A R T I C L E S
Jo et al.
structures that can be eventually dissembled after all NO has
been delivered.
block serves to obtain micellization rather than precipitation as
the hydrophilic precursor is reacted with NO to form the
hydrophobic poly(NONOate). Three poly(NONOate)s were
prepared from monomers in the family of piperazine (a
6-member ring) with different number of carbons and/or
substituents on the cyclic ring (see Scheme S1-2 in the
Supporting Information). The average conversion of amine
groups into NONOate groups was determined 71% by Griess
assay. The poly(NONOate) was then derived from the precursor
by reaction with NO (see Scheme S4 in the Supporting
Information); tert-Butoxycarbonyl (Boc) chemistry was used to
protect the secondary amine-containing monomer during RAFT
polymerization, which was later deprotected to restore the
secondary amine groups in the polymer. From solubility tests,
both poly(sodium 1-(N-acryloylpiperazin-1-yl)diazen-1-ium-1,2-
diolate) (PAZ ·NONOate, actual NO payload: 6.4 µmol/mg) and
poly(sodium 1-(N-acryloylhomopiperazin-1-yl)diazen-1-ium-
1,2-diolate) (PAZh ·NONOate, actual NO payload: 6.0 µmol/
mg) (see Chart 1 for structures) were soluble in water, yet
insoluble in any other organic solvent (see Table S1a-b in the
Supporting Information). Therefore, neither of PAZ ·NONOate
nor PAZh ·NONOate was considered as a suitable candidate
for the hydrophobe of self-assembled aggregates after
NONOation. In contrast, poly(sodium 1-(N-acryloyl-2,5-dim-
ethylpiperazin-1-yl)diazen-1-ium-1,2-diolate) (PAZd ·NONOate)
was insoluble in water in distinction from its water-soluble
precursor, PAZd (see Table S1c in the Supporting Information).
Thus, PAZd, selected as the pro-hydrophobic block NO accep-
tor, was copolymerized with PAM (see Scheme S3 in the
Supporting Information). The soluble poly[(N-acryloylmorpho-
line)-block-(N-acryloyl-2,5-dimethylpiperazine)] (PAM-PAZd)
diblock copolymer as prepared by deprotecting the precursor,
i.e. poly[(N-acryloylmorpholine)-block-(1-Boc-4-acryloyl-2,5-
dimethylpiperazine)] (PAM-BocPAZd), was reacted with NO
in deoxygenated water under a pressurized NO supply and in
the presence of base (see Scheme S5 in the Supporting
Information). As more amine groups of PAZd are gradually
converted into NONOate groups, the PAZd ·NONOate block
segregates more from the PAM block. In this way, in situ
formation of aggregates of poly[(N-acryloylmorpholine)-block-
(sodium 1-(N-acryloyl-2,5-dimethylpiperazin-1-yl)diazen-1-ium-
1,2-diolate)] (PAM-PAZd·NONOate) is affected in aqueous
media (Figure 1a). As a result, PAM-PAZd ·NONOate was self-
assembled into micelles, as shown in transmission electron
microscopy (TEM) images with 2% negative staining using
sodium phosphotungstate (Figure 2).
2. Experimental Section
2.1. Syntheses. Monomers were synthesized as described
elsewhere17,18 with modifications. Homo- and copolymerization
were carried out as described elsewhere.17
2.2. In situ Micelle Formation (Micellization). PAM-PAZd
was dissolved in Milli-Q water with adequate amount of base. After
degassing with Ar, NO was pressurized to 80-150 psi in an
autoclave. This reaction was continued for 5 d. Micelles were
dialyzed for 2 h and lyophilized or frozen and kept at -20 C° until
used.
2.3. NO Analysis. At 25 °C, dissociation of NONOate in PBS
(10 mM, pH 7.4) was monitored by UV spectrometry at 250 nm.
At 37 °C, NO radicals generated from poly(NONOate)s in PBS
(10 mM, pH 7.4) were recorded by a NO analyzer.
