Development of Fully Degradable Phosphonium-Functionalized Amphiphilic Diblock Copolymers for Nucleic Acids Delivery

Article abstract of DOI:10.1021/acs.biomac.8b00069

To expand the range of functional polymer materials to include fully hydrolytically degradable systems that bear bioinspired phosphorus-containing linkages both along the backbone and as cationic side chain moieties for packaging and delivery of nucleic acids, phosphonium-functionalized polyphosphoester-block-poly(l-lactide) copolymers of various compositions were synthesized, fully characterized, and their self-assembly into nanoparticles were studied. First, an alkyne-functionalized polyphosphoester-block-poly(l-lactide) copolymer was synthesized via a one pot sequential ring opening polymerization of an alkyne-functionalized phospholane monomer, followed by the addition of l-lactide to grow the second block. Second, the alkynyl side groups of the polyphosphoester block were functionalized via photoinitiated thiol-yne radical addition of a phosphonium-functionalized free thiol. The polymers of varying phosphonium substitution degrees were self-assembled in aqueous buffers to afford formation of well-defined core-shell assemblies with an average size ranging between 30 and 50 nm, as determined by dynamic light scattering. Intracellular delivery of the nanoparticles and their effects on cell viability and capability at enhancing transfection efficiency of nucleic acids (e.g., siRNA) were investigated. Cell viability assays demonstrated limited toxicity of the assembly to RAW 264.7 mouse macrophages, except at high polymer concentrations, where the polymer of high degree of phosphonium functionalization induced relatively higher cytotoxicity. Transfection efficiency was strongly affected by the phosphonium-to-phosphate (P⁺/P^-) ratios of the polymers and siRNA, respectively. The AllStars Hs Cell Death siRNA complexed to the various copolymers at a P⁺/P^- ratio of 10:1 induced comparable cell death to Lipofectamine. These fully degradable nanoparticles might provide biocompatible nanocarriers for therapeutic nucleic acid delivery.

Full text of DOI:10.1021/acs.biomac.8b00069

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Cite This: Biomacromolecules XXXX, XXX, XXX−XXX
Development of Fully Degradable Phosphonium-Functionalized
Amphiphilic Diblock Copolymers for Nucleic Acids Delivery
Yannick P. Borguet,† Sarosh Khan,† Amandine Noel,† Sean P. Gunsten,‡ Steven L. Brody,‡,§
Mahmoud Elsabahy,†,∥ and Karen L. Wooley*,†
†Departments of Chemistry, Chemical Engineering, and Materials Science & Engineering, and the Laboratory for Synthetic-Biologic
Interactions, Texas A&M University, College Station, Texas 77842, United States
‡Department of Medicine, Washington University, St. Louis, Missouri 63110, United States
§Department of Radiology, Washington University, St. Louis, Missouri 63110, United States
∥Department of Pharmaceutics, Faculty of Pharmacy, Assiut International Center of Nanomedicine, Alrajhy Liver Hospital, Assiut
University, Assiut 71515, Egypt
S
* Supporting Information
ABSTRACT: To expand the range of functional polymer
materials to include fully hydrolytically degradable systems that
bear bioinspired phosphorus-containing linkages both along
the backbone and as cationic side chain moieties for packaging
and delivery of nucleic acids, phosphonium-functionalized
polyphosphoester-block-poly(L-lactide) copolymers of various
compositions were synthesized, fully characterized, and their
self-assembly into nanoparticles were studied. First, an alkyne-
functionalized polyphosphoester-block-poly(L-lactide) copoly-
mer was synthesized via a one pot sequential ring opening
polymerization of an alkyne-functionalized phospholane
monomer, followed by the addition of L-lactide to grow the
second block. Second, the alkynyl side groups of the
polyphosphoester block were functionalized via photoinitiated thiol−yne radical addition of a phosphonium-functionalized
free thiol. The polymers of varying phosphonium substitution degrees were self-assembled in aqueous buffers to afford formation
of well-defined core−shell assemblies with an average size ranging between 30 and 50 nm, as determined by dynamic light
scattering. Intracellular delivery of the nanoparticles and their effects on cell viability and capability at enhancing transfection
efficiency of nucleic acids (e.g., siRNA) were investigated. Cell viability assays demonstrated limited toxicity of the assembly to
RAW 264.7 mouse macrophages, except at high polymer concentrations, where the polymer of high degree of phosphonium
functionalization induced relatively higher cytotoxicity. Transfection efficiency was strongly affected by the phosphonium-to-
phosphate (P+/P−) ratios of the polymers and siRNA, respectively. The AllStars Hs Cell Death siRNA complexed to the various
copolymers at a P+/P− ratio of 10:1 induced comparable cell death to Lipofectamine. These fully degradable nanoparticles might
provide biocompatible nanocarriers for therapeutic nucleic acid delivery.
polymers, although less efficient than viral vectors, are able to
protect and deliver genetic materials intracellularly via electro-
static interactions with the negatively charged nucleic acids to
form what so-called lipoplexes and polyplexes, respectively.
These cationic complexes can then undergo endocytosis and
ultimately release the genetic materials in the cytosol.2−6,10−14
While most of the cationic systems described in the literature
make use of ammoniums and other nitrogen-based functional
groups as charged centers, some recent studies performed
INTRODUCTION
■
Efficient delivery of genetic material intracellularly still
represents one of the major bottlenecks to the wide
applicability of nucleic acid-based therapies, despite progress
reported during the past decade.1−6 Intracellular delivery of
nucleic acid drugs, such as DNA and siRNA (short interfering
RNA), can either trigger or knockdown the expression of
specific proteins that are linked to several diseases. However,
the poor cellular uptake of genetic material, due to its overall
negative charge and rapid degradation of DNA/RNA by
nucleases in biological media, still represent major challenges
toward their successful cellular delivery.
́
independently in the Long15,16 and Frechet17 laboratories and
others18−20 have demonstrated the superiority of polymeric
Viral vectors have been used successfully to deliver genetic
materials to cells, but have raised many safety concerns.7−9 On
the other hand, nonviral vectors, such as cationic lipids and
Received: January 16, 2018
Revised: February 23, 2018
© XXXX American Chemical Society
A
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Dynamic light scattering (DLS) measurements were performed
using a Delsa Nano C (Beckman Coulter Inc., Fullerton, CA)
equipped with a laser diode operating at 658 nm. Scattered light was
detected at an angle of 165° and analyzed using a log correlator over
70 accumulations for a 0.5 mL sample in a glass sizing cell. Size
measurements were performed in microbiology-grade ultrapure water
at room temperature, unless otherwise specified. The calculations of
the particle sizes and distribution averages were performed using the
CONTIN routine using Delsa Nano 2.31 software. Unless otherwise
specified, the particle sizes are reported as the average number-
averaged hydrodynamic diameters and standard deviations from a
series of at least five measurements. The surface charge of the particles
phosphonium salts over their ammonium counterparts in head-
to-head comparison experiments. The increased efficiency of
phosphonium versus ammonium polycations was ascribed to
their efficient cationic charge distribution (centered on the
phosphorus atom, while distributed through adjacent carbons
on the ammonium moiety), as demonstrated by calcula-
tions,21,22 which lead to stronger and more stable interactions
with the negatively charged nucleic acids.
Our group has a long-standing interest in the design of
biocompatible nanomaterials for various biomedical applica-
tions via tailoring their polymeric structures and macro-
molecular assembly.23−25 In addition, cellular delivery of
nucleic acids has been achieved using shell-cross-linked
nanoparticles, and we have initiated a transition from
nondegradable polymer nanoparticles (e.g., poly-
(acrylamidoethylamine)-b-polystyrene))26−28 to partially de-
gradable polymer systems (e.g., poly(acrylamidoethylamine)-b-
polylactide).29 With the desire to move toward fully degradable
polymer nanoparticle systems, we have recently reported the
straightforward synthesis of versatile and degradable polymer
platforms making use of polyphosphoester (PPE)-based block
copolymers30−32 and demonstrated their potential use as drug
delivery vehicles in biomedical applications, such as the in vivo
delivery of paclitaxel33 and antimicrobial agents to the
lungs.34,35 We now report on the development of fully
degradable phosphonium-functionalized amphiphilic diblock
copolymers and their self-assembly into well-defined micellar
systems for siRNA intracellular delivery.
was evaluated through the measurement of their zeta potential (ζpot
)
by electrophoretic light scattering using a Delsa Nano C particle
analyzer equipped with a flow cell. Scattered light was detected at a 30°
angle. The zeta potential was measured in five different regions of the
flow cell, and a weighed mean was calculated to account for the
electroosmotic flow that is due to the surface charge of the cell walls.
