Intramolecular cyclization assistance for fast degradation of ornithine-based poly(ester amide)s

Article abstract of DOI:10.1002/pola.26788

Inspired by the spontaneous cyclization of ornithine in peptides, polyesters containing protected ornithine (Orn) side chains along the backbone were synthesized and shown to degrade rapidly upon deprotection through intramolecular cyclization. A new ornithine-based poly(ester amide) PEA 1 and a lysine-based control PEA 2, both bearing the light-sensitive protecting group o-nitrobenzyl alcohol (ONB), were synthesized. Tert-butyl carbamate (Boc)-protected versions 1-Boc and 2-Boc were also synthesized for proof of concept. GPC confirmed that 1-Boc degrades over 40 times faster than 2-Boc following deprotection into the designed intramolecular cyclization products. Finally, TEM visualization of particles made from 1 encapsulating iron oxide nanoparticles reveals complete disruption of nanoparticles and release of payload within a day upon UV irradiation. Copyright

Full text of DOI:10.1002/pola.26788

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Intramolecular Cyclization Assistance for Fast Degradation
of Ornithine-Based Poly(ester amide)s
Caroline de Gracia Lux,1 Jason Olejniczak,2 Nadezda Fomina,1
Mathieu L. Viger,1 Adah Almutairi1,3,4
1Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, California 92093
2Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, California 92093
3Department of NanoEngineering, University of California at San Diego, La Jolla, California 92093
4Department of Materials Science and Engineering, University of California at San Diego, La Jolla, California 92093
Correspondence to: A. Almutairi (E-mail: aalmutairi@ucsd.edu)
Received 29 March 2013; accepted 18 May 2013; published online 11 June 2013
DOI: 10.1002/pola.26788
ABSTRACT: Inspired by the spontaneous cyclization of ornithine
in peptides, polyesters containing protected ornithine (Orn)
side chains along the backbone were synthesized and shown
to degrade rapidly upon deprotection through intramolecular
cyclization. A new ornithine-based poly(ester amide) PEA 1 and
a lysine-based control PEA 2, both bearing the light-sensitive
protecting group o-nitrobenzyl alcohol (ONB), were synthe-
sized. Tert-butyl carbamate (Boc)-protected versions 1-Boc and
2-Boc were also synthesized for proof of concept. GPC con-
firmed that 1-Boc degrades over 40 times faster than 2-Boc
following deprotection into the designed intramolecular cycli-
zation products. Finally, TEM visualization of particles made
from 1 encapsulating iron oxide nanoparticles reveals complete
disruption of nanoparticles and release of payload within a day
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upon UV irradiation.
2013 Wiley Periodicals, Inc. J. Polym.
Sci., Part A: Polym. Chem. 2013, 51, 3783–3790
KEYWORDS: biomaterials; controlled release; depolymerization;
intramolecular cyclization; stimuli-sensitive polymers; photo-
chemistry; polyester amide; self-immolative; triggered release
and poly(ester amide)s. Currently, linear aliphatic polyesters27
including poly(lactic acid) (PLA), poly(glycolic acid) (PGA) and
copolymers (PLGA) and poly(E-caprolactone) (PCL), are
widely used in biomedical applications. Due to their bulk and
gradual degradation by hydrolysis of their ester linkages in
physiological conditions,28 they are particularly suitable for
long term implants and controlled release applications.29
However, this slow degradation also limits the range of appli-
cations for which they are useful; they could be made to
degrade faster by chemical modifications allowing on-demand,
stimulus-activated depolymerization.
INTRODUCTION Over the past several decades there has
been enormous progress in developing novel methods to
synthesize and control assembly of polymers, less work has
been done on designing novel mechanisms of controlled
depolymerization or polymer disassembly. However, this
trend is rapidly changing because of current technological
needs in both the medical sector and “green” industry, which
would be advanced by polymeric materials that can be disas-
sembled in a controlled fashion on demand.1 Such materials
find applications in the electronics industry,2,3 in patterning,4
in self-healing5 and reinforced composites,6 in tissue engi-
neering and implants,7 tissue adhesives,8 drug delivery9–12
and biosensors.13,14 Since the development of polyacetals
used as photoresists,3 designs of polymers intended to
degrade by particular mechanisms have grown more diverse.
