Synthesis and unimolecular micelles of amphiphilic dendrimer-like star polymer with various functional surface groups

Article abstract of DOI:10.1021/ma1021242

We report the synthesis and functionalization of amphiphilic dendrimer-like star polymers (DLSPs) with a hydrophobic star-shaped poly(l-lactide) (PLLA) core and a hydrophilic poly(amidoamine) (PAMAM) dendron shell. First, carboxylic acid-functionalized PL

Full text of DOI:10.1021/ma1021242

ARTICLE
pubs.acs.org/Macromolecules
Synthesis and Unimolecular Micelles of Amphiphilic Dendrimer-like
Star Polymer with Various Functional Surface Groups
Weiqiang Cao† and Lei Zhu*,†,§
†Polymer Program, Institute of Materials Science and Department of Chemical, Materials and Biomolecular Engineering, University of
Connecticut, Storrs, Connecticut 06269-3136, United States
§Department of Macromolecular Science and Engineering, Case Western Reserve University, Cleveland, Ohio 44106-7202, United
States
S Supporting Information
b
ABSTRACT: We report the synthesis and functionalization of
amphiphilic dendrimer-like star polymers (DLSPs) with a
hydrophobic star-shaped poly(L-lactide) (PLLA) core and a
hydrophilic poly(amidoamine) (PAMAM) dendron shell. First,
carboxylic acid-functionalized PLLA star polymer was synthe-
sized by ring-opening polymerization of L-lactide followed by
functionalization with succinic anhydride. Second, 1-, 2-, and
3-generation PAMAM dendrons with a primary amine at the
dendron root and benzyl ester protections at the periphery were
prepared via a divergent method. By amide coupling between
the carboxylic acid-terminated PLLA star polymer and six
PAMAM dendrons, amphiphilic DLSPs were successfully
synthesized. To enhance bioactivity and bioconjugation cap-
ability, the benzyl ester surface groups in these DLSPs were converted to carboxylic acid, primary amine, and triethylene glycol
functional groups, respectively. Nuclear magnetic resonance spectroscopy and size-exclusion chromatography were used to
confirm quantitative functionalization. These functional DLSPs exhibited a unique unimolecular micelle (14-28 nm) behavior
in aqueous solution with a small amount of aggregation (205-344 nm), as studied by dynamic light scattering. In addition, they
also exhibited large differences in thermal behaviors depending on the nature of different surface groups. Experimental results
showed that these DLSPs had good solubility in aqueous solutions (ca. 10-25 mg/mL) and could greatly enhance the water
solubility of hydrophobic drugs. Therefore, these amphiphilic DLSPs are promising candidates for controlled hydrophobic drug
delivery.
’ INTRODUCTION
side effects, prolong the circulation time, and reduce the uptake
by the reticuloendothelial system (RES).3,4 Typically, poly-
(lactic acid) or polylactides (PLA), poly(lactic-co-glycolic acid)
(PLGA), and poly(ε-caprolactone) (PCL) are copolymerized
with PEG to form either AB or ABA block copolymers and are
the most frequently used as biomaterials for drug delivery
because of their outstanding biodegradability and bio-
compatibility.5-10 These amphiphilic biodegradable (at least
partially) copolymers can easily self-assemble in aqueous solu-
tion to form nanoparticles with an average size ranging from 50
to 500 nm, and the self-assembled hydrophobic cores are used to
encapsulate hydrophobic drugs. In certain practical applications,
however, the size of nanoparticles is important to the biodis-
tribution in vivo.11 Previous studies using liposomes as drug
carriers have shown that the amount of sequestrated liposome
Even though many new anticancer drugs have been developed
by the pharmaceutical industry, the clinical use of new che-
motherapeutical drugs is still limited, and more than 6 million
people die from cancer around the world every year.1 New
experimental approaches are being actively sought and re-
searched to enhance delivery efficacy and reduce side effects
and systemic damage. One of many research tasks is to enhance
water solubility for anticancer drugs such as most alkylating
agents and plant alkaloids and terpenoids (e.g., paclitaxel and
docetaxel), while remaining anticancer potency. For example,
conjunction with surfactants or covalent conjugation with a
hydrophilic tail such as poly(ethylene glycol) (PEG) have been
attempted to improve water solubility.2 It is noted that a decrease
in efficacy and harmful side effects potentially exist for this
conjugation methodology.
Amphiphilic block or graft copolymers consisting of hydro-
philic and hydrophobic segments are considered as good candi-
dates for drug delivery because they may decrease the unwanted
Received: September 13, 2010
Revised:
January 27, 2011
Published: February 22, 2011
r
2011 American Chemical Society
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dx.doi.org/10.1021/ma1021242 Macromolecules 2011, 44, 1500–1512
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nanoparticles in spleen decreased with decreasing the particle
size12,13 and smaller nanoparticles (e.g., 8-20 nm) may deliver
drugs more effectively.14-16
use. Anhydrous toluene was dried over Na and distilled and stored under
a dry N2 atmosphere. Stannous(II) 2-ethylhexanoate [Sn(Oct)2],
ethylenediamine, methyl acrylate, and all other chemicals were pur-
chased from Sigma-Aldrich, Fluka, or Alfa Aesar and used without
further purification. The PLLA star polymer was prepared according to
procedures developed before.59 Its number-average molecular weight
was 9200 g/mol as determined by end-group analysis from proton
nuclear magnetic resonance (1H NMR) and molecular weight distribu-
tion was 1.09 as determined by size-exclusion chromatography (SEC).
Measurements and Instrumentation. 1H and 13C NMR
spectra were recorded on Bruker DRX-400 and DRX-500 (400 and
500 MHz, respectively), using either CDCl3 or DMSO-d6 as the solvent
depending on the solubility of compounds. Mass spectrometer (MS)
analysis of PAMAM dendrons with various generations was performed
on either a VG Quattro II mass spectrometer equipped with an
electrospray ionization source or a QSTAR Elite mass spectrometer.
Methanol/water (50/50 vol/vol) with 0.1% formic acid was used as the
solvent and eluent. The molecular weight distribution was determined
by SEC using a Waters 150C Plus gel permeation chromatograph
equipped with a Waters 410 differential refractometer. Dimethylaceta-
mide (DMAc) with 0.05 M LiBr was used as the mobile phase at a flow
rate of 1.0 mL/min, and poly(methyl methacrylate) standards were used
for conventional calibration. Differential scanning calorimetry (DSC)
was carried out on a TA Instruments DSC Q-20 with a scanning rate of
10 °C/min. The hydrodynamic sizes for micelles and aggregates in
aqueous solutions were determined by DLS using a Malvern Zetasizer
Nano S equipped with a 633 nm laser. The measurement was performed
at 20 °C with a detection angle of 90°. All polymer solutions (0.5 mg/
mL) were filtered through Millipore membranes with a pore size of
0.45 μm prior to measurements. The hydrodynamic diameters were calcu-
lated using the Zetasizer Nano Series software provided by the manu-
facturer. The morphology of the micelles was studied using transmission
electron microscopy (TEM) on a Tecnai T12 operating at an accelerat-
ing voltage of 120 kV. One drop of the aqueous solution (0.2 mg/mL) of
micelles was placed on a carbon-coated copper grid, which was irradiated
with UV light to increase the hydrophilicity of the carbon film for 1 min.
Excess solution was blotted away with a filter paper and dried in a
vacuum oven overnight before being stained with RuO4 for 15 min at
ambient temperature.
Synthesis of Carboxylic Acid-Functionalized PLLA Star
Polymer [G1-(COOH)6] 1. Succinic anhydride (1.304 g, 13.04
mmol) and 4-(dimethylamino)pyridine (DMAP) (0.08 g, 0.65 mmol)
were dissolved in a mixture solvent of 20 mL of dichloromethane and 5.0
mL of pyridine. After the reaction flask was flushed with dry N2, 15 mL of
dichloromethane solution containing 1.0 g (0.11 mmol) of G1-(OH)6
was added dropwise into the above solution at 0 °C. The mixture was
reacted for 2 days at room temperature. Afterward, the solvent was
removed under reduced pressure. The crude sample was redissolved in
dichloromethane and precipitated into methanol twice. The precipitate
was filtered and dried in a vacuum oven for 2 days to give 0.95 g of final
product, [G1-(COOH)6] 1 (yield: 89%). 1H NMR (CDCl3, δ): 1.41 (s,
9H, -CCH3(CH2O)2-), 1.49-1.60 (m, poly, -CH3), 2.12-2.15 (br,
3H, -C(CH3)(Ar)3), 2.63-2.71 (m, 24H, -COCH2-), 4.36-4.52
(m, 12H, -CCH3(CH2O-)2), 5.14-5.20 (m, poly, -CH-), 6.91-
7.06 (dd, 12H, Ar-). 13C NMR (CDCl3, δ): 16.76, 16.94, 17.78, 20.62,
28.74, 46.90, 65.86, 68.78, 69.13, 120.85, 129.85, 146.49, 148.66, 169.72,
170.24, 170.88, 171.70, 176.61. DSC results: a single melting temperature
at 107 °C upon the first heating and only a glass transition temperature
(Tg) at 54 °C upon the second heating. SEC results: PDI = 1.09.
