Aliphatic hyperbranched polyester: A new building block in the construction of multifunctional nanoparticles and nanocomposites

Article abstract of DOI:10.1021/la9037843

Herein we report the design and synthesis of multifunctional hyperbranched polyester-based nanoparticles and nanocomposites with properties ranging from magnetic, fluorescence, antioxidant and X-ray contrast. The fabrication of these nanostructures was achieved using a novel aliphatic and biodegradable hyperbranched polyester (HBPE) synthesized from readily available diethyl malonate. The polymer's globular structure with functional surface carboxylic groups and hydrophobic cavities residing in the polymer's interior allows for the formation of multifunctional polymeric nanoparticles, which are able to encapsulate a diversity of hydrophobic cargos. Via simple surface chemistry modifications, the surface carboxylic acid groups were modified to yield nanoparticles with a variety of surface functionalizations, such as amino, azide and propargyl groups, which mediated the conjugation of small molecules. This capability achieved the engineering of the HBPE nanoparticle surface for specific cell internalization studies and the formation of nanoparticle assemblies for the creation of novel nanocomposites that retained, and in some cases enhanced, the properties of the parental nanoparticle building blocks. Considering these results, the HBPE polymer, nanoparticles and composites should be ideal for biomedical, pharmaceutical, nanophotonics applications.

Full text of DOI:10.1021/la9037843

pubs.acs.org/Langmuir
©
2009 American Chemical Society
Aliphatic Hyperbranched Polyester: A New Building Block in the
Construction of Multifunctional Nanoparticles and Nanocomposites
†
†,‡
,†,‡,§
Santimukul Santra, Charalambos Kaittanis, and J. Manuel Perez*
†
‡
Nanoscience Technology Center, Burnett School of Biomedical Sciences, College of Medicine, and
Department of Chemistry, University of Central Florida, 12424 Research Parkway, Suite 400,
Orlando, Florida 32826
§
Received October 6, 2009. Revised Manuscript Received November 11, 2009
Herein we report the design and synthesis of multifunctional hyperbranched polyester-based nanoparticles and
nanocomposites with properties ranging from magnetic, fluorescence, antioxidant and X-ray contrast. The fabrication of
these nanostructures was achieved using a novel aliphatic and biodegradable hyperbranched polyester (HBPE)
synthesized from readily available diethyl malonate. The polymer’s globular structure with functional surface carboxylic
groups and hydrophobic cavities residing in the polymer’s interior allows for the formation of multifunctional polymeric
nanoparticles, which are able to encapsulate a diversity of hydrophobic cargos. Via simple surface chemistry
modifications, the surface carboxylic acid groups were modified to yield nanoparticles with a variety of surface
functionalizations, such as amino, azide and propargyl groups, which mediated the conjugation of small molecules. This
capability achieved the engineering of the HBPE nanoparticle surface for specific cell internalization studies and the
formation of nanoparticle assemblies for the creation of novel nanocomposites that retained, and in some cases
enhanced, the properties of the parental nanoparticle building blocks. Considering these results, the HBPE polymer,
nanoparticles and composites should be ideal for biomedical, pharmaceutical, nanophotonics applications.
9-13
Introduction
number of surface reactive groups.
Their easy one-step
synthesis, oftentimes involving the condensation of AB type
2
The design of multifunctional polymeric nanoparticles is an
emerging field with important applications in catalysis, sensing,
monomers, contrasts with the more complex synthesis of other
types of highly branched macromolecules, such as dendrimers.
One important feature of these HBPs, particularly those made
with a polyester backbone, is their high solubility in common
polar solvents (e.g., DMF, DMSO, acetone), which makes them
ideal for the development of hyperbranched polyester (HBPE)
nanostructures, such as polymeric nanoparticles, by the solvent
1-4
optics, material design, drug delivery and imaging.
Nanopar-
ticles that combine multiple materials or encapsulate a variety of
guest molecules can be used to incorporate unique properties to
5
these nanostructures. Furthermore, assembliesof multifunctional
nanoparticles can be engineered to obtain supramolecular assem-
blies with combined or enhanced properties, derived from both the
type and amount of the constituent polymeric nanoparticles used
1
4,15
diffusion method.
These HBPE nanoparticles would have the
6-8
benefits of being globular in nature, highly functionalized, biode-
gradable and able to encapsulate a variety of guest molecules in
their nanocavities located within the inner branches of the
polymeric coating.
in the assembly.
A variety of polymeric matrices has been used
to synthesize polymeric nanoparticles, but oftentimes the diversity
of the encapsulated molecules is compromised by the nature of the
polymeric matrix itself that limits the amount and kind of guest
molecule to be encapsulated. In addition, the surface functiona-
lization of the resulting nanoparticle is limited by the use of
complex and non-reproducible conjugation chemistries.
Acknowledging the benefits of HBPE and HBPE nanoparticles,
herein we report the design and synthesis of an aliphatic hyper-
branched polyester with hydrophobic cavities and hydrophilic
surface and its application in the development of multifunctional
nanoparticles and nanocomposites (Scheme 1). To the best of our
knowledge, this is the first time that this type of structure (4) has
been utilized to synthesize a novel HBPE polymer suitable for
the fabrication of theranostic polymeric nanoparticles with high
surface functional groups. This functional AB2 monomer is
designed in such a way that under polymerization conditions it
grows three dimensionally, forming a highly branched polymeric
structure. In addition, this is the first example of an aliphatic
Hyperbranched polymers (HBPs) composed of densely
branched structures adopt compact conformations with a large
*
Corresponding author. Telephone: 407-882-2843. Fax: 407-882-2819.
E-mail: jmperez@mail.ucf.edu.
1) Nayak, S.; Lee, H.; Chmielewski, J.; Lyon, L. A. J. Am. Chem. Soc. 2004,
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M. J. Am. Chem. Soc. 2009, 131, 1360–1361.
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(
1
(
(
and spherical-shaped hyperbranched polyester that utilizes the
(
14,15
solvent diffusion method
for the synthesis of multifunctional
3
(
(
(
(
6) Iyer, A. S.; Lyon, L. A. Angew. Chem., Int. Ed. Engl. 2009, 48, 4562–4566.
7) Nayak, S.; Lyon, L. A. Angew. Chem., Int. Ed. Engl. 2005, 44, 7686–7708.
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(11) Santra, S.; Kumar, A. Chem. Commun. 2004, 2126–2127.
(12) Marco Fischer, F. V. Angew. Chem., Int. Ed. 1999, 38, 884–905.
(13) Frechet, J. M. J.; Henmi, M.; Gitsov, I.; Aoshima, S.; Leduc, M. R.;
Grubbs, R. B. Science 1995, 269, 1080–1083.
Rotello, V. M. Nature 2000, 404, 746–748.
(
9) Duncan, R. Nat. Rev. Drug. Discov. 2003, 2, 347–360.
(14) McCarthy, J. R.; Perez, J. M.; Bruckner, C.; Weissleder, R. Nano. Lett.
(
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5
364 DOI: 10.1021/la9037843
Published on Web 12/03/2009
Langmuir 2010, 26(8), 5364–5373

Santra et al.
Article
Scheme 1. Schematic Representation of the Three Dimensional Structure of the Aliphatic Hyperbranched Polymer (HBPE 5) and Its HBPE
Nanoparticles
a
a
Monomer 4 has three-bond connectivity (the arrows) to grow three-dimensionally under polymerization conditions, resulting in a highly branched
polymer with hydrophobic cavities in the polymeric structure.
polymeric nanoparticles. Furthermore, our HBPE can be easily
synthesized in one-pot as opposed to dendrimers (e.g., PAMAM
dendrimers) that involve iterative synthetic steps. Our synthesized
new HBPE polyester can be easily modified to ultimately yield a
library of functional nanoparticles for the development of multi-
functional nanocomposites.