Several self-immolative dendrimers and polymers15 translate
cleavage of a triggering group into degradation by intramo-
lecular mechanisms, mainly by 1,6 and/or 1,4 elimination,
cyclization-elimination, and hemiacetal elimination.16–26
Among synthetic biodegradable polymers, PEAs recently
attracted interest as they combine the properties of amides
and ester moieties. PEAs’ amide-like properties include ther-
mal stability (thermal stability results from strong intermo-
lecular hydrogen bonding interactions between amide
groups), mechanical strength, and potential for conjugation
with drugs or bioactive compounds, while their polyester-
like properties include degradability and flexibility.30 PEAs
may be functionalized on pendant amine or carboxylic acid
functionalities. However, despite these advantages and their
We are interested in expanding the types of polymers that
degrade by intramolecular cyclization, including polyesters
Additional Supporting Information may be found in the online version of this article.
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biocompatibility, there are still few reports of PEAs contain-
ing reactive side chains.30–36 In addition, to our knowledge,
the degradation process reported so far for such hydropho-
bic polymeric materials (mainly micelles and films) depends
only on hydrolysis, driven by changes in solubility or enzy-
matic degradation of ester or amide linkages. A recent study
using the amino acid 2,4-diaminobutyric acid as a self-
immolative small molecule spacer in a PEA-based copolymer
asserts backbone degradation by cyclization of pendant
amine chains into a five-membered ring at 70 ꢀC over a 2
week period.37 Our current design of an amine-protected
PEA degrades rapidly via lactamization of the polymer itself
at physiologically relevant temperatures. Herein we charac-
terize both the chemistry and the degree of degradation.
pump, UV-vis 1260 DAD, 6120 Quadrupole LC/MS ESI source)
with a RP-18 column. HRMS measurements were done with
an Agilent 6230 ESI-TOF MS.
Polymer Synthesis
Polymers 1 and 2 were synthesized from commercially avail-
able Fmoc-Orn-Boc or Fmoc-Lys-Boc in a four-step procedure
(Fig. 1). A similar procedure was followed to synthesize the
Boc versions; in both cases, monomers were polymerized by
polycondensation of a diester amine bearing two functional-
ized a-amino acids, and a diacid chloride (Scheme S1). Syn-
thesis and characterization of the Boc analogues are
reported in the Supporting Information.
Compound 3a. Fmoc-Orn(Boc)OH (2.5 g, 5.5 mmol) was
stirred in 5 mL of DCM/TFA mixture (1/1) for 1 h. The sol-
vents were removed under reduced pressure. The residue was
dissolved in 100 mL toluene and added to a heterogeneous
mixture containing 4,5-dimethoxy-2-nitrobenzyl(4-nitrophe-
nyl)carbonate (2.08 g, 5.5 mmol) and DIEA (20 mL) in 30 mL
DMF. The reaction was stirred at room temperature overnight.
The solvents were removed and the product was isolated by
flash-chromatography on a reverse phase C18 column using a
gradient of water/acetonitrile. Yield: 1.850 g (57%).
Ornithine is known to undergo spontaneous lactamization in
peptides and activated esters and thus cannot be supported
by tRNA synthesis.38,39 Inspired by this we hypothesized that
a
related ornithine-based polymer bearing light-sensitive
groups would degrade rapidly upon irradiation. As a control,
we also synthesized lysine-based polymers; lysine’s longer
alkyl spacer between the amine and the acid makes it unreac-
tive enough for inclusion in proteins, as the only lactam it
could form would contain an unfavorable seven membered
ring. This control demonstrates that cyclization occurs only in
polymers with appropriately spaced amines and esters.