Synthesis of N-Boc-ethylenediamine 2. A dichloromethane
solution (100 mL) of di-tert-butyl dicarbonate (4.0 g, 18.33 mmol) was
added dropwise into 50 mL of dichloromethane solution of ethylene-
diamine (6.6 g, 110 mmol) at 0 °C. The reaction mixture was then stirred
at room temperature overnight. The solvent was removed using a rotary
To achieve smaller particle sizes, dendrimer, star, and hyper-
branched polymers are attractive as potential drug carriers. Com-
pared to linear polymers, dendrimers possess many unique
advantages, such as small particle size (e.g., only several
nanometers), large number of tunable surface functional groups,
high surface area-to-volume ratios, and small polydispersity indices
(PDI) with well-defined structures.17,18 Therefore, dendrimer-
based supermolecules are of widespread interest for many applica-
tions including drug delivery,19-21 gene delivery,22-25 tissue
sealants,26-28 catalysis,29-31 and light harvesting.32-34 In particu-
lar, the tunable surface functional groups allow a great opportunity
for further chemical conjugation with other molecules, such as
targeting molecules and polymers, fluorescence dye, or even drugs.
Research on biodegradable and amphiphilic dendrimers for drug
delivery has attracted substantial attention.35-41 Neverthe-
less, synthesis of these dendrimers, which involves either a
divergent42,43 or a convergent44-47 method, is nontrivial, and
especially, high generations are necessary to obtain large enough
unimolecular particles for drug encapsulation.
Alternatively, segment- and surface-block dendrimers and
dendritic-linear hybrids have been pursued to simplify the
synthetic steps.48-51 The amphiphilicity of these dendrimer-
based supermolecules can be easily tuned by introducing either a
hydrophobic or a hydrophilic linear chain. Depending on the
composition and hydrophobicity/hydrophilicity of the linear
polymer and the dendron, these supermolecules may still self-
assemble to form large aggregates in aqueous solution. Another
seemingly good candidate is the amphiphilic dendrimer-like star
polymer (DLSP), which has a similar structure to dendrimers but
the hydrophobic core is replaced by a star polymer.52-56 In
addition to the advantages of dendrimers, amphiphilic DLSPs
consisting of a hydrophobic core and a hydrophilic dendron shell
have several advantages for drug delivery. (1) Unimolecualr
micelles can be achieved and the particle size can be easily
adjusted by controlling the molecular weight of the star polymer.
(2) The amphiphilicity can be tuned by changing the ratio of the
hydrophobic core to the hydrophilic shell in order to achieve
good water solubility. (3) The synthesis can be easier because
high-generation dendrons are not necessary to obtain large enough
unimolecular micelles for hydrophobic drug encapsulation.
(4) In contrast to the rigid inner structure of dendrimers, the
flexible hydrophobic core in DLSP favors more encapsulation of
hydrophobic drugs.
Most amphiphilic DLSPs have so far been developed as new
materials for microelectronic industry.57,58 In this study, we
designed a novel DLSP comprised of a hydrophobic PLLA star
polymer and six hydrophilic PAMAM dendrons with different
generations. The surface of the DLSP could be quantitatively
functionalized with various groups such as carboxylic acid,
primary amine, and triethylene glycol. Their unimolecular mi-
celles in aqueous solution were studied by dynamic laser light
scattering (DLS). Intriguingly, these DLSPs had good solubility
in aqueous solutions and could greatly enhance the water
solubility of hydrophobic drugs.
’ EXPERIMENTAL SECTION
Materials. L-Lactide (Purac Inc.) was recrystallized from toluene
twice and dried in a vacuum oven at room temperature for 3 days before
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ARTICLE
evaporator, and the crude product was dissolved in 50 mL of doubly
distilled water, filtered, and then extracted with 100 mL of dichloro-
methane twice. The final product was obtained as a viscous and colorless
oil (2.5 g, yield: 85%). 1H NMR (CDCl3, δ): 1.41 (s, 9H, -C(CH3)3),
2.76-2.79 (br, 2H, -CH2NH2), 3.14-3.27 (br, 2H, -NHCH2-),
5.28 (br, 1H, BocNH-). 13C NMR (CDCl3, δ): 28.48, 41.84, 43.30,
79.15, 156.37. MS results for C7H16N2O2: calculated 160.2 and
found 160.9.
CH2CH2NH2), 3.06-3.20 (br, 2H, BocNHCH2-), 3.25-3.31 (q, 4H,
-CH2CH2NH2), 6.38 (br, 1H, BocNHCH2-), 7.23 (br, 2H, -
CONHCH2-). 13C NMR (CDCl3, δ): 28.60, 33.98, 38.48, 41.37,
41.97, 50.40, 53.28, 79.09, 156.51, 172.81. MS results for C17H36N6O4:
calculated 388.5 and found: 388.8.
g1-[Boc, (NH2)4] 7. 1H NMR (CDCl3, δ): 1.41 (s, 9H, -C(CH3)3),
2.25-2.40 (m, 12H, -CH2CONH-), 2.42-2.57 (t, 6H, -CH2N-
(CH2)2), 2.60-2.78 (t, 12H, -CH2N(CH2)2), 2.78-2.88 (t, 8H, -
CH2CH2NH2), 3.09-3.17 (br, 2H, BocNHCH2-), 3.17-3.32 (q,
12H, -CONHCH2-), 5.61 (br, 1H, BocNHCH2-), 7.45 (br, 4H, -
CONHCH2-), 7.86 (br, 2H, -CONHCH2-). 13C NMR (CDCl3, δ):
28.57, 33.95, 34.32, 37.86, 38.56, 41.53, 42.25, 50.11, 52.93, 78.14,
156.48, 172.95, 173.26. MS results for C37H76N14O8: calculated 845.1
and found: 845.2.
Synthesis of g0-[Boc, (COOCH3)2] 3 and General Synthesis
Procedure for Boc-Protected and Methyl Ester-Terminated
PAMAM Dendrons 4 and 5. A solution of 2.0 g (12.48 mmol) N-
Boc-ethylenediamine 2 in 7.0 mL of methanol was added dropwise to a
stirred solution of 4.33 g of methyl acrylate (49.92 mmol) in 8.0 mL of
methanol at 0 °C. The resulting solution was then allowed to warm up to
room temperature and stirred for 2 days. The solvent and the majority of
the excess methyl acrylate were removed under reduced pressure using a
rotary evaporator. The residue was further purified in a vacuum oven for
2 days to give the final product as a viscous yellowish oil 3 (4.11 g, yield:
99%). Similar procedures were used for the preparation of Boc-
protected and methyl ester-terminated PAMAM dendrons 4 and 5.
g2-[Boc, (NH2)8] 8. 1H NMR (DMSO-d6, δ): 1.36 (s, 9H, -
C(CH3)3), 2.14-2.26 (m, 28H, -CH2CONH-), 2.39-2.46 (m,
14H, -CH2N(CH2)2), 2.53-2.59 (m, 16H, -CH2CH2NH2), 2.60-
2.70 (m, 28H, -CH2N(CH2)2), 2.90-2.99 (br, 2H, BocNHCH2-),
3.00-3.14 (m, 28H, -CONHCH2-), 6.55 (br, 1H, BocNHCH2-),
7.88 (br, 14H, -CONHCH2-). 13C NMR (DMSO-d6, δ): 28.24,
33.25, 33.32, 36.92, 41.28, 42.15, 49.58, 49.68, 52.24, 77.52, 155.56,
171.26, 171.48. MS results for C77H156N30O16: calculated 1758.3 and
found: 1758.6.
Synthesis of g1-[Boc, (COOBn)4] 9 and General Synthesis
Procedure for Boc-Protected and Benzyl Ester-Terminated
PAMAM Dendrons 10 and 11. Amine-terminated PAMAM den-
dron, g0-[Boc, (NH2)2] 6 (1.0 g, 2.57 mmol), and benzyl acrylate (3.34
g, 20.59 mmol) were flushed with dry N2 and stirred at 80 °C for 5 days.
The reaction mixture was then cooled to room temperature. The crude
product was purified by column chromatography on silica gel with the
eluting solvent changing from ethyl acetate to methanol, yielding 2.32 g
(87%) of 9 in the form of a viscous and yellowish oil. Similar procedures
were used for the preparation of Boc-protected and benzyl ester-
terminated PAMAM dendrons 10 and 11.
1
g0-[Boc, (COOCH3)2] 3. H NMR (CDCl3, δ): 1.41 (s, 9H, -
C(CH3)3), 2.29-2.41 (t, 4H, -CH2CO2-), 2.41-2.50 (t, 2H, -
CH2N(CH2)2), 2.61-2.77 (t, 4H, -CH2N(CH2)2), 3.14-3.16 (br,
2H, BocNHCH2-), 3.64 (s, 6H, -OCH3), 5.28 (br, 1H, BocNHCH2-).
13C NMR (CDCl3, δ): 28.58, 32.79, 38.22, 49.34, 51.70, 53.24, 78.96,
156.22, 173.07. MS results for C15H28N2O6: calculated 332.4 and
found: 332.5.