The as-synthesized spherical-shaped, highly branched polymer
has internal hydrophobic cavities (polymer cavities) that provide
high affinity for the encapsulation of various hydrophobic guest
molecules and the polymer surface consists of polar carboxylic
acid groups that can be further functionalized to generate a
library of functional hyperbranched polymer using “click” chem-
istry and carbodiimide chemistry. Because of their surface func-
tionalities, the nanoparticles can be conjugated with functional
ligands to (i) target and (ii) self-assemble with other nanoparticles
to create multifunctional nanocomposites.
Precision Detector. Thermo gravimetric analyses (TGA) were
performed on a SETARAM, Mettler TC11 instrument with
sample sizes of 10-20 mg. All experiments were done using
a heating rate of 10 °C/min in air. The overall surface charge
(
ζ potential) of the HBPE nanoparticles was measured using
a Zetasizer Nano ZSfrom Malvern Instruments. Confocal images
were taken on a Zeiss Axioskop 2 mot plus confocal microscope.
Flow Cytometry experiments were performed using a BD FACS
Calibur multipurpose flow cytometer system from BD Bios-
ciences. MTT study has been done using the Biotek Synergy
HT multi-detection microplate reader. Dynamic light scattering
(DLS) studies were done using a PDDLS/CoolBatch 40T instru-
ment and a Precision Deconvolve 32 software. IVIS experiments
were done on an IVIS 50 imaging system from Xenogen imaging
technologies. Analytical Thin Layer Chromatography (TLC) was
performed on glass plates coated with silica gel GF 254 and
were visualized in iodine vapor. Column chromatography was
performed using silica gel (100-200 mesh), and the eluant is
mentioned in the procedures below for each case.
Experimental Section
Synthesis. Synthesis of 4-Bromobutyl Acetate (2). Tet-
rahydrofuran (12.2 mL, 148.4 mmol) and potassium bromide
(21.1 g, 176.5 mmol) were added into a 250 mL round-bottom
flask containing 150 mL of acetonitrile. The reaction mixture was
cooled to 0 °C, followed by dropwise addition of acetyl chloride
(11 mL, 155.1 mmol). Subsequently, the mixture was brought to
room temperature, where it was continuously stirred for 36 h. The
reaction mixture was poured in water and extracted with ethyl
acetate. The organic layer was washed with water, dried over
Materials. Dry DMF, DMSO, 3-(4,5-dimethylthiazol-2-yl)-
,5-diphenyltetrazolium bromide (MTT), 1,1 -carbonyldiimida-
0
2
zole (CDI), N-hydroxysuccinimide (NHS), AZT (azidothy-
midine), diethyl malonate, and other chemicals were purchased
from Sigma-Aldrich and used without further purification. Near
infrared dyes (DiI-D282, DiD-D7757, and DiR-D12731) and
0
4
,6-diamidino-2-phenylindole (DAPI-D1306) were purchased
from Invitrogen, whereas the EDC (1-ethyl-3-[3-(dimethy-
lamino)propyl] carbodiimide hydrochloride) was obtained from
Pierce Biotechnology. The human lung carcinoma cell line A549
Na
colorless liquid.
Yield: 24.3 g (85%). Bp: >250 °C. H NMR (300 MHz,
CDCl , δ ppm, J Hz): 1.79 (m, 2H), 1.92 (m, 2H), 2.03 (s, 3H),
3.46 (t, 2H, J = 7.6), 4.08 (t, 2H, J = 6.7). C NMR (75 MHz,
CDCl , δ ppm): 20.87, 27.36, 29.36, 33.03, 63.43, 170.95. IR
(CHCl
2 4
SO , and concentrated inorder toobtainthe pureproduct asa
1
(
CCL-185) and cardiomyocytes (H9c2) were obtained from
ATCC. Dialysis membranes were obtained from Spectrum La-
boratories. Acetonitrile, tetrahydrofuran, and other solvents were
purchased from Fisher Scientific and used as received, unless
otherwise stated.
3
1
3
3
-1
3
): 3038, 2926, 1352, 1243, 1052 cm .
Characterizations. Infrared spectra were recorded on a Per-
kinElmerSpectrum 100 FT-IR spectrometer. UV/vis spectra were
recorded using a CARY 300 Bio UV/vis spectrophotometer.
Fluorescence spectra were recorded on a NanoLog Horiba Jobin
Yvon fluorescence spectrophotometer. NMR spectra were
recorded on a MERCURY 300 MHz spectrometer using the
TMS/solvent signal as an internal reference. Gel permeation
chromatography (GPC) results were obtained using a JASCO
MD 2010 Plus instrument with a PD 2020 light scattering
Synthesis of 2-(4-Acetoxybutyl)malonic Acid Diethyl Ester (3).
Diethyl malonate (1) (10 g, 62.5 mmol) and 4-bromobutyl
acetate (2) (15.84 g, 81.3 mmol) were placed in a round-bottom
flask containing acetonitrile (120 mL) and stirred for 2 min
at room temperature. Then, potassium carbonate (34.5 g,
250.1 mmol) was added and refluxed for 36 h. Next, the mixture
was filtered and the filtrate was concentrated to obtain a yellow
liquid. This was extracted with ethyl acetate, and washed with
water. The organic layers were combined and dried over Na
SO ,
4
2
Langmuir 2010, 26(8), 5364–5373
DOI: 10.1021/la9037843 5365

Article
Santra et al.
and purified by column chromatography using 4% ethyl acetate
in petroleum ether as the eluent.
Synthesis of Hyperbranched Polyester Amine (HBPE-
EDA) 6. The polymer 5 (0.1 g, 0.0025 mmol) was dissolved in
dry DMF (1 mL) and CDI (0.041 g, 0.25 mmol) in dry DMF
(0.1 mL) was added dropwise. The reaction mixture was
incubated for 2 h at room temperature. Ethylenediamine
1
Yield: 13.02 g (76%). Bp: 250 °C. H NMR (300 MHz, CDCl ,
3
δ ppm, J Hz): 1.28 (t, 6H, J = 7.6), 1.38 (m, 2H), 1.62 (q, 2H, J =
7
(
.2), 1.98 (q, 2H, J = 7.7), 2.05 (s, 3H), 3.34 (t, 1H, J = 7.7), 4.09
t, 2H, J = 6.6), 4.22 (q, 4H, J = 7.2). C NMR (75 MHz,
1
3
(
0.015 g, 0.25 mmol) in dry DMF (0.4 mL) was then added
CDCl , δ ppm): 14.06, 20.79, 23.74, 28.25, 28.25, 51.84, 61.27,
3
dropwise to the reaction mixture and incubated at room tempera-
ture for 24 h. The resulting reaction mixture was then precipitated
in methanol, centrifuged and dried in a vacuum pump to get the
6
1
3
3.89, 169.31, 171.11. IR (CHCl ): 2982, 1728, 1463, 1367, 1233,
-1
151, 1029, 860 cm
Synthesis of 2-(4-Hydroxybutyl)malonic Acid (4). 2-(4-
.
pure aminated polymer.
1
Acetoxybutyl)malonic acid diethyl ester (3) (5.0 g, 18.25 mmol,
see Section S1 in the Supporting Information for the synthesis of
compound 3) was taken in a 100 mL round-bottom flask contain-
ing methanol (50 mL) and stirred at room temperature for 2 min.
