1-CPTROS (Figure 4a) was
achieved through an in-situ reaction between the
salicylhydroxamate group of the polymer P1 and boronic acid
group of prodrug in aqueous media (see SI for details). The
resultant polymer-drug conjugate self-assembled to nanosized
particles with a diameter of ~70 nm (Figure 4c and 4d).
Transmission electron microscopy (TEM) studies suggest that the
nanoaggregate had a spherical shape with the size of ~50 nm
(Figure 4e). The smaller size in TEM, compared to that found with
dynamic light scattering (DLS), is likely due to the shrinkage of the
particles upon drying in the former technique. Here too, note that
the polymer itself exhibits ill-defined aggregates, as ascertained
by the bad correlation function (Figure 2d). Again, this suggests
that the in-situ conjugation-induced self-assembly of polymeric
Figure 4. Polymer-prodrug conjugates formation. a) structure of ROS sensitive
polymer-drug conjugates (P1-CPTROS), b) structure of redox sensitive polymer-
drug conjugates (P1-CPTredox), c) DLS of the assemblies from polymer-drug
conjugates, d) correlation functions for these assemblies from DLS data, e) TEM
image of in-situ formed assembly of ROS sensitive polymer-drug conjugates
(P1-CPTROS), f) TEM image of in-situ formed assembly of redox sensitive
polymer-drug conjugates (P1-CPTredox).
Following the formation of the nanoassembly, we monitored the
drug release under different pH values. The P1-BTZ conjugates
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nanoparticles can be achieved. The characteristic absorption
peak of CPT at ~366 nm (Figure S7) further confirmed the drug
conjugation. This distinct peak allowed for the quantification of the
drug loading, which was found to be as high as ~70% for this post-
polymerization conjugation method (Figure S7 and S8). In
comparison, the physical encapsulation of CPT drug without
boronic acid functionalization exhibited very poor loading capacity
mM DTT (mimics intracellular reduction conditions) and the
release kinetics was monitored (Figure 5d). The drug can be
efficiently released, as all of the prodrug can be transformed into
CPT within 1 h. In comparison, there is negligible release amount,
when 10 µM DTT (mimics extracellular conditions) was used
(Figure S25). Further, this linker is also hydrolytically stable under
different pH values (7.4 and 5.0) (Figure S27). These results
suggest that the CPT drug can be selectively released in
response to an intracellular biological trigger. Further, cell toxicity
for the nanoassembly of this polymer-drug conjugate was also
studied toward three different cell lines (HeLa, MDA-MB-231,
MCF-7). The polymer-drug conjugates can efficiently kill the
cancer cells with dosage and time dependence (Figure 5f, Figure
S28 and Table S2).
(~2%)
and
loading
efficiency
(~5%)
(Figure
S9), further demonstrating the advantages of the RISA process.
Further, we tested the scope of the methodology by tuning the
ratio of the SaH functional group to PEG unit in polymers (10%,
25% and 75% SaH group). The reaction between polymers and
boronic acid containing prodrug (CPTROS) resulted in good
nanosized assemblies for conjugates, except P3-CPTROS with the
smallest amount of the hydrophobic functional group (10%). Also,
all polymers (P1-P5), by themselves, exhibited ill-defined
aggregates (Figure S10-S12). These results were consistent with
the RISA process for nanoassembly formation, through
alterations in the polymer HLB. It is also to be noted that all these
polymers exhibited very high loading drug efficiency (70-90%)
(Figure S13).
We hypothesized that the linker would offer to release CPT in
its pristine form, upon encountering the ROS stimulus. To
investigate this possibility, the nature of the released molecules
was analyzed in the presence of H2O2, using LC-MS/MS (see SI
for details, Figure S14-S23). The elution profile shows the
evolution of the release of pristine drug, suggesting that the self-
immolation reaction indeed causes traceless drug release. In
control, the linker is hydrolytically stable under different pH values
(7.4 and 5.0) (Figure S24). Also, the drug release kinetics was
understandably dependent on the ROS concentration (Figure 5c
and S25). Due to the easy formation of polymer-drug conjugates
with well-defined nanostructure and triggerable release profile,
the cytotoxicity of the drug conjugates was assessed toward
different cancer cell lines. The polymer-drug conjugate also
exhibited good cell killing ability with
a
concentration
(encapsulated CPT drug) and time-dependent profile (Figure 5e),
which is comparable to the free CPT (Figure S28 and Table S2).
To further expand the scope of this approach, we were
interested in developing a strategy where the conjugating boronic
acid moiety itself is not involved in the stimulus-induced release
of the drug. To this end, we targeted a glutathione (GSH)
responsive linker, as this tripeptide concentration is ~103 times
higher inside cells, compared to the extracellular environment.[54-
56] The linker is based on a disulfide bond, which is connected to
the β-position of the carbonate functionality that connects to the
drug, CPT. The relative positioning of these two functionalities
would ensure a self-immolative cyclization, [48,57] when the
disulfide bond is reductively cleaved to produce the
corresponding thiol (Figure 4b and 5b). The other terminus of the
linker contains the boronic acid moiety that would connect with
the polymer P1 to cause the in-situ reaction induced self-
assembly. Similar to the ROS sensitive CPT-based
nanoassembly above, a ~70 nm spherical assembly was
observed, as discerned from DLS and TEM studies (Figure 4d-4f).
The drug loading efficiency was found to be ~74% (Figure S8).
The traceless release of CPT drug from the redox sensitive drug
conjugates was also demonstrated by LC-MS/MS (details in
Supporting Information, Figure S14-S26) in the presence of 10
Figure 5. Drug release under stimuli. a) schematic illustration of the drug
release from ROS sensitive polymer-drug conjugates, b) schematic illustration
of the drug release from redox sensitive polymer-drug conjugates, c) LC-MS/MS
MRM chromatogram for monitoring CPT release from CPTROS under 1 mM H2O2,
d) LC-MS/MS MRM chromatogram for monitoring CPT release from CPTredox
under 10 mM DTT, e) cytotoxicity studies for ROS sensitive polymer-drug
conjugates toward different cells after different time incubation, f) cytotoxicity
studies for redox sensitive polymer-drug conjugates toward different cells after
different time incubation. The concentration is based on the equivalent CPT
drug.
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10.1002/anie.202004017
Angewandte Chemie International Edition
COMMUNICATION
Entry for the Table of Contents
COMMUNICATION
Bin Liu, Ruiling Wu, Shuai Gong, Hang
Xiao, S. Thayumanavan*
Page No. – Page No.
In Situ Formation of Polymeric
Nanoassemblies Using an Efficient
Reversible Click Reaction
Superfast reversible
In-situ reversible click reaction induced polymer-drug conjugates and formation of nanoassembly with stimuli-induced pristine
drug release process.
This article is protected by copyright. All rights reserved.