The zeta potentials are reported as the average and standard deviations
from a series of at least three measurements.
Transmission electron microscopy (TEM) images were collected on
a JEOL 1200 EX (Tokyo, Japan) operating at 100 kV and micrographs
were recorded at calibrated magnifications using a SLA-15C CCD
camera. Samples for TEM were prepared as follows: 10 μL of polymer
or particle solution (1 mg·mL−1) was deposited onto a carbon-coated
copper grid, and after 2 min, the excess solution was blotted with a
piece of filter paper. The samples were then negatively stained with an
aqueous solution of phosphotungstic acid (PTA, 1 wt%). After 20 s,
the excess staining solution was blotted with a piece of filter paper.
The samples were then dried under a gentle stream of dry nitrogen
and analyzed within 24 h.
Representative Synthesis of a Clickable Phosphonium Salt.
Synthesis of (2-Hydroxyethyl)trimethylphosphonium Chloride (1a).
A 50 mL flame-dried round-bottomed flask was filled with 6.6 mL of 2-
chloroethanol (98 mmol) and capped with a rubber septum. The
reagent was deoxygenated by bubbling nitrogen for 30 min.
Trimethylphosphine (5.0 g, 70 mmol) was added dropwise under an
inert atmosphere to the stirred 2-chloroethanol on an ice−water bath.
The solution was stirred at 0 °C for 30 min; then, a glass stopper was
fitted and maintained in position with electrical tape and the vial was
heated at 50 °C for 2 d. The volatiles were evaporated in vacuo, and
the white solid that formed was washed repeatedly with diethyl ether
and finally dried under vacuum overnight to form the targeted
structure as a white powder (5.5 g, 53%). 1H NMR (500 MHz,
DMSO-d6, ppm) δ 5.69 (s, 1H), 3.82−3.67 (m, 2H), 2.44 (dt, J = 13.6,
6.1 Hz, 2H), 1.89 (d, J = 15.1 Hz, 9H). 13C NMR (126 MHz, DMSO-
d6, ppm) δ 54.2 (d, J = 7 Hz), 26.2 (d, J = 53 Hz), 8.3 (d, J = 54 Hz).
31P NMR (202 MHz, DMSO-d6, ppm) δ 28.6. FTIR (ATR) 3205,
EXPERIMENTAL SECTION
■
Materials. Dichloromethane, tetrahydrofuran (THF), and N,N-
dimethylformamide (DMF) were purified by passage through a
solvent purification system (JC Meyer Solvent Systems) and used as
anhydrous solvents. The deuterated solvents (CDCl3, CD2Cl2,
DMSO-d6) were products of Cambridge Isotopes Laboratories. All
other reagents and solvents, tributylphosphine (PBu3, Alpha Aesar),
trimethylphosphine (PMe3, Alpha Aesar), 2-chloroethanol (Alpha
Aesar), thionyl chloride (SOCl2, Aldrich), potassium thioacetate
(KSAc, Alpha Aesar), anhydrous methanol (Alpha Aesar), 2,2-
dimethoxy-2-phenylacetophenone (DMPA, Aldrich), anhydrous di-
methyl sulfoxide (ACROS, DMSO), Alexa Fluor 488 azide (A488, Life
Technologies), copper bromide (CuBr, Aldrich), N,N,N′,N′,N″-
pentamethyldiethylenetriamine (PMDETA, Aldrich), and ultrapure
water (molecular biology grade, Quality Biological, Inc.) were used as
received.
2987−2809, 1287, 1056, 973, 856 cm−1.
(2-(Acetylthio)ethyl)trimethylphosphonium Chloride (2a). In the
glovebox, (2-hydroxyethyl)trimethylphosphonium chloride (2.0 g, 13
mmol) was weighed in a Schlenk flask containing a stir bar and capped
with a septum. Thionyl chloride (4.6 mL, 64 mmol) was added
dropwise at 0 °C under a flow of nitrogen with stirring. The resulting
homogeneous solution was stirred for an additional 15 min at 0 °C
and then heated to 75 °C for 2 h with frequent venting under a flow of
nitrogen. The excess of SOCl2 was evaporated under vacuum at 75 °C,
and the resulting white solid was dried for one additional hour under
vacuum at 75 °C. The resulting alkyl chloride was directly converted to
the thioacetate without intermediate purification. Dry dichloro-
methane (50 mL) was added to the flask, and the formed suspension
was transferred under nitrogen via cannula to a 250 mL round-
bottomed flask containing a suspension of potassium thioacetate (5.5
g, 48 mmol) in 100 mL of dry dichloromethane. The reaction mixture
was stirred at room temperature for 24 h. The yellow supernatant was
filtered (0.45 μm, NYLON), and the residual solid was extracted with
dichloromethane (2 × 25 mL). The organic fractions were combined
and the volatiles were evaporated under reduced pressure. The
resulting yellow solid was then washed with diethyl ether (3 × 50 mL)
and dried under high vacuum overnight to form an off-white
1
Characterization. H, 13C{1H},31P{1H}, and DQF COSY NMR
spectra were recorded on Varian Mercury 300 (300 MHz) or Varian
Inova 500 (500 MHz) spectrometers. The spectra were analyzed with
MestReNova 6.0.2. Chemical shifts are listed in parts per million
downfield from TMS and are referenced from the solvent peaks (1H,
13C{1H} NMR) or from H3PO4 (85 wt%; 31P{1H} NMR). IR spectra
were recorded on a Shimadzu IR Prestige attenuated total reflectance
Fourier-transform infrared spectrometer (ATR-FTIR) and analyzed
using IR solution v.1.40 software.
Glass transition temperatures (Tg) and melting temperatures (Tm)
were measured by differential scanning calorimetry (DSC) on a
Mettler-Toledo DSC822 (Mettler-Toledo, Inc., Columbus, OH)
under nitrogen with temperature gradients of 10 °C·min−1. Measure-
ments were analyzed using Mettler-Toledo Stare v.10.00 software. The
Tg was taken as the midpoint of the inflection tangent upon the second
heating scan (−50 to 100 °C), and the Tm is reported as the onset of
the transition during the first heating cycle. Thermogravimetric
analyses (TGA) were performed under an Ar atmosphere using a
Mettler-Toledo model TGA/DSC 1, with a heating rate of 10 °C·
min−1.
B
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hygroscopic solid (2.37 g, 87%). 1H NMR (500 MHz, CDCl3, ppm) δ
3.19−3.09 (m, 2H), 2.87−2.77 (m, 2H), 2.34 (s, 3H), 2.24 (d, J = 14.4
Hz, 9H). 13C NMR (126 MHz, CDCl3, ppm) δ 195.0 (s), 30.6 (s),
23.4 (d, J = 49 Hz), 20.9 (s), 7.2 (d, J = 53 Hz). 31P NMR (202 MHz,
CDCl3, ppm) δ 27.4. HRMS (ESI, m/z): 179.0665 (found), 179.0654
(calcd for C7H16OPS+). FTIR (ATR) 2965−2896, 1693, 1424, 1290,
1134, 981, 624 cm−1.
of 0.5 mg·mL−1 in a buffered aqueous solution (MOPS, 50 mM, pH =
7.4, or acetate, 50 mM, pH = 5.0) and sonicated for 1 h over an ice
water bath (0 °C). The particle stock solution was split into 10 vials
that were kept in a shaker at 37 °C. Vials were removed from the
shaker at timed intervals, and the samples were characterized in terms
of their average number-averaged size distributions, scattered light
intensities (attenuation corrected), and zeta potentials, as determined
by DLS.