To this solution, NaOH (2.1 g, 54.74 mmol) in water (7 mL) was
added and stirred at 90 °C for 8 h. The reaction mixture was
brought to room temperature and acidified (pH 2-3) with the
dropwise addition of dilute hydrochloric acid at room tempera-
ture with constant stirring. The mixture was then concentrated
using rotary evaporator and applying vacuum. To the concen-
tratedreactionmixturechloroform(50 mL) wasadded and Argon
gas was bubbled through the solution at 60 °C to remove excess
HCl. The mixture was filtered and the filtrate was concentrated.
This was then purified by column chromatography using 35%
Yield: 88%. H NMR (300 MHz, DMSO-d , δ ppm): 1.27 (m,
6
2H), 1.55 (m, 2H), 1.74 (m, 2H), 2.26 (m, 4H), 2.88 (m, 4H), 3.34
(
m, 1H), 3.63 (m, 4H), 4.04 (m, 2H). IR: 3245, 2940, 2864, 1725,
1
659, 1534, 1435, 1240, 1159, 1062, 1021, 952, 929, 826, 749, 704,
-1
663 cm
.
Synthesis of Alkynated Hyperbranched Polyester (HBPE-
PA) 7. A similar procedure has been followed as described
for the synthesis of polymer 6. Instead of ethylenediamine,
propargylamine (0.014 g, 0.25 mmol) was used as the starting
material.
1
Yield: 80%. H NMR (500 MHz, DMSO-d
6
, δ ppm): 1.28 (m,
2
(
1
H), 1.54 (m, 2H), 1.75 (m, 2H), 2.25 (m, 2H), 3.42 (bs, 1H), 3.96
m, 4H), 4.03 (m, 2H). IR: 3121, 2954, 2698, 2215, 1728, 1705,
664, 1530, 1458, 1437, 1326, 1260, 1165, 1094, 1065, 827,
ethyl acetate in petroleum ether as eluent.
Yield:2.31g (72%). H NMR (300MHz, CDCl
-1
1
748 cm .
3
, δ ppm, J Hz):
Synthesis of Azide-Functionalized Hyperbranched Polye-
ster (HBPE-APA) 8. Similar procedure has been followed as
described for the synthesis of polymer 6. Instead of ethylenedia-
mine, aminopropyl azide (0.025 g, 0.25 mmol) was used as the
starting material. Aminopropyl azide was prepared using the
1
(
.41 (m, 2H), 1.59 (m, 2H), 1.91 (q, 2H, J
t, 1H, J = 7.4), 3.64 (t, 2H, J = 6.5), 5.54 (bs, 1H). C NMR (75
MHz, CDCl , δ ppm): 23.53, 28.52, 31.75, 52.64, 62.11, 170.55. IR
CHCl ): 3507, 2941, 1710, 1626, 1459, 1438, 1391, 1198, 1157,
050, 947, 772, 741, 664 cm
Synthesis of Hyperbranched Polyester (HBPE) 5. The
1
= 7.3, J = 7.8), 3.37
2
1
3
3
(
1
3
-
1
.
2
7
previously reported method.
1
Yield: 84%. H NMR (500 MHz, DMSO-d , δ ppm): 1.29 (m,
6
monomer 4 and the catalyst p-toluenesulfonic acid (100:1 molar
ratio) were taken into a 10 mL round-bottom flask and dried
under high vacuum followed by the release of vacuum using dry
argon gas. Then theflask was slowly heatedto 150 °C under argon
atmosphere using an oil bath, and it was kept at this temperature
for 2 h. The evolution of the byproduct (water vapor) was clearly
visible after the sample was heated at 150 °C. The melted reaction
mixture was evacuated at 0.2 mm/Hg for 1 h, while maintaining
the same polymerization temperature. The polymer was purified
by dissolving in DMF and reprecipitating in methanol. This was
then centrifuged, washed with methanol and dried in a high
6
H), 1.49 (m, 6H), 1.80 (m, 2H), 3.21 (m, 4H), 3.39 (bs, 1H), 4.01
(
1
m, 2H). IR (Neat): 3217, 2949, 2098, 1730, 1669, 1599, 1391,
260, 1165, 1066, 834, 757, 665 cm .
Synthesis and Characterizations of Cargo Encapsulated
-1
HBPE Nanoparticles: Water-Based Solvent Diffusion
Method. Synthesis of Cargo-Encapsulating Polymeric
Nanoparticles (9-12). Here, 5 μL solutions of different near
IR dyes (DiI, DiR or DiD, 10 μg/μL), chelated metals
(DOTA-Gd, 25 μg/μL), and iodinated molecules (5-amino-
2,4,6-triiodoisophthalic acid, 50 μg/μL) in 250 μL of DMF were
separately mixed into 250 μL of DMF solution containing
hyperbranched polymers (5, 6, 7, and 8, 0.025 g) and vortexed.
The resulting polymer-cargos mixture in DMF was added drop-
wise to deionized water (5 mL) with continuous stirring at room
temperature forming cargos encapsulating HBPE nanoparticles.
The synthesized nanoparticles (9-12) were purified using a
PD-10 column and finally dialyzed (MWCO 6-8K) against
PBS (pH = 7.4).
vacuum pump to get pure polymer.
Yield: 65%. H NMR (300 MHz, DMSO-d
1
6
, δ ppm): 1.25 (m,
2
(
H), 1.52 (m, 2H), 1.67 (m, 2H), 3.38 (m, 1H), 3.58 (m, 2H), 5.28
1
m, 1H). C NMR (75 MHz, DMSO-d
3
6
, δ ppm): 23.82, 28.23,
5
1
2
1.85, 52.63, 65.37, 170.45. IR: 2954, 1727, 1458, 1436, 1343, 1218,
-1
152, 1054, 943, 858, 743, 694 cm . TGA: 10% weight loss at
50 °C.
Gel Permeation Chromatography (GPC). The molecular
weight of the resulting polymer was determined using Gel Per-
meation Chromatography (GPC). The average molecular weight
was calculated against a polystyrene standard, using HPLC-grade
DMF as the mobile phase. For a comparative study between the
Synthesis of Taxol and DiI Coencapsulating Polymeric
Nanoparticles (9, 11). Taxol (5 μL, 1 μg/μL) and DiI dye
(5 μL, 10 μg/μL) were taken in a tube containing either carboxy-
lated (5) or alkynated (7) polymer (0.025 g) in 500 μL of DMF,
and the solvent diffusion method was followed as described
above.
average molecular weight (M ) and the polymerization time at
w
1
50 °C, the samples were taken from the reaction mixture
periodically and analyzed by GPC. With increase in time there
was an increase in the molecular weight, whereas a dramatic
increase in the polymer’s molecular weight was observed when
high vacuum was applied. The average molecular weight of the
Synthesis of AzT and DiI Coencapsulating Polymeric
Nanoparticles (9, 11). AzT (azidothymidine, 5 μL, 1 μg/μL)
and DiI dye (5 μL, 10 μg/μL) were taken in an Eppendorf
tube containing either carboxylated (5) or alkynated (7) polymer
(0.025 g) in 500 μL of DMF, and the solvent diffusion method was
followed as described above.
w
polymer 5 was M = 42000, PD = 1.6, whereas only oligomers
and low molecular weight polymers were obtained before apply-
ing vacuum.
Post-functionalizations of HBPE 5: Carbodiimide Chem-
Synthesis of Functional HBPE Nanoparticles 10, 11, and 12:
Water-Soluble EDC Chemistry. Alternatively, different sur-
face functional HBPE nanoparticles (10, 11 and 12) can be
prepared from carboxylated HBPE nanoparticles (9, Suppor-
ting Information Scheme S1) using water-soluble EDC
[1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide hydrochloride]
0
istry. The water insoluble carbodiimide 1,1 -carbonyldiimida-
zole (CDI) was used for the synthesis of aminated (6), alkynated
(7), and azide-functionalized (8) hyperbranched polymers. The
resulting polymers were found to be soluble in DMF and DMSO.
