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U. Wais et al. / Journal of Controlled Release 222 (2016) 141–150
We previously reported the use of emulsion freeze-drying to form
2.2. Synthesis of crosslinked branched poly(ethylene glycol)-b-poly(N-
isopropylacrylamide) (PEG-PNIPAM)
organic or drug nanoparticles in situ in water-soluble porous polymer.
The polymer scaffold prevents the nanoparticles from aggregation in
the solid state, ensuring a long storage time. The nanoparticles can be
readily released by dissolving the polymer scaffold in water to produce
aqueous nanoparticle dispersion [38]. Both polymer (e.g., poly(vinyl al-
cohol)) and surfactant (e.g., sodium dodecyl sulphate) are required to
form the emulsions, produce the porous scaffold, and stabilize the nano-
particles in aqueous suspensions. It is possible to generate porous poly-
mer by freeze drying and then employ a solvent evaporation approach
to form organic/drug nanoparticles directly in the porous polymeric
scaffold [39,40]. Aqueous nanoparticles dispersion can be prepared sim-
ilarly. In both approaches, the use of both polymer and surfactant is im-
portant in forming stable aqueous nanoparticle dispersions. This,
however, can result in low loading of drug compounds in the formula-
tions. A formulation that utilizes a biocompatible polymer acting both
as scaffold and surfactant would be advantageous in improving drug
loading and reducing the formulation complexity (e.g., in assessing
biocompatibility).
2.2.1. Synthesis of ethylene diacrylamide
Ethylene diamine (1.2 g, 20 mmol, 1 eq) and sodium acetate (3.6 g,
40 mmol, 2 eq) were dissolved in CHCl3 (50 mL) and the solution cooled
to 0 °C in an ice bath. Acryloyl chloride (3.6 g, 44 mmol, 2.2 eq) in CHCl3
(50 mL) was then added dropwise over 20 min. The reaction was left to
stir for 1 h at 0 °C. The reaction was then refluxed for 1 h at 60 °C and the
solution filtered while hot, upon cooling a white precipitate formed
which was isolated by filtration. The crude white solid was further pu-
rified by recrystallization in hot CHCl3 to afford the desired product eth-
ylene diacrylamide as a white solid (1.2 g, 36%). 1H NMR (DMSO-d6): δ
8.19 (s, 2H), 6.19 (m, 2H), 6.07 (m, 2H), 5.58 (m, 2H), 3.21 (m, 4H). 13C
NMR (DMSO-d6): δ 164.9, 131.8, 125.2, 38.4. HRMS (ESI) m/z: [M + H]+
calculated for C8H13N2O2, 169.0972; found: 169.0980 (ppm 4.77).
2.2.2. Synthesis of PEG-PNIPAM (1–3) and the corresponding nanoparticle
dispersions
We have also reported synthesis of super-lightly crosslinked
branched copolymers and a new direct monomer-to-particle synthetic
strategy based on these copolymers, which could be applied in drug de-
livery [41–43]. After a simple dialysis process to generate isolated mac-
romolecular species, well-defined uni-molecular polymer nanoparticles
can be obtained directly. This de novo synthetic approach differs signif-
icantly from the reported arm-first or core-first core-crosslinked star-
polymer synthesis where the core is effectively a highly cross-linked
microgel formed by the addition of a large volume of cross-linkers
such as divinylbenzene at the end of the polymerization [44–46]. In
contrast, polymer nanoparticles were prepared from discrete soluble
molecular species (soluble branched copolymers) which have been syn-
thesized by a controlled branching strategy. Utilizing this strategy, it
was possible to prepare amphiphilic materials with defined nanoparti-
cle shape by a one-pot, concerted growth process rather than joining of
pre-formed spheres [41,42]. The lightly crosslinked core could offer the
obtained polymer nanoparticles with larger loading capacity of guest
compounds. And the stability of the nanoparticles was very high (e.g.,
up to one year maintaining the size and shape). This synthetic method-
ology may be easily scaled-up as we demonstrated previously, even
with the possibility to be extended in the synthesis of hyperbranched
polydendrons [47].