(2-Mercaptoethyl)trimethylphosphonium Chloride (3a). In the
glovebox, (2-(acetylthio)ethyl)trimethylphosphonium chloride (500
mg, 2.33 mmol) was introduced into a 20 mL glass vial equipped with
a stir bar and capped with a septum. The vial was removed from the
glovebox and degassed HCl (4 mL, aqueous, ca. 6 M) was added. The
resulting clear yellow solution was stirred for 3 h at 65 °C after which
the byproducts and water were removed under high vacuum. The
resulting solid was washed with dry THF (3 × 10 mL) and finally
dried under high vacuum to form the final product as a pale-yellow
Cell Viability Assay. RAW 264.7 cells, a transformed mouse
macrophage cell line, were purchased from the American Type Culture
Collection (ATCC, Manassas, VA) and cultured in RPMI-1640
medium (Cellgro, Mediatech, Manassas, VA) supplemented with 10%
fetal calf serum (Sigma-Aldrich) and 2 mM L-glutamine. Cells were
seeded (2.5 × 104 cells/well) on black-walled, clear-bottomed, 96-well
plates (BD Falcon, Franklin Lakes, NJ). After 24 h, diblock
copolymers were diluted in media, added to the cells, and then
incubated at 37 °C and 5% CO2 for 24 h. Cells were then equilibrated
to room temperature for 30 min and washed three times with
phosphate-buffered saline (PBS). To detect ATP activity, Cell-Titer-
Glo Reagent (Promega, Madison, WI) was added to cells and mixed
on an orbital shaker for 2 min at room temperature. Cells were then
incubated at room temperature for an additional 10 min and
luminescence was detected with a Molecular Devices SpectraMax
Gemini Microplate Spectrofluorometer (Sunnyvale, CA). Control cells
treated with media only were used as the denominator to calculate
percent of cell viability. Lipofectamine LTX (Invitrogen, Carlsbad,
CA) was used according to the manufacturer instructions (0.2 μL/
well) and its effect on cell viability was measured following the same
procedures as that of the copolymers.
1
solid (386 mg, 96%). H NMR (500 MHz, CD3OD, ppm) δ 2.93−
2.80 (m, 2H), 2.71−2.59 (m, 2H), 2.00−1.92 (m, 9H). 13C NMR
(126 MHz, CD3OD, ppm) δ 29.2 (d, J = 50 Hz), 17.6 (d, J = 4 Hz),
8.5 (d, J = 55 Hz). 31P NMR (202 MHz, CD3OD) δ 27.6. HRMS
(ESI, m/z): 137.0541 (found), 137.0548 (calcd for C5H14PS+). FTIR
(ATR) 2966−2895, 2388, 1296, 1145, 980, 887, 778 cm−1.
Representative Photoinitiated Thiol−yne “Click” Function-
alization of PBYP50-b-PLLA50 with (2-Mercaptoethyl)-
trimethylphosphonium Chloride. The polymer (PBYP50-b-
PLLA50, 40 mg, 125 μmol of alkyne groups) and the photoinitiator
(DMPA, 20 mg, 78 μmol) were solubilized in dry DMSO (1.0 mL),
and the resulting solution was deoxygenated by bubbling with a stream
of nitrogen for 15 min in the dark. A solution of (2-mercaptoethyl)-
trimethylphosphonium chloride 3a (43 mg, 250 μmol, 2 equiv vs
alkynes) in dry methanol (1 mL) was prepared in the glovebox and
added to the polymer solution to form a light-yellow solution. The
homogeneous reaction mixture was irradiated with a hand-held UV
lamp (365 nm, 6W) for 1 h in a cold room (4 °C) and precipitated in
diethyl ether (40 mL) twice from methanol (2 mL). The polymer was
purified by dialysis against nanopure water (regenerated cellulose
(RC) dialysis tubing, MWCO = 12−14 kDa) for 8 h in a cold room (4
°C) with frequent solvent change. The resulting aqueous polymer
solution was lyophilized to form the polymer as a white to light-yellow
hygroscopic solid. All polymers were stored at −20 °C and handled
under a dry atmosphere. Polymer Me60 (60% phosphonium
Flow Cytometry. RAW 264.7 cells were seeded (5 × 104 cells/
well) on 48-well plates. After 24 h, diblock copolymers labeled with
Alexa Fluor 488 dyes were diluted in media, added to the cells, and
then incubated at 37 °C and 5% CO2 for 2 or 24 h. Cells were scraped
from the plates and washed three times with flow cytometry buffer
composed of PBS (pH 7.4) containing 2% fetal bovine serum. Samples
in triplicates were analyzed (10000 events per sample) using a
FACSCalibur flow cytometer (BD Biosciences, San Jose, CA). Alexa
Fluor 488 signal in cells incubated with copolymers was compared to
cells alone to determine the percent of positive cells. Mean
fluorescence intensity (MFI) was calculated using the geometric
mean of the fluorescence distribution minus background. Both values
were determined with CELLquest software (BD Biosciences).
Fluorescence Imaging. RAW 264.7 cells were seeded (9 × 104
cells/well) in a Lab-Tek, 8-well chamber slide (Nalge Nunc,
Rochester, NY) that was previously coated with filter-sterilized 50
μg/mL Type I rat tail collagen (BD Bioscience) in 0.02 N acetic acid.
After 24 h, diblock copolymers labeled with Alexa Fluor 488 dyes were
diluted in media, added to the cells, and then incubated at 37 °C and
5% CO2 for 4 h. The cells were washed with PBS, fixed with 4%
paraformaldehyde, covered with antifade medium (Vectashield, Vector
Laboratories, Burlingame, CA), and mounted under a coverslip.
Confocal microscopy imaging was performed using a Zeiss LSM 510
META laser scanning confocal instrument (Zeiss, Thornwood, NY).
Cells were scanned along the z-axis to collect 10 μm thick sections.
Fluorescence and differential interference contrast (DIC) images were
overlaid. Photomicrographs were globally adjusted for contrast and
brightness using Photoshop (Adobe Systems, San Jose, CA).
Gel Shift Assays. The siRNA binding affinity of the different
copolymers, Me40, Me60, and Me80 (40, 60, and 80% phosphonium
functionalization, respectively), was investigated by gel shift assay.
Agarose gels (1 wt%) were prepared in Tris-acetate-EDTA buffer (Bio-
Rad Laboratories, Inc., Hercules, CA). The siRNA (5′-Cy3-(sense
strand)-GGCCACAUCGGAUUUCACU, Mw = 13814 g/mol, Dhar-
macon, Chicago, IL), either free or complexed to phosphonium-
functionalized amphiphilic diblock copolymers (total molar concen-
tration of the phosphonium in the polymers to phosphate groups in
the siRNA (P+/P−) ratios ranging from 0.5 to 20 (1.3 μg siRNA/25
μL/well)), were mixed with glycerol (20% v/v) prior to the
electrophoresis. Gel electrophoresis was carried out using a horizontal
apparatus at 100 V for 20 min, and fluorescence imaging of the
1
functionalization): yield 64.2 mg. H NMR (300 MHz, DMSO-d6,
ppm) δ 7.42 (s, 0.1 H), 6.51−6.20 (m, 0.4 H), 5.77−5.55 (m, 0.4 H),
5.20 (q, J = 7.0 Hz, 2.0 H), 4.47−4.05 (m, 6.0 H), 3.27−2.55 (m, 6.1
H), 2.42 (d, J = 12.0 Hz, 0.74 H), 1.97 (d, J = 12.0 Hz, 12.0 H), 1.46
(d, J = 7.0 Hz, 6.3 H). 13C NMR (126 MHz, DMSO-d6, ppm) δ 169.2,
127.0, 125.3, 124.8, 124.3, 68.7, 68.6, 66.34, 65.4, 63.1, 41.1, 36.9, 33.5,
33.1, 29.9, 24.7, 23.5, 23.4, 23.0, 21.8, 16.5, 7.5 (d, J = 52 Hz). 31P
NMR (121 MHz, DMSO-d6, ppm) δ 28.1, 27.9, 27.7, −1.2, −1.3. Tm =
10%
69 °C, Tg = 54 °C, Td
remaining at 500 °C.
= 237 °C, Tp = 300 °C, 32% weight
Self-Assembly of the Amphiphilic Diblock Copolymers. The
amphiphilic diblock copolymers were weighed (ca. 5.0 mg) under a
dry nitrogen atmosphere, suspended into ultrapure water, or any
otherwise specified buffers, to yield a final concentration of 1 mg·
mL−1, and sonicated for 60 min over an ice−water bath (0 °C) to form
clear solutions. The assemblies were then characterized by TEM and
DLS.
