Disassembly of polymeric nanoparticles with enzyme-triggered polymer unzipping: Polyelectrolyte complexes: Vs. amphiphilic nanoassemblies

Article abstract of DOI:10.1039/d0cc03257c

Alkaline phosphatase (ALP) responsive polymers, which can unzip from head to tail are reported. Hydrophilic and hydrophobic modification of the polymer was carried out for the formation of a polyelectrolyte complex and an amphiphilic nanoassembly, respect

Full text of DOI:10.1039/d0cc03257c

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DOI: 10.1039/D0CC03257C
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Disassembly of Polymeric Nanoparticles with Enzyme-Triggered
Polymer Unzipping: Polyelectrolyte Complexes vs. Amphiphilic
Nanoassemblies
Received 00th January 20xx,
Accepted 00th January 20xx
Vikash Kumar a, Oyuntuya Munkhbat a, Hatice Secinti a, S. Thayumanavana, b,c*
DOI: 10.1039/x0xx00000x
Alkaline phosphatase (ALP) responsive polymers, which can unzip
from head to tail are reported. Hydrophilic and hydrophobic
modification of polymer was carried out for polyelectrolyte
complex and amphiphilic nanoassembly formations, respectively,
which offered distinct enzyme-triggered disassembly kinetics.
Dysfunctional enzymes are the primary culprits in many
pathological conditions.1,2 Therefore, developing enzyme
responsive nanomaterials that exhibit programmable and
functional response are attractive in applications in many areas
including therapeutic delivery, diagnostics and sensing.3–7 To
this end, several platforms involving polymeric8,9, oligomeric10
and dendritic11,12 assemblies have been developed, in which a
desired response is achieved through substrate-specific activity
of enzymes. A major limitation with these amphiphilic self-
assembling systems is their slow response to enzymatic
treatment, compared to their small molecule substrate
counterparts. This is primarily because the substrate
functionalities are often buried in the hydrophobic core of these
self-assembled structures, thus limiting accessibility for
enzymes.13–16
Scheme 1: (A) Structure of ALP triggerable polymer, P0; its hydrophilic and
hydrophobic modification into P1 and P2; (B) Proposed schematic of
particle formulation using P1 and P2 and its triggered disassembly in
response to ALP.
hydrophilic side of the polymer to the rest of the assembly. We
envisioned an alkaline phosphatase (ALP) triggerable polymer,
represented by the poly(benzyl carbamate) P0 and its
hydrophilic and hydrophobic analogues P1 and P2 respectively
(Scheme 1A). The phosphate terminus in these polymers are
substrate functionalities for ALP, which undergo a cleavage
reaction to set-off the depolymerization cascade, because of
the revealed phenolic functionality. When these polymers are
processed to form nanoparticles, we envisaged that the
depolymerization process would cause an enzyme-induced
disassembly and a molecular release of the non-covalently
bound guest molecules (Scheme 1B).
Poly(benzyl carbamate) backbone was synthesized using a
previously reported procedure24 and its chain terminus was
capped using a derivative of benzyl ether protected phosphate
trigger. Deprotection of benzyl ether groups from the trigger,
and t-butyl ester groups from the side chain of polymer was
carried out to synthesize ALP-responsive P0 (see SI). Average
number of repeating units in the polymer was ⁓12. Further, the
terminal alcohol group from P0 was used for the hydrophilic and
Responsive depolymerization has attracted recent focus,
because it offers
a
convenient pathway for signal
amplification.17–20 In these cases, a single stimulus induced
activation event at a polymer chain terminus leads to the
unzipping of polymer chain from head to tail.18,21,22 Utilizing a
specific enzyme as an input signal to trigger the disassembly of
nanoparticles formulated using depolymerizable polymers is a
promising, yet relatively less explored, approach to address the
slow kinetics of enzyme-triggerable materials.23 Specifically, we
were interested in utilizing the depolymerization pathway as
the mechanism to transduce an enzymatic reaction on the
a. Department of Chemistry, University of Massachusetts, Amherst, MA-01003, USA.
b. Centre for Bioactive Delivery, Institute for Applied Life Science, University of
Massachusetts, Amherst, MA-01003, USA.
c. Molecular and Cellular Biology Program, University of Massachusetts, Amherst,
MA-01003, USA.
Electronic Supplementary Information (ESI) available: [details of any supplementary
information available should be included here]. See DOI: 10.1039/x0xx00000x
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Next, the self-assembly behaviour of P0 was studied to test
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DOI: 10.1039/D0CC03257C
if the enzyme triggered molecular-scale depolymerization
behaviour can be translated to nanomaterials with similar
kinetics. The aqueous phase solubility of P0 by itself was
deemed to be relatively poor. Therefore, we modified P0 with a
hydrophilic PEG chain using its terminal alcohol group to react
with poly(ethylene glycol)isocyanate. Excess poly(ethylene
glycol)isocyanate was removed through multiple washings of
the reaction mixture with methanol (Figure S7). The resultant
polymer P1 was indeed found to be more water soluble.
