Selective N -alkylation of β-alanine facilitates the synthesis of a poly(amino acid)-based theranostic nanoagent

Article abstract of DOI:10.1021/bm2009334

The development of functional amino acid-based polymeric materials is emerging as a platform to create biodegradable and nontoxic nanomaterials for medical and biotechnology applications. In particular, facile synthetic routes for these polymers and their corresponding polymeric nanomaterials would have a positive impact in the development of novel biomaterials and nanoparticles. However, progress has been hampered by the need to use complex protection-deprotection methods and toxic phase transfer catalysts. In this study, we report a facile, single-step approach for the synthesis of an N-alkylated amino acid as an AB-type functional monomer to generate a novel pseudo-poly(amino acid), without using the laborious multistep, protection-deprotection methods. This synthetic strategy is reproducible, easy to scale up, and does not produce toxic byproducts. In addition, the synthesized amino acid-based polymer is different from conventional linear polymers as the butyl pendants enhance its solubility in common organic solvents and facilitate the creation of hydrophobic nanocavities for the effective encapsulation of hydrophobic cargos upon nanoparticle formation. Within the nanoparticles, we have encapsulated a hydrophobic DiI dye and a therapeutic drug, Taxol. In addition, we have conjugated folic acid as a folate receptor-targeting ligand for the targeted delivery of the nanoparticles to cancer cells expressing the folate receptor. Cell cytotoxicity studies confirm the low toxicity of the polymeric nanoparticles, and drug-release experiments with the Taxol-encapsulated nanoparticles only exhibit cytotoxicity upon internalization into cancer cells expressing the folate receptor. Taken together, these results suggested that our synthetic strategy can be useful for the one-step synthesis of amino acid-based small molecules, biopolymers, and theranostic polymeric nanoagents for the targeted detection and treatment of cancer.

Full text of DOI:10.1021/bm2009334

Article
pubs.acs.org/Biomac
Selective N-Alkylation of β-Alanine Facilitates the Synthesis of a
Poly(amino acid)-Based Theranostic Nanoagent
Santimukul Santra*,† and J. Manuel Perez*,†,‡
‡
†NanoScience Technology Center and Burnett School of Biomedical Sciences, College of Medicine, Chemistry Department,
University of Central Florida, 12424 Research Parkway, Suite 400, Orlando, Florida 32826, United States
S
* Supporting Information
ABSTRACT: The development of functional amino acid-
based polymeric materials is emerging as a platform to create
biodegradable and nontoxic nanomaterials for medical and
biotechnology applications. In particular, facile synthetic routes
for these polymers and their corresponding polymeric
nanomaterials would have a positive impact in the develop-
ment of novel biomaterials and nanoparticles. However,
progress has been hampered by the need to use complex
protection−deprotection methods and toxic phase transfer
catalysts. In this study, we report a facile, single-step approach
for the synthesis of an N-alkylated amino acid as an AB-type
functional monomer to generate a novel pseudo-poly(amino acid), without using the laborious multistep, protection−
deprotection methods. This synthetic strategy is reproducible, easy to scale up, and does not produce toxic byproducts. In
addition, the synthesized amino acid-based polymer is different from conventional linear polymers as the butyl pendants enhance
its solubility in common organic solvents and facilitate the creation of hydrophobic nanocavities for the effective encapsulation of
hydrophobic cargos upon nanoparticle formation. Within the nanoparticles, we have encapsulated a hydrophobic DiI dye and a
therapeutic drug, Taxol. In addition, we have conjugated folic acid as a folate receptor-targeting ligand for the targeted delivery of
the nanoparticles to cancer cells expressing the folate receptor. Cell cytotoxicity studies confirm the low toxicity of the polymeric
nanoparticles, and drug-release experiments with the Taxol-encapsulated nanoparticles only exhibit cytotoxicity upon
internalization into cancer cells expressing the folate receptor. Taken together, these results suggested that our synthetic strategy
can be useful for the one-step synthesis of amino acid-based small molecules, biopolymers, and theranostic polymeric nanoagents
for the targeted detection and treatment of cancer.
diblock copolymer structures.17−19 However, the presence of
hydrophobic linear pendants in these polymeric structures
INTRODUCTION
■
Biodegradable amphiphilic polymers self-assemble into poly-
increases the cavity size of the polymeric core, thereby
meric nanoparticles (PNPs) and polymersomes in aqueous
enhancing the loading of therapeutic cargos. For instance,
solution and can potentially be used in catalysis, sensing,
parenteral drug delivery, and imaging.1−6 In order to introduce
unique properties into these polymeric nanoparticles, a variety
of therapeutic cargos have been encapsulated within the
polymeric nanocavities.7−10 Often times, the diversity of the
encapsulated cargos is compromised by the nature of the
polymeric nanocavities that limits the amount and type of cargo
that is encapsulated. Most polymeric matrixes are composed of
a hydrophobic core (polymeric backbone) and a large number
of polar reactive surface groups.11−13 The hydrophobic core
serves as a reservoir for hydrophobic cargos, whereas the
hydrophilic surface can be functionalized for targeting.14 In
particular, aliphatic polymeric nanoparticles have received much
attention due to their solubility and biodegradability.15,16 For
example, aliphatic linear polyesters such as poly(L-lactic acid)
(PLA), poly(DL-lactic-co-glycolic acid) (PLGA), and poly(ε-
caprolactone) (PCL) have been combined with hydrophilic
poly(ethylene glycol) (PEG) segments to produce AB-type (A:
acidic or electrophilic center; B: basic or nucleophilic center) of
poly(aspartic acid)−PEG diblock copolymer, an amino acid-
based biopolymer, exhibited promising results in encapsulating
or chemically conjugating cargos within the polymeric core and
has resulted in an effective suppression of tumor growth when
injected in vivo.20 This is due to the presence of pendant
carboxylated aliphatic chains in the polymer structure, creating
large polymeric cavities in the nanostructure for the effective
preservation of various cargos. In addition, other amino acid-
based biopolymers, such as poly(glutamic acid),21 poly(aspartic
acid),22 and polyproline dendrimers,23 are also widely used in
biomedical applications due to their enhanced solubility,
biocompatibility, and low profile cytotoxicity. Syntheses of
such biomacromolecules involve polymerization of an appro-
priate cyclic monomer22 or selective −NH2 group protection
Received: July 5, 2011
Revised: September 15, 2011
Published: September 30, 2011
© 2011 American Chemical Society
3917
dx.doi.org/10.1021/bm2009334|Biomacromolecules 2011, 12, 3917−3927

Biomacromolecules
Article
and deprotection of the corresponding amino acid. This is due
to the fact that direct polymerization of an amino acid often
results in the formation of cyclic monomers and cross-linked
low molecular weight poly(amino acid)s and oligomers.
Furthermore, the interest in developing new protocols for the
selective syntheses of mono- and di-N-alkylated amino acids
and its derivatives is increasing due to their potential
applications in creating small molecule-based therapeutic agents
and biopolymers.24,25 Hence, the design and synthesis of
functional amino acids for the synthesis of biodegradable
branched polymers would provide an ideal platform to develop
novel theranostic nanoagents for the effective encapsulation
and delivery of therapeutic drugs.