Degradation Study Monitored by NMR. The amphiphilic
diblock copolymer Me60 (ca. 6 mg) was solubilized at a concentration
of 10 mg·mL−1 in a buffered D2O solution (MOPS, 50 mM, pH = 7.4,
or acetate, 50 mM, pH = 5.0) and sonicated for 1 h over an ice−water
bath (0 °C). The resulting solution was introduced in an NMR tube
containing an internal reference insert (PPh3 in DMSO-d6). The tubes
were maintained at the desired temperature (37 or 4 °C), and 1H and
31P spectra were recorded at timed intervals at room temperature. The
signals arising from PPh3 were used as chemical shift and integration
1
standards for the analysis of all H and 31P spectra.
Degradation Study Monitored by DLS. The amphiphilic
diblock copolymer Me60 (ca. 6 mg) was solubilized at a concentration
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separated siRNA bands was performed using a ChemiDoc XRS imager
and the data were analyzed by using Image Lab software (Bio-Rad
Laboratories, Inc., Hercules, CA).
Scheme 1. Clickable (2-Mercaptoethyl)trialkylphosphonium
Salts were Synthesized in Three Steps from Chloroethanol
and the Parent Phosphine
Death-siRNA Transfection Assays. OVCAR-3 cells (5 × 103
cells/well) were plated in 96-well plate in RPMI-1640 medium (20%
fetal bovine serum, and 1% penicillin/streptomycin). Cells were
incubated at 37 °C in a humidified atmosphere containing 5% CO2 for
24 h to adhere. Then, the medium was replaced with a fresh medium 1
h prior to the addition of the siRNA (100 nM final concentrations of
AllStars Hs Cell Death- or negative control-siRNA (Qiagen, Valencia,
CA)) mixed with copolymers, Me40 (25 and 50 μg/mL for P+/P−
ratios of 5 and 10, respectively), Me60 (18 and 36 μg/mL for P+/P−
ratios of 5 and 10, respectively), and Me80 (15 and 30 μg/mL for P+/
P− ratios of 5 and 10, respectively), or Lipofectamine 2000. The cells
were incubated with the various formulations for 48 h and then the
medium was replaced with 100 μL of fresh medium prior to the
addition of 20 μL of MTS combined reagent to each well (Cell Titer
96 Aqueous Non-Radioactive Cell Proliferation Assay, Promega Co.,
Madison, WI). The cells were incubated with the reagent for 2 h at 37
°C in a humidified atmosphere containing 5% CO2 protected from
light. Absorbance was measured at 490 nm using SpectraMax M5
(Molecular Devices, Sunnyvale, CA). The cell viability was calculated
by estimating the relative cell viability of the cells treated with death-
siRNA to the negative control siRNA-loaded formulations. The
Lipofectamine−siRNA complexes were prepared according to the
manufacturer instructions and the transfection efficiency was measured
following the same procedures of the siRNA complexes.
The hydrophobic PBYP50-b-PLLA50 diblock copolymer was
modified by photoinitiated thiol−yne addition of thiol-
functionalized trialkylphospohonium salts 3a or 3b to form
the corresponding phosphonium-functionalized amphiphilic
diblock copolymers (Scheme 2). A preliminary experiment
was conducted in the presence of 2 equiv of trimethylphos-
phonium salt 3a and 2,2-dimethoxy-2-phenylacetophenone
(DMPA, 50 wt% vs the polymer) as a photoinitiator in a
mixture of dimethyl sulfoxide and methanol (1:1, v/v) to
ensure the solubility of all the components, under UV
irradiation (365 nm) in a cold room for 1 h. The product
was purified by precipitation in diethyl ether followed by
dialysis against nanopure water (MWCO = 12−14 kDa) in the
cold room to minimize any possible degradation (vide supra)
and finally lyophilized to form a hygroscopic off-white solid that
was stored under dry atmosphere at low temperature (−20 °C).
Proton and phosphorus NMR spectroscopies both indicate
the successful attachment of trimethylphosphonium side groups
onto the PBYP backbone (Figure 1 and Supporting
Information). As mostly broad and overlapping signals were
observed in the 1H NMR spectrum of the copolymer in
DMSO, a two-dimensional 1H NMR experiment (COSY-
DQF) was performed that allowed for the unambiguous
attribution of the resolved signals in Figure 1 (Supporting
Information, Figure S2). The intense signal centered at 1.97
ppm was attributed to the methyl protons of the
trimethylphosphonium side groups and integrated for about
11H instead of an expected value of 18H when the integration
of the methylene protons α to the phosphate group was set at
6H. This observation denotes a less than quantitative
functionalization of the PBYP block, which could be attributed
either to the relatively low thiol-to-alkyne feed ratio of 2:1 that
was initially investigated or to the low reactivity of the
trimethylphosphonium-functionalized thiol during the click
reaction caused by steric hindrance, or both. The two broad
peaks centered around 6.30 and 5.60 ppm (alkene region),
integrating for approximately 0.40 each versus the methylene
protons of the polymer backbone, indicate the presence of vinyl
thioether substituents resulting from the addition of only one
thiol molecule across the triple bond of the PBYP block. The
1H NMR spectrum did not provide evidence for the presence
of unreacted triple bonds along the PBYP backbone due to
signal overlap; however, the two-dimensional spectrum
RESULTS AND DISCUSSION
■
Synthesis of Degradable Phosphonium-Function-
alized Amphiphilic Diblock Copolymers. The degradable
phosphonium-functionalized polyphosphoester-b-poly(L-lac-
tide) amphiphilic copolymers were obtained in two steps
using the strategy previously described by our group.32 First, an
alkyne-functionalized polyphosphoester-b-poly(L-lactide) co-
polymer (PBYP50-b-PLLA50) was synthesized via a one-pot
sequential ring-opening polymerization of an alkyne-function-
alized phospholane monomer (BYP30,31), followed by the
addition of L-lactide to grow the second block (details are
included in the Supporting Information and Figure S1).
Subsequently, the functionalization of the alkynyl side groups
of the PBYP block was performed via the photoinitiated thiol−
yne radical addition of a phosphonium-functionalized free thiol.
This sequential approach allowed the elaboration of a small
library of amphiphilic diblock copolymers that share a common
backbone.
The mercaptoethyl-functionalized trialkylphosphonium salts
(3a,b) required for the thiol−yne click modification of the
polyphosphoester block were synthesized in three steps from
chloroethanol and the corresponding trialkylphosphines
(Scheme 1). The nucleophilic substitution of trialkylphosphines
with chloroethanol afforded the formation of the (2-
hydroxyethyl)trialkylphosphonium chloride salts that were
converted to their thioacetates 2a or 2b by activation with
thionyl chloride and subsequent substitution with potassium
thioacetate. Finally, aqueous acidic treatment of the thioacetates
under inert atmosphere formed the desired (2-mercaptoethyl)-
trialkylphosphonium salts (3a, 3b) in good overall yields and
purities. The tributylphosphonium derivative (3b) was isolated
as an oil and was soluble in a number of organic solvents
(chloroform, tetrahydrofuran, methanol, dimethyl sulfoxide),
whereas the trimethylphosphonium salt 3a was obtained as a
white crystalline solid with a melting point of approximately
129 °C and was mostly soluble in methanol and water.