Carboxylic acid functionalities from the side chain of P1 were
then used to form
a
polyelectrolyte complex with
stoichiometric amount of positively charged poly
(diallyldimethylammonium chloride) (PDADMAC). Average
diameter of the polyelectrolyte complex particles was found to
be ~183 nm in dynamic light scattering (DLS) (Figure S8). Particle
morphology was studied using transmission electron
microscopy (TEM) which revealed spherical solid nanoparticle
morphology (Figure S9). The particles were also used to non-
covalently encapsulate a fluorescent hydrophobic guest, DiI.
Subsequently, the effect of ALP on the polyelectrolyte
complex particles was studied. The nanoparticle disassembly
hypothesis here is that the ALP-induced decrease in the
Figure 1: (A) Depolymerization of P0 in presence of ALP to form R1; (B)
UV-Vis of P0 solution at different time with ALP incubation; (C) UV-Vis of
P0 without ALP incubation; (D) UV-Vis comparison of (P0 + ALP) solution
and small molecule reporter, R1; (E) Unzipping % of P0 with time.
hydrophobic modification using poly(ethylene glycol) polymer chain length would weaken the polyvalent interaction
isocyanate (Mn ~5000) and octadecylisocyanate to achieve P1 between P1 and PDADMAC. Indeed, the polyelectrolyte
and P2 respectively (see SI for synthetic scheme and details).
nanoparticles revealed the formation of small molecule
Prior to nanoparticle formulation, the enzyme triggered reporter, R1, with time in the presence of ALP. However, the
depolymerization process was studied using the non- kinetics was found to be substantially slower, compared to the
assembling polymer P0 in bicarbonate buffer (250 mM, unzipping of P0 in solution (Figure 2A, 2B, S10).
pH=8.5). Briefly, the phosphate group is cleaved by ALP to
generate a phenolic chain terminus, which triggers the chain
unzipping process to afford monomer-like products, including
the amino-cinnamic acid derivative R1 (Figure 1A and S1). The
distinct UV-absorption features of R1 at 348 nm served as the
characterization handle to monitor the chain unzipping process
(Figure 1B). First, the initial step of phosphate group cleavage
was studied using 31P NMR, which showed the formation of
phosphoric acid within 10 min after addition of 3 nM ALP to 0.2
mM concentration of P0 (Figure S2). Then, the temporal
evolution of the absorbance peak at 348 nm (Figure 1B),
corresponding to the formation of R1, was monitored (the small
molecule R1 was independently synthesized to confirm the
peak at 348 nm, as shown in Figure 1D). While there is a clear
increase in the intensity of this peak with time in the ALP-
incubated sample, no such peak was observed without the
enzyme (Figure 1C). Formation of R1 was also confirmed using
1H NMR (Figure S3). As an additional control, the enzyme ALP
itself also did not show any absorbance peak at 348 nm (Figure
S4). To further test if the polymer unzipping event is specific
only to ALP-triggering, the polymer solution was incubated with
a non-specific enzyme, porcine liver esterase. Here, no change
was observed in the absorbance profile (Figure S5), confirming
that the depolymerization is indeed specifically triggered by
Figure 2: (A) UV-Vis of P1 + PDADMAC complex after ALP incubation; (B)
ALP. Using the absorbance calibration curve of R1 (Figure S6),
Kinetics of evolution of absorbance at 348 nm after incubation of P1 +
we estimated that ~80% of capped polymer undergoes
depolymerization in 90 min (Figure 1E).
PDADMAC complex with ALP; (C) 31P NMR study of P1 + PDADMAC after
ALP incubation at different time intervals.
2 | J. Name., 2012, 00, 1-3
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Depolymerization of P0 reached saturation within ~60 min,
while the unzipping of P1 in the P1-PDADMAC complex took ~24
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Characterization of the P2 nanoparDtiOclIe: 1s0.w10a3s9/Dd0oCnCe03u2s5in7Cg
h. Also, monitoring the release of the encapsulated microscopy imaging techniques and dynamic light scattering. In
hydrophobic dye molecule showed that molecular release from TEM, spherical morphology with homogeneous distribution of
these nanoparticles is negligible over the 24 h time period the particles was observed (Figure 3A). Since, we used Nile red
(Figure 2A). We surmised that the slow response of the complex as a hydrophobic guest molecule, red fluorescence was
could be due to the phosphate groups being buried in the observed in fluorescence microscopy image of the particles
polyelectrolyte nanoparticle complex, limiting accessibility to
ALP enzyme.23 To test this possibility, time-dependent 31P NMR
study of the complex was carried out. NMR peak, corresponding
to the phosphate group was not observed initially, likely due to
the restricted segmental mobility in the polyelectrolyte
complex. However, after incubating the complex particles with
ALP, formation of the liberated phosphoric acid was indeed
observed, but this appearance was significant only after 20 h
(Figure 2C). In contrast to the free polymer P0, the ALP-induced
cleavage of the phosphate group itself was substantially slower,
supporting the assertion that the availability of phosphate
moieties for the enzymes is limited in the polyelectrolyte
nanoparticles from P1.