1
g1-[Boc, (COOCH3)4] 4. H NMR (CDCl3, δ): 1.41 (s, 9H, -
C(CH3)3), 2.31-2.37 (t, 4H, -CH2CONH-), 2.37-2.43 (t, 8H, -
CH2CO2-), 2.47-2.59 (t, 6H, -CH2N(CH2)2), 2.68-2.81 (m, 12H,
-CH2N(CH2)2), 3.09-3.21 (br, 2H, BocNHCH2-), 3.25-3.33 (q,
4H, -CONHCH2-), 3.64 (s, 12H, -OCH3), 5.43 (br, 1H,
BocNHCH2-), 6.96 (br, 2H, -CONHCH2-). 13C NMR (CDCl3,
δ): 28.30, 32.48, 33.63, 36.92, 38.25, 49.05, 49.68, 51.45, 52.42, 52.83,
78.60, 155.93, 172.08, 172.85. MS results for C33H60N6O12: calculated
732.9 and found 732.8.
g1-[Boc, (COOBn)4] 9. 1H NMR (CDCl3, δ): 1.41 (s, 9H, -
C(CH3)3), 2.31-2.37 (m, 4H, -CH2CONH-), 2.37-2.43 (m, 8H, -
CH2CO2-), 2.47-2.59 (m, 6H, -CH2N(CH2)2), 2.68-2.81 (m,
12H, -CH2N(CH2)2), 3.09-3.21 (br, 2H, BocNHCH2-), 3.25-
3.33 (m, 4H, -CONHCH2-), 5.10 (s, 8H, -CH2Ph), 5.42 (br, 1H,
BocNHCH2-), 6.93 (br, 2H, -CONHCH2-), 7.25-7.38 (m, 20H, -
CH2Ph). 13C NMR (CDCl3, δ):28.58, 32.78, 33.79, 37.24, 38.52, 49.26,
49.90, 52.64, 53.06, 66.47, 78.97, 128.37, 128.67, 135.90, 156.18, 172.29,
172.50. MS results for C57H76N6O12: calculated 1037.2 and found:
1037.2.
1
g2-[Boc, (COOCH3)8] 5. H NMR (CDCl3, δ): 1.41 (s, 9H, -
C(CH3)3), 2.28-2.42 (m, 12H, -CH2CONH-), 2.42-2.51 (t, 16H,
-CH2CO2-), 2.51-2.65 (m, 14H, -CH2N(CH2)2), 2.70-2.84 (m,
28H, -CH2N(CH2)2), 3.09-3.17 (br, 2H, BocNHCH2-), 3.19-3.34
(q, 12H, -CONHCH2-), 3.64 (s, 24H, -OCH3), 5.63 (br, 1H,
BocNHCH2-), 6.98 (br, 4H, -CONHCH2-), 7.64 (br, 2H, -
CONHCH2-). 13C NMR (CDCl3, δ): 28.52, 32.72, 33.82, 37.25,
37.51, 38.50, 49.29, 49.84, 50.03, 51.68, 52.58, 52.63, 52.96, 78.87,
156.26, 172.48, 172.56, 173.11. MS results for C69H124N14O24: calcu-
lated 1533.8 and found 1534.2.
1
g2-[Boc, (COOBn)8] 10. H NMR (CDCl3, δ): 1.41 (s, 9H, -
C(CH3)3), 2.28-2.42 (m, 12H, -CH2CONH-), 2.42-2.51 (m, 16H,
-CH2CO2-), 2.51-2.65 (m, 14H, -CH2N(CH2)2), 2.70-2.84 (m,
28H, -CH2N(CH2)2), 3.09-3.17 (br, 2H, BocNHCH2-), 3.19-3.34
(m, 12H, -CONHCH2-), 5.10 (s, 16H, -CH2Ph), 5.63 (br, 1H,
BocNHCH2-), 6.96 (br, 4H, -CONHCH2-), 7.25-7.38 (m, 40H, -
CH2Ph), 7.65 (br, 2H, -CONHCH2-). 13C NMR (CDCl3, δ): 28.65,
32.91, 33.87, 37.41, 37.59, 38.63, 49.37, 49.93, 50.12, 52.58, 52.73, 53.03,
66.48, 78.91, 128.41, 128.71, 135.97, 156.31, 172.45, 172.55. MS results
for C117H156N14O24: calculated 2142.6 and found: 2143.2.
Synthesis of g0-[Boc, (NH2)2] 6 and General Synthesis
Procedure for Boc-Protected and Amine-Terminated PA-
MAM Dendrons 7 and 8. Methyl ester-terminated PAMAM den-
dron g0-[Boc, (COOCH3)8] 3 (4.0 g, 12.03 mmol) was dissolved in 20
mL of methanol and was then added dropwise to a cold (0 °C) 30 mL
methanol solution of 43.4 g (0.722 mol) of ethylenediamine over a
period of 1 h. The reaction mixture was allowed to warm up to room
temperature and stirred for 5 days. Afterward, the solvent was removed
under reduced pressure using a rotary evaporator. The excess ethylene-
diamine was distilled off as an azeotrope with toluene/methanol, and the
remaining toluene was removed by vacuum distillation. The residue was
dried in a vacuum oven for 2 days to give the amino-terminated product
as a honey-like oil 6 (4.58 g, yield: 98%). Similar procedures were used
for the preparation of Boc-protected and amine-terminated PAMAM
dendrons 7 and 8.
1
g3-[Boc, (COOBn)16] 11. H NMR (CDCl3, δ): 1.41 (s, 9H, -
C(CH3)3), 2.25-2.40 (m, 28H, -CH2CONH-), 2.40-2.49 (m, 32H,
-CH2CO2-), 2.49-2.62 (m, 30H, -CH2N(CH2)2), 2.65-2.84 (m,
60H, -CH2N(CH2)2), 3.09-3.19 (br, 2H, BocNHCH2-), 3.19-3.34
(m, 28H, -CONHCH2-), 5.10 (s, 32H, -CH2Ph), 5.73 (br, 1H,
BocNHCH2-), 6.99 (br, 8H, -CONHCH2-), 7.25-7.38 (m, 80H, -
CH2Ph), 7.59 (br, 4H, -CONHCH2-), 7.75 (br, 2H, -CONHCH2-).
13C NMR (CDCl3, δ): 28.4, 32.63, 33.68, 37.15, 37.40, 49.11, 49.70, 49.86,
52.35, 52.71, 66.16, 78.58, 128.11, 128.43, 135.72, 156.13, 172.28, 172.43.
MS results for C237H316N30O48: calculated 4353.2 and found: 4354.0.
g0-[Boc, (NH2)2] 6. 1H NMR (CDCl3, δ): 1.41 (s, 9H, -C(CH3)3),
2.25-2.40 (t, 4H, -CH2CONH-), 2.41-2.55 (t, 2H, -CH2N-
(CH2)2), 2.57-2.72 (t, 4H, -CH2N(CH2)2), 2.72-2.88 (t, 4H, -
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Synthesis of g1-[NH2, (COOBn)4] 12 and General Synthe-
sis Procedure for Deprotection of Boc Groups for 13 and
14. Boc-protected and benzyl ester-terminated PAMAM dendron
g1-[Boc, (COOBn)4] 9 (1.0 g, 0.964 mmol) was dissolved in 3.0 mL
of dichloromethane, and the solution was cooled to 0 °C. 1.5 mL of
trifluoroacetic acid (TFA) was added, and the reaction mixture was
stirred at room temperature for 12 h. The solvent and the majority of the
excess TFA were removed under reduced pressure using a rotary
evaporator. The dried solid was redissolved in 30 mL of dichloro-
methane and extracted with 10 mL of 25% aqueous solution of sodium
carbonate. The organic phase was dried by anhydrous MgSO4. The
product was dried in a vacuum oven for 2 days to give a viscous and
yellowish oil (0.81 g, yield: 90%). Similar deprotection procedures were
used to prepare benzyl ester-terminated PAMAM dendrons 10 and 11
with primary amine at the root.
-CCH3(CH2O-)2), 5.09 (s, 48H, -CH2Ph), 5.14-5.20 (m, poly, -
CH-), 6.80-6.89 (m, 12H, -CONHCH2-), 6.92-7.05 (dd, 12H,
Ar-), 7.16-7.23 (m, 6H, -CONHCH2-), 7.25-7.35 (m, 120H, -
CH2Ph). 13C NMR (CDCl3, δ): 16.69, 16.84, 17.71, 20.60, 29.27, 30.49,
32.85, 33.90, 37.28, 37.70, 46.84, 49.28, 49.86, 52.83, 52.95, 65.81, 66.42,
68.37, 69.06, 120.78, 128.32, 128.64, 129.78, 135.87, 146.43, 148.60,
169.66, 170.38, 170.82, 171.24, 172.51. DSC results: Tg at 19 °C upon
second heating. SEC results: PDI = 1.12.