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Scheme 2. Photoinitiated Thiol-yne Addition of (2-Mercaptoethyl)trialkylphosphonium Salts to PBYP50-b-PLLA50 Copolymers
Yields Phosphonium-Functionalized Amphiphilic Diblock Copolymers Presenting Varied Substitution Patterns
easy comparison between the samples, the notion of
phosphonium content (%P+) was introduced. The %P+
parameter describes the actual extent of functionalization of
the polyphosphoester block relative to a hypothetical fully
functionalized one. The phosphonium content was thus
calculated as follows: %P+ = (x/2 + z) × 100, where x and z
are the relative ratios of mono- and bis-adducts across the triple
1
bonds of the PBYP block (Scheme 2), as determined by H
NMR spectroscopy (Table 1), based on the signal attribution
presented above. In the case of the trimethylphosphonium
derivative, we were able to achieve a rather high degree of
functionalization (%P+ = 80%) by increasing both the reaction
time and the thiol-to-alkyne feed ratio to 2.5:1, while a
reduction of the feed ratio to 1:1 led to a phosphonium content
of 40% (entries 1−3). Conversely, the reaction with (2-
1
Figure 1. H NMR spectra of the diblock copolymer before (upper
mercaptoethyl)tributylphosphonium 3b led to only 55%
panel) and after (lower panel) thiol−yne functionalization of the
functionalization of the PBYP block, despite the use of a
polyphosphoester block with a clickable phosphonium salt. W stands
rather high thiol-to-alkyne feed ratio of 5:1. This observation
for water and S stands for solvent (signal of the residual hydrogenated
further highlights the sensitivity of thiol−yne/ene click
fraction present in DMSO-d6). The labels of the proton peaks are
explained in Scheme 2.
reactions to steric constraints when used as a strategy for
postpolymerization modification.36
Thermogravimetric analysis (TGA) of the phosphonium-
functionalized diblock copolymers showed a concomitant
increase in the thermal stability of the samples with the
phosphonium content in the polyphosphoester block within
the trimethylphosphonium functionalized series (1−3, Table 1,
and Supporting Information, Figure S3). All three polymers
from the trimethylphosphonium series (Me80, Me60, and Me40)
appeared as semicrystalline powders that exhibited a clear
melting transition (Tm) on the first heating cycle during
differential scanning calorimetry (DSC) measurements (Sup-
porting Information, Figure S4). The increase in the %P+
correlated to an increase in the observed melting transition
temperature. It should also be noted that a second melting
exhibited a cross correlation peak between the signals at 4.15
and 2.60 ppm, which is characteristic of the butynyl side groups
found in the starting diblock copolymer (Supporting
Information, Figure S2). As expected, the proton-decoupled
31P NMR spectrum displayed a new set of signals centered at
about 28.0 ppm, typical of a trimethylphosphonium derivative,
along with another signal at about −1.2 ppm that was attributed
to the polyphosphoester backbone (Supporting Information).
We further investigated the thiol−yne modification step by
varying the reaction conditions to elaborate a small library of
phosphonium-functionalized amphiphilic block copolymers
with varied degrees of phosphonium functionalization along a
shared polyphosphoester backbone (Table 1). To allow for an
Table 1. Characterization and Thermal Properties of Phosphonium-Functionalized Amphiphilic Diblock Copolymers
Generated by Photoinitiated Thiol-yne Addition
a
b
b
b
c
d
e
f
10%
polymer
R
thiol/alkyne
x
y
z
%P+
Tg (°C)
Tm (°C)
Td
(°C)
Me80
Me60
Me40
Bu55
Me
2.5
2.0
1.0
5.0
0.2
0.4
0.4
0.4
0.1
0.7
80
60
40
55
59
54
49
6
80
69
61
37
247
237
219
234
0.2
0.4
0.4
0.2
Bu
0.25
0.35
a
b
Thiol/alkyne refers to the molar ratio of thiol reagent to alkynes from the PBYP block. The x and z ratios were determined by integration of the
1
characteristic signals in the H NMR spectra and represent the relative ratios of mono- and bis-adducts across the triple bonds of the PBYP block,
c
and y was determined as follows: y = 1 − x − z. The phosphonium content (%P+) of the polyphosphoester block was defined as follows: %P+ = (z +
d
e
f
x/2) × 100. Determined by DSC analysis during the second heating ramp. Determined by DSC during the first heating ramp. Determined by
TGA.
E
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transition could be observed during the last heating cycle (ca.
120−130 °C), but was relatively ill-defined because of the
concomitant thermal decomposition of the sample (supported
by discoloration of the samples). The glass transition (Tg)
observed during the second heating cycle followed a trend
similar to the Tm, with Tg values comprised between 49 and 59
°C for Me40 and Me80, respectively. The melting transition was
not easily identifiable in the tributylphosphonium-function-
alized polymer, which could possibly be attributed to the
relatively long alkyl chains leading to a poor packing of the
polymer chains in the solid state.
Self-Assembly of the Amphiphilic Diblock Copoly-
mers. The four amphiphilic diblock copolymers presented in
Table 1 were hygroscopic and presented good solubility in
nanopure water and phosphate-buffered saline (PBS) solutions.
The polymer solutions were homogenized by sonication for 1 h
over an ice−water bath (0 °C) to minimize degradation, and
clear solutions were formed for all three trimethylphosphonium
functionalized copolymers. The tributylphosphonium-function-
alized copolymer Bu55 afforded a cloudy suspension even upon
extended sonication. The ability of this polymer to form well-
defined assemblies in aqueous solutions was, thus, not
investigated further.
ions due to the high salt concentration (150 mM NaCl).
Contrary to the nanopure water solutions, a nice correlation
between the %P+ and the zeta potentials was observed in PBS
solutions (Figure 2, green triangles).
Hydrolytic Degradation of the Diblock Copolymers.
Polyphosphoesters are susceptible to degradation at the
phosphoester linkages between the repeat units in the polymer
backbone, leading to either main chain or side group cleavages.
The degradation of polyphosphoesters has been studied
extensively in the literature and occurs spontaneously in
aqueous buffers34,37 but can also be mediated by enzymes.38
Recent reports from our laboratory have investigated the
degradation of self-assembled amphiphilic diblock copolymers
made up of at least one polyphosphoester-based block using a
range of characterization techniques (NMR spectroscopy, DLS,
and mass spectrometry) and shown that the degradation rate
was highly dependent on the nature of the side chain,34,39 as
previously reported in the literature. In this work, the hydrolytic
stability of polymer assemblies generated from phosphonium-
functionalized amphiphilic diblock copolymers was evaluated in
detail by multinuclear NMR spectroscopy, dynamic light
scattering, and zeta potential measurements at acidic and
neutral pH values (pH = 5.0 and pH = 7.4, at 37 °C) using
polymer Me60 as a model compound.
The hydrodynamic sizes and surface charges of the
assemblies formed in aqueous solutions from the trimethyl-
phosphonium-functionalized diblock copolymers Me40, Me60,
and Me80 were assessed by dynamic light scattering (DLS) and
the morphology of the assemblies was evaluated by bright-field
transmission electron microscopy (TEM; Figure 2, and
For the NMR study, polymer solutions (10 mg·mL−1) were
prepared by direct dissolution of copolymer Me60 in deuterated
aqueous buffers (MOPS, 50 mM, pH = 7.4, or acetate, 50 mM,
pH = 5.0) and incubated at the desired temperature (4 or 37
°C). The extent of hydrolysis of the charged block was
evaluated at timed intervals by 1H and 31P NMR spectroscopies
at room temperature (Supporting Information, Figure S6). The
relative integration of the signals attributed to the methylene
protons connected to the phosphate group (4.5−4.0 ppm, in
1H NMR spectra) or the signal attributed to the phosphorus
nuclei in the polymer backbone (−1.2 ppm, in 31P NMR
spectra) were monitored versus an external chemical shift
calibration (31P NMR spectroscopy) and integration standard
(Figure 3).
The degradation proceeded fast at 37 °C, independent of the
pH values investigated (7.4 or 5.0), as evidenced by the rapid
drop in the integration of the signals that are characteristic to
the polyphosphoester block (both in 1H and 31P NMR
spectra), with a half-life of about 24 h, before reaching a
plateau. Despite the apparent similarity in behavior at both pH
values, it should be noted that macroscopic precipitation was
observed within 20 h upon incubation at pH = 7.4, whereas it
took 5 d at pH = 5.0. The major effect exerted by the pH of the
incubation medium over the kinetics of degradation of
polyphosphoesters has been previously reported in the
literature34,37,39−43 and is discussed further below, in light of
the DLS data. At lower temperature (4 °C) the polyphos-
phoester backbone was substantially more stable at both pH
values and most of the degradation that occurred was imputable
to the fact that the NMR experiments were run at room
temperature, which led to a faster apparent degradation.
The degradation process was also monitored by dynamic
light scattering and zeta potential measurements. Polymer Me60
was incubated in buffered aqueous solutions (50 mM, MOPS
or acetate, pH = 7.4 or 5.0, respectively) at 37 °C, and the
normalized scattered intensities (corrected for attenuation),
zeta potentials, and number-averaged hydrodynamic size
distributions were monitored over time (Figure 4). The
degradation profiles obtained using light scattering techniques
Figure 2. Effect of the phosphonium content (%P+) in the
polyphosphoester block on the sizes of the assemblies in water
(Dh,water(n), red circle) and their surface charges in nanopure water or
PBS (blue square and green triangle, respectively).