Selective mono- and di-N-alkylation of amino acid is an
approach that typically is used to functionalize amino acids for
further applications. Various methods for the selective N-
alkylation of amino acids have been reported; however, they
often involve multiple reaction steps, resulting in a process that
is difficult to scale-up, with low yields and the generation of
toxic byproducts that hamper their clinical applications.26−28 In
addition, this process is limited to some amino acids29 and
typically requires the use of time-consuming protection−
deprotection protocols26,30−38 and toxic phase transfer
catalysts.39 On the other hand, N-alkylated amino acid
facilitates the formation of soluble, high molecular weight
polymers upon polymerization, whereas cyclization of the
starting monomers, low-molecular weight oligomers, and cross-
linked byproducts resulted when a simple amino acid is
polymerized. Therefore, there is a great need for a facile, one-
step synthetic strategy for the synthesis of selective N-alkylated
amino acids.
treatment of cancer. More importantly, the synthesized
functional AB monomer is designed in such a way that, when
polymerized, the resulting pseudo-poly(amino acid) (5)
contains an amphiphilic polyester backbone and hydrophobic
butyl pendants in the structure. These aliphatic butyl pendants
played an important role in creating hydrophobic nanocavities
when solvent diffusion method14,42 was used to synthesize
these polymeric nanoparticles (PNPs). Therefore, the PNP’s
hydrophobic cavity provides high affinity for the encapsulation
of hydrophobic dyes and therapeutic drugs. The nanoparticles’
surface carboxylic acid groups can be further functionalize to
target and induce delivery of the encapsulated therapeutic
agents. Taken together, our one-step green synthetic approach
has several advantages: (i) selective mono- and di-N-alkylation
of amino acids, (ii) synthesis of amino acid-based functional
molecules and biodegradable polymers, (iii) easy fabrication of
theranostic PNPs in aqueous medium, (iv) “click” chemistry-
based surface functionalizations (folate ligand) for cancer
targeting, (v) hydrophobic dyes encapsulations for optical
detection of cancer, and (vi) therapeutic agent (Taxol)43−45
encapsulation and delivery for cancer treatment.
EXPERIMENTAL SECTION
■
Materials. Propylene oxide (PO), butyl bromide (BuBr), β-
alanine, potassium hydroxide (KOH), tetrahydrofuran (THF), acetyl
chloride, acetonitrile, potassium bromide (KBr), p-toluenesulfonic acid
(pTSA), N,N′-dimethylformamide (DMF), Taxol, 3-(4,5-dimethylth-
iazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), N-hydroxysucci-
nimide (NHS), propargylamine, folic acid, porcine liver esterase,
ethylenediamine, sodium azide, and other chemicals were purchased
from Sigma-Aldrich and used without further purification. Hydro-
phobic dye (DiI: D282) and 4′,6-diamidino-2-phenylindole (DAPI:
D1306) were purchased from Invitrogen, whereas the EDC (1-ethyl-3-
[3-(dimethylamino)propyl]carbodiimide hydrochloride) was obtained
from Pierce Biotechnology. PD-10 columns (Sephadex G-25M) were
obtained from GE Healthcare Biosciences. The human lung carcinoma
cell line A549 and cardiomyocytes H9c2 were obtained from ATCC.
Dialysis cups were obtained from Spectrum Laboratories.
In this paper, we report a facile, one-step strategy for the
selective synthesis of mono- and di-N-alkylated amino acids,
poly(amino acid)s, and Taxol-loaded theranostic polymeric
nanoagents for the selective delivery of Taxol for the treatment
of cancer. Our strategy is facile, easy to scale-up, and involves
no conventional protection−deprotection methods26,30−38 or
phase transfer catalysts.39 In addition to the one-step selective
synthesis of mono-N-alkylated amino acids (3, Scheme 1), the
Synthesis and Characterizations of Mono-N-butylated β-
Alanine (3-(Butylamino)propionic Acid, 3). β-Alanine (1, 1.0 g,
11.23 mmol), ethanol (15 mL), and KOH (1.26 g, 22.47 mmol) were
taken in a 100 mL round-bottom flask and stirred at room
temperature. Water was added to the flask in a dropwise fashion
until the reaction mixture became homogeneous. Butyl bromide (2,
1.39 g, 10.11 mmol) was added dropwise to the reaction mixture and
stirred at room temperature. The progress of the reaction was
monitored by thin layer chromatography (TLC). The reaction mixture
was concentrated using a rotary evaporator under reduced pressure.
The product was then purified by column chromatography using 12%
methanol in chloroform as eluent to get pure colorless viscous liquid.
Scheme 1. Synthesis of Selective Mono- and Di-N-alkylated
β-Alanine and Its Functional Poly(amino acid)
1
Yield: 1.19 g (73%). H NMR (400 MHz, CDCl3, δ ppm, J Hz): 0.94
(t, 3H, J = 7.3), 1.42 (m, 2H), 1.73 (m, 2H), 2.53 (t, 2H, J = 6.1), 2.92
(t, 2H, J = 7.6), 3.09 (t, 2H, J = 5.8), 8.85 (bs, 1H). 13C NMR (100.56
MHz, CDCl3, δ ppm): 13.49, 19.82, 28.04, 31.84, 44.46, 46.57, 176.74.
IR (CHCl3): 3414, 2961, 2870, 2448, 1711, 1578, 1464, 1394, 1304,
1198, 1137, 1037, 932, 847, 692, 597 cm−1. Q-tof high-resolution mass
spectrometry (HRMS): Calculated mass for C7H16NO2 is 146.1181.
Found: 146.1184.
Synthesis and Characterizations of di-N-butylated β-Alanine
(3-(Dibutylamino)propionic Acid, 3A). β-Alanine (1, 1.0 g, 11.23
mmol), ethanol (15 mL), and KOH (1.89 g, 33.71 mmol) were taken
in a 100 mL round-bottom flask and stirred at room temperature.
Water was added to the flask in a dropwise fashion until the reaction
mixture became homogeneous. Butyl bromide (2, 3.54 g, 25.84 mmol)
was added dropwise to the reaction mixture and stirred at room
temperature. The progress of the reaction was monitored by TLC. The
reaction mixture was concentrated using rotary evaporator under
present synthetic strategy is also capable of synthesizing novel
di-N-alkylated functional molecules (4), using a different
alkylating agent. Here, we have used this functional molecule
(4) as an AB-type linear monomer for the synthesis of a novel
pseudo-poly(amino acid) (5).40,41 Our present study further
demonstrates the application of our current strategy by
synthesizing novel theranostic nanoagents (6−11) from the
resulting polymer and their potential use in the detection and
3918
dx.doi.org/10.1021/bm2009334|Biomacromolecules 2011, 12, 3917−3927

Biomacromolecules
Article
reduced pressure. The product was then purified by column
chromatography using 10% methanol in chloroform as eluent to get
pure colorless viscous liquid. Yield: 1.46 g (65%). 1H NMR (400 MHz,
CDCl3, δ ppm, J Hz): 0.95 (t, 6H, J = 7.4), 1.37 (m, 4H), 1.60 (m,
4H), 2.50 (t, 2H, J = 6.4), 2.78 (t, 4H, J = 8.2), 2.98 (t, 2H, J = 6.4),
10.14 (bs, 1H). 13C NMR (100.56 MHz, CDCl3, δ ppm): 13.55, 20.07,
25.79, 30.42, 49.70, 51.56, 174.78. IR (CHCl3): 3399, 2944, 2831,
2516, 2226, 2044, 1708, 1606, 1450, 1202, 1119, 1027, 714 cm−1. Q-
tof high-resolution mass spectrometry (HRMS): Calculated mass for
C11H24NO2 is 202.1807. Found: 202.1814.
Thermogravimetric Analysis (TGA). The thermal stability of the
synthesized pseudo-poly(amino acid) (5) was measured using
thermogravimetric analysis. This experiment was 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.
Synthesis and Characterizations of DiI Dye-Encapsulating
Polymeric Nanoparticles (6). Water-Based Solvent Diffusion
Method. 1.2 mg of DiI dye in 250 μL of DMF was mixed in 250 μL of
DMF solution containing polymer 5 (25 mg) and vortexed. The
resulting polymer−DiI mixture in DMF was added dropwise to
deionized water (5 mL) with continuous stirring at room temperature
forming DiI-encapsulating PNPs (6). The synthesized PNPs (6) were
purified using a PD-10 column and finally dialyzed (MWCO 6K−8K)
against PBS (pH = 7.4).
Synthesis of Taxol and DiI Coencapsulating PNPs (7).