G1-g2-(COOBn)48 16. 1H NMR (CDCl3, δ): 1.41 (s, 9H, -
CCH3(CH2O)2-), 1.49-1.60 (m, poly, -CH3), 2.12-2.15 (br, 3H,
-CCH3(Ar)3), 2.23-2.31 (m, 72H, -CH2CONH-), 2.34-2.44 (m,
96H, -CH2CO2Bn), 2.44-2.52 (m, 96H, -OCOCH2CH2CO-, -
CH2N(CH2)2), 2.61-2.81 (m, 180H, -OCOCH2CH2CO-, -
CH2N(CH2)2), 3.13-3.33 (m, 84H, -CONHCH2-), 4.36-4.52
(m, 12H, -CCH3(CH2O-)2), 5.09 (s, 96H, -CH2Ph), 5.14-5.20
(m, poly, -CH-), 6.95-6.97 (m, 30H, -CONHCH2-, Ar-), 7.08-
7.10 (d, 6H, Ar-), 7.25-7.36 (m, 240H, -CH2Ph), 7.46-7.49 (m, 6H,
-CONHCH2-), 7.55-7.57 (m, 12H, -CONHCH2-). 13C NMR
(CDCl3, δ): 16.72, 16.88, 17.73, 20.60, 29.31, 30.45, 32.85, 33.91, 34.03,
37.35, 37.60, 37.92, 46.86, 49.31, 49.87, 50.15, 52.67, 52.96, 53.52, 65.81,
66.42, 68.37, 69.08, 120.80, 128.34, 128.65, 129.80, 135.91, 146.44,
148.62, 169.68, 170.41, 170.84, 171.33, 172.50. DSC results: Tg at 12 °C
upon second heating. SEC results: PDI = 1.15.
g1-[NH2, (COOBn)4] 12. 1H NMR (CDCl3, δ): 2.23-2.35 (m, 4H,
-CH2CONH-), 2.36-2.43 (m, 8H, -CH2CO2-), 2.43-2.54 (m,
4H, -CH2N(CH2)2), 2.54-2.67 (m, 6H, -CH2N(CH2)2), 2.68-2.81
(m, 8H, -CH2N(CH2)2), 2.96-3.16 (br, 2H, H2NCH2-), 3.13-3.26
(m, 4H, -CONHCH2-), 5.10 (s, 8H, -CH2Ph), 6.93 (br, 2H, -
CONHCH2-), 7.25-7.38 (m, 20H, -CH2Ph). 13C NMR (CDCl3, δ):
32.90, 33.67, 37.38, 37.55, 48.95, 49.35, 51.21, 52.89, 66.57, 128.46,
128.76, 135.99, 172.78. MS results for C52H68N6O10: calculated 937.1
and found: 937.2.
G1-g3-(COOBn)96 17. 1H NMR (CDCl3, δ): 1.41 (s, 9H, -CCH3-
(CH2O)2-), 1.49-1.60 (m, poly, -CH3), 2.12-2.15 (br, 3H, -
CCH3(Ar)3), 2.23-2.31 (m, 168H, -CH2CONH-), 2.34-2.44 (m,
192H, -CH2CO2Bn), 2.44-2.52 (m, 192H, -OCOCH2CH2CO-, -
CH2N(CH2)2), 2.61-2.81 (m, 372H, -OCOCH2CH2CO-, -CH2N-
(CH2)2), 3.13-3.33 (m, 180H, -CONHCH2-), 4.36-4.52 (m, 12H,
-CCH3(CH2O-)2), 5.09 (s, 192H, -CH2Ph), 5.14-5.20 (m, poly, -
CH-), 6.92-6.98 (m, 54H, -CONHCH2-, Ar-), 7.04-7.06 (d, 6H,
Ar-), 7.18-7.20 (m, 24H, -CONHCH2-), 7.25-7.30 (m, 480H, -
CH2Ph), 7.50-7.55 (m, 12H, -CONHCH2-), 7.64-7.66 (m, 6H, -
CONHCH2-). 13C NMR (CDCl3, δ): 16.83, 17.01, 17.85, 20.72, 29.38,
30.50, 32.96, 33.86, 37.47, 37.67, 37.98, 46.97, 49.42, 50.00, 50.18, 52.61,
53.05, 65.92, 66.52, 68.49, 69.19, 120.91, 128.45, 128.76, 129.92, 136.04,
146.56, 148.74, 169.79, 170.53, 170.95, 172.50, 172.61. DSC results: Tg
at -4 °C upon second heating. SEC results: PDI = 1.16.
1
g2-[NH2, (COOBn)8] 13. H NMR (CDCl3, δ): 2.28-2.42 (m,
12H, -CH2CONH-), 2.42-2.51 (m, 16H, -CH2CO2-), 2.51-2.65
(m, 14H, -CH2N(CH2)2), 2.70-2.84 (m, 28H, -CH2N(CH2)2),
3.09-3.19 (br, 2H, H2NCH2-), 3.19-3.34 (m, 12H, -CONHCH2-),
5.10 (s, 16H, -CH2Ph), 7.23 (br, 4H, -CONHCH2-), 7.25-7.38
(m, 40H, -CH2Ph), 7.54 (br, 2H, -CONHCH2-). 13C NMR
(CDCl3, δ): 32.87, 33.37, 34.20, 36.56, 37.27, 37.46, 49.37, 49.89,
50.11, 52.82, 53.54, 66.43, 128.34, 128.67, 135.94, 172.54, 172.94. MS
results for C112H148N14O22: calculated 2042.4 and found: 2043.3.
1
g3-[NH2, (COOBn)16] 14. H NMR (CDCl3, δ): 2.24-2.40 (m,
28H, -CH2CONH-), 2.40-2.51 (m, 32H, -CH2CO2-), 2.51-2.64
(m, 30H, -CH2N(CH2)2), 2.64-2.86 (m, 60H, -CH2N(CH2)2),
3.09-3.18 (br, 2H, H2NCH2-), 3.19-3.34 (m, 28H, -CONHCH2-),
5.10 (s, 32H, -CH2Ph), 6.99 (br, 8H, -CONHCH2-), 7.25-7.38
(m, 80H, -CH2Ph), 7.59 (br, 4H, -CONHCH2-), 7.75 (br, 2H, -
CONHCH2-). 13C NMR (CDCl3, δ): 32.8, 33.56, 36.75, 37.36, 48.57,
49.28, 49.86, 50.08, 52.33, 52.85, 66.37, 128.30, 128.62, 135.90, 172.39,
172.49, 172.89. MS results for C232H308N30O46: calculated 4253.1 and
found: 4254.0.
Synthesis of G1-g1-(COOBn)24 15 and General Synthesis
Procedure for Conjugation of the PLLA Core and Different
PAMAM Dendrons. Carboxylic acid-functionalized PLLA core 1 (1.0
g, 0.102 mmol) was dissolved in 30 mL of dichloromethane, and then 0.25
g (2.45 mmol) of triethylenamine (TEA) and 0.64 g (1.23 mmol) of
(benzotriazol-1-yloxy)tripyrrolidinophosphonium hexafluorophosphate
(PyBOP) were added. The reaction solution was stirred at room
temperature for 1 h, and the amine-functionalized PAMAM dendron 12
(0.86 g, 0.92 mmol) was added. The reaction mixture was stirred at room
temperature for 20 h before precipitation in methanol. The crude product
was redissolved in dichloromethane and precipitated in methanol again.
The precipitated solid was collected and washed carefully with fresh
methanolseveraltimes. The finalproduct was obtained asa yellowishsolid
15 (1.28 g, yield: 82%). Similar procedures were carried out for the
synthesis of G1-g2-(COOBn)48 16 and G1-g3-(COOBn)96 17.
G1-g1-(COOBn)24 15. 1H NMR (CDCl3, δ): 1.41(s, 9H, -CCH3-
(CH2O)2-), 1.49-1.60 (m, poly, -CH3), 2.12-2.15 (br, 3H, -
CCH3(Ar)3), 2.23-2.31 (m, 24H, -CH2CONH-), 2.34-2.44 (m,
48H, -CH2CO2Bn), 2.44-2.52 (m, 48H, -OCOCH2CH2CO-, -
CH2N(CH2)2), 2.61-2.81 (m, 84H, -OCOCH2CH2CO-, -CH2N-
(CH2)2), 3.13-3.33 (m, 36H, -CONHCH2-), 4.36-4.52 (m, 12H,
Synthesis of G1-g1-(COOH)24 18 and General Procedure
for Deprotection of Benzyl Ester Terminal Groups. G1-
g1-(COOBn)24 15 (0.5 g, 32.65 μmol) was dissolved in 15 mL of TFA,
and the solution was cooled to 0 °C. 6.0 mL of methanesulfonic acid
(MsOH) and 1.5 mL of anisole were added slowly, and the resulting
solution was kept stirring at 0 °C for 2 h. Once the reaction was complete,
the polymer was precipitated in diethyl ether and dried in a vacuum oven.
The dried sample was redissolved in 10 mL of DMSO, and 0.5 mL of TEA
was added to neutralize the excess acid. The sample was then purified by
dialysis in DMSO using a dialysis tube with a molecular weight cutoff
(MWCO) of 6000 g/mol (regenerated cellulose dialysis tube from Fisher
Scientific). The final product was precipitated in diethyl ether and was
obtained as a slightly yellow solid 18 (0.34 g, yield: 79%). Similar procedures
were used to synthesize G1-g2-(COOH)48 19 and G1-g3-(COOH)96 20.