Supporting Information, Figure S5). DLS demonstrated
unimodal distributions with average number-averaged hydro-
dynamic radii (Dh(n)) ranging between 38 to 50 nm in water
and slightly smaller in PBS (Supporting Information, Figure
S5). As seen in Figure 2, no marked effect of the %P+ over the
sizes of the aggregates was observed in aqueous solutions (red
circles). The surface charges of the assemblies were evaluated in
terms of their zeta potentials (ζpot), as determined by
electrophoretic light scattering and was positive in all cases in
nanopure water with values in the 50−55 mV range and
appeared to be only slightly affected by %P+ (Figure 2, blue
squares). In PBS, the particle surface charge was greatly
diminished, with zeta potentials ranging from about 5 to 25
mV, which could be explained by the screening effect of the
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Figure 3. Evaluation of the degradation kinetics of diblock copolymer Me60 at pH = 7.4 (50 mM MOPS buffer in D2O, squares) or pH = 5.0 (50
mM acetate buffer in D2O, triangles) at 37 and 4 °C (red and blue, respectively). The reactions were monitored by integrating the signals of the
methylene protons connected to the phosphate groups on the polyphosphoester block (left panel) or, alternatively, the signals arising from the
phosphorus nuclei of the polyphosphate backbone (right panel) vs an internal reference (PPh3 in DMSO-d6 capillary insert) against time.
Figure 4. Evaluation of the hydrolytic stability of the micelles upon incubation in aqueous buffers (50 mM, MOPS or acetate, pH = 7.4 or 5.0,
respectively) at 37 °C as monitored by DLS: (A) scattered light intensities, (B) zeta potentials, (C) average number-averaged hydrodynamic
diameters and their standard deviations. Macroscopic precipitation was observed within 20 h when the degradation was performed at pH = 7.4, as
denoted by *.
displayed a dichotomous behavior that contrasts starkly with
the degradation profiles that were observed by NMR
spectroscopy, in spite of similar macroscopic behavior in both
sets of experiments. A precipitate formed within 16 h upon
incubation at pH = 7.4, while incubation at pH = 5.0 did not
lead to any precipitate over the course of the entire study (70
h). On the basis of the DLS data, it is hypothesized that the loss
of solubility of the micellar assemblies upon incubation at pH =
7.4 might be attributed to cleavages along the polyphosphoester
main chain. Such cleavages would lead to a rapid loss of
electrostatic stabilization and also expose hydrophobic patches
from the PLLA core and destabilize the assemblies that would
then aggregate and ultimately precipitate. Interestingly, our
group has recently reported a similar behavior for the hydrolytic
degradation of a related carboxylate-functionalized PPE-b-
PLLA copolymer upon incubation in buffered aqueous
systems.34 In the present case, the fast drop in surface charge
(Figure 4B) would support the hypothesis that main chain
scissions are occurring predominantly over side chain cleavage.
In a seminal study, Baran and Penczek showed, using NMR
spectroscopy and titration techniques, that incubation pH value
exerts a profound influence on the relative rate of hydrolysis of
main chain versus side groups in methoxy-substituted
polyphosphoesters.40 Importantly, they concluded that main
chain cleavages were favored at higher pH values over side
group cleavages. In the present work, at pH = 5.0, no
macroscopic precipitation was noticed over the course of the
study, while high scattering intensities and nearly constant
particle sizes were observed, denoting the overall colloidal
stability of the assemblies in solution. Interestingly, the surface
charge of the assemblies decreased monotonously over time to
attain neutral values within 72 h of incubation. Based on the
above discussion and the early work from Baran and Penczek,
we hypothesize that, at near-neutral pH values, the polymer
assemblies degraded mostly through main chain cleavages
leading to a rapid loss of colloidal stability and resulting in
macroscopic precipitation, whereas under more acidic con-
ditions (pH = 5.0), cleavage of the side groups occurred faster.
This cleavage pattern resulted in the formation of a negatively
charged and hydrolytically more stable40,44 polyphosphate
backbone that was still able to stabilize the assemblies in
solution but led to a structure presenting an overall decrease in
surface charge. Unfortunately, signal overlaps in the NMR
spectra prevented a more in-depth investigation and no other
meaningful information could be extracted from them to
further support this hypothesis.
Feasibility of Utilizing the Diblock Copolymers as
Carriers for Nucleic Acids (for Example, siRNA). The
initial ability of nanoparticles toward nucleic acids delivery was
tested via evaluating their cytotoxicity, cellular uptake, ability to
electrostatically bind to a model siRNA, and finally by
measuring the cell transfection efficiency of AllStars Hs Cell
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Death siRNA when complexed to the nanoparticles versus the
Lipofectamine 2000 commercial transfection reagent. The
effect of the phosphonium-functionalized diblock copolymers
on viability of mouse macrophages (RAW 264.7) was evaluated
at increasing polymer concentrations after incubation with the
cells for 24 h (Figure 5). Worth mentioning is that the
Figure 6. Concentration- and time-dependent cellular uptake of a
Me60−Alexa Fluor 488 dye conjugated diblock copolymer in RAW
264.7 cells. The cells were incubated with the polymer at varied
concentrations for 2 or 24 h and analyzed by flow cytometry (median
fluorescence intensity, MFI, upper panel; percent cellular uptake,
lower panel).
Figure 5. Cytotoxicity of the phosphonium-functionalized diblock
copolymers toward mouse macrophage (RAW 264.7) cell line after 24
h incubation time. Control is Lipofectamine LTX, 0.2 μL/well.
negatively charged nucleic acids, due to the efficient cationic
charge distribution (i.e., centered on the phosphorus
atom).17,21,22 The transfection efficiencies of AllStars Hs Cell
Death siRNA, a set of siRNAs that target cell survival genes,
complexed to the copolymers Me40, Me60, and Me80 were
evaluated by measuring the effect of the complexes on the
viability of human ovarian carcinoma cells (OVCAR-3). The
cell survival was compared to each of the individual copolymers
complexed with negative-control siRNA and Lipofectamine
2000 controls (Figure 7). The effect of the P+/P− ratio on the
transfection efficiency of AllStars Hs Cell Death siRNA was also
studied in OVCAR-3 cells. Complexes prepared at a P+/P−
ratio of 10 showed significantly higher transfection as compared
to the transfection obtained at P+/P− ratio of 5. The AllStars Hs
Cell Death siRNA complexed to copolymers Me40, Me60, and
Me80 at P+/P− ratio of 10 each induced similar levels of cell
death that were slightly lower as compared to Lipofectamine
2000, which might be related to the higher stability of the
complexes, partially retarding the release of siRNA intra-
cellularly.48,49 We have previously demonstrated comparable
transfection efficiency for the AllStars Hs Cell Death siRNA
when it was complexed to nondegradable hierarchically
assembled theranostic nanostructures.47,49 The polymer−
siRNA polyplexes were prepared immediately prior to the
cellular transfection studies. As can be seen from Figure 6,
uptake of the nanoparticles occurs mostly within the first 2 h of
incubation with the cells and that the cellular uptake after 2 h
was almost similar to the uptake after 24 h. Based on the
degradation studies, it is expected that the nanoparticles were
able to deliver the complexed siRNA before significant
degradation occurs. The phosphonium-functionalized fully
degradable nanoparticles developed in the current study may
have great potential for delivery of nucleic acid-based
therapeutics by achieving transfection efficiency comparable
to nondegradable commercially available transfection reagents
and multifunctional nanoparticles previously developed by our
group while being designed to degrade over time and to possess
higher cellular biocompatibility.
polymers might partially degrade over the 24 h period of
incubation with the cells. An effect of the phosphonium content
(%P+) over the cytotoxicity was particularly obvious at high
polymer concentration (200 μg·mL−1), where Me40 displayed
no toxicity, while Me80 led to about 30% cellular death under
identical conditions. The low cytotoxicity pattern of the
polymers is comparable to what has been previously observed
with cationic degradable polyphosphoester micelles and, thus,
confirms the higher biocompatibility of degradable polymers
than the commercially available reagents (e.g., IC50 values for
Lipofectamine and polyethylenimine were 31.4 6 and 4.3
1
μg/mL, when evaluated under identical conditions).45
To evaluate the cellular uptake of the polymers, an azido-
functionalized Alexa Fluor 488 dye was conjugated to the Me60
polymer via copper-assisted azide−alkyne reaction, and the
conjugation was confirmed by UV−vis and fluorescence
spectroscopies and gel electrophoresis (Supporting Informa-
tion, Figure S7). The cellular uptake of the cationic micelles
was then investigated by flow cytometry and fluorescence
imaging (Figure 6 and Supporting Information, Figure S8). The
uptake of the Me60-Alexa Fluor 488 dye conjugated copolymer
by RAW 264.7 mouse macrophages increased upon increasing
the concentration of the polymers and incubation time with the
cells (Figure 6). The uptake of the polymer was also confirmed
and visualized using laser scanning confocal microscopy, where
the images demonstrated an uptake (green fluorescence) after 4
h of incubation of the RAW 264.7 mouse macrophages with the
Me60−Alexa Fluor 488 conjugate (Supporting Information,
Figure S8).