1H NMR (600 MHz, DMSO-d6, d): 7.88 (d, J 5 7.8 Hz, 2H; Ar
H), 7.72 (d, J 5 6.6 Hz, 2H; Ar H), 7.69 (s, 1H, Ar H), 7.61 (d,
J 5 7.2 Hz, 1H; NH), 7.47 (t, J 5 6.0 Hz, 1H; NH), 7.41 (t, J
5 7.2 Hz, 2H; Ar H), 7.32 (t, J 5 7.2 Hz, 2H; Ar H), 7.17 (s,
1H, Ar H), 5.33 (s, 2H; CH2), 4.28–4.20 (m, 3H; CH2, CH),
3.97–3.90 (m, 1H; CH), 3.88 (s, 3H; CH3), 3.86 (s, 3H; CH3),
3.01 (dd, J 5 12.6 Hz, J 5 6.6 Hz, 2H; CH2), 1.79–1.66 (m,
1H; CH), 1.62–1.55 (m, 1H, CH), 1.54–1.42 (m, 2H; CH2); 13C
NMR (150 MHz, CDCl3, d): 173.9, 156.2, 155.7, 153.4, 147.7,
143.9, 140.8, 139.3, 128.1, 127.7, 127.1, 125.3, 120.1, 110.4,
108.1, 65.7, 62.3, 56.2, 56.1, 55.0, 53.7, 46.7, 28.2, 26.3;
HRMS (ESI, m/z): [M1Na]1 calcd C30H31N3O10Na, 616.1902;
found, 616.1900.
EXPERIMENTAL
Materials
Unless otherwise noted, all chemicals were obtained from
commercial sources and were used without further purifica-
tion. All reactions were carried out under a nitrogen atmos-
phere in oven dried glassware unless otherwise noted.
Characterization and Measurements
Flash column chromatography purification was performed
using a Teledyne Isco Combiflash Companion with RediSep Rf
prepacked silica or C18 columns. Thin layer chromatography
was performed with EMD TLC Silica gel 60 F254 glass plates.
1H NMR spectra were acquired using a Varian 400 MHz, a
Bruker 600 MHz or JEOL 500 MHz NMR spectrometer and
13C NMR spectra were acquired using a Varian NMR spec-
trometer at 100 MHz or a Bruker 150 MHz NMR spectrome-
ter. Molecular weights were determined by gel permeation
chromatography, performed with a Waters e2695 instrument
with a series of Styragel HR4 and Styragel HR2 columns in
Compound 4a. 3a (0.4 g, 0.67 mmol), hexanediol (0.026 g,
0.22 mmol) and DMAP (0.027 g, 0.22 mmol) were dissolved
in 3.8 mL of DCM and DMF (5/1) under argon atmosphere.
A solution of DCC (0.153 g, 0.741 mmol) in 1 mL DCM was
added to the reaction mixture dropwise. The reaction mix-
ture was stirred overnight at room temperature. The solvent
were removed and the product was purified by silica column
using a linear gradient of DCM/methanol (100%/0%—10%/
90%). Yield: 0.372 g (87%).