Similarly, 1.5 mg of Taxol and 0.25 mg of DiI dye in 250 μL of DMF
were mixed with carboxylated polymer 5 (25 mg) in 250 μL of DMF
and followed the solvent diffusion method as described above.
Synthesis of Propargylated Polymeric Nanoparticles (8, 9).
Carbodiimide chemistry was used following the previously reported
method.14,46,47 Briefly, to a solution of carboxylated PNPs (6 or 7, 1.0
mmol) in PBS (pH = 7.4), a solution of EDC (10 mmol) and NHS
(10 mmol) in MES buffer (pH = 6.0) was added followed by 3 min
incubation at room temperature. Propargylamine (10 mmol) in DMF
was then added dropwise and continued for 3 h to obtain 8 or 9,
respectively. The synthesized functional PNPs were purified using a
PD-10 column and finally dialyzed (MWCO 6K−8K) against PBS
buffer (pH = 7.4). They were stored in refrigerator for further
characterization.
Synthesis of Folate-Conjugated Polymeric Nanoparticles
(10, 11). “Click” chemistry was used following previously reported
methods.14,46,47 Briefly, the alkynated PNPs 8 or 9 (6 × 10−3 mmol) in
bicarbonate buffer (pH = 8.5) were taken to an Eppendorf containing
catalytic amount of CuI (6 × 10−10 mmol) in 250 μL of bicarbonate
buffer (pH = 8.5) and vortexed. To the resulting solution, azide-
functionalized folic acid14,46,47 (6 × 10−2 mmol) in DMSO was added,
and the reaction was incubated at room temperature for 12 h. The
synthesized PNPs (10, 11) were purified using a PD-10 column and
finally dialyzed (MWCO 6K−8K) against PBS solution (pH = 7.4).
The number of folate molecules per nanoparticle was found to be 23 ±
4, following a protocol described before.14 They were stored in
refrigerator for further characterization.
Drug and Dye Encapsulation Efficiency. The nanoparticles
were digested in a slightly acidic DI water (pH = 6.0−6.5) to release
encapsulated cargos. The presence of a trace amount of diluted HCl
was removed by applying nitrogen flow overnight. The analysis on the
assays of Taxol and DiI dye were determined by high-performance
liquid chromatography (HPLC, PerkinElmer 200 series) at the
detection wavelength of 227 nm and by standard UV/vis spectroscopic
method at the detection wavelength of 553 nm, respectively. Standard
calibration curves for free Taxol and DiI dye were plotted by
performing HPLC and UV/vis experiments with different concen-
trations of Taxol and DiI dye, respectively. HPLC chromatograms of
free Taxol and Taxol-encapsulating nanoparticles presented in the
Supporting Information (Figure S8) showed the successful encapsu-
lation of the drug. The Taxol and DiI dye encapsulation efficiencies
(EE%) in polymeric nanoparticle (11) were found to be 68.5 ± 3.2%
and 21.2 ± 1.3%, respectively.
Synthesis and Characterizations of 3-[Butyl-(2-
hydroxypropyl)amino]propionic Acid (4). 3-(Butylamino)-
propionic acid (3, 2.0 g, 13.79 mmol) and commercial ethanol (20
mL) were taken in a round-bottom flask and stirred at room
temperature. Propylene oxide (1.6 g, 27.58 mmol) was added
dropwise to the reaction mixture and stirred at room temperature.
The progress of the reaction was monitored by TLC. The reaction
mixture was evaporated using rotary evaporator under reduced
pressure to get the pure compound as a viscous liquid. Yield: 1.90 g
1
(68%). H NMR (400 MHz, CDCl3, δ ppm, J Hz): 0.95 (t, 3H, J =
7.4), 1.21 (d, 3H, J = 6.4), 1.37 (m, 2H), 1.67 (m, 2H), 2.00 (s, 1H),
2.59 (m, 2H), 2.95 (m, 2H), 3.07 (m, 2H), 3.26 (m, 2H), 4.18 (m,
1H), 9.19 (bs, 1H). 13C NMR (100.56 MHz, CDCl3, δ ppm): 13.64,
19.99, 21.10, 25.41, 30.83, 51.16, 53.22, 60.63, 61.65, 175.58. IR
(CHCl3): 3414, 2965, 2935, 2876, 2571, 2239, 1715, 1593, 1459,
1382, 1196, 1141, 1088, 1009, 912, 845, 732, 644, 600 cm−1. Q-tof
high-resolution mass spectrometry (HRMS): Calculated mass for
C10H22NO3 is 204.1600. Found: 204.1601.
Synthesis and Characterizations of the Polymer 5. 3-[Butyl-
(2-hydroxypropyl)amino]propionic acid (4) and the catalyst p-
toluenesulfonic acid (100:1 molar ratio) were taken in a polymer-
ization vessel, and the polymerization reaction was carried out under
dry argon gas atmosphere. Then the reaction vessel was slowly heated
to 160 °C using an oil bath and kept at this temperature for 4 h. The
evolution of the byproduct, water vapor, was clearly visible from the
reaction vessel, confirming the progression of the polymerization
reaction. The reaction mixture was then evacuated by applying vacuum
at 0.2 mm/Hg for 2 h while maintaining the same polymerization
temperature. The resulting polymer was found to be soluble in DMF,
DMSO, chloroform, and insoluble in water and acetone. The polymer
was purified by precipitating into acetone from its chloroform solution.
This was then centrifuged, washed with solvent, and dried in a vacuum
1
oven to get pure polymer. Yield: 56%. H NMR (400 MHz, CDCl3, δ
ppm): 0.90 (m, 3H), 1.28 (m, 5H), 1.46 (m, 2H), 2.07 (s, 1H), 2.52
(m, 2H), 2.82 (m, 2H), 3.52 (m, 2H), 4.72 (m, 1H). 13C NMR
(100.56 MHz, CDCl3, δ ppm): 13.66, 19.82, 20.59, 21.79, 25.82,
29.24, 30.82, 31.58, 49.06, 50.69, 52.65, 54.77, 61.61, 67.78, 170.88. IR
(CHCl3): 3300, 2960, 2933, 2873, 1731, 1638, 1563, 1457, 1379,
1239, 1187, 1122, 1034, 919, 817, 682, 644, 568 cm−1. SEC (CHCl3):
Mw = 58 700, PDI = 1.6.
Characterization of Synthesized Monomers and Polymer.
Infrared spectra were recorded on a PerkinElmer Spectrum 100 FT-IR
spectrometer in chloroform. UV/vis spectra were recorded using
CARY 300 Bio UV/vis spectrophotometer. Fluorescence spectra were
recorded on a NanoLog Horiba jobin Yvon fluorescence spectropho-
tometer. NMR spectra were recorded on a Varian 400 MHz
spectrometer using the TMS/solvent signal as an internal reference.
Q-tof Micro mass experiments (HRMS) were carried out using Waters
Q-Tofmicro-YA-105.
Size Exclusion Chromatography (SEC). The molecular weight
of the resulting polymer (5) was determined using size exclusion
chromatography (SEC), using a JASCO MD 2010 Plus instrument
with a PD 2020 light scattering precision detector. The average
molecular weight was calculated against a polystyrene standard, using
HPLC-grade chloroform as the mobile phase. The average molecular
weight of the polymer 5 was Mw = 58 700, PDI = 1.6. Degradation
experiment of this polymer was performed using porcine liver esterase
in solution. After 24 h of incubation at 37 °C, the average molecular
weight of the resulting low molecular weight polymers and oligomers
were found to be Mw = 21 300; PDI = 2.3.
Size and Zeta Potential of PNPs. The size and dispersity of the
synthesized functional PNPs was measured using dynamic light
scattering (DLS) using PDDLS/CoolBatch 40T instrument with
Precision Deconvolve 32 software. The overall surface charge (zeta
potential) of functional PNPs was measured using a Zetasizer Nano
ZS from Malvern Instruments.