5
366 DOI: 10.1021/la9037843
Langmuir 2010, 26(8), 5364–5373

Santra et al.
Article
Scheme 2. Schematic Representation of the Syntheses of Functional Hyperbranched Polyesters (5-8) and HBPE Nanoparticles (HBPE-NPs
a
9
-12) Using the Melt Polymerization Technique and the Solvent Diffusion Method, Respectively
a
“Click” and carbodiimide chemistry has been used for the syntheses of the library of functional HBPE nanoparticles.
2
7
chemistry, following previously reported method. Briefly, to a
solution of carboxylated HBPE nanoparticles (9, 1.0 mol) in MES
buffer, a solution of EDC (10 mmol) and NHS (10 mmol) was
added followed by 3 min incubation at room temperature. Ethy-
lenediamine, propargylamine or aminopropyl azide (10 mmol) in
DMF were then added dropwise and continued for 3 h to obtain
finally dialyzed (MWCO 6-8K) against PBS solution (pH =
7.4). Nanocomposites were stored in a refrigerator for further
characterization.
In Vitro Cell Studies. Cell Cultures. Human lung cancer
cells (A549) and rat cardiomyocytes (H9c2, noncancer) were
obtained from ATCC, and maintained in accordance to the
supplier’s protocols. Briefly, the A549 cells were maintained in a
5%-FBS-containing DMEM medium supplemented with anti-
mycotic/antibiotic, whereas the H9c2 cells were propagated in a
10%-FBS-containing MEM medium containing antimycotic/
antibiotic. Cells were grown in a humidified incubator at 37 °C
1
0, 11, or 12, respectively.
Synthesis of Folate-Functionalized Polymeric Nanoparticle
1
3: “Click” Chemistry. The alkynated HBPE nanoparticles 11
3
-
(
taken to an Eppendorf containing catalytic amount of CuI
0.025 g, 6 ꢀ 10 mmol) in bicarbonate buffer (pH = 8.5) were
-10
(
8
0.11 μg, 6 ꢀ 10
mmol) in 250 μL of bicarbonate buffer (pH =
.5), vortexed for 30 s (Supporting Information Scheme S2).
To this, azide-functionalized folic acid (0.003 g, 6 ꢀ10 mmol)
in DMSO was added and the reaction was incubated at room
temperature for 12 h. The synthesized HBPE nanoparticles (13)
were purified using a PD-10 column and finally dialyzed (MWCO
2
under 5% CO atmosphere.
Confocal Laser-Scanning Microscopy. H9c2 and A549 cells
were grown overnight on culture dishes, before treatment. After
incubation with the nanoparticles, the cells were washed three
times with 1X PBS, fixed with 5% formalin solution, and stained
with DAPI (Molecular Probes). Subsequently, the cells were
examined with a Zeiss LSM 510 confocal microscope equipped
with a 40ꢀ objective.
-2
6
-8K) against PBS solution (pH = 7.4). They were stored in
refrigerator for further characterization.
MTT Assay. A549 and H9c2 cells (2,500 cells/well) were
seeded in 96-well plates, incubated with HBPE nanoparticles for
Synthesis of Nanocomposites: “Click” Chemistry. Nano-
composites were synthesized following “click” chemistry as de-
scribed earlier. Briefly, the equivalent amount of alkynated
metallic (cerium oxide and iron oxide) or HBPE nanoparticles
6
h at 37 °C. Then, each well was washed three times with 1ꢀ PBS
and treated with 20 μL of MTT (5 μg/μL) for 2 h. The resulting
formazan crystals were dissolved in acidified isopropanol (0.1 N
HCl) and the absorbance was recorded at 570 and 750 nm
(
11) were mixed with equivalent amount of azide-functionalized
HBPE nanoparticles (12) in the presence of catalytic amount of
CuI and bicarbonate buffer (pH = 8.5), incubated at room
temperature for 12 h. The resulting fluorescent HBPE nanocom-
posite and nanocomposite containing cerium oxide were purified
using a PD-10 column, whereas magnetic nanocomposite con-
taining iron oxide were purified using a magnetic column and
(
background), using a Synergy HT multidetection microplate
reader (Biotek). These experiments were performed in triplicates.
Flow Cytometry. A549 cells were grown until reaching con-
fluence and then incubated with HBPE nanoparticles. After
detachment and centrifugation at 1000 rpm, the cell pellets were
Langmuir 2010, 26(8), 5364–5373
DOI: 10.1021/la9037843 5367

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Santra et al.
Figure 1. Characterization of HBPE 5 and HBPE nanoparticles 9. (a) GPC traces of HBPE 5, showing the formation of (i) oligomers and
low-molecular weight polymers before applying vacuum and (ii) high molecular weight polymer after applying vacuum. (b) Determination of
the hydrodynamic diameter of HBPE nanoparticles 9 using DLS, showing an average diameter of 88 nm. (c) Fluorescence emission spectra
of different dye (DiI, DiD, and DiR)-encapsulated HBPE nanoparticles (9) in PBS buffer. (d) Comparing the fluorescence emission spectrum
ofDiI-HBPENPs(9) withthatoffreeDiIdye insolution. Inset:imagesshowing(A)DiI dyealoneinDMFsolutionand(B)DiI encapsulating
HBPE nanoparticles (9) in PBS.
collected and resuspended in 1ꢀ PBS and the cellular suspensions
were examined using a FACSCalibur flow cytometer (BD
Biosciences).
obtained, which is unsuitable for further polymerization as it will
eventually form undesired cross-linked byproducts. Next, the
monosubstituted product 3 was subjected to basic hydrolysis
using NaOH as a base and a mixed solvent (methanol/water)
approach. Note that use of water should be avoided in the work-
up stage as the hydrolyzed product 4 was highly soluble in water.
IVIS Analysis. A total of 10 000 lung carcinoma cells were
incubated for 6 h with the corresponding HBPE nanoparticles,
and the supernatant was collected in Eppendorf tubes. Cells were
thoroughly washed with 1X PBS, and detached as stated
above. The resulting pellets were resuspended in 1 mL culture
media. All Eppendorf tubes were examined simultaneously on a
Xenogen IVIS system, using the ICG filter for DiR dye. All
experiments were performed in triplicates.
The resulting novel AB monomer (4) has three-bond connectiv-
2
ity with two carboxylic acid groups and one hydroxyl group in the
structure (Scheme 1), which upon melt-polymerization produced
a three-dimensional water-insoluble HBPE polymer 5 (see Ex-
perimental Section and the Supporting Information, section S1,
for detailed synthesis protocols and characterizations). The as-
synthesized HBPE polymer is highly branchedand the presenceof
ester linkages in the three-dimensional polymeric backbone
In Vitro Drug/Dye Release. The in vitro drug/dye release
studies were carried out using a dynamic dialysis technique
at 37 °C. Briefly, 100 μL of HBPE nanoparticles (13) were in-
cubated with porcine liver esterase (20 μL) inside a dialysis bag
1
0,16,17
(
(
MWCO 6-8 K), which was then placed in a PBS solution
pH 7.4). The amount of guest (dye or drug) molecules released
makes this polymer biodegradable.