1
G1-g1-(COOH)24 18. H NMR (DMSO-d6, δ): 1.33 (s, 9H, -
CCH3(CH2O)2-), 1.41-1.47 (m, poly, -CH3), 2.15-2.22 (m, 27H,
-CCH3(Ar)3, -CH2CONH-), 2.32-2.36 (m, 48H, -CH2CO2Bn),
2.48-2.52 (m, 48H, -OCOCH2CH2CO-, -CH2N(CH2)2), 2.67-
2.76 (m, 84H, -OCOCH2CH2CO-, -CH2N(CH2)2), 3.06-3.17 (m,
36H, -CONHCH2-), 4.32-4.43 (m, 12H, -CCH3(CH2O-)2),
5.08-5.19 (m, poly, -CH-), 7.03-7.10 (dd, 12H, Ar-), 7.69-7.97
(m, 18H, -CONHCH2-). 13C NMR (DMSO-d6, δ): 16.45, 16.54,
16.89, 20.32, 28.65, 29.67, 31.58, 32.96, 36.32, 36.82, 46.44, 48.81, 49.42,
51.93, 52.06, 65.71, 67.83, 68.67, 120.99, 129.33, 146.22, 148.31, 169.19,
169.81, 170.30, 171.29, 171.86, 173.59. DSC results: Tg at 45 °C upon
second heating. SEC results: PDI = 1.10.
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a
Scheme 1. Synthesis of Carboxylic Acid-Functionalized G1-(COOH)6
a Reagents and conditions: (a) DMAP, pyridine, CH2Cl2, 2 days.
1
G1-g2-(COOH)48 19. H NMR (DMSO-d6, δ): 1.33 (s, 9H, -
CCH3(CH2O)2-), 1.41-1.47 (m, poly, -CH3), 2.15-2.27 (m, 75H,
-CCH3(Ar)3, -CH2CONH-), 2.29-2.38 (s, 96H, -CH2CO2H),
2.45-2.52 (m, 96H, -OCOCH2CH2CO-, -CH2N(CH2)2), 2.62-
2.78 (m, 180H, -OCOCH2CH2CO-, -CH2N(CH2)2), 3.06-3.18
(m, 84H, -CONHCH2-), 4.32-4.43 (m, 12H, -CCH3(CH2O-)2),
5.08-5.19 (m, poly, -CH-), 7.03-7.10 (dd, 12H, Ar-), 7.43-7.48
(m, 42H, -CONHCH2-). 13C NMR (DMSO-d6, δ): 16.45, 16.55,
16.89, 20.32, 28.63, 29.65, 31.57, 32.93, 36.27, 36.66, 36.81, 46.43, 48.84,
49.41, 49.46, 51.88, 51.98, 52.10, 65.71, 67.82, 68.66, 120.98, 129.33,
146.22, 148.30, 169.18, 169.81, 170.31, 171.28, 171.86, 173.64. DSC
results: Tg at 51 °C upon second heating. SEC results: PDI = 1.12.
(s, 144H, -CH2CH2OCH3), 3.37-3.44 (m, 192H, -CON-
HCH2CH2O-, -CH2CH2OCH3), 3.47-3.52 (m, 288H, -
CH2OCH2CH2O-), 4.32-4.43 (m, 12H, -CCH3(CH2O-)2),
5.08-5.19 (m, poly, -CH-), 7.03-7.10 (dd, 12H, Ar-), 7.71-7.82
(m, 42H, -CONHCH2-), 7.82-7.93 (m, 48H, -CONHCH2-).
DSC results: Tg at -8 °C upon second heating. SEC results: PDI =
1.27.
Synthesis of G1-g2-(NHBoc)48 22. According to the above
procedure, G1-g2-(COOH)48 19 (50 mg, 2.82 μmol) was reacted with
N-Boc-ethylenediamine (44 mg, 0.27 mmol) to give the final product as
a yellowish solid (63 mg, yield: 91%). 1H NMR (DMSO-d6, δ): 1.33 (s,
9H, -CCH3(CH2O)2-), 1.37 (s, 432H, -C(CH3)3), 1.41-1.47 (m,
poly, -CH3), 2.15-2.27 (m, 171H, -CCH3(Ar)3, -CH2CONH-),
2.35-2.43 (m, 96H, -OCOCH2CH2CO-, -CH2N(CH2)2), 2.58-
2.70 (m, 180H, -OCOCH2CH2CO-, -CH2N(CH2)2), 2.90-3.06
(m, 96H, -CH2CH2NHBoc), 3.06-3.18 (m, 180H, -CONHCH2-,
-CH2CH2NHBoc), 4.32-4.43 (m, 12H, -CCH3(CH2O-)2), 5.08-
5.19 (m, poly, -CH-), 6.70-6.78 (m, 48H, -CH2NHBoc), 7.03-
7.10 (dd, 12H, Ar-), 7.71-7.82 (m, 42H, -CONHCH2-), 7.82-7.93
(m, 48H, -CONHCH2-). DSC results: Tg at 43 °C upon second
heating. SEC results: PDI = 1.21.
Synthesis of G1-g2-(NH2)48 23. G1-g2-(NHBoc)48 22 (50 mg,
2.04μmol) wasdissolvedin1.0 mLofTFAandstirredat0°C for 3 h. TFA
was evaporated by using a rotary evaporator, and the solid was redissolved
in1.0mLofDMSO. TEA(25mg) was added toneutralizethe excessacid.
The sample was then purified by precipitation in diethyl ether twice. The
final product was obtained as a yellowish solid 23 (34 mg, yield: 85%). 1H
NMR (DMSO-d6, δ): 1.33 (s, 9H, -CCH3(CH2O)2-), 1.41-1.47 (m,
poly, -CH3), 2.15-2.27 (m, 171H, -CCH3(Ar)3, -CH2CONH-),
2.35-2.43 (m, 96H, -OCOCH2CH2CO-, -CH2N(CH2)2), 2.58-2.70
(m, 180H, -OCOCH2CH2CO-, -CH2N(CH2)2), 2.78-2.90 (m,
96H, -CH2CH2NH2), 3.06-3.18 (m, 180H, -CONHCH2-, -
CH2CH2NH2), 4.32-4.43 (m, 12H, -CCH3(CH2O-)2), 5.08-5.19
(m, poly, -CH-), 7.03-7.10 (dd, 12H, Ar-), 7.71-7.82 (m, 42H, -
CONHCH2-), 7.82-7.93 (m, 48H, -CONHCH2-). DSC results: Tg
at 46 °C upon second heating.
1
G1-g3-(COOH)96 20. H NMR (DMSO-d6, δ): 1.33 (s, 9H, -
CCH3(CH2O)2-), 1.41-1.57 (m, poly, -CH3), 2.15-2.29 (m, 171H,
-CCH3(Ar)3, -CH2CONH-), 2.32-2.40 (m, 192H, -CH2CO2Bn),
2.45-2.52 (m, 192H, -OCOCH2CH2CO-, -CH2N(CH2)2), 2.64-
2.80 (m, 372H, -COCH2CH2CO-, -CH2N(CH2)2), 3.04-3.21 (m,
180H, -CONHCH2-), 4.32-4.43 (m, 12H, -CCH3(CH2O-)2),
5.13-5.23 (m, poly, -CH-), 7.03-7.10 (dd, 12H, Ar-), 7.74-8.01
(m, 90H, -CONHCH2-). 13C NMR (DMSO-d6, δ): 16.46, 16.57,
16.90, 20.32, 28.63, 29.66, 31.51, 32.81, 36.19, 36.58, 36.87, 46.44, 48.89,
49.39, 51.85, 52.09, 65.71, 67.84, 68.67, 121.00, 129.34, 146.23, 148.32,
169.20, 169.83, 170.36, 171.30, 171.88, 173.69. DSC results: Tg at 61 °C
upon second heating. SEC results: PDI = 1.13.
Synthesis of G1-g2-(TEG)48 21. G1-g2-(COOH)48 19 (50 mg,
2.82 μmol) and N-hydroxysulfosuccinimide (NHS, 47 mg, 408 μmol)
were dissolved in 1.0 mL of dry DMSO, and then 279 mg (1.35 mmol) of
N,N-dicyclohexylcarbodiimide (DCC) was added. The reaction solution
was left stirring at room temperature overnight and then filtered to
remove the white precipitate, N,N-dicyclohexylurea (DCU). Amine-
functionalized TEG (44 mg, 0.27 mmol) was added to the activated
solution and stirred at room temperature overnight. Once the reaction
was complete, DCU was removed by filtration. The crude product was
precipitated in diethyl ether, dried, redissolved in dichloromethane, and
finally precipitated in diethyl ether. The final product was obtained as a
yellowish solid 21 (64 mg, yield: 92%). 1H NMR (DMSO-d6, δ): 1.33 (s,
9H, -CCH3(CH2O)2-), 1.41-1.47 (m, poly, -CH3), 2.14-2.25 (m,
171H, -CCH3(Ar)3, -CH2CONH-), 2.35-2.43 (m, 96H, -
OCOCH2CH2CO-, -CH2N(CH2)2), 2.58-2.70 (m, 180H, -
OCOCH2CH2CO-, -CH2N(CH2)2), 3.02-3.13 (m, 84H, -
CONHCH2-), 3.13-3.21 (m, 96H, -CONHCH2CH2O-), 3.23
’ RESULTS AND DISCUSSION
Synthesis of PLLA-Based DLSPs. The hexahydroxy-function-
alized ring-opening polymerization (ROP) initiator based on
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Scheme 2. Divergent Synthesis of Amine-Functionalized PAMAM Dendronsa
a Reagents and conditions: (a) (Boc)2O, CH2Cl2, overnight; (b) methyl acrylate, MeOH, 2 days; (c) excess ethylenediamine, MeOH, 5 days; (d) benzyl
acrylate, 80 °C, 5 days; (e) TFA, CH2Cl2, overnight.