ꢀ
DMF with 0.01% LiBr at 37 C. The instrument was calibrated
with monodisperse polystyrene standards. Irradiation with
350 nm UV light was performed using a Luzchem LZC-ORG
photoreactor equipped with 8UV-A lamp (8W maximum inten-
sity). Polymer degradation was monitored by gel permeation
chromatography using an Agilent 1100 aqueous system with
acetonitrile/phosphate buffer (0.2 M) (8/2). The instrument
was calibrated with monodisperse polyethylene glycol stand-
ards. Particles were imaged by with a FEI Spirit TEM used at
120 kV. Mass determination and dimer degradation studies
were performed with a HPLC-MS Agilent 160 Infinity (binary
1H NMR (600 MHz, DMSO-d6, d): 7.87 (d, J 5 7.2 Hz, 4H; Ar
H), 7.76 (d, J 5 7.8 Hz, 2H; NH), 7.71–7.67 (m, 6H; Ar H),
7.45 (t, J 5 5.4 Hz, 2H; NH), 7.39 (t, J 5 7.2 Hz, 4H; Ar H),
7.31 (t, J 5 7.2 Hz, 4H; Ar H), 7.16 (s, 1H; Ar H), 5.32 (s,
4H; CH2) 4.28–4.20 (m, 6H; CH2, CH), 4.00–3.90 (m, 6H; CH2,
CH), 3.87 (s, 6H; CH3), 3.85 (s, 6H; CH3), 3.02 (dd, J 5 12.6
Hz, J 5 6.6 Hz, 4H; CH2), 1.75–1.67 (m, 2H; CH2), 1.65–1.56
(m, 2H; CH2), 1.54–1.41 (m, 8H; CH2), 1.27–1.19 (m, 4H;
CH2); 13C NMR (150 MHz, CDCl3, d): 172.1, 156.5, 156.0,
155.6, 153.3, 147.7, 143.7, 140.6, 139.3, 127.8, 127.5, 126.9,
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FIGURE 1 (a) Structure and mechanism of light-induced 1 degradation: removal of the light-sensitive groups followed by a combina-
tion of amino-assisted ester hydrolysis and aminolysis. (b) Synthesis of polymers 1 and 2. (a) (i) TFA/DCM (1/1), (ii) 4,5-dimethoxy-2-
nitrobenzyl (4-nitrophenyl) carbonate, DIEA, toluene, DMF (3a, 57%; 3b, 78%), b) 1,6-hexanediol, DCC, DMAP, DCM (4a, 87%; 4b,
80%), (c) piperidine, DMF, (d) adipoyl chloride, DCM (1, 48%; 2, 60%). Control polymer 2 does not undergo backbiting cyclizations.
125.0, 119.9, 110.6, 108.2, 66.6, 64.2, 62.1, 56.1, 56.0, 53.7,
47.4, 46.6, 33.2, 27.8, 24.7, 24.3; HRMS (ESI, m/z): [M1Na]1
calcd C66H72N6O20Na, 1291.4594; found, 1291.4685.
dissolved in 60 mL toluene and added to a heterogeneous mix-
ture containing 4,5-dimethoxy-2-nitrobenzyl(4-nitrophenyl)car-
bonate (1.26 g, 3.34 mmol) and DIEA (12 mL) in 20 mL DMF.
The reaction was stirred at room temperature overnight. The
solvents were removed and the product was isolated by flash-
chromatography on a reverse phase C18 column using a gradi-
ent of water/ acetonitrile. Yield: 1.590 g (78%).
Polymer 1. 4a (0.38 g, 0.299 mmol) was deprotected in 1.2
mL piperidine/DMF (5/95). The solvents were removed
under reduced pressure. The residue was dissolved in 1.5
mL DCM under nitrogen atmosphere and pyridine (0.145
mL, 1.79 mmol) was added. Adipoyl chloride (0.043 mL,
0.299 mmol) was added to the reaction mixture dropwise.
The reaction was stirred at room temperature overnight. The
polymer was isolated by precipitation in 20 mL of chilled
methanol. The low molecular weight fraction was removed
by precipitating the polymer into methanol from DCM three
times. Yield: 0.134 g (48%). Molecular weight (relative to
polystyrene standards): Mw 5 37 500 Da (PDI 5 1.4).