The amphiphilic nature
of the HBPE polymer 5 derives from the presence of hydrophobic
moieties in the inner polymeric backbone, whereas polar groups
are found at the surface. Upon dispersion in an aqueous environ-
ment, this HBPE polymer self-assembles, bringing the hydro-
phobic areas together to minimize contact with the aqueous
environment, while exposing the hydrophilic segments containing
the polar carboxylic acid groups to the aqueous media. These
properties make our HBPE polymer important and attractive,
particularlyfor biomedicalapplications, asthis branched polymer
is suitable for the fabrication of functional polymeric nanoparti-
cles via the water-based solvent diffusion method (Schemes 1
and 2). This is contrary to microemulsion techniques utilized for
the synthesis of nanoparticles made of commercially available
polymers. In addition, our synthesized biodegradable branched
polymer can be easily fine-tuned for the construction of multi-
functional HBPE nanoparticles with diverse surface functionali-
zation and encapsulating payloads. Our HBPE polymer, contrary
to the conventional linear polymers, is amorphous, highly
from the nanoparticle into the PBS solution was determined at
regular time intervals by taking 1 mL aliquots from the PBS
solution and measuring the fluorescence intensity at 582 nm for
DiI and 378 nm for Taxol. The concentration of the either dye or
drug was calculated using a standard calibration curve. The
cumulative fraction of release versus time was calculated using
the following equation:
cumulative release ð%Þ ¼ ½guestꢁ =½guestꢁ
ꢀ 100
t
total
where [guest]
guest]total is the total guest present in the guest encapsulated
HBPE nanoparticles.
t
is the amount of guest released at time t, and
[
Results and Discussion
Synthesis of the HBPE polymer (5) was achieved by the
selective mono-C-alkylation of a readily available diethyl malo-
nate 1 with bromobutyl acetate 2 to yield the monosubstituted
compound 3 (Scheme 2). This monosubstitution reaction was
controlled by using a weak base, such as potassium carbonate,
and equivalent amount of starting materials. It should be noted
that when the weak base was substituted with a strong base, like
KOH or NaOH, the corresponding disubstituted product was
(16) Kaihara, S.; Matsumura, S.; Mikos, A. G.; Fisher, J. P. Nat. Protoc. 2007, 2,
767–2771.
2
(
17) (a) Wang, C.; Ge, Q.; Ting, D.; Nguyen, D.; Shen, H. R.; Chen, J.; Eisen, H.
N.; Heller, J.; Langer, R.; Putnam, D. Nat. Mater. 2004, 3, 190–196. (b) Li, S.-D.;
He, J.-D.; Yu, P. H.; Cheung, M. K. J. Appl. Polym. Sci. 2003, 89, 1530–1536.
5
368 DOI: 10.1021/la9037843
Langmuir 2010, 26(8), 5364–5373

Santra et al.
Article
a
Table 1. Determination of Hydrodynamic Diameter (D) of Functional HBPE Nanoparticles (NPs) 13, Immediately after Synthesis
NPs
D (nm)
Dil/NPs
DiD/NPs
DiR/NPs
Taxol/NPs
AzT/NPs
I
2
-mol/NPs
Gd-DOTA/NPs
NP-DiI
88 ( 2
89 ( 4
91 ( 1
92 ( 1
90 ( 3
91 ( 3
89 ( 1
67 ( 4
NP-DiD
59 ( 4
NP-DiR
53 ( 2
NP-Tax þ DiI
47 ( 2
44 ( 3
24 ( 3
NP-AzT þ DiI
29 ( 4
NP-I
2
-mol
58 ( 3
NP-Gd-DOTA
69 ( 6
a
Quantitative estimation of amount of dyes (DiI, DiD, and DiR), Taxol, AzT, iodinated molecule and Gd-DOTA per HBPE nanoparticles (NPs)
were performed using UV/vis spectroscopy. Folate per HBPE nanoparticle was 74 ( 5 in folate decorated NPs (D = 93 ( 2 nm).
branched, soluble, and biocompatible and has numerous surface
functional groups for targeting and hydrophobic cavities for
effective encapsulation of guest molecules, suggesting its versati-
lity in biomedical applications.
Our first generation HBPE polymer 5 can be used as is, in order
to make HBPE nanoparticles with carboxylic groups on their
surface (9). Also 5 can be further post-functionalized (6-8) to
yield surface aminated (10), alkynated (11) or azide-functiona-
lized (12) HBPE nanoparticles (Scheme 2) using carbodiimide
chemistry. The molecular weight of HBPE 5 was determined
using Gel Permeation Chromatography (GPC) and showed
the formation of high molecular weight polymer (M =42000,
w
PD=1.6) after applying vacuum (Figure 1a). All monomers and
1
13
polymers were further characterized by H and C NMR, Mass
and FTIR spectroscopies (Supporting Information Figures S1
and S2). Thermal gravimetric analysis (TGA) showed moderate
thermal stability (10% weight loss at 250 °C in air) of the polymer,
an excellent property for a biodegradable polymer (Supporting
Information Figure S3). The resulting HBPE polymers (5-8)
were utilized to synthesize a series of functionalized HBPE
nanoparticles (9-12) containing various types of cargos.
Figure 2. Assessment of DiI-encapsulating HBPE nanoparticles’
cellular uptake via confocal laser-scanning microscopy. (a) No
internalization was observed in cells incubated with carboxylated
HBPE nanoparticles (9); (b) enhanced internalization was ob-
served with folate-clicked HBPE nanoparticles (13). Cells incu-
bated with(c) Taxol-encapsulatingfolateHBPE nanoparticles (13)
induced Taxol-mediated mitotic arrest, leading to cell death, with
the (d-f) corresponding bright field confocal images of the
A549 cells treated with the corresponding functional HBPE nano-
particles. Nuclei stained with DAPI (blue). These observations
confirmed the targeting capability and receptor-mediated interna-
lization of our theranostic HBPE nanoparticles.
1
7
In our first set of experiments, we have encapsulated a series of
hydrophobic fluorescent dyes (DiI, DiD and DiR, 10 μg/μL),
drugs (Taxol and AzT, 1 μg/μL), chelated metals (Gd-DOTA,
2
5 μg/μL) and iodinated molecules (50 μg/μL) in the HBPE
nanoparticles (9-12, Scheme 2). In brief, the polymer (20 mg) and
cargo (5 μL) weredissolved inDMF (500μL), a polar solvent, and
added dropwise to the stirring DI water (5 mL), driving both the
self-assembly and encapsulation processes and resulting in the
formation of functional HBPE nanoparticles. Upon dispersion in
an aqueous environment, the synthesized HBPE polymer self-
assemble bringingthe hydrophobic areas and hydrophobic cargos
together to minimize contact with the aqueous environment,
while exposing the hydrophilic segments containing carboxylic
groups to the aqueous solution. These properties make our HBPE
polymers suitable for the fabrication of HBPE nanoparticles via
the solvent diffusion method. In this method, both the polymer
and hydrophobic cargos to be encapsulated are dissolved in
water-miscible organic solvents (DMF or DMSO) and the solu-
tion is added dropwise to an Eppendorf tube containing water
under constant stirring. Under these conditions, the miscible
solvent rapidly diffuses into the water, causing the polymer to
self-assemble by bringing the hydrophobic areas together, there-
fore creating hydrophobic nanocavities that encapsulate the
hydrophobic molecule, while exposing hydrophilic segments to
the aqueous solution. This procedure results in the formation
of carboxyl-functionalized spherical polymeric nanoparticles
suspended in an aqueous environment that can be easily
functionalized using facile carbodiimide chemistry. Scanning
electron microscopy of the HBPE nanoparticles confirms the
spherical geometry of the nanoparticles (Supporting Information,
Scheme S1).
All HBPE nanoparticles were purified using a PD-10 column
and dialysis (MWCO 6-8K) against PBS solution (pH = 7.4).