2,2-bis(hydroxymethyl)propionic acid (bis-MPA) and 1,1,1-tris-
(hydroxyphenyl)ethane (THPE) was prepared according to the
literature.55 Well-defined PLLA star polymer, G1-(OH)6, was
obtained by ROP of L-lactide from the initiator in bulk at
110 °C, using Sn(Oct)2 as the catalyst, and the targeted degree
of polymerization for each arm was 12. It was confirmed that the
polymerization had been initiated from all six hydroxyl groups in
the initiator by Sn(Oct)2.59 The six hydroxyl end groups of
G1-(OH)6 were then functionalized with a large excess of succinic
anhydride in the presence of DMAP and pyridine to form
carboxylic acid-terminated G1-(COOH)6 1 (Scheme 1) with a
89% yield.
The PAMAM dendrons with different generations were
synthesized using a divergent method, i.e., the Michael addition
of monoprotected ethylenediamine with methyl acrylate, fol-
lowed by exhaustive amidation of the resulting esters with a large
excess of ethylenediamine to give an amine-terminated dendron
in a nearly quantitative yield.60,61 Boc anhydride was selected as
the protecting group for the starting ethylenediamine because it
could be easily deprotected by TFA to form free amine. The
Michael addition and amidation were allowed to repeat a couple
times in methanol to form amine-terminated dendrons with
various generations, denoted as g0-[Boc, (NH2)2] 6, g1-[Boc,
(NH2)4] 7, and g2-[Boc, (NH2)8] 8 (Scheme 2). The resulting
amine-terminated PAMAM dendrons were then reacted with
excess benzyl acrylate at 80 °C for 5 days, and the products were
isolated by column chromatography on fumed silica to give the
benzyl ester-terminated PAMAM dendrons, denoted as g1-[Boc,
(COOBn)4] 9, g2-[Boc, (COOBn)8] 10, and g3-[Boc,
(COOBn)16] 11, respectively. Here, benzyl acrylate was selected
as the final Michael acceptor because it allowed formation of
carboxylic acid groups after deprotection. Compounds 9, 10, and
11 were thus orthogonally protected; the amine group at the root
was Boc-protected, and the carboxylic acid terminal groups were
protected by benzyl esters. These orthogonally protected PA-
MAM dendrons were then deprotected by TFA in dichloro-
methane at room temperature to provide amine-functionalized
PAMAM dendrons in excellent yields, denoted as g1-[NH2,
(COOBn)4] 12 (90% yield), g2-[NH2, (COOBn)8] 13 (93%
yield), and g3-[NH2, (COOBn)16] 14 (95% yield). In this
deprotection, the Boc group was selectively removed by acid-
olysis with TFA without cleavage of the benzyl ester groups. The
reaction sequence is shown in Scheme 2.
These benzyl ester-protected PAMAM dendrons with a
primary amine at the root allowed for conjugation to the
carboxylic acid-functionalized PLLA core, G1-(COOH)6 1, by
the amide coupling to provide amphiphilic benzyl ester-pro-
tected DLSPs, G1-g1-(COOBn)24 15, G1-g2-(COOBn)48 16,
and G1-g3-(COOBn)96 17 in good yields (82% for 15, 89%
for 16, and 91% for 17). PyBOP was found more efficient in
the amidization reaction in organic solvents than 1-ethyl-
3-(3-(dimethylamino)propyl)carbodiimide (EDC), DCC, and
other commercially available peptide coupling reagents. To
obtain carboxylic acid-functionalized DLSP, the benzyl ester
groups were deprotected by acidolysis with TFA/MsOH/anisole
at 0 °C for a short period of time followed by precipitation in
diethyl ether (Scheme 3). Since the tertiary amines in the
PAMAM dendrons could be easily protonated by excess MsOH
or TFA to form salts, the precipitate was dissolved in a small
amount of DMSO and treated with excess TEA to neutralize the
protonated tertiary amines in the PAMAM dendrons. Finally, the
pure carboxylic acid-terminated DLSPs, G1-g1-(COOH)24 18,
G1-g2-(COOH)48 19, and G1-g3-(COOH)96 20 were obtained
by dialysis in DMSO and subsequent precipitation in diethyl
ether with good yields (79% for 18, 84% for 19, and 85% for 20).
The terminal carboxylic acid groups in the PAMAM dendrons
rendered us an opportunity to further functionalize the DLSP
surface with other groups, including TEG and primary amine.
Here, we chose G1-g2-(COOH)48 as an example. First, G1-
g2-(COOH)48 was activated in dry DMSO in the presence
of DCC and NHS. Excess DCC was used because G1-
g2-(COOH)48 was hygroscopic and could contain some moist-
ure and excess DCC could scavenge possible moisture. This
activated compound then reacted with amine-functionalized
TEG or Boc-protected ethylenediamine in good yields, denoted
as G1-g2-(TEG)48 21 and G1-g2-(NHBoc)48 22, respectively.
This amide coupling reaction was found to be much more
efficient than the alternative method, where PyBOP was used
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Scheme 3. Synthesis of Carboxylic Acid-Functionalized DLSPsa
a Reagents and conditions: (a) PyBOP, TEA, CH2Cl2, 24 h; (b) TFA/MsOH/anisole, 0 °C, 2 h.
Scheme 4. Synthesis of DLSPs with TEG and Amine Surface Groupsa
a Reagents and conditions: (a) DCC, NHS, DMSO, 2 days; (b) TFA, overnight, and excess TEA.
as the coupling reagent, because of the lower steric hindrance of
DCC. The Boc-protected DLSP G1-g2-(NHBoc)48 was further
deprotected by TFA at room temperature to provide the amine-
functionalized PAMAM dendrons in an excellent yield (85%),
denoted as G1-g2-(NH2)48 23 (Scheme 4). Again, the primary
and tertiary amines in the PAMAM dendrons could be easily
protonated by TFA to form salts. These protonated amines were
neutralized with excess TEA in DMSO to provide pure G1-
g2-(NH2)48 23.
resonance peak at 1.4 ppm, which was assigned to the Boc
protecting group, was observed (Figure 1A), indicating that the
Boc group was successfully removed after treatment with TFA.
This was also verified by 13C NMR result in Figure 2A, where the
Boc peaks at 28.6 and 156.3 ppm (data range not shown)
completely disappeared.
1
Figures 1B and 2B show H and 13C NMR spectra for
the succinic anhydride-functionalized PLLA star polymer,
G1-(COOH)6 1. New peaks (g/g0) appeared around 2.7 ppm,
which could be assigned to the methylene proton from the
succinic acid chain ends. In the 13C NMR spectrum (Figure 2 B),
a new peak (a, b) was found at 28.7 ppm, which could be assigned
to the methylene carbons from succinic acid end groups. The
complete disappearance of the resonance at 66.6 ppm (data
range not shown), which is associated with the last methine
carbon in the polymer chain, suggested the complete functiona-
lization by succinic acid. SEC chromatograms in Figure 3 show
Molecular Characterization. The molecular structure of
1
each compound was confirmed by H and 13C NMR spectros-
copy in conjunction with SEC. Figures 1 and 2 show 1H and 13
C
NMR spectra for G1-g2-(COOH)48 19 and its intermediates,
including g2-(NH2, (COOBn)8) 13, G1-(COOH)6 1, and G1-
g2-(COOBn)48 16. Figures 1A and 2A show 1H and 13C NMR
spectra for g2-(NH2, (COOBn)8) 13 with corresponding peak
assignments for the deprotected PAMAM dendron. No
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Figure 1. 1H NMR spectra of (A) g2-[NH2, (COOBn)8] 13, (B)
G1-(COOH)6 1, (C) G1-g2-(COOBn)48 16 in CDCl3, and (D) G1-
g2-(COOH)48 19 in DMSO-d6.
Figure 3. SEC curves of G1-(OH)6, G1-(COOH)6 1, G1-g2-(COOBn)48
16, and G1-g2-(COOH)48 19.
The benzyl ester-protected DLSP was synthesized by the
amide coupling between the carboxylic acid-functionalized PLLA
core and amine-terminated PAMAM dendron. Both 1H and 13
C
NMR were used to investigate whether all six carboxylic acid
chain ends were reacted or not (Figures 1C and 2C). In the 1H
NMR (Figure 1C), the peak h0 at 3.1 ppm, which is assigned to
the methylene protons adjacent to the amine group at the root
(see Figure 1A), shifted downfield and overlapped with peak h
after the coupling reaction. In the 13C NMR spectra (Figure 2C),
the peak at 28.7 ppm from succinic acid chain ends shifted
downfield and split into two peaks, a and b, where peak a was the
methylene carbon adjacent to ester group and peak b was the
methylene carbon adjacent to amide group. Meanwhile, peaks c
and d, which were assigned to the methylene carbons adjacent to
the amine and amide groups in g2-[NH2, (COOBn)8] 13 (see
Figure 1A), also shifted downfield. The SEC trace in Figure 3
shows that the overall molecular weight increased for the G1-
g2-(COOBn)48 16, but the molecular weight distribution was
still fairly narrow with PDI = 1.15. The above results clearly
indicated that the carboxylic acid chain ends in G1-(COOH)6 1
was fully substituted by six benzyl ester-protected PAMAM
dendrons. The G1-g2-(COOBn)48 16 was soluble in most
organic solvents, such as chloroform, THF, and DMSO, but
not in hexane.
