Dual responsive polymeric nanoparticles prepared by direct functionalization of polylactic acid-based polymers via graft-from ring opening metathesis polymerization

Article abstract of DOI:10.1039/c5cc07882b

Polylactic acid (PLA) has found widespread use in plastics and in biomedical applications due to its biodegradability into natural benign products. However, PLA-based materials remain limited in usefulness due to difficulty of incorporating functional groups into the polymer backbone. In this paper, we report a strategy for PLA functionalization that establishes the preparation of highly derivatized materials in which ring opening metathesis polymerization (ROMP) is employed as a graft-from polymerization technique utilizing a norbornene-modified handle incorporated into the PLA backbone. As a demonstration of this new synthetic methodology, a PLA-derived nanoparticle bearing imidazole units protected with a photolabile group was prepared. The morphology of this material could be controllably altered in response to exposure of UV light or acidic pH as a stimulus. We anticipate that this graft-from approach to derivatization of PLA could find broad use in the development of modified, biodegradable PLA-based materials.

Full text of DOI:10.1039/c5cc07882b

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Dual responsive polymeric nanoparticles
prepared by direct functionalization of polylactic
acid-based polymers via graft-from ring opening
metathesis polymerization†
Cite this:DOI: 10.1039/c5cc07882b
Received 20th September 2015,
Accepted 22nd October 2015
Kate M. Veccharelli, Venus K. Tong, Jennifer L. Young, Jerry Yang* and
Nathan C. Gianneschi*
DOI: 10.1039/c5cc07882b
www.rsc.org/chemcomm
Polylactic acid (PLA) has found widespread use in plastics and in challenging to produce high molecular weight polymers with a
biomedical applications due to its biodegradability into natural large weight percentage of the added functionality. This is likely
benign products. However, PLA-based materials remain limited in due to decreased rates of polymerization resulting from the
usefulness due to difficulty of incorporating functional groups into steric bulk of functionalized lactides, leading to incomplete
the polymer backbone. In this paper, we report a strategy for PLA polymerization of the substituted monomer.11,16 Therefore,
functionalization that establishes the preparation of highly deriva- ring substitution has a major impact on the polymerizability
tized materials in which ring opening metathesis polymerization of functionalized lactide monomers.11,16 To generate PLA poly-
(ROMP) is employed as a graft-from polymerization technique mers with a higher degree of functionality, post polymerization
utilizing a norbornene-modified handle incorporated into the PLA modification strategies via a graft-to approach have been
backbone. As a demonstration of this new synthetic methodology, a demonstrated. For example ‘‘Click’’ chemistry allows functionality
PLA-derived nanoparticle bearing imidazole units protected with a to be incorporated into the polymer relatively easily since the
photolabile group was prepared. The morphology of this material backbone and the side chains can be prepared separately prior to
could be controllably altered in response to exposure of UV light or coupling.12,15,17–20 However, efforts to increase graft densities are
acidic pH as a stimulus. We anticipate that this graft-from approach usually limited as a result of steric repulsion between the bulky
to derivatization of PLA could find broad use in the development of side chains.21–24 Another way to prepare functionalized PLA
modified, biodegradable PLA-based materials.
materials is to utilize a graft-through polymerization method. In
this scenario, a macromonomer is polymerized via polymerizable
Polymers with hydrolyzable backbones such as polylactic acid end groups to create a brush polymer. PLA polymers have been
(PLA) have opened new avenues for research and development synthesized via this method by coupling the PLA polymer to a
with their enhanced biodegradability and resulting low toxicity strained olefin and subsequently polymerizing the olefin, resulting
for in vivo use.1 PLA has been utilized heavily as a biodegradable in bottle-brush PLA polymers.25–28 Utilizing this grafting-through
polymer in numerous in vivo applications ranging from surgical technique is attractive for polymer synthesis because it does not
sutures,2 surgical implants3 and drug delivery systems.4,5 However, entail orthogonal chemistries for grafting various side chains.