Cell Cultures. The human lung carcinoma (A549) and
cardiomyocyte (H9c2) cells were obtained from ATCC and
maintained in accordance to the supplier’s protocols. Briefly, the
lung carcinoma cells were grown in a 5%-FBS-containing DMEM
medium supplemented with L-glutamine, streptomycin, amphotericin
B, and sodium bicarbonate. The H9c2 cells were propagated in a 10%
3919
dx.doi.org/10.1021/bm2009334|Biomacromolecules 2011, 12, 3917−3927

Biomacromolecules
Article
FBS-containing MEM medium containing penicillin, streptomycin,
and bovine insulin (0.01 mg/mL). Cells were grown in a humidified
incubator at 37 °C under 5% CO2 atmosphere.
1). In a typical reaction, β-alanine (1) and KOH were taken in
ethanol at room temperature, and a minimum amount of water
was added in a dropwise fashion with continuous stirring until
all reactants got solubilized in the reaction medium. Afterward,
butyl bromide (2) was added slowly to the resulting
homogeneous reaction mixture and the stirring continued
until the desired reaction time at room temperature. By
systematically changing the reaction time and equivalent ratios
of the starting materials, we obtained mono- and disubstituted
products in different ratios. Therefore, to optimize this facile
and one-step synthetic strategy, reactions were carried out
under various reaction conditions (Scheme 2). We noticed that
Cytotoxicity Assay. H9c2 and A549 cells (2500 cells/well) were
seeded in 96-well plates, incubated with the corresponding PNPs (35
μL, 5.8 nM, in PBS pH = 7.4) at 37 °C. After 24 h of incubation, each
well was washed three times with 1X PBS and treated with 30 μL
MTT (2 μg/μL) for 2 h. The resulting formazan crystals were
dissolved in acidic isopropanol (0.1 N HCl), and the absorbance was
recorded at 570 and 750 nm (background), using a Synergy μQuant
microtiter plate reader (Biotek). These experiments were performed in
triplicates.
Fluorimetric Assessment of in Vitro Cellular Internalization
and Apoptosis. Folate-receptor positive cells (A549) and negative
cells (H9c2) were seeded to reach confluency in a Costar’s black
polystyrene 96-well plate. The cells were treated with functional PNPs
(35 μL, 5.8 nM) in PBS (pH = 7.4), and after 24 h of incubation, the
cells were washed three times with 1X PBS. Subsequently, 1 mL of 1X
PBS was added to each well and fluorescence emission acquisition
using the TECAN’s infinite M200 PRO fluorescence plate reader.
Confocal Laser-Scanning Microscopy. H9c2 and A549 cells
were grown overnight on culture dishes, before treatment. Cells (10
000 cells/well) were incubated with the corresponding functional
PNPs (100 μL, 5.8 nM, in PBS pH = 7.4) in a humidified incubator
(37 °C, 5% CO2). After 24 h of incubation, the cells were thoroughly
washed three times with 1X PBS and fixed with 10% formalin solution,
followed by nuclear staining with DAPI, which was performed as
recommended by the supplier. Then, multiple confocal images were
obtained using a Zeiss LSM 510 confocal microscope equipped with a
40× objective.
Scheme 2. Selective Mono- and Di-N-alkylation of β-Alanine
with Butyl Bromide under Various Conditions
In Vitro Drug Release from Polymeric Nanoparticles. The in
vitro drug release studies were carried out using a dynamic dialysis
technique at 37 °C. Briefly, 100 μL of Taxol-encapsulating PNPs (11,
5.8 nM) were incubated with porcine liver esterase (20 μL) inside a
small dialysis cup (MWCO 6000−8000), which was then placed in a
PBS solution (pH 7.4). The amount of Taxol molecules released from
the nanoparticles 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 378 nm for Taxol. The cumulative
fraction of release versus time was calculated using the following
equation:
when 2 equiv of KOH and 0.9 equiv of 2 reacted with 1 equiv
of 1, monosubstituted product 3 resulted as a major product
(73%), after 16 h of incubation and performing flash column
chromatography using 10% methanol in chloroform as eluent
(entry I). This is an optimized reaction condition for the
synthesis of mono-N-butylated β-alanine (3), minimizing the
production of di-N-butylated β-alanine (3A) as a side product.
Similarly, when the equivalent ratio of 2 and KOH increased to
2.3 and 3.0, respectively, disubstituted product 3A was obtained
as a major yield (65%), after 36 h of incubation (entry V) and
purifications. The formation of either mono- or di-N-
substituted β-alanine as a side product was observed and easily
purified using flash column chromatography. The synthesis of
pure mono- and di-N-butylated β-alanine was confirmed by FT-
IR, NMR, and mass spectroscopic methods (Figure 1 and
Figures S1−S3). In addition, when excess of starting materials
were used, the tri-N-substituted β-alanine salt (3B) yielded as a
major product (entry VI). These results further indicated that
the product’s selectivity depends on the equivalent ratios of the
starting materials and reaction time. Taken together, our one-
step method is facile, easy to scale-up, reproducible, and uses a
simple mixed-solvent approach to eliminate the use of toxic
phase transfer catalysts (PTC). In addition, this strategy does
not use any conventional protection−deprotection method, as
the selectivity of the N-substitution reaction (mono- or di-) was
monitored with the equivalent ratios of the starting materials.
This synthetic strategy was used for the synthesis of functional
di-N-alkylated β-alanine (4), capable of producing a pseudo-
poly(amino acid) polymer (5, Scheme 1).
where [Taxol]t is the amount of Taxol released at time t and
[Taxol]total is the total Taxol present in the Taxol-encapsulating PNPs.
Flow Cytometry. Flow cytometry was done to assess the
fluorescence enhancement of our PNPs when folate-receptor-
expressing A549 cells were incubated with the functional PNPs in a
humidified incubator at 37 °C under 5% CO2 atmosphere. Cells were
seeded to reach confluency in Petri dishes and treated with functional
PNPs (100 μL, 5.8 nM), and after 24 h of incubation the cells were
harvested after trypsinization and centrifugation at 1000 rpm for 8
min. Subsequently, the cell pellets were resuspended in 1 mL of 1X
PBS, and flow cytometry was done using a BD FACSCalibur flow
cytometer from BD Biosciences.
RESULTS AND DISCUSSION
■
Syntheses of Selective Mono- and Di-N-alkylated
Amino Acids and Functional Poly(amino acid). In our
novel one-step method, we used two different miscible solvents,
water and ethanol, to facilitates the formation of a
homogeneous reaction mixture containing the starting materials
(mixed-solvent approach).48 This eliminates the use of a toxic
phase transfer catalyst (PTC) required to bring the starting
materials to one phase for the reaction to take place. We
selected a water-soluble amino acid, β-alanine (1), and a water-
insoluble alkylating agent, butyl bromide (2), as starting
materials for the selective N-alkylation of β-alanine (Scheme
Acknowledging the benefit of our one-step method, we
designed a synthetic strategy to prepare novel di-N-alkylated β-
alanine using two different types of alkylating agents, without
using any protection−deprotection steps. We hypothesized that
3920
dx.doi.org/10.1021/bm2009334|Biomacromolecules 2011, 12, 3917−3927

Biomacromolecules
Article
1
Figure 1. H NMR spectra of the monomers (3 and 4) and the pseudo-poly(amino acid) (5).