The deprotection of benzyl ester group was first attempted by
hydrogenolysis using Pd/C as the catalyst, and the complete
elimination of the benzyl ester protecting groups was not
achieved. Instead, TFA/MsOH/anisole was used as the catalyst
to remove the benzyl ester groups at 0 °C. 1H and 13C NMR were
used to quantify the complete removal of the benzyl ester groups
(see Figures 1D and 2D). The peaks at 5.1 and 7.3 ppm in 1H
NMR spectra (Figure 1D) and the peaks at 128.3 and 128.6
ppm in 13C NMR spectra (Figure 2D) from benzyl ester groups
disappeared after the deprotection reaction. The number-aver-
age molecular weight determined by 1H NMR end-group
analysis and the molecular weight distribution measured by
SEC analysis (see Figure 3) were unaffected by the acidolysis,
indicating that no degradation occurred under the reaction
conditions. Ouchi also found that no degradation was observed
on PLLA after acidolytic deprotection at 0-5 °C using TFA/
MsOH/anisole.63 The carboxylic acid-functionalized DLSP was
Figure 2. 13C NMR spectra of (A) g2-[NH2, (COOBn)8] 13, (B)
G1-(COOH)6 1, (C) G1-g2-(COOBn)48 16 in CDCl3, and (D) G1-
g2-(COOH)48 19 in DMSO-d6.
that the molecular weight and its distribution (PDI = 1.09) did
not change before and after functionalization of G1-(OH)6 with
succinic anhydride, indicating that no cross-link occurred during
the reaction. Note that there were small shoulder peaks at
27.9 mL retention volume for both G1-(OH)6 and G1-(COOH)6
in Figure 3. By using both UV (wavelength at 254 nm) and
differential refractive index (RI) detectors (see Figure S1A in the
Supporting Information), we determined that these small
shoulder peaks were attributed to the PLLA homopolymer
contamination initiated from a trace amount of adventitious
co-initiators present in the reaction system rather than the
hexahydroxyl co-initiator purposely added.62 These adventitious
co-initiators could include any hydroxyl-containing compounds
capable of forming -SnOR species from Sn(Oct)2 in the
carboxylate-alkoxide exchange reactions in solvent, monomer,
or even Sn(Oct)2 itself. Because the reaction kinetics for L-lactide
initiated from the adventitious co-initiators was much slower
than that for L-lactide initiated from the added hexahydroxyl co-
initiator,62 the molecular weight of the PLLA homopolymer
contamination was lower than that of the G1-(OH)6 star
polymer.
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Figure 4. 1H NMR spectra of (A) G1-g3-(COOH)96 20, (B) G1-
g2-(COOH)48 19, and (C) G1-g1-(COOH)24 18 in DMSO-d6.
Figure 6. SEC curves of G1-g3-(COOH)96 20, G1-g2-(COOH)48 19,
and G1-g1-(COOH)24 18.
Figure 5. 13C NMR spectra of (A) G1-g3-(COOH)96 20, (B) G1-
g2-(COOH)48 19, and (C) G1-g1-(COOH)24 18 in DMSO-d6.
Figure 7. 1H NMR spectra of (A) G1-g2-(TEG)48 21, (B) G1-
g2-(NHBoc)48 22, and (C) G1-g2-(NH2)48 23 in DMSO-d6.
soluble in DMSO, DMF, and DMAc, but not in most other
organic solvents. In aqueous solution, their solubilities were
around 10 mg/mL (below 10 mg/mL the solution was trans-
parent and above 10 mg/mL the solution showed some
turbidity). From SEC results in Figure 3, the small shoulder
peaks at a higher retention volume disappeared for both G1-
g2-(COOBn)48 16 and G1-g2-(COOH)48 19. It was likely that
the linear PLLA-dendron conjugates had different solubilities in
methanol than the DLSP during repeated dissolution (in
dichloromethane) and precipitation (in methanol) purification
cycles. In other words, the linear PLLA-dendron conjugates
were successfully fractionated out in the final products.
SEC curves of DLSPs with different generations of PAMAM
dendrons are shown in Figure 6, where narrow and nearly
symmetric peaks are shown [PDI = 1.10 for G1-g1-(COOH)24,
1.12 for G1-g2-(COOH)48, and 1.13 for G1-g3-(COOH)96].
Meanwhile, the DLSPwith a higher generation PAMAM dendrons
had a smaller elution volume reflecting a higher molecular weight.
The carboxylic acid-terminated DLSP allowed other types of
surface functionalization. As shown in Scheme 4, carboxylic acid
end groups have been successfully and quantitatively converted
to either TEG or primary amine groups. The chemical struc-
tures of G1-g2-(TEG)48 21, G1-g2-(NHBoc)48 22, and G1-
1H and 13C NMR spectra of the carboxylic acid-functionalized
DLSPs with different generations of PAMAM dendrons are
shown in Figures 4 and 5, respectively. Peak b from the PAMAM
dendron increased with an increase in generation after normal-
izing peak a, which is assigned to the methine proton from PLLA
in Figure 4. The significantly different chemical shifts from the
methylene carbons in PAMAM dendrons clearly reflected the
generation number in 13C NMR spectra in Figure 5. The
resonances from the methylene groups (a-l) were well sepa-
rated up to the second generation. In the third-generation DLSP,
the resonances from the first- and second-generation methylene
groups (b and c, f and g, j and k) became inseparable.
1
g2-(NH2)48 23 were confirmed by H NMR (see Figure 7).
Four new peaks from TEG, denoted as e-h, were observed in
Figure 7A after coupling the G1-g2-(COOH)48 19 with amine-
terminated methoxy-TEG. Furthermore, peak d0 originating
from G1-g2-(COOH)48 19 shifted upfield and overlapped with
peak d after terminal amidization. The peak around 3.3
ppm originated from HOD in DMSO-d6. The coupling of Boc-
protected ethylenediamine was verified by the appearance of
peaks e and f in Figure 7B. After treating with TFA, peak f almost
disappeared and peak e shifted upfield, indicating the removal of
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Unimolecular Micelles and Aggregates in Aqueous Solu-
tions. The dilute aqueous solution properties of the above
amphiphilic DLSPs were studied by DLS. The dilute aqueous
solution at 0.05% was prepared by slow addition of doubly
distilled water, using a syringe pump, into a 0.05% DMF solution
of the corresponding DLSP, followed by dialysis with a dialysis
tube having a MWCO of 3000 g/mol (regenerated cellulose
dialysis tube from Fisher Scientific) to completely remove DMF.
The DLS results for G1-g2-(COOH)48 19, G1-g2-(TEG)48 21,
and G1-g2-(NH2)48 23 are summarized in Figure 9; Figure 9A
shows the intensity distribution, and Figure 9B shows the volume
distribution. From the intensity distribution in Figure 9A, two
distinct peaks for the mean hydrodynamic diameter (Dh) were
observed: (i) 28 and 205 nm for G1-g2-(COOH)48 19, (ii) 15
and 295 nm for G1-g2-(TEG)48 21, and (iii) 14 and 344 nm for
G1-g2-(NH2)48 23. The smaller mean Dh around 15 nm for G1-
g2-(TGE)48 21 and G1-g2-(NH2)48 23 could be assigned to
unimolecular micelles, while the larger mean Dh around 295-
344 nm should result from their aggregates in the aqueous
solution. We speculate that the hydrophilic dendron shell might
not be enough to fully cover the hydrophobic DLSP core, and the
interaction among the DLSP cores was the driving force for the
large aggregates. Note that the scattered light intensity from
the aggregates of G1-g2-(TEG)48 21 was weaker than that from the
aggregates of G1-g2-(NH2)48 23, suggesting less aggregation for
the G1-g2-(TEG)48 21 due to the repulsive TEG surface groups.
For G1-g2-(COOH)48 19, the smaller mean Dh was around 28
nm, almost twice the size of unimolecular micelles (∼15 nm).
Meanwhile, different from the cases for G1-g2-(TEG)48 21 and
G1-g2-(NH2)48 23, the smaller Dh peak significantly overlapped
with the larger Dh peak. We speculate that the strong hydrogen
bonding among the surface carboxylic acid groups in G1-
g2-(COOH)48 19 resulted in the formation of dimers or even
trimers of the unimolecular micelles. Judging from the highest
scattering light intensity for the aggregate at 295 nm for G1-
g2-(COOH)48 19, the amount of aggregates was the highest
among three samples and might result from both hydrogen
bonding in the dendron shells and hydrophobic interaction
among the PLLA cores.
Note that light scattering intensity is not a linear function of
the concentration of different species in the solution, and large
aggregates have much higher scattering power than individual
molecules. Therefore, the intensity distribution in Figure 9A did
not represent the population distribution of unimolecular mi-
celles and their aggregates in the aqueous solution. Instead, the
information on the population distribution could be obtained
from the volume distribution results in Figure 9B. We can see
that the amount of the large aggregates for three DLSP samples
could nearly be ignored and most population was the un-
imolecular micelles of G1-g2-(TEG)48 21 and G1-g2-(NH2)48
23 and dimers of G1-g2-(COOH)48 19. The unimolecular
micelle morphology for G1-g2-(TEG)48 21, G1-g2-(NH2)48
23, and G1-g2-(COOH)48 19 was also studied by TEM, and
results are shown in Figure S2 of the Supporting Information.