the utility of PLA is limited as the polymer backbone is difficult to However, this polymerization technique can be challenging due
chemically functionalize. As such, significant effort has been to the increased steric hindrance of the propagating polymer
expended to increase functionalization of PLA polymers and to chain. As a result, polymerizations can be slow and may not
tune its mechanical properties.6–9
proceed to complete conversion.21–24
Current strategies for functionalizing PLA typically involve
An alternative technique utilizes a graft-from polymerization
copolymerization of D,L-lactide with functionalized lactides in strategy to incorporate initiation groups into ROP monomers, such
the presence of a catalyst and an initiator to generate func- as cyclic esters and carbonates, prior to ROP. This novel technique
tionalized PLA copolymers via ring opening polymerization has been demonstrated in the literature only a handful of times.29–33
(ROP).10–15 While this technique has led to the development of In this method, polymer chains are grown from the polymer as a
a range of functionalized PLA polymers, it remains synthetically macroinitiator, with multiple initiation sites located along its back-
bone. This technique should allow for greater graft densities along
the polymer backbone since only small monomers are added to the
Department of Chemistry and Biochemistry, University of California,
growing polymer chain, mitigating steric repulsion.21–24
San Diego 9500 Gilman Drive, La Jolla, CA 92093, USA.
Here we report the use of a graft-from polymerization tech-
E-mail: jerryyang@ucsd.edu, ngianneschi@ucsd.edu
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5cc07882b nique to generate chemical functionality onto a PLA backbone
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via ring opening metathesis polymerization (ROMP). ROMP
was chosen as the grafting technique because it can be initiated
by catalysts with known tolerance to a range of functional
groups and results in polymers with narrow dispersity.34 In this
method, PLA polymers containing ROMP-reactive norbornene
handles were first prepared. The norbornene units on this
starting polymer were then used to prepare an initiator, which
could be readily functionalized by reaction with substituted
norbornene monomers. As a demonstration of the utility of
this strategy, nanoparticles that change morphology in response
to both pH and UV light were prepared.
Fig. 1 Synthesis of functionalized PLA-based polymers utilizing a graft-from
ROMP strategy. (A) ROP synthesis: 15 mol% of monomer 1 was copolymerized
with lactide in the presence of Sn(Oct)2 as the catalyst and 4-tert-butylbenzyl
alcohol as the initiator (B) ROMP polymer synthesis: (i) polymer 2 was mixed
with Ru initiator. (ii) Polymer 2 with catalyst loaded was then mixed with
norbornene monomers as shown. (iii) Termination of polymerization using
ethyl vinyl ether to generate final polymers 3 and 4.
Preparation of a norbornene-functionalized PLA polymer
began with the synthesis of the bifunctional lactide 1, which
was prepared in five steps from commercially available exo-5-
norbornenecarboxylic acid in 14% overall yield (see ESI,† for
experimental details). Norbornene-substituted PLA polymers
were then synthesized by copolymerizing lactide with 15 mol%
of monomer 1, utilizing 0.7 mol% of stannous octoate as
the catalyst and 4-tert-butylbenzyl alcohol as an initiator.11 added to the catalyst-loaded polymer 2 and allowed to stir
The resulting polymers were then characterized by size exclu- for 1 h before quenching with ethyl vinyl ether, generating
sion chromatography coupled with multiangle light scattering polymer 3 (Fig. 1).
1
(SEC-MALS) and H-NMR to determine the percent conversion
This technique allowed for the preparation of polymer 3
of lactides to polymer, the number-average molecular weight without generating any free ROMP polymer, as verified by
(Mn), and the dispersity (Ð or Mw/Mn) of the copolymers the presence of a monomodal distribution in the SEC-MALS
(Table 1). ROP of the lactides afforded a 98% conversion of chromatogram (Fig. S2B, ESI†). SEC-MALS also verified the
monomers to norbornene functionalized polymer 2.