These HBPE nanoparticles were highly stable in aqueous buffered
solutions for more than a year, without significant precipitation
or reduction in the fluorescence emission when concentrated
(
Supporting Information, Figure S4). Dynamic light scattering
experiment confirmed the presence of stable and monodispersed
HBPE nanoparticles with hydrodynamic diameter of 88 ( 2 nm
(
Figure 1b). The presence of different surface functional groups
and encapsulation of cargos in the HBPE nanoparticles hydro-
phobic cavities was confirmed by FTIR, ζ-potential, spectro-
scopic analysis (Supporting Information, Figures S5-S8), and
fluorescence experiments where the presence of fluorescence
emission maxima at 570, 675, and 780 nm confirmed the en-
capsulation of DiI, DiD and DiR dyes inside the cavities
(
Figure 1c). Encapsulation of cargos inside the HBPE nanopar-
1
8,19
ticles cavity was further confirmed by observing a shift
of
23 nm (Figure 1d) in the HBPE nanoparticle encapsulating DiI as
compared to the free dye in solution. Amount of dyes, drugs,
iodinated molecule, and Gd-DOTA per HBPE nanoparticle
(18) Shi, M.; Wosnick, J. H.; Ho, K.; Keating, A.; Shoichet, M. S. Angew.
Chem., Int. Ed. Engl. 2007, 46, 6126–6131.
(
19) Imaz, I.; Hernando, J.; Ruiz-Molina, D.; Maspoch, D. Angew. Chem., Int.
Ed. 2009, 48, 2325–2329.
Langmuir 2010, 26(8), 5364–5373
DOI: 10.1021/la9037843 5369

Article
Santra et al.
Figure 3. Assessment of HBPE nanoparticles’ cell association
via flow cytometry (FACS). (a) Absence of fluorescence emission
was observedincontrol untreated A549cells (1XPBS), whereas (b)
partial association was observed for carboxylated HBPE nanopar-
ticles (9). (c) Aminated (10) and (d) folate-clicked (13) nanoparti-
cles interacted more profoundly with the cells, as indicated by
higher levels of fluorescence emission.
and hydrodynamic diameters were calculated, as previously
2
0,21
described
(Table 1).
Figure 4. Determination of the theranostic HBPE nanoparticles’
toxicity through the MTT assay. (a) The folate-decorated Taxol-
and AzT-encapsulating HBPE nanoparticles induced a significant
reduction in cell (A549) viability. (b) Determination of the speci-
ficity of the theranostic HBPE nanoparticles’ toxicity using H9c2
cells. Results indicated no toxicity of the HBPE nanoparticles in
H9c2 cells, does not overexpress folate receptor. Control cells were
treated with 1ꢀ PBS. Average values of four measurements are
depicted ( standard error.
The resulting small library of functional polymeric nanoparti-
cles with diverse surface functionalities, as well as different cargos,
can be readily used or further functionalized with targeting
ligands for the design of targeted therapeutics, imaging and
2
2
sensing applications. Furthermore, they can be used in the
formation of supramolecular assemblies (nanocomposites) to
design novel materials with multifunctional properties for appli-
cations in tissue engineering, sensing devices, photonics and
4
,23
catalysis.
29,30
which overexpress the folate receptor,
significant fluorescence
First, we examined the surface-charged-dependent cell inter-
nalization and cytotoxicity of our nanoparticles. The HBPE
nanoparticles 9-12 (3.5 mg/mL) containing fluorescent DiI
molecules as cargos were incubated with A549 lung carcinoma
or H9c2 human cardiomyocyte cells (10,000 cells) for 6 h and
confocal microscopy studies were performed. Results showed no
internalization of the carboxylated (9, Figure 2a), alkynated (11)
or azide-derivatized (12) HBPE nanoparticles and no associated
toxicity, which was evaluated through the MTT assay
in the cytoplasm was observed (Figure 2b). This contrasts with the
results obtained with the corresponding parent nanoparticles
(
9 and 11) (Figure 2a, Supporting Information, Figure S10),
which do not internalize in A549 cells. Flow cytometric (FACS)
analysis corroborated these results, showing limited cell-asso-
ciated fluorescence emission from cells treated with carboxylated
HBPE 9 (Figure 3b), whereas a 3-fold increase in cell-associated
fluorescence emission was observed in cells treated with the
aminated HBPE nanoparticles 10 (Figure 3c) and folate HBPE
nanoparticles 13 (Figure 3d) when compared to the control
nontreated cells (Figure 3a). In addition, similar results were
obtained in experiments using non-fixed A549 cells, where inter-
nalization of carboxylated (9) and folate-conjugated (13) HBPE
nanoparticles were monitored using epifluorescence microscopy
(
Supporting Information, Figures S9 and S10). In contrast,
cellular internalization of the aminated HBPE (10) nanoparticles
was observed, indicated by the presence of strong fluorescence in
the cytoplasm of both A549 and H9c2 cells (Supporting Informa-
tion, Figure S11).
However, when we conjugated the carboxylated (9) or alky-
(
Supporting Information, Figure S14). Experiments using live
2
4-28
nated (11) nanoparticles with folic acid via “click” chemistry
Supporting Information, Scheme S2, Figure S12) and incubated
the resulting folate HBPE nanoparticles (13) with A549 cells,
cells are important, as these studies confirmed that the observed
enhanced fluorescence was due to the folate-conjugated fluores-
cence nanoparticles’ internalizations in live cells and not a
potential artifact due to cell-fixing. Meanwhile, internalization
of 13 was nominal in A549 cells preincubated with free folic acid
(
(
20) Koch, A. M.; Reynolds, F.; Kircher, M. F.; Merkle, H. P.; Weissleder, R.;
Josephson, L. Bioconjug. Chem. 2003, 14, 1115–1121.
21) Shen, T.; Weissleder, R.; Papisov, M.; Bogdanov, A., Jr.; Brady, T. J.
Magn. Reson. Med. 1993, 29, 599–604.
(
Supporting Information, Figure S15), as well as in studies using
(
H9c2 cardiomyocytes, a cell line that does not express the folate
3
1
(
22) Kim, I. B.; Bunz, U. H. J. Am. Chem. Soc. 2006, 128, 2818–2819.
receptor (Supporting Information, Figure S13b). These experi-
ments confirmed the folate-receptor-mediated internalization of
the folate-conjugated HBPE nanoparticles (13), whereas the
aminated HBPE nanoparticles (10) showed nonspecific cellular
internalization.
(
23) Kim, I. B.; Dunkhorst, A.; Gilbert, J.; Bunz, U. H. F. Macromolecules 2005,
38, 4560–4562.
(
(
24) Sun, E. Y.; Josephson, L.; Weissleder, R. Mol. Imaging. 2006, 5, 122–128.
25) Bachovchin, D. A.; Brown, S. J.; Rosen, H.; Cravatt, B. F. Nat. Biotechnol.
2
009, 27, 387–394.
26) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed. Engl.
001, 40, 2004–2021.
(
2
(
(
27) Santra, S.; Kaittanis, C.; Grimm, J.; Perez, J. M. Small 2009, 5, 1862–1868.
28) Englert, B. C.; Bakbak, S.; Bunz, U. H. F. Macromolecules 2005, 38, 5868–
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Chem. 2004, 47, 3887–3891.
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877.
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29) Yuan, H.; Miao, J.; Du, Y. Z.; You, J.; Hu, F. Q.; Zeng, S. Int. J. Pharm.
2008, 348, 137–145.
P. Anal. Biochem. 2005, 338, 284–293.
5
370 DOI: 10.1021/la9037843
Langmuir 2010, 26(8), 5364–5373

Santra et al.
Article
Figure 5. Drug and dye release profiles of Taxol and DiI coencapsulating HBPE nanoparticles (13) in PBS (pH = 7.4) at 37 °C. Release of
Taxol (a and b) and DiI (c and d) were observed in the presence of an esterase enzyme (a and c) and at pH 4.0 (b and d).