1H NMR (600 MHz, DMSO-d6, d): 7.88 (d, J 5 7.8 Hz, 2H; Ar
H), 7.72 (d, J 5 6.6 Hz, 2H; Ar H), 7.69 (s, 1H, Ar H), 7.61 (d,
J 5 7.2 Hz, 1H; NH), 7.47 (t, J 5 6.0 Hz, 1H; NH), 7.41 (t, J
5 7.2 Hz, 2H; Ar H), 7.32 (t, J 5 7.2 Hz, 2H; Ar H), 7.17 (s,
1H, Ar H), 5.32 (s, 2H; CH2), 4.27–4.21 (m, 3H; CH2, CH),
3.90–3.88 (m, 1H; CH), 3.88 (s, 3H; CH3), 3.86 (s, 3H; CH3),
3.01 (dd, J 5 12.6 Hz, J 5 6.6 Hz, 2H; CH2), 1.70–1.61 (m,
2H; CH2), 1.42–1.34 (m, 4H; CH2); 13C NMR (150 MHz,
CDCl3, d): 173.8, 156.1, 155.6, 153.3, 147.7, 143.8, 140.7,
139.3, 127.9, 127.6, 127.0, 125.2, 120.1, 110.5, 108.1, 65.5,
62.2, 56.2, 56.1, 53.8, 46.6, 40.1, 30.5, 28.9, 22.8; HRMS (ESI,
m/z): [M2H]2 calcd C31H33N3O10, 606.22; found, 606.3.
1H NMR (500 MHz, DMSO-d6, d): 8.13 (d, J 5 7.0 Hz, 2H;
NH), 7.66 (s, 2H; Ar H), 7.45 (d, J 5 6.5 Hz, 2H; NH), 7.14 (s,
2H; Ar H), 5.27 (s, 4H; CH2), 4.18 (dd, J 5 13.5 Hz, J 5 8.0
Hz, 2H; CH), 4.02–3.92 (m, 4H; CH2), 3.87 (s, 6H; CH3), 3.84
(s, 6H; CH3), 3.00 (d, J 5 5.5 Hz, 4H; CH2), 2.1 (s, 4H; CH2),
1.71–1.63 (m, 2H; CH2), 1.56-1.45 (m, 14H, CH2), 1.25 (s, 4H,
CH2); 13C NMR (100 MHz, CDCl3, d): 172.4, 172.3, 172.2,
155.7, 153.3, 147.7, 139.3, 139.2, 128.0, 110.4, 108.1, 108.1,
64.2, 62.3, 56.0, 56.2, 51.7, 34.7, 28.3, 28.0, 26.0, 24.9 ppm.
Compound 4b. 3b (1.36 g, 2.24 mmol), hexanediol (0.088 g,
0.75 mmol) and DMAP (0.136 g, 10 %) were dissolved in 17
mL of DCM and DMF (7/1) under argon atmosphere. A solu-
tion of DCC (0.508 g, 2.46 mmol) in 3 mL DCM was added
to the reaction mixture dropwise. The reaction mixture was
stirred overnight at room temperature. The solvent were
removed and the product was purified by silica column
using a linear gradient of hexane/ethyl acetate (100%/0%—
0%/100%). Yield: 0.790 g (80%).
Compound 3b. Fmoc-Lys(Boc)OH (1.56 g, 3.34 mmol) was
stirred in 4 mL of DCM/TFA mixture (1/1) for 1 h. The sol-
vents were removed under reduced pressure. The residue was
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FIGURE 2 (a) Products formed by photolysis (15 min irradiation, 350 nm) of the light-cleavable monomer 4a (n 5 1) used in the
synthesis of 1 in acetonitrile/phosphate buffer pH 7.4 (1/2). (b) Integration of the Fmoc-lactam product HPLC-MS peak of ornithine-
based dimers as a function of incubation time, either unirradiated (red) or after 15 min UV irradiation (blue).
1H NMR (600 MHz, DMSO-d6, d): 7.87 (d, J 5 7.2 Hz, 4H; Ar
H), 7.75 (d, J 5 7.8 Hz, 2H; NH), 7.71–7.67 (m, 6H; Ar H),
7.45 (t, J 5 5.4 Hz, 2H; NH), 7.39 (t, J 5 7.2 Hz, 4H; Ar H),
7.31 (t, J 5 7.2 Hz, 4H; Ar H), 7.16 (s, 2H; Ar H), 5.31 (s,
4H; CH2) 4.27–4.20 (m, 6H; CH2, CH), 4.00–3.91 (m, 6H; CH2,
CH), 3.87 (s, 6H; CH3), 3.85 (s, 6H; CH3), 3.00 (s, 4H; CH2),
1.66–1.61 (m, 4H; CH2), 1.48–1.24 (m, 16H; CH2); 13C NMR
(150 MHz, CDCl3, d): 172.4, 156.1, 155.6, 153.3, 143.8, 143.7,
140.7, 139.3, 127.9, 127.8, 127.0, 120.1, 110.5, 108.1, 99.5,
65.6, 64.2, 62.2, 59.8, 56.1, 56.0, 46.6, 30.3, 28.9, 27.9, 24.8,
22.8, 20.8.