Figure 2. Characterizations of polymer 5 and polymeric nanoparticles. (A) SEC trace of polymer 5 in chloroform, showing formation of high
molecular weight polymer. (B) DLS measurement determines the average hydrodynamic diameter (101 ± 2 nm) of PNPs 10. Inset: scanning
transmission electron microscopy (STEM) image of PNPs 10. Scale bar 100 nm. (C) UV/vis spectrum indicating the presence of DiI and folic acid
and (D) comparing fluorescence emission spectra of DiI in PNPs 10 with that of free DiI dye in PBS (pH = 7.4).
the design and synthesis of functional di-N-alkylated β-alanine
monomer would be a versatile precursor for the synthesis of
novel biopolymers. In addition, the presence of a butyl pendant
in the monomer will enhance the solubility of the resulting
polymer in common organic solvents. Furthermore, the butyl
pendant will create a pseudo-branched structure upon
3921
dx.doi.org/10.1021/bm2009334|Biomacromolecules 2011, 12, 3917−3927

Biomacromolecules
Article
a
Scheme 3. Synthesis of Functional Poly(amino acid)-Based Theranostic Polymeric Nanoparticles
a
i. Solvent diffusion method, DMF, H2O, DiI, Paclitaxel. ii. Carbodiimide chemistry, propargylamine, EDC, NHS. iii. Click chemistry, Folate∼N3,
CuI.
polymerization due to the hydrophobic−hydrophobic inter-
polymer was further confirmed by SEC experiment after
incubation with porcine liver esterase that resulted in the
formation of low molecular weight polymers and oligomers
(Figure S6). In addition, compared to the conventional linear
polymers, our pseudo-poly(amino acid) is amorphous, soluble,
biocompatible, containing numerous functional groups, and
possessing the ability to form hydrophobic cavities important
for effective encapsulation of therapeutic agents, indicated its
versatility in targeted drug delivery. Therefore, the synthesized
pseudo-poly(amino acid) (5) was used to create a series of
functional PNPs (6−11) containing various cargos and
functional groups.
Syntheses and Characterizations of Cargos-Encapsu-
lating Functional PNPs. For the synthesis of our cargo-
encapsulating PNPs, we used a surfactant-free, water-based
solvent diffusion method14,42 (Scheme 3). In this method, we
coencapsulated a hydrophobic fluorescent dye (DiI, 0.25 mg)
and a therapeutic drug (Taxol, 1.5 mg) during the formation of
PNPs, in one pot. Briefly, the polymer (5, 25 mg) and cargos
were dissolved in DMF and added dropwise to the stirring DI
water (5 mL), driving both the self-assembly of hydrophobic
butyl pendants and encapsulation process in one-pot, creating
cargo-encapsulating PNPs (6, 7). In this process, the polymer 5
self-assembles to form nanoparticles, bringing the hydrophobic
butyl pendants together to minimize the contact with water,
while exposing the polar carboxylic acid groups toward the
aqueous media for further functionalizations. Next, the resulting
carboxylated PNPs (6, 7; Scheme 3) were functionalized to
yield propargylated nanoparticles (8, 9) using water-soluble
carbodiimide chemistry.46 Then, highly selective “click”
chemistry50−54 was used to generate folate-functionalized
multimodal PNPs (10, 11), using azide-derivatized folic
acid.46 These PNPs (6−11) were purified using PD-10 column
followed by dialysis (MWCO 6K−8K) against PBS buffer (pH
= 7.4). Nanoparticles were found to be highly stable in aqueous
buffer solution (pH = 7.4), without significant precipitation or
actions, hinting the formation of hydrophobic nanocavities in
the polymeric nanostructure. As hypothesized, we started with
monobutylated β-alanine (3) as an important ethanol soluble
precursor and a typical nucleophilic substitution reaction was
performed with the addition of a different alkylating agent,
propylene oxide, in ethanol. The reaction was continued for 30
h at room temperature to obtain the pure di-N-alkylated β-
alanine (4, Scheme 1), the AB-type of butyl-functional
monomer in high yield (68%). Importantly, this reaction did
not require any work-up step or column purification due to the
evaporation of low-boiling (34 °C) propylene oxide from the
reaction mixture, while concentrating the reaction mixture. In
addition, this step neither uses water as a cosolvent since the
reactants were soluble in ethanol nor uses any bases for the
reaction to proceed, as the starting material (3) contains a
highly nucleophilic secondary amine group. The resulting
bifunctional derivative (4) was polymerized using the melt-
polymerization technique14,42 in the presence of p-toluenesul-
fonic acid (pTSA) as a catalyst at 160 °C. Importantly, the
melt-polymerization technique does not require any surfactants,
used for microemulsion polymerization,49 indicating the
formation of pure polymer. As hypothesized, the resulting
amino acid-based biodegradable13,15 polymer (5) was found to
be soluble in chloroform, DMF, and DMSO and insoluble in
water and acetone. The polymer was purified by precipitating in
acetone from a chloroform solution and isolated as pure
polymer with high yield (56%). The synthesized amino acid
derivatives and polymer were characterized using spectroscopic
methods (Figure 1 and Figures S1−S4 ), and the formation of
polymer was further confirmed by size exclusion chromatog-
raphy (SEC: Mw = 58 700, PDI = 1.6; Figure 2A). Thermal
gravimetric analysis (TGA) showed the moderate thermal
stability of the synthesized polymer (10% weight loss at 253 °C
in air), indicating the formation of a potentially degradable
polymer16 (Figure S5). In addition, the degradability of our
3922
dx.doi.org/10.1021/bm2009334|Biomacromolecules 2011, 12, 3917−3927

Biomacromolecules
Article
Figure 3. Drug release profiles of folate-conjugated Taxol-encapsulating PNPs (11, 100 μL, 5.8 nM) at 37 °C. Release of Taxol were observed in the
presence of an esterase enzyme (A, ■) and in PBS at pH 5.0 (B, ▼), whereas the encapsulating drug was found to be stable in PBS at pH 7.4 (A
and B, ●).
Figure 4. Determination of cytotoxicity of functional PNPs (35 μL, 5.8 nM in PBS, pH = 7.4): (A, B) Time-dependent and (C, D) dose-dependent
viabilities of A549 cells (A, C) and H9c2 cells (B, D) treated with the functional PNPs. Carboxylated (6, ■), Taxol-encapsulating carboxylated (7,
▲), and folate-conjugated (10, ●) nanoparticles showed biocompatibility with nominal toxicity in both the cell lines. The Taxol-encapsulating
folate-conjugated PNPs (11, ▼) showed more than 90% reduction in cell viability when treated with A549 cells (A) and not with H9c2 cells (B),
confirming folate-receptor mediated internalizations. Cells were incubated with PNPs 11 and Taxol (1:1 equiv of Taxol concentrations) for 24 h (C,
D), and average values of four measurements are depicted ± standard errors.
reduction in the fluorescence emission when concentrated
(Table S1).
confirmed by measuring the overall surface charge (ζ-potential,
Figure S7) and by UV/vis spectroscopy showing an absorption
maximum at 352 nm (Figure 2C) for the conjugated folic acid.
The presence of encapsulated Taxol (EE = 68.5 ± 3.2%), DiI
dye (EE = 21.2 ± 1.3%),17,57−63 and surface-clicked folic acid
(23 ± 4/PNP) in the PNPs 11 was further confirmed by HPLC
(Figure S8) and spectroscopic techniques (Figure S9).
In Vitro Release of Taxol from Nanoparticles. To
investigate the potential therapeutic applications of our
functional PNPs, which rely on their biodegradability, the
rate of release of the encapsulated drug (Taxol) from the
polymeric nanocavities was studied. To evaluate the PNPs’ (11,
100 μL, 5.8 nM) drug release profile, we performed enzymatic
Dynamic light scattering (DLS) experiments confirmed the
formation of stable and monodispersed PNPs (10 in PBS, pH =
7.4) with an average diameter (D) of 101 ± 2 nm (Figure 2B).
The presence of an absorption maximum at 553 nm in the UV/
vis spectrum (Figure 2C) and a corresponding fluorescence
emission peak at 568 nm (Figure 2D) confirmed the effective
encapsulation of DiI dye (EE = 59.2 ± 5.8%) in the PNPs 10.