Internalization of either aminated (10) or folate (13) HBPE
nanoparticles in A549 lung carcinoma exhibited nominal cyto-
toxicity (Supporting Information, Figure S9), demonstrating
their biocompatibility and non-toxicity even when internalized
by the cells. However, when folate-conjugated HBPE nanoparti-
cles (13) were loaded with Taxol, a cell microtubule synthesis
its delivery, in order to prevent damage of nontransformed cells
and healthy tissue. In another set of experiments, as most lung
carcinomas exhibit aberrant telomerase activity, we found that
the reverse transcriptase inhibitor AzT, encapsulated in HBPE
nanoparticles (13), induced significant cell death, presumably via
inhibition of telomerase activity (Supporting Information, Figure
S16). Taken together, these findings support the principle that
folate-decorated HBPE nanoparticles can target and deliver
chemotherapeutic agents to folate-receptor-overexpressing carci-
nomas, while visualizing the drug’s homing.
3
2-34
inhibitor commonly used in cancer chemotherapy,
75% cell
death was observed in A549 cells (MTT assay, Figure 4a).
3
5
Meanwhile, when AzT, a viral reverse transcriptase and human
3
6
telomerase inhibitor, was encapsulated into the nanoparticles
13), 55% cell death was observed(A549 cells, Figure 4a). Overall,
(
The therapeutic applications of our HBPE nanoparticles rely
on the biodegradability and biocompatibility of the nanoparti-
cles, and most importantly, on the rate of release of the encapsu-
lated drug from the polymeric nanocavities. To evaluate the
HBPE nanoparticles’ (13) drug release profile, we performed
enzymatic (esterase from porcine liver) and low-pH degradation
experiments using a dynamic dialysis technique at 37 °C. Results
indicated that Taxol (Figure 5a,b) was released faster than the dye
(Figure 5c,d) when the nanoparticles were incubated with either
an esterase or at low pH 4.0. These differential release profiles
may be attributed to the drug’s size and its hydrophobic nature.
As nominal cargo release was observed at physiological pH, we
deduced that the HBPE nanoparticles should be stable under
these conditions, whereas they are readily biodegraded upon
enzymatic and intracellular environmental triggers, such as loca-
lization in acidified lysosomal compartments. Taken together,
these results indicate the efficient drug release capability of our
theranostic HBPE nanoparticles, rendering targeted HBPE na-
noparticles useful for potential in vivo applications.
these data suggested that the induction of cell death was mainly
mediated by either Taxol or AzT and not by the fluorophores. In
contrast, no significant cytotoxicity was observed when H9c2
cells, were treated with the Taxol- or AzT-encapsulating nano-
particles (13), confirming their exclusive folate-receptor-mediated
internalization (Figure 4b). Furthermore, these results suggest
that the coencapsulation of a fluorophore and a therapeutic agent
in our HBPE nanoparticles can be utilized for cellular targeting,
þ
such as in cancer or anti-HIV CD4 -specific therapeutic regimes
and visualization of tumor regression in clinical studies. The
internalization and cytotoxicity effects of either the Taxol or AzT
HBPE nanoparticles (13) were confirmed using confocal micro-
scopy, showing the induction of significant mitotic arrest, leading
to dramatic morphological changes and cell death (Taxol,
Figure 2c). This observation indicated that Taxol’s therapeutic
efficacy was preserved, despite its HBPE nanoparticle encapsula-
tion and strongly supports the importance of encapsulating this
potent antitumor agent within the polymeric cavity and targeting
For in vivo imaging applications, nanoparticles with excitation
and emission in the near-infrared region (650-900 nm) are
(
82.
(
(
(
32) Davis, M. E.; Chen, Z. G.; Shin, D. M. Nat. Rev. Drug. Discov. 2008, 7, 771–
3
7
needed for deep tissue fluorescence imaging. Toward this end,
DiR (excitation/emission: 751/780 nm) encapsulating carboxy-
lated (9) and folate (13) HBPE nanoparticles were incubated with
7
33) Fonseca, C.; Simoes, S.; Gaspar, R. J. Controlled Release 2002, 83, 273–286.
34) Gupte, A.; Ciftci, K. Int. J. Pharm. 2004, 276, 93–106.
35) Shen, L.; Peterson, S.; Sedaghat, A. R.; McMahon, M. A.; Callender, M.;
Zhang, H.; Zhou, Y.; Pitt, E.; Anderson, K. S.; Acosta, E. P.; Siliciano, R. F. Nat.
Med. 2008, 14, 762–766.
(
36) Datta, A.; Nicot, C. Oncogene 2008, 27, 1135–1141.
(37) Weissleder, R.; Ntziachristos, V. Nat. Med. 2003, 9, 123–128.
Langmuir 2010, 26(8), 5364–5373
DOI: 10.1021/la9037843 5371

Article
Santra et al.
Figure 6. Fluorescence images of the cellular uptake of DiR-
encapsulating (a) carboxylated (9) and (b) folate-derivatized (13)
HBPE nanoparticles taken from a Xenogen IVIS system.
A549 cells for 6 h and imaged with an in vivo imaging system
(
IVIS), using an ICG filter set (Figure 6). No cell-associated
fluorescence was observed in cells treated with carboxylated
HBPE nanoparticles 9 (Figure 6a), whereas a dose-dependent
increase in fluorescence was observed in cells treated with the
folate HBPE nanoparticles 13 (Figure 6b), demonstrating recep-
tor-mediated internalization and in vivo imaging capability.
Taken together, these findings support the principle that our
HBPE nanoparticles can be used in imaging and therapeutic
applications in targeted delivery of chemotherapeutic agents,
visualization of drug’s homing and monitoring of drug release.
The unique properties of the HBPE nanoparticles, such as (a)
encapsulation of diverse cargos, (b) different surface functiona-
lization, and (c) stability at physiological pH with selective
biodegradability and cargo release upon enzymatic or low pH
triggers, make them ideal nanomeric units for the creation of
supramolecular assemblies and nanocomposites. In recent years
there has been an interest in the synthesis of polymeric nanocom-
posites with unique magnetic, luminescent and radiation-protec-
tion properties that are different from the bulk properties of the
same material. Particularly, the creation of multifunctional im-
plantable materials for application in tissue engineering or im-
plantable devices with signal-driven cargo release is an emerging
field. Therefore, we hypothesized whether our HBPE nanoparti-
cles, particularly the ones conjugated with azide or alkyne surface
moieties (clickable polymeric nanoparticles) would self-assemble
among themselves or with other metallic nanoparticles (using
Figure 7. Schematic representation of the “click” chemistry
mediated water-based syntheses of an X-ray blocking (a) and
highly fluorescent (b) polymeric nanocomposite (NCs) encapsulat-
ing a tri-iodinated molecule and DiR, respectively. Nanocompo-
sites without iodinated molecule (a: X-i and Z-i) and DiR (b: X-i
and Z-i) showed no X-ray blocking and fluorescence properties,
respectively. Y-I and Y-ii contained PBS.
“
click” chemistry) to create novel polymeric orpolymeric-metallic
nanocomposites with enhanced magnetic or radiopacity proper-
ties for MRI and X-ray-CT applications, enhanced fluorescence,
catalytic activities, radical scavenging and anti-inflammatory
properties for tissue engineering.