Typically, the radical macro-initiator poly(ethylene glycol) dimer
(12 kDa, 1.2 g, 0.1 mmol, 1 eq), N-isopropylacrylamide (0.56 g,
5 mmol, 25 eq per PEG chain), ethylene diacrylamide (10.1 mg,
0.06 mmol, 0.3 eq per PEG chain) and dodecanethiol (10.1 mg,
0.05 mmol, 0.25 eq per PEG chain) were transferred into a small schlenk
tube fitted with a magnetic stirrer bar and N,N′-dimethylformamide
(DMF, 7 mL) added. The reaction mixture was degassed and the vessel
was backfilled with N2. The reaction mixture was then placed in an oil
bath at 70 °C and the polymerization was quenched by rapid cooling
after 16 h. The reaction mixture was dissolved in a minimal amount of
tetrahydrofuran (THF) and added dropwise to a large excess of ice-
cold diethyl ether. The precipitation was repeated once more before
the desired branched copolymer was obtained as a white solid
(0.94 g). The molar ratio of ethylene diacrylamide per PEG chain was
varied as 0.3 (1), 0.6 (2), and 0.9 (3) eq per PEG change for the PEG-
PNIPAM branched block copolymers. Corresponding nanoparticle aque-
ous suspension can be prepared by a simple solvent-removal process.
Typically, 10 mg of branched block copolymer was dissolved in 5 mL
of acetone, followed by addition of 5 mL of water and stirred for 0.5 h
at room temperature. Acetone was removed by evaporation at room
temperature, and final transparent nanoparticles aqueous suspension
was obtained.
Herein, we demonstrated for the first time that the branched copol-
ymer nanoparticles (BCN) could be used to form stable emulsions with-
out other additives. The branched copolymers applied here were the
biocompatible poly(ethylene glycol)-b-(N-isopropylacrylamide) (PEG-
PNIPAM). The formed oil-in-water (O/W) emulsions with hydrophobic
dyes or drug compounds dissolved in the oil-droplet phase were freeze-
dried to form nanoparticles in situ within the PEG-PNIPAM scaffold,
which can then be readily dissolved in water to produce aqueous nano-
particles dispersions.
2.3. Formulation of nanoparticles by emulsion-freeze-drying approach
Stock solutions of 2 wt.% branched block polymer PEG-PNIPAM (0.3,
0.6 and 0.9 cross-linkages as synthesized by ethylene diacrylamide of
0.3, 0.6, 0.9 eq per PEG chain) in deionized water and 0.5 wt.% Oil Red
O (or indomethacin, ibuprofen, ketoprofen) in cyclohexane (o-xylene)
solutions were prepared. Cyclohexane and o-xylene were chosen as
the organic solvents to dissolve the hydrophobic dye/drugs because
they are Class 2 solvents for pharmaceuticals with high concentration
limits (3880 ppm and 2170 ppm, respectively) [48]. Both solvents are
volatile with high melting points (~4 °C for cyclohexane and −25 °C
for o-xylene), which makes them suitable for a freeze-drying process.
Furthermore, both solvents could be readily emulsified to form stable
oil-in-water emulsions [38,49]. Solvent residuals after freeze-drying
could be within the limit as defined by the International Council for Har-
monization of Technical Requirements for Pharmaceuticals for Human
Use (ICH) as shown by freeze-drying organic solvents with similar va-
pour pressure [50]. Under stirring at 1000 rpm with an overhead stirrer
(Eurostar digital, IKA-WERKE), the cyclohexane solution was added
dropwise over a period of 2 min to the aqueous PEG-PNIPAM solution
at room temperature (also once at 50 °C to investigate the temperature
effect because the NIPAM block is known to be temperature sensitive).
The emulsions with the volume ratios of aqueous phase to organic
phase (W/O) of 1:4; 1:3 and 1:2 were prepared. After continuously
2. Experimental
2.1. Chemicals and reagents
Deionized water was prepared using an AquaMAX-Basic 321 DI
water purification system. Oil Red O (OR) dye content ≥75%, ketoprofen
≥98% (TLC), ibuprofen ≥98% (HPLC), indomethacin ≥99% (TLC), o-
xylene ≥98% (GC), sodium acetate, N-isopropylacrylamide (NIPAM,
97%), and dodecanethiol (98%) were purchased from Sigma-Aldrich.
Macro-azo poly(ethylene glycol) initiator was obtained from Wako
Pure Chemical Industries Ltd. (Osaka, Japan). Cyclohexane (extra
pure) and o-xylene were purchased from Fisher scientific and VWR in-
ternational respectively. All other solvents were reagent grade and pur-
chased from Sigma-Aldrich. All chemicals were used as received.