groups, and model Boc-protected polymers (1-Boc, 2-Boc)
to study the mechanism of degradation. Light-sensitive poly-
mer 1 was formulated into nanoparticles encapsulating
model payloads to examine the kinetics of release. Polymer
2 serves as a control; only 1 should degrade by lactamiza-
tion because the formation of a 6-membered ring is favor-
able while seven-membered rings have greater ring strain
and thus are unfavorable. Accordingly, ornithine undergoes
spontaneous cyclization, and thus cannot be supported on
tRNA.40,41
Only Ornithine-Based Dimers 4a Degrade by
Polymer 2. 4b (0.307 g, 0.237 mmol) was deprotected in
1.2 mL piperidine/DMF (5/95). The solvents were removed
under reduced pressure. The residue was dissolved in 1.5
mL DCM under nitrogen atmosphere and pyridine (0.115
mL, 1.42 mmol) was added. Adipoyl chloride (0.035 mL,
0.237 mmol) was added to the reaction mixture dropwise.
The reaction was stirred at room temperature overnight. The
polymer was isolated by precipitation in 20 mL of chilled
methanol. Low molecular weight fraction was removed by
precipitating the polymer into methanol from DCM three
times. Yield: 0.144 g (60%). Molecular weight (relative to
polystyrene standards): Mw 5 41 500 Da (PDI 5 1.7).
Lactamization
Prior to investigation of polymer degradation, lysine- and
ornithine-based dimers were exposed to UV light to test our
hypothesis concerning the formation of lactam derivatives
(Fig. 2). The presence of four different intermediates was con-
firmed over time by integrating the HPLC-MS peaks of the sin-
gle ions (m/z 5 791 (for IM 1, [M1H], z 5 1), m/z 5 455
(for IM 2, [M1H], z 5 1), m/z 5 355 [for Fmoc-Orn, [M1H],
z 5 1), and m/z 5 359 (for Fmoc-lactam, [M1H1Na], z 5 1)
respectively]. All HPLC-MS graphs for both ornithine-based
dimers (4a) and lysine-based dimers (4b) are presented in
Supporting Information Figures S1–S2. The lactam derivative
was formed from ornithine-based dimers and not from lysine-
based dimers (Fig. S2, Supporting Information). Only
ornithine-based dimers (4a) degrade by lactamization (Fig. 2).
Non-irradiated dimers did not degrade.
1H NMR (500 MHz, DMSO-d6, d): 8.10 (d, J 5 7.0 Hz, 2H; NH),
7.65 (s, 2H; Ar H), 7.42 (d, J 5 6.5 Hz, 2H; NH), 7.14 (s, 2H;
Ar H), 5.27 (s, 4H; CH2), 4.19–4.10 (m, 2H; CH), 4.04–3.92 (m,
4H; CH2), 3.87 (s, 6H; CH3), 3.84 (s, 6H; CH3), 3.04–2.93 (m,
4H; CH2), 2.09 (s, 4H; CH2), 1.64–1.26 (m, 24H; CH2).