success of the grafting technique by showing an increase in
The norbornene units on polymer 2 were then prepared as molecular weight of the PLA polymer 3 from Mn = 32 000 (Ð 1.4)
initiation sites by addition of polymer 2 dropwise to a pyridine- to Mn = 51 000 (Ð 1.8) after the addition of the phenyl monomer
modified variant of Grubbs’ second generation catalyst, (IMesH2)- (see Fig. S2a and b, ESI†). As a negative control, unfunctionalized
(C5H5N)2(Cl)2RuCHPh.35 This catalyst was chosen due to its PLA polymers were exposed to the same solution conditions and
exceptional functional group tolerance and favorable rates of reagents including the Ru-initiator that were used for the prep-
initiation and propagation to afford well-defined polymers with aration of polymer 3. This treatment yielded unfunctionalized
low dispersity. Polymer 2 was pre-loaded with 1.1 equivalents PLA and no free homopolymer of monomer 3, based on
of Grubbs’ catalyst with respect to the norbornene units for 1H-NMR and SEC-MALS, demonstrating the reliability of the
10 min. The polymer was precipitated with methanol and precipitation step to eliminate any free, unreacted initiator
excess/unreacted catalyst washed away to avoid competing from solution (see ESI† and Fig. S3 for more experimental
polymerization events not originating from the PLA polymer- detail). In order to demonstrate that no undesired cross linking
bound initiation sites. Following polymer resuspension in of the PLA polymers were taking place, another control reaction
methylene chloride, phenyl-modified monomer 3 (5 equivalents was performed. Polymer 2 was resynthesized and this polymer
with respect to norbornene units on the PLA backbone) was was added to the Grubbs’ catalyst. The solution was let to
stir for 1 h and then a large excess of ethyl vinyl ether
(30 equivalents with respect to norbornene backbone units)
Table 1 Physical parameters of the PLA polymers
was added to the reaction to produce a cross metathesis
product and to terminate the opened olefin. Polymer 2 was
Mn (kg molÀ1
)
Mw (kg molÀ1
)
Ðc
DPd
a
b
Polymer
analyzed by SEC-MALS before the reaction (Mn = 44 000 Ð = 1.1)
and after the reaction (Mn = 46 000 and Ð = 1.1). The Mn did not
increase a substantial amount, confirming no crosslinked
product. (See Fig. S4 ESI,† for more information).To demon-
strate graft-from polymers could be prepared as higher molecular
PLA
13
32
51
45
59
15
45
93
—
—
1.2
1.4
1.8
—
—
—
5
Polymer 2
Polymer 3
Polymer 4
Polymer 4b
5
—
10e
All data was obtained by SEC-MALS, except for those describing the weight assemblies, the polymers were allowed to assemble by
imidazole containing polymers, which did not elute on the SEC-MALS
dissolution in THF followed by slow addition to water and concen-
tration in vacuo to form polymeric nanoparticles. Transmission
a
column and were instead characterized by 1H-NMR. Number-average
b
molecular weight from light scattering or 1H-NMR. Weight average
molecular weight from light scattering. c The dispersity of each polymer. electron microscopy (TEM) was then used to verify the formation
d
Experimentally determined degree of polymerization of ROMP block via
of these nanoscale assemblies. Samples for TEM were prepared on
carbon TEM grids by drop deposition followed by staining with
1H-NMR. Polymer 4b was polymerized to a degree of 10: DP of 5 for
e
monomer 4 and DP of 5 for the TEG monomer to be able to be utilized for
nanoparticle synthesis.
1% uranyl acetate solutions. Comparison of these TEM images
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Fig. 2 TEM images of nanoparticles comprised of (A) unfunctionalized
PLA, (B) polymer 2, and (C) polymer 3 with phenyl functionality. Stained
with 1% uranyl acetate. Scale bars = 200 nm. These nanospheres were
prepared by the solvent evaporation method by dissolving each polymer in
THF and adding DI water dropwise. The polymers formed nanospheres
after concentrating the solutions in vacuo.
(Fig. 2) and dynamic light scattering (DLS) data (Fig. S5, ESI†) of
unfunctionalized PLA, polymer 2 and polymer 3, indicated that
each material had very similar morphology, consisting of spherical
nanoparticles with diameters of 100–200 nm.
To demonstrate one potential application of this graft-from
polymerization technique, graft polymers featuring a stimuli-
responsive functional moiety were synthesized. In this example,
a grafted PLA polymer was synthesized by incorporating a norbor-
nene monomer functionalized with a 5-dimethoxy-2-nitrobenzyl
caged imidazole moiety. This moiety was chosen due to its
potential to change its physical properties in response to two
separate stimuli; UV light (via cleavage of the 2-nitrobenzyl
cage) and pH (by way of protonation of the imidazole unit).
Moreover, imidazole, in particular, was of interest since its
protonation (pKa of the immidazolium ion is B7) is within a
biologically relevant range (pH 5.0–7.4) and would demonstrate
the possibility of triggering a morphology change in this
material upon exposure to mild changes in pH environments.