To address this accessibility issue, we targeted
a
nanoparticle formulation approach that does not involve
electrostatic complexation. In addition to P0 being limited in
aqueous solubility, we envisaged that it also lacked hydrophilic-
lipophilic balance needed to form amphiphilic nanoassemblies.
Therefore, a hydrophobic modification of P0 was carried out
using octadecyl isocyanate, to form the polymer P2. This
polymer is reasonably soluble in volatile organic solvents, such
as chloroform. Hence, an oil-in-water emulsion methodology
was used for assembly formation.25 Briefly, suspension of P2
and a hydrophobic guest, Nile red, in chloroform (oil phase) was
prepared and added to an aqueous solution of poly(vinyl
alcohol) co-surfactant (water phase). Introduction of
mechanical force using sonication, led to the formation of oil
droplets containing polymer and guest molecules, stabilized by
poly(vinyl alcohol) at the interface. Evaporation of the organic
solvent and subsequent washing of excess surfactant molecules
led to the formation of P2-based nanoparticles with
encapsulated Nile red.
Figure 4: (A) UV-Vis of P2 based emulsion nanoparticles after incubation
with ALP; (B) Kinetics of evolution of absorbance at 348 nm after
incubating P2 emulsion nanoparticles with ALP; (C) Guest molecule
release profile from the particle (red- with ALP; black- without ALP); (D)
TEM image of P2 based emulsion nanoparticles after ALP addition.
(Figure 3B). This revealed the confined location of guest
molecules in the particle interior. Further, scanning electron
microscopy (SEM) was also used to confirm the spherical
morphology of nanoparticles (Figure 3C). Average diameter of
the particles was found to be ⁓230 nm, as measured using DLS
(Figure 3D).
After the successful formation and characterization of
nanoparticles using polymer P2, particle disassembly was
studied in the presence of ALP. Colloidal suspension of particles
was prepared in 2.5 mM bicarbonate buffer (pH=8.5). After
incubating the particles with ALP, we observed the formation of
small molecule reporter, R1, because of polymer chain
unzipping (Figure 4A, 4B, S11). It is noteworthy that significant
amount of the unzipping product appeared within just 1h,
compared to the formation of the product requiring 24 h in the
case of P1-PDADMAC complex. Concurrent with the polymer
unzipping, release of encapsulated guest molecules was also
observed (Figure 4A, 4C). In the control solution, without ALP,
no evolution of the small molecule reporter formation was
noted (Figure S12). Additionally, there was no guest release in
the absence of enzyme, implying that the particles were stable
hosts in the absence of enzyme. After ALP addition, spherical
morphology of the particles was also lost, and the resultant
particles were ill-defined, as evidenced by TEM studies (Figure
4D). The kinetics of P2 unzipping is in fact comparable to the
Figure 3: Characterization of emulsion nanoparticles using (A)
Transmission electron microscopy (TEM); (B) Fluorescence microscopy; (C)
Scanning electron microscopy (SEM); (D) Dynamic light scattering (DLS)
This journal is © The Royal Society of Chemistry 20xx
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7
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hydrophilic terminus of the amphiphilic polymer ensures that
this moiety is exposed and available for processing by the
enzyme in the amphiphilic assembly. This results in a rapid and
amplified response leading to nanoparticle disassembly and
release of the encapsulated guest molecules.
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slower in response, compared to the free polymer, while the
kinetics of unzipping of the polymer in the amphiphilic
assemblies was comparable to that of the free polymer. This
molecular scale difference also translated to differences in
kinetics of disassembly of the nanomaterials, where the host-
guest properties of these materials were compromised by the
presence of enzyme. The difference is attributed to the
variations in the degree of accessibility of the enzyme-
responsive functionalities in the context of their orientations
within the nanoparticle. Results outlined in this work are
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Conflicts of interest
22 A. P. Esser-Kahn, N. R. Sottos, S. R. White and J. S. Moore, J. Am.
Chem. Soc., 2010, 132, 10266–10268.
The authors declare no conflicts of interest.
23 J. Zhuang, H. Seçinti, B. Zhao and S. Thayumanavan, Angew.
Chem. Int. Ed., 2018, 57, 7111–7115.
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0568) is gratefully acknowledged.
25 N. Fomina, C. McFearin, M. Sermsakdi, O. Edigin and A.
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