Mechanism of Polymer Degradation
Our strategy for the degradation of these new polymers is
not to rely only on protonation of pendant amine groups to
increase hydrophilicity and facilitate hydrolysis of the poly-
mer, but to tune the chemical structure of the materials to
enable additional, rapid mechanisms of degradation. The
length of the alkyl spacer between the neighboring protected
amine and the polymer backbone dictates the possible mech-
anism and rate of degradation. Degradation of polymers 1
RESULTS AND DISCUSSION
Design and Strategy
Here, we report a new light-sensitive polymer: PEAs based
on ornithine (PEA-Orn 1) and lysine amino acids (PEA-Lys
2). We synthesized both light-sensitive versions (1, 2), in
which amine groups were protected with o-nitrobenzyl
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FIGURE 3 (a) Deprotection procedure to unmask the amine: exposure to TFA/DCM (1:1) mixture and purification on a PL-10 desalt-
ing column. (b) GPC traces of 1-NH2 (main graph) and 2-NH2 (inset) after 0, 2, 5, 24, 50 and 150 h of incubation at pH 7.4. (c) Loss
of MW (relative to PEO standards) over time.
upon deprotection of the amine moieties includes intramo-
lecular general base catalysis, general acid-specific base
catalysis (assisted by intramolecular H-bonding), and intra-
molecular nucleophilic cyclization40,41 (Fig. 1), while degra-
ꢁ150 days. After Boc deprotection, 2 is much more stable
than 1 at both pH values, so lysine-based polymers can be
considered control polymers regardless of the protecting
group. A solution of 1-Boc in 0.2 M phosphate buffer/aceto-
nitrile (3.5/6.5) was also incubated at 37 ꢀC without being
deprotected. Unfortunately, 1-Boc is not soluble in aqueous
solution and needs a mixture of organic and aqueous sol-
vents to be dissolved. However, aliquots removed periodi-
cally did not show any loss in molecular weight, thus
validating the controlled intramolecular depolymerization
mechanism (Fig. S3a–b, Supporting Information).
dation of polymer
cyclization.
2 does not involve intramolecular
Ornithine-Based Polymer 1-Boc, and Not Lysine-Based
Polymer 2-Boc, Degrades Rapidly
To determine whether lactamization affects the polymer deg-
radation rate, we first monitored the degradation of Boc-
deprotected polymers (1-Boc and 2-Boc) at pH 6.0 and 7.4
by GPC (Fig. 3). As expected, degradation was faster at neu-
tral versus slightly acidic pH, as both lactamization (intramo-
lecular aminolysis) and NH2-catalyzed hydrolysis are more
favorable at pH 7.4. Boc-protected rather than light-sensitive
polymers were chosen for proof of concept because of the
ease of complete removal of Boc protecting groups through-
out the backbone, eliminating the effect of deprotection effi-
ciency. At pH 7.4, t1/2 (2-NH2) ꢁ 140 h, while t1/2 (1-NH2)
ꢁ 3 h, revealing that lactamization allows faster degradation
than ester hydrolysis. Similar experiments at pH 6.0 show
that t1/2 (1-Boc) ꢁ 150 h, while t1/2 (2-Boc) is estimated at
1-Boc, and Not 2-Boc, Degrades by Lactamization
Depolymerization of 1-Boc and 2-Boc was also observed by
NMR in deuterium phosphate buffer (0.2 M) at pH 7.4. NMR
spectra confirmed degradation rates and the identity of the
by-products (Fig. 4). New chemical shifts appear around 4.2
and 3.3 ppm following irradiation of 1, but not of 2, indicat-
ing the formation of lactam derivatives only for 1. The chem-
ical shift at 3.5 ppm, found for both 1 and 2, indicates
cleavage of ester bonds to form alcohols (ester hydrolysis).
After 96 h, almost 80% of the amine and ester functions of
polymer
1
undergo lactamization and hydrolysis,
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FIGURE 4 1H NMR spectra of deprotected polymer 1-Boc (a) and 2-Boc (b) in deuterium phosphate buffer (0.2 M) at pH 7.4, as a
function of incubation time (37 ꢀC).
respectively. For the same incubation time, hydrolysis of 2
does not exceed 15%. Complementary MALDI (Fig. S4, Sup-
porting Information for 1-NH2 at pH 7.4 after 24 h of incu-
bation) validated that the by-products obtained when the
polymer is partially degraded were the expected lactam.