The caged imidazole-containing norbornenyl monomer 4
readily polymerized, yet the resulting polymer 4 formed only
amorphous aggregates rather than well dispersed spherical
particles. As such, this monomer was copolymerized with
tetra(ethylene glycol) norbornene monomer 5 to increase
the water solubility of the resulting polymer (4b, Fig. 3).
Fig. 3 Responsive imidazole derivatized PLA particles. Polymers were
dialyzed from DMSO to buffered water at a concentration of 1 mg mLÀ1
.
Spherical nanoparticles of 100 nm diameter were formed at both pH 7.4
(A) and 5.5 (C). After exposing to 350 nm UV light, particles in pH 7.4 buffer
remained discrete nanostructures (B) and particles at pH 5.5 transformed
from nanospheres to micron scale aggregates (D). This suggests that
protonation of the liberated imidazole moiety is necessary for the shape
change, due to putative electrostatic repulsion of the positively charged
imidazolium ions at low pH. Stained with 1% uranyl acetate. Scale bar =
200 nm.
morphology of 4b after exposure to UV light is likely due
to electrostatic repulsion of the positively charged imidazolium
groups and also to the removal of the potentially self-
associating 2-nitrobenzyl moieties. Imidazole-containing nano-
particles generated by removal of the 2-nitrobenzyl group from
polymer 4b in pH 7.4 buffered water (Fig. 3A) could be made to
form the same aggregated structures as shown in Fig. 3D by
decreasing the pH of the solution via slow addition of HCl to
pH 5.5 (Fig. 3B), indicating the necessity of both stimuli, a
reduction in pH and the application of UV light, to facilitate the
morphology changed observed for polymer 4b (Fig. S8, ESI†).
Likewise, note that alkyl imidazoles, such as 2-nitrobenzyl
protected polymer 4b, could also become protonated in acidic
solutions, however, this material does not aggregate until
exposed to UV-light, indicating that a decrease in pH is not
alone sufficient for the morphology change (Fig. 3C).
1
The polymers were characterized by H-NMR to determine the
degree of polymerization (DP) (Fig. S6, ESI†). Note: a detailed
characterization of the polymer’s molecular weight was not
possible by SEC-MALS due to aggregation on the SEC column.
Following nanoparticle formation by dialysis from DMSO into
buffered water of polymer 4b, spherical nanoparticles of
100 nm diameter were formed at both pH 7.4 and 5.5 as
evidenced by TEM (Fig. 3) and DLS (Fig. S7, ESI†).
Deprotection of the nitrobenzyl caged-imidazole via treat-
ment of the nanoparticles with 350 nm UV light for three
minutes at either pH 5.5 or 7.4 resulted in a change in shape
and diameter of the nanoparticles. At pH 5.5, we observed a
change to micron-sized aggregates from discrete spherical
structures as evidenced by TEM (Fig. 3D) and DLS (Fig. S7,
ESI†). We attribute this change in shape of polymer 4b to both
removal of the bulky nitrobenzyl group and to protonation of
the newly liberated imidazole moiety after exposure to UV light
in an acidic environment. The disruption of the nanoparticle
In summary, we have synthesized novel PLA-based materials
via graft-from ROMP to install a high density of functionality
from relativity low density branch points along the PLA back-
bone. As a proof-of-principle, we synthesized polynorbornyl grafts
derivatized with a phenyl group along the PLA backbone and
generated well-defined polymers and polymeric nanoparticles.
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´
13 W. W. Gerhardt, D. E. Noga, K. I. Hardcastle, A. J. Garcıa,
We then synthesized 2-nitrobenzyl-protected imidazole graft
variants to illustrate that chemical handles with increasingly
useful functionality can be incorporated into PLA via this method
by capitalizing on the high functional group tolerance of the
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materials that are potentially useful for a wide range of biome-
dical or materials applications.
This work was partially supported by the NSF (CHE-0847530)
and the AFOSR (FA9550-12-1-0435). We thank Dr Matt Thompson
for his technical assistance with polymer characterization. The
authors also thank Dr Angela Blum for her critical reading of the
manuscript and helpful suggestions. We also acknowledge the
UCSD Cryo-Electron Microscopy Facility and Norm Olsen for TEM
training and assistance.
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