Encapsulation of this cargo inside the PNP’s cavity was further
confirmed by observing a blue shift14,55,56 of 22 nm (Figure
2D) after encapsulating DiI dye as compared to the free dye
(590 nm) in PBS (pH = 7.4). Surface functionalization was
3923
dx.doi.org/10.1021/bm2009334|Biomacromolecules 2011, 12, 3917−3927

Biomacromolecules
Article
Figure 5. Time-dependent intracellular differential fluorescence emissions from A549 cells (A) and H9c2 cells (B) treated with novel functional
PNPs (35 μL, 5.8 nM in PBS, pH = 7.4).
Figure 6. In vitro assessment of amino acid-based functional PNP’s (100 μL, 5.8 nM, in PBS pH = 7.4, 24 h) cellular uptake via confocal microscopy
using A549 cells (10 000 cells/well). (A, B) Very nominal internalization was observed for carboxylated PNPs (6). (C, D) Enhanced internalization
was indicated for folate-conjugated PNPs (10). (E, F) Induced cell death was observed when treated with Taxol-encapsulating PNPs (11). (G, H)
No significant internalization was observed when A549 cells were preincubated with excess of free folic acid, before incubation with folate-conjugated
PNPs 10. Nucleus stained with DAPI (blue).
3924
dx.doi.org/10.1021/bm2009334|Biomacromolecules 2011, 12, 3917−3927

Biomacromolecules
Article
(esterase from porcine liver) and low-pH (pH = 5.0)
degradation experiments using a dynamic dialysis techni-
que14,46,47 at 37 °C. Results indicated that Taxol was released
faster in the case of low-pH release method (▼, Figure 3B),
when compared to the enzymatic release (■, Figure 3A).
These differential release profiles may be attributed to the faster
rate of hydrolysis (degradation) of the ester linkages present in
the polymeric backbone of the polymer 5 at low pH.64 As
nominal drug release was observed at physiological pH 7.4 (●),
we deduced that our functional PNPs should be stable under
these physiological conditions (Table S1), whereas they are
readily biodegraded upon enzymatic and intracellular environ-
mental triggers, such as localization in acidified lysosomal
compartments. Taken together, these results confirm the
efficient drug release capability of our theranostic PNPs,
rendering targeted PNPs’ useful for potential in vivo
applications.
In Vitro Cytotoxicity of Cargo-Encapsulating Func-
tional PNPs. After evaluating the rate of release of the
encapsulating drug from the polymeric nanocavities, we
examined the in vitro cytotoxicity of the functional PNPs (35
μL, 5.8 nM in PBS, pH = 7.4) using MTT assay. In this
experiment, we used lung carcinoma cells (A549, 2500 cells/
well) expressing folate receptor (FR +)14,65−67 to compare the
cytotoxicities of our functional PNPs. Results confirmed that a
time-dependent decrease in the number of viable A549 cells,
when incubated with folate-decorated Taxol-encapsulating
PNPs (11, ▼, Figure 4A). Within 48 h of incubation, the
folate-decorated Taxol-encapsulating PNPs 11 (▼) showed
more than 90% reduction in cell viability whereas, PNPs 6 (■),
7 (▲), and 10 (●) exhibited nominal cytotoxicities. In
contrast, when incubated with cardiomyocytes (H9c2 cells,
2500 cells/well), which do not overexpress folate receptor (FR
−),68 no significant cell death was observed with all the
functional PNPs including folate-conjugated Taxol-encapsulat-
ing PNPs 11 (▼, Figure 4B). These results demonstrate the
biocompatibility, folate-receptor-mediated internalization and
the potential application of our theranostic nanoagents for the
targeted treatment of folate receptor-expressing tumors. In
another set of experiments, dose-dependent cell viability
experiments confirmed the higher cytotoxicity of our folate-
decorated Taxol-encapsulating PNPs 11 (▼, IC50 = 2.1 nM)
when compared with the similar concentrations of free Taxol
(⧫, IC50 = 5.2 nM) in A549 cells (Figure 4C). This is due to
the higher rate of folate receptor-mediated internalization of
our folate-decorated Taxol-encapsulating PNPs 11, when
compared to the nonspecific internalization of free Taxol. On
the other hand, the folate-decorated Taxol-encapsulating PNPs
11 (▼) showed very nominal dose-dependent cytotoxicity in
H9c2 cells, as these cells lack folate receptor (Figure 4D). In
contrast, Taxol exhibits nonselective cytotoxicity (⧫, IC50 = 6.9
nM, Figure 4D), irrespective of the presence of folate receptor
for cellular internalization. Overall, these findings support the
principle that folate-decorated Taxol-encapsulating PNPs 11
(▼) can target and deliver chemotherapeutic agents to folate
receptor-expressing carcinomas, while visualizing the drug’s
homing.
emissions were measured at different time points using a
fluorescence microtiter plate reader. Results showed faster
internalizations and enhanced fluorescence emissions from
A549 cells incubated with folate-conjugated PNPs 10 (●,
Figure 5A), reaching a maximum cell-associated fluorescence
intensity after 24 h. On the other hand, when treated with
folate-decorated Taxol-encapsulating PNPs 11 (▼), a faster
increase in cell-associated fluorescence emission was observed,
reaching a plateau within 12 h of incubation. Then, the
fluorescence intensity rapidly decreases after 12 h of incubation
due to the selective induction of apoptosis and therefore
reduction in the number of viable fluorescent cells. In contrast,
when A549 cells treated with non-folate PNPs (6, ■; 7, ▲)
exhibited a nominal increase in cell-associated fluorescence as
expected (Figure 5A). In another set of experiments, minimal
fluorescence emission was observed when H9c2 cells (FR −)
were treated with our functional PNPs (6, ■; 7, ▲; 10, ●; 11,
▼; Figure 5B). These control experiments further confirmed
the targeted internalizations and selective fluorescence
emissions of our theranostic PNPs. Taken together, these
findings support the principle that folate-decorated PNPs can
target and deliver chemotherapeutic agents to folate-receptor-
expressing carcinomas, in order to prevent damage of
nontransformed cells and healthy tissues.
In Vitro Cellular Uptake of Functional PNPs. Next, we
further investigated the folate receptor-mediated selective
uptake and cytotoxicity of our functional PNPs (100 μL, 5.8
nM in PBS, pH = 7.4) by A549 cells (10 000 cells/well) using
confocal microscopy. As expected, nominal internalizations
were observed when A549 cells were incubated with
carboxylated PNPs (6, 24 h of incubation, Figure 6A,B) due
to its negatively charged carboxylated surface (zeta potential ζ
= −49 mV, Figure S7 ); however, significant internalization of
the folate-conjugated PNPs (10, Figure 6C,D) was indicated as
an enhanced fluorescence emission observed from the cell
cytoplasm. Interestingly, when A549 cells were incubated with
folate-conjugated Taxol-encapsulating PNPs (11), mitotic
arrest was observed, leading to a dramatic cellular morphology
changes and cell death (Figure 6E,F). Importantly, these
confocal microscopic results directly corroborated with our in
vitro cytotoxicity (Figure 4) and fluorescence spectroscopic
(Figure 5) results. In another set of experiments, no
internalization of folate-conjugated PNPs 10 was observed
(Figure 6G,H), when A549 cells were preincubated with excess
of free folic acid, confirming folate receptor-mediated internal-
izations of our folate-conjugated nanoparticles. In addition, no
significant cell-associated fluorescence was observed when
H9c2 cells (FR −) were incubated with folate-conjugated
PNPs 10 (Figure S10), further confirming folate receptor-
mediated internalizations of our functional nanoparticles.
Flow cytometric experiments (FACS, Figure 7) were
performed using lung carcinomas (A549 cells: 10 000 cells/
well), showing direct corroboration with our previous findings.
Results showed nominal cell-associated fluorescence emissions
from the A549 cells when incubated with carboxylated PNPs
(6, 100 μL, 5.8 nM, in PBS pH = 7.4, 24 h of incubation, Figure
7A), as compared to the control A549 cells (inset, Figure 7A).