However, no degradation products were detected for 2-NH2,
suggesting that 24 h incubation was not sufficient to form
any significant amount of low molecular weight oligomers.
efficiency obtained by this technique was confirmed by TEM:
no SPIONs remain unencapsulated.
Electrosprayed particles formulated from polymer 1 encapsulat-
ing SPIONs were characterized by transmission electron micros-
copy (TEM) upon degradation (Fig. 5). Unirradiated particles or
particles irradiated for 5 min with 350 nm UV light were exam-
ined after 1 day incubation at 37 ꢀC in dilute triethylamine/
water solution (pH 8.0; used instead of PBS to minimize wash-
ing necessary to remove phosphate salts). A disruption in the
morphology of the nanoparticles is observed by TEM after
irradiation.
Nanoparticles of Polymer 1 Fall Apart Upon Irradiation;
Remain Intact While Unirradiated
We formulated particles from polymer 1 encapsulating 10
nm super paramagnetic iron oxide (Fe3O4) nanoparticles
(SPIONs) by electrospray as 1 did not form nanoparticles by
single emulsion because of its low solubility in organic sol-
vent. SPIONs were chosen as payload to allow tracking of
release by TEM. This formulation method employs high volt-
age to inject charge into DMF/CHCl3 solution containing
polymer 1 and SPIONs, which causes the liquid to break into
a jet of very fine aerosol nanodroplets propelled toward the
glass slide collector. Dense, solid particles are generated as
the solvent evaporates in flight. Optimal encapsulation
Irradiated particles appear only as free SPIONs and chunks
of aggregated material [Fig. 5(b) and Fig. S6, Supporting
Information). In contrast, when samples are not irradiated,
no evidence of degradation nor aggregation is observed; only
intact particles are visible [Fig. 5(a) and Fig. S5, Supporting
Information). In parallel, particles were also examined after
15 min irradiation and 1 or 4 days incubation in order to
mimic the NMR degradation study. Similar results were
obtained concerning both integrity and degradation (Figs.
S7–S8,
Supporting
Information).
Complete
particle
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FIGURE 5 Representative TEM images of nanoparticles; either unirradiated (a) or irradiated for 5 min with 350 nm UV light (b)
after 1 day incubation at 37 ꢀC in dilute triethylamine/ water solution (pH 8.0).
degradation within 1 day, much faster than the 4 days
required to observe 80% of lactam derivatives by NMR,
implies that the carrier is falling apart by other processes in
addition to cyclization and hydrolysis. This is not surprising;
the huge change of hydrophilicity upon removal of the pho-
tolabile group is likely to contribute to particle breakdown.
deprotection. Furthermore, these polymers switch solubility in
aqueous solution, which speeds cyclization reactions as
observed in the faster degradation at physiologically relevant
temperatures. This work helps open up new possibilities for
rapid release upon triggered degradation of poly(ester
amide)s.
CONCLUSIONS
ACKNOWLEDGMENTS
A novel stimuli-responsive PEA containing protected ornithine
self-immolative side chains was synthesized and employed to
prepare nanoparticles that degrade upon irradiation. H NMR
The authors thank the NIH New Innovator Award (DP
2OD006499) and KACST (through the KACST-UCSD Center of
Excellence in Nanomedicine) for funding. They also thank Minh
C. Nguyen for his help in the formulation of nanoparticles. NMR
data was acquired at the UCSD Skaggs School of Pharmacy and
Pharmaceutical Sciences NMR facility.
1
and GPC reveal the degree of degradation through intramolec-
ular cyclization forming lactam derivatives. As expected, once
formulated into light-sensitive nanoparticles, both degradation
and release occur only upon irradiation, with a synergistic
effect of cyclization and solubility switch. Our polymer
degrades completely and rapidly at physiologically relevant
temperatures. This is in contrast to recently puꢀblished studies
on similar polymers that require heating to 70 C for a period
of 2 weeks to degrade.37 This difference stems from our poly-
mer design that allows each monomer to be degradable on
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