Complexation between the cationic polymers and siRNA
allows for greater enzymatic stability and higher transfection
efficiency, as compared to the free siRNA.46,47 The ability of
copolymers Me40, Me60, and Me80 to efficiently complex Cy3-
labeled siRNA (molecular weight ca. 13814.3 g/mol) was
investigated at a wide range of phosphonium-to-phosphorus
(P+/P−, polymer/siRNA) ratios. At a P+/P− ratio of 2, the three
copolymers were able to completely bind siRNA, as confirmed
by the gel retardation assay (Supporting Information, Figure
S9). Phosphonium-functionalized polymers were reported to
have an increased efficiency of strong and stable binding to
CONCLUSIONS
■
A series of phosphonium-functionalized polyphosphoester-b-
poly-L-lactide copolymers with increasing degrees of function-
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Figure 7. Transfection efficiency of cell-death siRNA complexed with Lipofectamine 2000, and copolymers Me40, Me60, and Me80 into OVCAR-3
cells at P+/P− ratios of 5 and 10 (mean SD, n = 3). The effect of the P+/P− ratio on the transfection efficiency was determined by comparing the
viabilities of cells treated with cell-death siRNA complexes vs negative control−siRNA complexes.
Notes
alization of the hydrophilic backbone with quaternary
phosphonium salts were synthesized and fully characterized.
The diblock copolymers were shown to self-assemble in
aqueous buffers to form spherical nanosized assemblies with a
core−shell structure as supported by DLS and TEM measure-
ments. Detailed NMR spectroscopic and DLS studies revealed
the putative degradation mechanism of those assemblies under
hydrolytic degradation conditions. The positive surface charge
of the assemblies as evaluated by zeta potentials allowed for the
efficient complexation of nucleic acids (i.e., siRNA). The
copolymer assemblies exhibited low toxicity on RAW 264.7
mouse macrophages, particularly, at low degrees of phospho-
nium functionalization. Transfection efficiency was strongly
affected by the phosphonium-to-phosphate ratios (P+/P−) in
OVCAR-3 cells and the AllStars Hs Cell Death siRNA
complexed to the various copolymers at a P+/P− ratio of 10
was able to induce comparable cell death to Lipofectamine.
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We gratefully acknowledge financial support from the National
Heart Lung and Blood Institute of the National Institutes of
Health as a Program of Excellence in Nanotechnology
(HHSN268201000046C), the National Science Foundation
(DMR-1507429), and the Welch Foundation through the W.
T. Doherty-Welch Chair in Chemistry (A-0001). The
Microscopy and Imaging Center (MIC), the Laboratory for
Biological Mass Spectrometry (LBMS/TAMU), and the
Laboratory for Synthetic−Biologic Interactions (LSBI) at
Texas A&M University are also gratefully acknowledged.
REFERENCES
■
(1) Wu, S. Y.; Lopez-Berestein, G.; Calin, G. A.; Sood, A. K. RNAi
Therapies: Drugging the Undruggable. Sci. Transl. Med. 2014, 6,
240ps7.
ASSOCIATED CONTENT
* Supporting Information
■
S
(2) Wu, Y.; Smith, A. E.; Reineke, T. M. Lipophilic Polycation
Vehicles Display High Plasmid DNA Delivery to Multiple Cell Types.
Bioconjugate Chem. 2017, 28, 2035−2040.
(3) Jiang, Y.; Sprouse, D.; Laaser, J. E.; Dhande, Y.; Reineke, T. M.;
Lodge, T. P. Complexation of Linear DNA and Poly(styrenesulfonate)
with Cationic Copolymer Micelles: Effect of Polyanion Flexibility. J.
Phys. Chem. B 2017, 121, 6708−6720.
(4) Dhande, Y. K.; Wagh, B. S.; Hall, B. C.; Sprouse, D.; Hackett, P.
B.; Reineke, T. M. N-Acetylgalactosamine Block-co-Polycations Form
Stable Polyplexes with Plasmids and Promote Liver-Targeted Delivery.
Biomacromolecules 2016, 17, 830−840.
(5) Morsi, N. G.; Ali, S. M.; Elsonbaty, S. S.; Afifi, A. A.; Hamad, M.
A.; Gao, H.; Elsabahy, M. Poly(glycerol methacrylate)-based
degradable nanoparticles for delivery of small interfering RNA.
Pharm. Dev. Technol. 2017, 1−13.
(6) Elzeny, H.; Zhang, F.; Ali, E. N.; Fathi, H. A.; Zhang, S.; Li, R.;
El-Mokhtar, M. A.; Hamad, M. A.; Wooley, K. L.; Elsabahy, M.
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.bio-
mac.8b00069.
Detailed protocols for the synthesis of the phosphonium
salts, block copolymer synthesis, modification, assembly,
and characterization, as well as biological experiments
(PDF).
AUTHOR INFORMATION
Corresponding Author
*E-mail: wooley@chem.tamu.edu.
■
ORCID
Karen L. Wooley: 0000-0003-4086-384X
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Article
Polyphosphoester nanoparticles as biodegradable platform for delivery
of multiple drugs and siRNA. Drug Des., Dev. Ther. 2017, 11, 483−496.
(7) Giacca, M.; Zacchigna, S. Virus-mediated gene delivery for
human gene therapy. J. Controlled Release 2012, 161, 377−388.
(8) Wang, J.; Huang, S.-W.; Zhang, P.-C.; Mao, H.-Q.; Leong, K. W.
Effect of side-chain structures on gene transfer efficiency of
biodegradable cationic polyphosphoesters. Int. J. Pharm. 2003, 265,
75−84.
(9) Vannucci, L.; Lai, M.; Chiuppesi, F.; Ceccherini-Nelli, L.; Pistello,
M. Viral Vectors: a look back and ahead on gene transfer technology.
New Microbiol 2013, 36, 1−22.
(10) Yue, Y.; Wu, C. Progress and perspectives in developing
polymeric vectors for in vitro gene delivery. Biomater. Sci. 2013, 1,
152−170.
(11) Ibraheem, D.; Elaissari, A.; Fessi, H. Gene therapy and DNA
delivery systems. Int. J. Pharm. 2014, 459, 70−83.
(12) Wagner, E. Polymers for siRNA Delivery: Inspired by Viruses to
be Targeted, Dynamic, and Precise. Acc. Chem. Res. 2012, 45, 1005−
1013.
(13) Liu, X.-Q.; Sun, C.-Y.; Yang, X.-Z.; Wang, J. Polymeric-Micelle-
Based Nanomedicine for siRNA Delivery. Part Part Syst. Charact 2013,
30, 211−228.
(14) Islam, M. A.; Park, T. E.; Singh, B.; Maharjan, S.; Firdous, J.;
Cho, M.-H.; Kang, S.-K.; Yun, C.-H.; Choi, Y. J.; Cho, C.-S. Major
degradable polycations as carriers for DNA and siRNA. J. Controlled
Release 2014, 193, 74−89.
gene and oligonucleotide transfection of mammalian cells. Biomaterials
2009, 30, 968−977.
(27) Zhang, K.; Fang, H.; Wang, Z.; Li, Z.; Taylor, J.-S. A.; Wooley,
K. L. Structure-activity relationships of cationic shell-crosslinked
knedel-like nanoparticles: Shell composition and transfection
efficiency/cytotoxicity. Biomaterials 2010, 31, 1805−1813.