However, an enhanced cell-associated fluorescence emission
was observed for folate-conjugated PNPs (10, Figure 7B),
demonstrating folate receptor-targeted cell binding of our
theranostic PNPs. In contrast, reduced cell-associated fluo-
rescence was observed when A549 cells were incubated with
folate-conjugated Taxol-encapsulating PNPs (11, Figure 7C)
Intracellular Fluorescence Emissions and Selective
Cytotoxicity. To further demonstrate the targeted internal-
ization of our theranostic PNPs and its internalization causing
cell death, A549 (FR +) and H9c2 (FR −) cells (2500 cells/
well) were treated with functional PNPs (35 μL, 5.8 nM in
PBS, pH = 7.4). The resulting differential fluorescence
3925
dx.doi.org/10.1021/bm2009334|Biomacromolecules 2011, 12, 3917−3927

Biomacromolecules
Article
drug to the folate-expressing tumors and induction of apoptosis
was monitored using confocal microscopy and flow cytometric
analysis. We anticipate that our synthetic strategy may lead to
many exciting opportunities for the synthesis of biologically
active small molecules, amino acid-based biodegradable
polymers, and theranostic polymeric nanoparticles for the
targeted delivery of therapeutic agents in clinical settings.
ASSOCIATED CONTENT
■
S
* Supporting Information
Characterizations of the synthesized amino acid-based small
molecules, polymer, and theranostic nanoparticles; experimen-
tal results using living cells. This material is available free of
charge via the Internet at http://pubs.acs.org.
Figure 7. In vitro flow cytometry analysis of A549 cells incubated with
functional PNPs (100 μL, 5.8 nM, in PBS pH = 7.4) for 24 h. (A)
Nominal fluorescence emission was observed when incubated with
carboxylated PNPs (6) compared to the control A549 cells with no
treatment (inset). (B) Enhanced fluorescence emission observed for
folate-conjugated PNPs (10) whereas, (C) reduced fluorescence
emission observed for folate-conjugated Taxol-encapsulating PNPs
(11) due to induction of apoptosis. (D) Nominal fluorescence
emission was observed for folate-conjugating PNPs (10) in the
presence of excess of free folic acid.
AUTHOR INFORMATION
■
Corresponding Author
*Tel 313-530-4366, e-mail santra@ucf.edu (S.S.); Tel 407-882-
2843, Fax 407-882-2819, e-mail jmperez@ucf.edu (J.M.P.).
ACKNOWLEDGMENTS
■
We thank Prof. Anil Kumar, Chemistry Department, IIT
Bombay, India, for his advice and help with the organic
syntheses and instrumentation facilities. This work was
supported by NIH Grant GM 084331 and UCF Start Up
Fund, all to J.M.P.
for 24 h, indicating target-specific cell binding and delivery of
therapeutic drug inducing apoptosis. These results further
corroborated with our previous findings: the cytotoxicity
(Figure 4), fluorescence spectroscopic (Figure 5), and confocal
microscopic (Figure 6) results. In another set of experiments,
nominal cell-associated fluorescence emission was observed
when A549 cells were preincubated with excess of free folic acid
before incubating with folate-conjugating PNPs (10, Figure
7D), indicating exclusive folate receptor-mediated cell binding
of our theranostic PNPs. Overall, these results further support
the hypothesis that our newly developed theranostic PNPs can
target folate receptor-expressing tumors in order to prevent
healthy tissues and only induce apoptosis upon receptor-
mediated drug delivery.
REFERENCES
■
(1) Kim, C. K.; Ghosh, P.; Pagliuca, C.; Zhu, Z. J.; Menichetti, S.;
Rotello, V. M. J. Am. Chem. Soc. 2009, 131, 1360−1361.
(2) Marder, S. R.; Kippelen, B.; Jen, A. K. Y.; Peyghambarian, N.
Nature 1997, 388, 845−851.
(3) Nayak, S.; Lee, H.; Chmielewski, J.; Lyon, L. A. J. Am. Chem. Soc.
2004, 126, 10258−10259.
(4) Yokoyama, M.; Kwon, G. S.; Okano, T.; Sakurai, Y.; Seto, T.;
Kataoka, K. Bioconjugate Chem. 1992, 3, 295−301.
(5) You, C. C.; Miranda, O. R.; Gider, B.; Ghosh, P. S.; Kim, I. B.;
Erdogan, B.; Krovi, S. A.; Bunz, U. H.; Rotello, V. M. Nature
Nanotechnol. 2007, 2, 318−323.
(6) Cheng, Z.; Tsourkas, A. Langmuir 2008, 24, 8169−8173.
(7) Yadav, S.; van Vlerken, L. E.; Little, S. R.; Amiji, M. M. Cancer
Chemother. Pharmacol. 2009, 63, 711−722.
(8) Jabr-Milane, L.; van Vlerken, L.; Yadav, S.; Amiji, M. M. Cancer
Treat. Rev. 2008, 34, 592−602.
(9) Peer, D.; Karp, J. M.; Hong, S.; Farokhzad, O. C.; Margalit, R.;
Langer, R. Nature Nanotechnol. 2007, 2, 751−760.
(10) Nayak, S.; Lyon, L. A. Angew. Chem., Int. Ed. 2005, 44, 7686−
7708.
(11) Duncan, R. Nat. Rev. Drug Discovery 2003, 2, 347−360.
(12) Frechet, J. M. J.; Henmi, M.; Gitsov, I.; Aoshima, S.; Leduc, M.
R.; Grubbs, R. B. Science 1995, 269, 1080−1083.
(13) Lee, C. C.; MacKay, J. A.; Frechet, J. M.; Szoka, F. C. Nature
Biotechnol. 2005, 23, 1517−1526.
(14) Santra, S.; Kaittanis, C.; Perez, J. M. Langmuir 2010, 26, 5364−
5373.
(15) Kaihara, S.; Matsumura, S.; Mikos, A. G.; Fisher, J. P. Nature
CONCLUSIONS
■
In conclusion, we have introduced a single-step synthetic
strategy for the selective N-alkylation of amino acids, capable of
generating biologically active small molecules. This simple
strategy, as opposed to the conventional multistep protection−
deprotection method, is facile, highly economic, reproducible,
and amenable to scale-up. The resulting functional molecule
was used as an AB-type of monomer for the synthesis of
biopolymer. The synthesized novel pseudo-poly(amino acid) is
different from available linear polymers as its butyl pendants
create hydrophobic nanocavities for effective encapsulation of
hydrophobic cargos and enhance polymer’s solubility in
common organic solvent. Utilizing these properties, cargos-
encapsulating functional PNPs were synthesized from the
resulting amino acid-based polymer, showing promising results
in targeted optical detection and treatment of cancer.
Particularly, the synthesized Taxol-encapsulating and folate-
decorated (using “click” chemistry) theranostic PNPs provided
targeted detection and delivery of the therapeutic drug to the
folate receptor-expressing cancer cells, resulting in apoptosis,
while avoiding healthy, nontransformed tissues. The intra-
cellular fluorescence experiments, cytotoxicity assays, and drug
release studies further confirmed for the targeted detection and
treatment of cancer. The targeted delivery of the therapeutic
Protoc. 2007, 2, 2767−2771.
(16) Wang, C.; Ge, Q.; Ting, D.; Nguyen, D.; Shen, H. R.; Chen, J.;
Eisen, H. N.; Heller, J.; Langer, R.; Putnam, D. Nature Mater. 2004, 3,
190−196.
(17) Jin, C.; Bai, L.; Wu, H.; Song, W.; Guo, G.; Dou, K. Pharm. Res.
2009, 26, 1776−1784.
(18) Yuan, M. L.; Wang, Y. H.; Li, X. H.; Xiong, C. D.; Deng, X. M.
Macromolecules 2000, 33, 1613−1617.
(19) Langer, R. Nature 1998, 392, 5−10.