From these TEM observations, the unimolecular micelles seen in
DLS were further confirmed in real space.
Figure 8. SEC curves of G1-g2-(TEG)48 21, G1-g2-(NHBoc)48 22,
and G1-g2-(NH2)48 23.
Boc protection groups and the formation of primary amine
surface groups.
Figure 8 shows SEC curves for three DLSPs with different
surface groups. Apparently, no degradation or cross-linking
occurred during the synthesis of G1-g2-(TEG)48 21 and G1-
g2-(NHBoc)48 22. Comparing with the SEC result for G1-
g2-(COOH)48 19 in Figure 6, the retention volumes for G1-
g2-(TEG)48 21 and G1-g2-(NHBoc)48 22 decreased from 26.0 mL
for G1-g2-(COOH)48 19 to ca. 25.7 mL, indicating slight
increases in hydrodynamic volume and thus molecular weight.
After deprotecting the Boc groups in G1-g2-(NHBoc)48 22, a
broad peak at a higher retention volume was observed for G1-
g2-(NH2)48 23, although no obvious degradation was observed
based on 1H NMR end-group analysis. From the previous
discussion, we knew that TFA should not degrade the PLLA
backbone at 0 °C. Combining the above evidence, we speculated
that the terminal primary amine groups in G1-g2-(NH2)48 23
stuck onto the SEC columns and resulted in a broad peak at a
higher retention volume. To confirm this, SEC measurements
were performed for two PAMAM dendron samples, g2-[Boc,
(COOCH3)8] 5 and g2-[Boc, (NH2)8] 8, to prove whether
primary amine-terminated samples would adsorb onto the
column or not. It was found that the primary amine-terminated
PAMAM dendron, g2-[Boc,(NH2)8] 8, eluted out at a much
higher retention volume than that of the ester-terminated
dendron, g2-[Boc, (COOCH3)8] 5, even though the molecular
weight increased after amidation with ethylenediamine (see
Figure S1B in Supporting Information). Cao et al. also found
that the molecular weight and molecular weight distribution of
the primary amine-terminated PAMAM could not be determined
by SEC due to its strong absorption to the column.64 Therefore,
we concluded that the interaction between the primary amine-
terminated sample, G1-g2-(NH2)48 8, and the column led to
the broadened SEC peak at a higher retention volume with a
broad distribution. Note that both G1-g2-(TEG)48 and G1-
g2-(NH2)48 DLSPs were readily soluble in organic solvents such
as chloroform, THF, DMSO, DMF, etc. In aqueous solution,
their solubilities were around 25 and 15 mg/mL, respectively
(again, below these values the solutions appeared transparent
and above these values the solutions were slightly turbid).
Thermal Properties of DLSPs with Different Surface
Groups. The nature of the surface groups played an important
role in both solution and bulk properties of the correspond-
ing DLSP. Figure 10A shows DSC thermograms for G1-
g2-(COOH)48 19 and its intermediates. Although the precursor
PLLA star polymers, G1-(OH)6 and G1-(COOH)6, could
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Figure 9. Dynamic light scattering (DLS) (A) intensity and (B) volume distributions for G1-g2-(COOH)48 19, G1-g2-(TEG)48 21, and G1-
g2-(NH2)48 23.
Figure 10. (A) Differential scanning calorimetry (DSC) thermograms of G1-g2-(COOH)48 19 and its intermediates, G1-(OH)6, G1-(COOH)6 1, and
G1-g2-(COOBn)48 16. (B) DSC thermograms of G1-g2-(TEG)48 21, G1-g2-(NHBoc)48 22, and G1-g2-(NH2)48 23.
crystallize after precipitation into methanol (see crystal melting
peaks during the first heating process in Figure S3 of the
Supporting Information), most samples were completely
amorphous during the second heating process. Note that the
amorphous state might be more beneficial for drug loading,
delivery, and biodegradation than the crystalline state. It was
reported that the Tg of dendritic polymers depended mainly on
the internal monomer units, number of end groups, and the
interaction between the main branch and end groups, such as
hydrogen bonding.46,65,66 This is also found in our DLSP
samples. For example, G1-(OH)6 had a Tg around 46 °C, while
G1-(COOH)6 1 had a Tg of 54 °C, suggesting that hydrogen
bonding among the end COOH groups promoted the Tg. After
conjugated with the PAMAM dendron, g2-[NH2, COOBn]48
13, the Tg for G1-g2-(COOBn)48 16 decreased to 12 °C,
indicating that the PAMAM dendron was miscible with the
PLLA core. Finally, after deprotection G1-g2-(COOH)48 19
exhibited a Tg around 51 °C. Again, hydrogen bonding among
the terminal COOH groups increased the Tg. Figure 10B
shows the DSC thermograms for G1-g2-(TEG)48 21, G1-
g2-(NHBoc)48 22, and G1-g2-(NH2)48 23. Apparently, attach-
ing TEG to the G1-g2-(COOH)48 19, the Tg decreased to -
8 °C, again indicating that TEG end groups were miscible with
the DLSP and decreased the Tg. However, after converting the
COOH end groups to NHBoc and NH2 groups, the Tg increased
to 43 °C for G1-g2-(NHBoc)48 22 and 46 °C for G1-g2-(NH2)48
23. This was possibly due to the hydrogen bonding among the
terminal amide groups. Comparing the Tg values for DLSP
samples with different end groups, the lower Tg of G1-
g2-(NH2)48 23 suggested that the hydrogen-bonding interaction
was weaker than that in G1-g2-(COOH)48 19. This was con-
sistent with the DLS results discussed before, where G1-
g2-(COOH)48 19 formed dimers and G1-g2-(NH2)48 23 only
formed unimolecular micelles.
Hydrophobic Drug Encapsulation. Although it is well-
known that PLLA is hydrophobic and PAMAM is hydrophilic,
to unambiguously determine the amphiphilicity of these DLSPs, a
hydrophobic drug molecule, doxorubicin (DOX),67 is encapsu-
lated into G1-g2-(TEG)48 unimolecular micelles. Our result
showed that the maximum loading of DOX into the G1-
g2-(TEG)48 21 was ca. 11.5 wt % without significant precipitation
for a prolonged preriod. Therefore, the solubility of DOX in
aqueous solution at pH 7.4 could increase from 0.0625 mg/mL
without DLSP encapsulation to as much as 0.272 mg/mL with
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DLSP encapsulation. Detailed drug encapsulation and an example
release profile of DOX from the G1-g2-(TEG)48 DLSP at 37 °C
and pH = 7.4 are shown in Figure S5 of the Supporting
Information.
highly appreciated. We also thank Dr. Junfu Yu from the mass
spectroscopy facility in the Department of Chemistry at UConn
for helping with MS measurements.
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’ CONCLUSIONS
A novel amphiphilic DLSP was synthesized by amide coupling
between a carboxylic acid-functional PLLA star polymer and a
benzyl ester-terminated PAMAM dendron with a primary amine
at the root, using PyBOP as the catalyst. Deprotection of the
benzyl ester groups by TFA/MsOH/anisole successfully ren-
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functionalized quantitatively with TEG and primary amine
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aqueous solutions, as studied by DLS. The smaller size peaks in
DLS (14-28 nm) were attributed to unimolecular micelles for
TEG and amine-terminated DLSPs and dimers (and/or trimers)
for the carboxylic acid-terminated DLSP. The large size peaks
(205-344 nm) were attributed to the aggregates due to hydro-
gen-bonding and/or hydrophobic/hydrophilic interactions. Judg-
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unimolecular micelles or dimers instead of aggregates. All the
DLSPs were amorphous because of the low degree of polymer-
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dendrons. The observation of the single Tg for all DLSP samples
suggested that the PLLA core and PAMAM dendrons were
miscible. The Tg values were largely dependent on the nature and
specific interactions among the end groups; by increasing the
hydrogen-bonding interactions among the surface functional
groups, the Tg increased. Experimental results showed that these
DLSPs had good solubility in aqueous solutions (ca. 10-25 mg/
mL) and could greatly enhance the water solubility of a hydro-
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S
Supporting Information. UV and RI SEC curves for
b
G1-(OH)6; RI SEC curves for g2-[Boc, (COOCH3)8] 5 and
g2-[Boc, (NH2)8] 8; TEM micrographs for G1-g2-(COOH)48
19, G1-g2-(TEG)48 21, and G1-g2-(NH2)48 23; DSC first
heating and cooling cycles for G1-g2-(COOH)48 19 and its
intermediates; DSC first heating and cooling cycles for G1-
g2-(TEG)48 21, G1-g2-(NHBoc)48 22, and G1-g2-(NH2)48 23;
hydrophobic drug (DOX) encapsulation and an example release
profile at 37 °C and pH = 7.4. This material is available free of
charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail lxz121@case.edu, Tel (216) 368-5861.
’ ACKNOWLEDGMENT
This work was supported by NSF DMR-0705716. Helpful
discussions with Professor Yong Wang and assistance from Mr.
Alexander Mann at University of Connecticut (UConn) are
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