Toward this goal, we first facilitated the self-assembly of two
sets of HBPE nanoparticles, one encapsulating a hydrophobic
iodinated molecule (5-amino-2,4,6-triiodoisophthalic acid, Sup-
porting Information, Figure S17) with alkyne-functionalized
surface (11) and a second one encapsulating a fluorescent dye
Figure 8. Schematic representation of the syntheses of a magnetic
and fluorescent (a) and free radical scavenging (b) polymeric-
metallic nanocomposite (NCs) containing iron oxide and cerium
oxide nanoparticles, respectively, using “click” chemistry.
or in phosphate buffered solution (Figure 7a: Y-i, ii). Similarly, no
fluorescence was observed in nanoassemblies without DiR dye
(
DiR, Supporting Information, Figure S18) with azide-functio-
nalized surface (12). We have chosen the triiodinated compound,
(
(
Figure 7b: X-i and Z-i) and in phosphate buffered solution
Figure 7b: Y-i, ii). In addition, nanocomposites containing a
because it is an important synthon for the synthesis of radiopaque
3
8
molecules for X-ray CT imaging, is used for radio labeling of
fluorescent dye and a chelated metal (Gd-DOTA) were devel-
oped, demonstrating the capability of our system to create HBPE
nanocomposites containing chelated metals for applications in
imaging and catalysis.
Similarly, a nanocomposite can be engineered with a mixture of
HBPE and metallic nanoparticles. For these studies clickable iron
oxide and cerium oxide nanoparticle derivatized with alkyne
groups were prepared, as previously reported, and conjugated with
azide-functionalized HBPE nanoparticle encapsulating DiR (12).
3
9
proteins and can be easily encapsulated inside the polymeric
nanocavities due to the presence of an extensive hydrogen-
bonding network. The resulting two sets of clickable nanoparti-
cles self-assemble upon addition of CuI as catalyst, forming a
HBPE nanocomposite with both radiopaque (Figure 7a) and
fluorescent (Figure 7b) properties. Absence of X-ray contrast was
observed in control nanoparticle assemblies composed of HBPE
nanoparticles without iodinated molecule (Figure 7a: X-i and Z-i)
2
7
40
(
(
38) Beck, T.; Sheldrick, G. M. Acta Crystallogr. 2008, E64, o1286.
39) Ogan, M.; Tomasella, F. P.; Tu, J.-I. US patent 5463080, 1992.
(40) Asati, A.; Santra, S.; Kaittanis, C.; Nath, S.; Perez, J. M. Angew. Chem., Int.
Ed. Engl. 2009, 48, 2308–2312.
5
372 DOI: 10.1021/la9037843
Langmuir 2010, 26(8), 5364–5373

Santra et al.
Article
after addition of H O ; green, after 48 h). Upon addition of H O ,
2
2
2
2
a yellowcolor was observedrapidly, corresponding to thered shift
in UV/vis transmittance spectrum (red curve), compared to the
control (black curve). We observed a gradual shift of the
transmittance curve to its original control value, demonstrating
the reversibility and autocatalytic activity of the nanocomposite
under normal physiological condition.
Summary
In summary, we have introduced a new class of hyperbranched
polymer capable of forming a library of functional polymeric
nanoparticles with dual diversity in the nanoparticle surface and
cargo. The hyperbranched polymer was new in its class and used
for the first time to synthesize theranostic nanoparticles and
nanocomposites for biomedical applications. The presence of
hydrophobic pockets insidethe three-dimensional hyperbranched
structures played an important role in the encapsulation of a
variety of hydrophobic cargos such as fluorescent dyes, thera-
peutic agents, chelated metals and iodinated molecules using the
solvent diffusion method. The resulting nanoparticles showed
high solubility and stability of encapsulation of their respective
cargos in aqueous buffered solutions, at physiological pH. Only
an enzyme (esterase) or acidic pH triggered the release of the
cargos at different rates (for example, drug vs dye). Furthermore,
the resulting functional hyperbranched nanoparticles can be
surface-modified with a variety of ligands for targeting and sens-
ing applications. Also, the nanoparticles’ surface functionality
allows the formation of nanoparticle assemblies (nanocom-
posites) and multifunctional materials for the creation of smart
responsive materials. These polymeric nanocomposites showed
excellent magnetic, fluorescent, MRI imaging, X-ray contrast and
catalytic activity when conjugated with dye encapsulating HBPE
nanoparticles, iron oxide and cerium oxide nanoparticles. Our
studies demonstrated the importance of our newly synthesized
HBPE polymers, nanoparticles and nanocomposite as they are
biodegradable, targetable, nontoxic, and have capability to
encapsulate a wide range of guest molecules. This offers the
possibility to create novel multifunctional materials composed of
hyperbranched polymeric nanoparticles with unique properties
that can be tailored by the nature of their cargo, their surface
functionality, and the formation of supramolecular assemblies.
Figure 9. Autocatalytic activity of nanocomposite containing cer-
ium oxide nanoparticles. Upon addition of hydrogen peroxide, a
rapid color change from clear (a) to yellow (b) was observed,
corresponding to a concomitant red shift in the UV/vis transmit-
tance spectrum. Gradually, as the system regenerated a shift in the
transmittance curve toward its original control value was seen.
When alkynated iron oxide nanoparticles were assembled via
“click” chemistry with the azide HBPE fluorescent nano-
particle, a nanocomposite with dual fluorescent and magnetic
properties was obtained (Figure 8a). By incorporating magnetic
nanoparticles into the HBPE composite, a fluorescent material
that can be controlled by an external magnetic field is obtained. It is
also worth noticing that the localization, and integrity of an
implantable material made with this composite could be poten-
tially visualized in deep tissue through magnetic resonance imaging
(MRI) and optical imaging. Subsequently, when alkynated cerium
oxide (ceria) nanoparticles were used to create the nanoassembly,
a composite with enhanced fluorescent properties and resistant to
UV-induced photobleaching was obtained (Figure 8b). Results
using this nanocomposite showed enhanced photostability of the
DiR’s near-infrared fluorescence within the nanocomposite
(
DiR-Ce-NCs, Figure 8b) as compared to the photostability
of the dye in the fluorescent nanoparticles (DiR-NPs) or DiR dye
in solution. The dye’s photobleaching in the absence of ceria may
be attributed to the generation of reactive oxygen species (ROS)
during UV irradiation. Therefore, in the cerium oxide-containing
nanocomposite the photobleaching of the dye is prevented via
a ceria-mediated mechanism, as cerium oxide nanoparticles have
Acknowledgment. We thank Dr. Annette Khaled (UCF) and
Mounir Chehtane (UCF) for assistance with FACS analysis, as
well as Atul Asati (UCF) for assistance with the cerium oxide nano-
particle. This work was supported by NIH Grants CA101781 and
GM084331 and UCF NSTC Start Up Fund, all to J.M.P.
41-43
the ability to selectively scavenge ROS.
In addition, this nanocomposite showed excellent autocatalytic
and antioxidant activity (Figure 9) as expected due to the presence
of cerium oxide nanoparticles. The ability of our cerium oxide
3þ
4þ
Supporting Information Available: (1) synthesis and char-
acterizations of monomers and polymers, (2) nanoparticle
characterizations, (3) receptor-mediated cellular internaliza-
tions and determination of cytotoxicity using confocal mi-
croscopy and MTT assay, incuding figures showing NMR
spectra, a TGA thermogram, structures and photos of the
dyes, ζ-potential of 9 and 10, FT-IR spectra, fluorescence
emission spectra, determination of the HBPE nanoparticles’
toxicity, microscopic images of cells, and UV/vis images, and
schemes showing synthetic representations of the syntheses.
This material is available free of charge via the Internet at
http://pubs.acs.org.
containing nanocomposite to reversibly switch from Ce to Ce
is a key factor for its catalytic and biological applications as
antioxidants. We examined whether the formation of a nanoas-
sembly compromised the ceria nanoparticles’ autocatalytic beha-
vior. Inthe UV/vis experiments, the transmittance spectra showed
reversible autocatalytic behavior when incubated with H O at
2
2
pH 7.4 (black, control, before addition of H O ; red, immediately
2
2
(
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Langmuir 2010, 26(8), 5364–5373
DOI: 10.1021/la9037843 5373