3926
dx.doi.org/10.1021/bm2009334|Biomacromolecules 2011, 12, 3917−3927

Biomacromolecules
Article
(20) Yokoyama, M.; Miyauchi, M.; Yamada, N.; Okano, T.; Sakurai,
Y.; Kataoka, K.; Inoue, S. Cancer Res. 1990, 50, 1693−1700.
(21) Mathews, B. T.; Beezer, A. E.; Snowden, M. J.; Hardy, M. J.;
Mitchell, J. C. New J. Chem. 2001, 25, 807−818.
(22) Nita, L. E.; Chiriac, A. P.; Popescu, C. M.; Neamtu, I.; Alecu, L.
J. Optoelectron. Adv. Mater. 2006, 8, 663−666.
(23) Crespo, L.; Sanclimens, G.; Royo, M.; Giralt, E.; Albericio, F.
Eur. J. Org. Chem. 2002, 1756−1762.
(57) Koch, A. M.; Reynolds, F.; Kircher, M. F.; Merkle, H. P.;
Weissleder, R.; Josephson, L. Bioconjugate Chem. 2003, 14, 1115−
1121.
(58) Shen, T.; Weissleder, R.; Papisov, M.; Bogdanov, A. Jr.; Brady,
T. J. Magn. Reson. Med. 1993, 29, 599−604.
(59) Chan, J. M.; Zhang, L.; Yuet, K. P.; Liao, G.; Rhee, J.-W.;
Langer, R.; Farokhzad, O. C. Biomaterials 2009, 30, 1627−1634.
(60) Sun, B.; Ranganathan, B.; Feng, S.-S. Biomaterials 2008, 29,
475−486.
(61) Danhier, F.; Vroman, B.; Lecouturier, N.; Crokart, N.;
Pourcelle, V.; Freichels, H.; Jerome, C.; Marchand-Brynaert, J.;
Feron, O.; Preat, V. J. Controlled Release 2009, 140, 166−173.
(62) Yu, D.-H.; Lu, Q.; Xie, J.; Fang, C.; Chen, H.-Z. Biomaterials
2010, 31, 2278−2292.
(24) Modica, E.; Zenaletti, R.; Freccero, M.; Mella, M. J. Org. Chem.
2001, 66, 41−52.
(25) Garcia-Santos, M. P.; Calle, E.; Casado, J. J. Am. Chem. Soc.
2001, 123, 7506−7510.
(26) Adlington, R. M.; Mantell, S. J. Tetrahedron 1992, 48, 6529−
6536.
(63) Guo, J.; Gao, X.; Su, L.; Xia, H.; Gu, G.; Pang, Z.; Jiang, X.; Yao,
L.; Chen, J.; Chen, H. Biomaterials 2011, 32, 8010−8020.
(64) Jung, J. H.; Ree, M.; Kim, H. Catal. Today 2006, 115, 283−287.
(65) Yoon, S.-A.; Choi, J. R.; Kim, J.-O.; Shin, J.-Y.; Zhang, X.-H.;
Kang, J.-H. Cancer Res. Treat. 2010, 42, 163−171.
(27) Fukuyama, T.; Jow, C. K.; Cheung, M. Tetrahedron Lett. 1995,
36, 6373−6374.
(28) Trost, B. M.; Calkins, T. L.; Oertelt, C.; Zambrano, J.
Tetrahedron Lett. 1998, 39, 1713−1716.
(29) Ebata, M.; Takahashi, Y.; Otsuka, H. Bull. Chem. Soc. Jpn. 1966,
39, 2535−2538.
(66) You, J.; Li, X.; Cui, F.; Du, Y.-Z.; Yuan, H.; Hu, F.
Nanotechnology 2008, 19, 1−9.
(30) Bar-Haim, G.; Kol, M. Org. Lett. 2004, 6, 3549−3551.
(31) Bhatt, U.; Mohamed, N.; Just, G.; Roberts, E. Tetrahedron Lett.
1997, 38, 3679−3682.
(67) Yuan, H.; Miao, J.; Du, Y. Z.; You, J.; Hu, F. Q.; Zeng, S. Int. J.
Pharm. 2008, 348, 137−145.
(68) Parker, N.; Turk, M. J.; Westrick, E.; Lewis, J. D.; Low, P. S.;
Leamon, C. P. Anal. Biochem. 2005, 338, 284−293.
(32) Bowman, W. R.; Coghlan, D. R. Tetrahedron 1997, 53, 15787−
15798.
(33) Domling, A.; Ugi, I. I. Angew. Chem., Int. Ed. 2000, 39, 3168−
3210.
(34) Ohfune, Y.; Kurokawa, N.; Higuchi, N.; Saito, M.; Hashimoto,
M.; Tanaka, T. Chem. Lett. 1984, 441−444.
(35) Ramanjulu, J. M.; Joullie, M. M. Synth. Commun. 1996, 26,
1379−1384.
(36) Reinhold, V. N.; Ishikawa, Y.; Melville, D. B. J. Med. Chem.
1968, 11, 258−260.
(37) Song, Y. T.; Sercel, A. D.; Johnson, D. R.; Colbry, N. L.; Sun, K.
L.; Roth, B. D. Tetrahedron Lett. 2000, 41, 8225−8230.
(38) Tietze, L. F. Chem. Rev. 1996, 96, 115−136.
(39) Albanese, D.; Landini, D.; Lupi, V.; Penso, M. Eur. J. Org. Chem.
2000, 1443−1449.
(40) Kwon, H. Y.; Langer, R. Macromolecules 1989, 22, 3250−3255.
(41) Yu, J. H.; Quan, J. S.; Huang, J.; Wang, C. Y.; Sun, B.; Nah, J.
W.; Cho, M. H.; Cho, C. S. Acta Biomater. 2009, 5, 2485−2494.
(42) McCarthy, J. R.; Perez, J. M.; Bruckner, C.; Weissleder, R. Nano
Lett. 2005, 5, 2552−2556.
(43) Davis, M. E.; Chen, Z. G.; Shin, D. M. Nat. Rev. Drug Discovery
2008, 7, 771−782.
(44) Fonseca, C.; Simoes, S.; Gaspar, R. J. Controlled Release 2002,
83, 273−286.
(45) Gupte, A.; Ciftci, K. Int. J. Pharm. 2004, 276, 93−106.
(46) Santra, S.; Kaittanis, C.; Grimm, J.; Perez, J. M. Small 2009, 5,
1862−1868.
(47) Santra, S.; Kaittanis, C.; Perez, J. M. Mol. Pharmaceutics 2010, 7,
1209−1222.
(48) Du, X.; He, J. H. Mater. Res. Bull. 2009, 44, 1238−1243.
(49) Texter, J.; Ge, L.; Mourey, T. H.; Bryan, T. G. Langmuir 2004,
20, 11288−11292.
(50) Bachovchin, D. A.; Brown, S. J.; Rosen, H.; Cravatt, B. F. Nature
Biotechnol. 2009, 27, 387−394.
(51) Englert, B. C.; Bakbak, S.; Bunz, U. H. F. Macromolecules 2005,
38, 5868−5877.
(52) Kolb, H. C.; Finn, M. G.; Sharpless, K. B. Angew. Chem., Int. Ed.
2001, 40, 2004−2021.
(53) Sun, E. Y.; Josephson, L.; Weissleder, R. Mol. Imaging 2006, 5,
122−128.
(54) Thorek, D. L. J.; Elias, D. R.; Tsourkas, A. Mol. Imaging 2009, 8,
221−229.
(55) Imaz, I.; Hernando, J.; Ruiz-Molina, D.; Maspoch, D. Angew.
Chem., Int. Ed. 2009, 48, 2325−2329.
(56) Shi, M.; Wosnick, J. H.; Ho, K.; Keating, A.; Shoichet, M. S.
Angew. Chem., Int. Ed. 2007, 46, 6126−6131.
3927
dx.doi.org/10.1021/bm2009334|Biomacromolecules 2011, 12, 3917−3927