2.4. Ex vivo Infusion of Micelles in the Rabbit Carotid
Artery. Carotid arteries from male New Zealand white rabbits
weighing 3 to 3.5 kg were obtained from a local slaughterhouse
immediately upon sacrifice. Vessels were stored in a PBS solution
and put on ice for transport. Excess tissue and adventitia were
removed, and a 2 cm-long arterial segment was mounted on 2.5
mm diameter cannula. The arteries were then stretched longitudi-
nally to their in ViVo length, submerged in a Krebs buffer solution
and kept at 37 °C for 1 h. Following this, a Krebs solution filling
the artery was replaced by approximately 1 mL of fluorescent-
labeled micelle solution. A Millar Mikro-Tip Catheter Transducer
was inserted through one cannula while a 20 mL syringe full of air
was attached to the other cannula. Experimental conditions were
achieved by depressing the plunger of the syringe and fixing the
transmural pressure at 1 atm (for 1 min) or repeatedly depressing
and releasing the plunger of the syringe, creating a pulsating
pressure ranging from ambient to 1 atm every 10 s (×10). After
the experiment the artery was rinsed for one minute in Krebs
solution, fixed in tissue freezing medium (Tissure-Tek O.C.T.) and
kept at -20 °C.
3. Results and Discussion
We first hypothesized that the hydrophobic microenvironment
within a micelle core can protect a reservoir of NONOate from
protons diffusing from the surroundings and thus delay proton-
catalyzed NO liberation. To form a self-assembled core-shell
structure, a pro-amphiphilic diblock copolymer was designed.
Reversible addition-fragmentation transfer (RAFT) polymer-
ization, a living radical polymerization, was employed to
synthesize well-defined block copolymers. As reported earlier
in our group,17 various analogues of acrylamides with cyclic
secondary amine side chains have been successfully homo- and
copolymerized using RAFT polymerization with excellent
control and low polydispersity. We sought to form a hydro-
phobic poly(N-diazeniumdiolate) (poly(NONOate)) by reacting
NO with precursors; poly(N-acryloylpiperazine) (PAZ) or
poly(N-acryloylhomopiperazine) (PAZh) or poly(N-acryloyl-2,5-
dimethylpiperazine) (PAZd) block, which are all water-soluble
but yielding an insoluble poly(NONOate)s.
The morphology of the formed micelles depends on the block
length ratio between the two polymer blocks, higher hydropho-
bic block ratios giving bigger hydrodynamic diameters than
lower ones. Longer hydrophilic blocks yielded longer worm-
like micelles: PAM146-PAZd·NONOate57 being ca. 110 nm (see
Figure S9a in the Supporting Information) whereas PAM146
-
PAZd·NONOate23 being ca. 80 nm (see Figure S9b in the
Supporting Information). Interestingly, despite the copolymer
comprising a smaller fraction of hydrophobe, PAZd·NONOate23,
than hydrophile, PAM146, spherical micelles were not the
dominant in morphology, but rather worm-like micelles. How-
Homopolymeric poly(NONOate)s were synthesized to de-
termine (in)solubility, and then an amphiphilic diblock copoly-
mer containing poly(NONOate) was synthesized with poly(N-
acryloylmorpholine) (PAM) as a hydrophilic block. The PAM
ever, with an excess amount of base, ca. 10-20 times, PAM142
-
PAZd·NONOate23 tended to form spherical micelles (Figure
2) with approximately 50 nm average diameter, as confirmed
by dynamic light scattering (DLS) and TEM. The critical micellar
concentration (cmc) of the PAM142-PAZd·NONOate23 spherical
micelles was determined to be 2.2 × 10-4 M by the pyrene
(17) Jo, Y. S.; van der Vlies, A. J.; Gantz, J.; Antonijevic, S.; Demurtas,
D.; Velluto, D.; Hubbell, J. A. Macromolecules 2008, 41, 1140.
(18) Zheng, H.; Weiner, L. M.; Bar-Am, O.; Epsztejn, S.; Cabantchik, Z. I.;
Warshawsky, A.; Youdim, M. B. H.; Fridkin, M. Bioorg. Med. Chem.
2005, 13, 773.
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14414 J. AM. CHEM. SOC. VOL. 131, NO. 40, 2009