(28) Shrestha, R.; Elsabahy, M.; Florez-Malaver, S.; Samarajeewa, S.;
Wooley, K. L. Endosomal escape and siRNA delivery with cationic
shell crosslinked knedel-like nanoparticles with tunable buffering
capacities. Biomaterials 2012, 33, 8557−8568.
(29) Samarajeewa, S.; Ibricevic, A.; Gunsten, S. P.; Shrestha, R.;
Elsabahy, M.; Brody, S. L.; Wooley, K. L. Degradable Cationic Shell
Cross-Linked Knedel-like Nanoparticles: Synthesis, Degradation,
Nucleic Acid Binding, and in Vitro Evaluation. Biomacromolecules
2013, 14, 1018−1027.
(30) Zhang, S.; Li, A.; Zou, J.; Lin, L. Y.; Wooley, K. L. Facile
Synthesis of Clickable, Water-Soluble, and Degradable Polyphos-
phoesters. ACS Macro Lett. 2012, 1, 328−333.
(31) Zhang, S.; Zou, J.; Zhang, F.; Elsabahy, M.; Felder, S. E.; Zhu, J.;
Pochan, D. J.; Wooley, K. L. Rapid and Versatile Construction of
Diverse and Functional Nanostructures Derived from a Polyphos-
phoester-Based Biomimetic Block Copolymer System. J. Am. Chem.
Soc. 2012, 134, 18467−18474.
(32) Lim, Y. H.; Heo, G. S.; Cho, S.; Wooley, K. L. Construction of a
Reactive Diblock Copolymer, Polyphosphoester-block-Poly(l-lactide),
as a Versatile Framework for Functional Materials That Are Capable of
Full Degradation and Nanoscopic Assembly Formation. ACS Macro
Lett. 2013, 2, 785−789.
(33) Zhang, F.; Zhang, S.; Pollack, S. F.; Li, R.; Gonzalez, A. M.; Fan,
J.; Zou, J.; Leininger, S. E.; Pavía-Sanders, A.; Johnson, R.; Nelson, L.
D.; Raymond, J. E.; Elsabahy, M.; Hughes, D. M. P.; Lenox, M. W.;
Gustafson, T. P.; Wooley, K. L. Improving Paclitaxel Delivery: In Vitro
and In Vivo Characterization of PEGylated Polyphosphoester-Based
Nanocarriers. J. Am. Chem. Soc. 2015, 137, 2056−2066.
(34) Lim, Y. H.; Tiemann, K. M.; Heo, G. S.; Wagers, P. O.;
Rezenom, Y. H.; Zhang, S.; Zhang, F.; Youngs, W. J.; Hunstad, D. A.;
Wooley, K. L. Preparation and in Vitro Antimicrobial Activity of Silver-
Bearing Degradable Polymeric Nanoparticles of Polyphosphoester-
block-Poly(l-lactide). ACS Nano 2015, 9, 1995−2008.
(35) Zhang, F.; Smolen, J. A.; Zhang, S.; Li, R.; Shah, P. N.; Cho, S.;
Wang, H.; Raymond, J. E.; Cannon, C. L.; Wooley, K. L. Degradable
polyphosphoester-based silver-loaded nanoparticles as therapeutics for
bacterial lung infections. Nanoscale 2015, 7, 2265−2270.
(36) Lowe, A. B. Thiol-ene ″click″ reactions and recent applications
in polymer and materials synthesis. Polym. Chem. 2010, 1, 17−36.
(37) Lim, Y. H.; Heo, G. S.; Rezenom, Y. H.; Pollack, S.; Raymond, J.
E.; Elsabahy, M.; Wooley, K. L. Development of a Vinyl Ether-
Functionalized Polyphosphoester as a Template for Multiple
Postpolymerization Conjugation Chemistries and Study of Core
Degradable Polymeric Nanoparticles. Macromolecules 2014, 47, 4634−
4644.
(38) Wachiralarpphaithoon, C.; Iwasaki, Y.; Akiyoshi, K. Enzyme-
degradable phosphorylcholine porous hydrogels cross-linked with
polyphosphoesters for cell matrices. Biomaterials 2007, 28, 984−993.
(39) Shen, Y.; Zhang, S.; Zhang, F.; Loftis, A.; Pavía-Sanders, A.; Zou,
J.; Fan, J.; Taylor, J.-S. A.; Wooley, K. L. Polyphosphoester-Based
Cationic Nanoparticles Serendipitously Release Integral Biologically-
Active Components to Serve as Novel Degradable Inducible Nitric
Oxide Synthase Inhibitors. Adv. Mater. 2013, 25, 5609−5614.
(40) Baran, J.; Penczek, S. Hydrolysis of Polyesters of Phosphoric
Acid. 1. Kinetics and the pH Profile. Macromolecules 1995, 28, 5167−
5176.
(15) Hemp, S. T.; Smith, A. E.; Bryson, J. M.; Allen, M. H.; Long, T.
E. Phosphonium-Containing Diblock Copolymers for Enhanced
Colloidal Stability and Efficient Nucleic Acid Delivery. Biomacromo-
lecules 2012, 13, 2439−2445.
(16) Hemp, S. T.; Allen, M. H.; Green, M. D.; Long, T. E.
Phosphonium-Containing Polyelectrolytes for Nonviral Gene Deliv-
ery. Biomacromolecules 2012, 13, 231−238.
́
(17) Ornelas-Megiatto, C.; Wich, P. R.; Frechet, J. M. J.
Polyphosphonium Polymers for siRNA Delivery: An Efficient and
Nontoxic Alternative to Polyammonium Carriers. J. Am. Chem. Soc.
2012, 134, 1902−1905.
(18) Loczenski Rose, V.; Shubber, S.; Sajeesh, S.; Spain, S. G.; Puri,
S.; Allen, S.; Lee, D. K.; Winkler, G. S.; Mantovani, G. Phosphonium
Polymethacrylates for Short Interfering RNA Delivery: Effect of
Polymer and RNA Structural Parameters on Polyplex Assembly and
Gene Knockdown. Biomacromolecules 2015, 16, 3480−3490.
(19) Rose, V. L.; Mastrotto, F.; Mantovani, G. Phosphonium
polymers for gene delivery. Polym. Chem. 2017, 8, 353−360.
(20) Jangu, C.; Long, T. E. Phosphonium cation-containing
polymers: From ionic liquids to polyelectrolytes. Polymer 2014, 55,
3298−3304.
(21) Wang, S.-W.; Liu, W.; Colby, R. H. Counterion Dynamics in
Polyurethane-Carboxylate Ionomers with Ionic Liquid Counterions.
Chem. Mater. 2011, 23, 1862−1873.
(22) Liang, S.; O’Reilly, M. V.; Choi, U. H.; Shiau, H.-S.; Bartels, J.;
Chen, Q.; Runt, J.; Winey, K. I.; Colby, R. H. High Ion Content
Siloxane Phosphonium Ionomers with Very Low Tg. Macromolecules
2014, 47, 4428−4437.
(23) Elsabahy, M.; Wooley, K. L. Data Mining as a Guide for the
Construction of Cross-Linked Nanoparticles with Low Immunotox-
icity via Control of Polymer Chemistry and Supramolecular Assembly.
Acc. Chem. Res. 2015, 48, 1620−1630.
(24) Elsabahy, M.; Li, A.; Zhang, F.; Sultan, D.; Liu, Y.; Wooley, K. L.
Differential immunotoxicities of poly(ethylene glycol)- vs. poly-
(carboxybetaine)-coated nanoparticles. J. Controlled Release 2013,
172, 641−652.
(25) Elsabahy, M.; Samarajeewa, S.; Raymond, J. E.; Clark, C.;
Wooley, K. L. Shell-crosslinked knedel-like nanoparticles induce lower
immunotoxicity than their non-cross-linked analogs. J. Mater. Chem. B
2013, 1, 5241.
(26) Zhang, K.; Fang, H.; Wang, Z.; Taylor, J.-S. A.; Wooley, K. L.
Cationic shell-crosslinked knedel-like nanoparticles for highly efficient
(41) Wen, J.; Zhuo, R.-X. Preparation and characterization of
poly(D,L-lactide-co-ethylene methyl phosphate). Polym. Int. 1998, 47,
503−509.
(42) Iwasaki, Y.; Nakagawa, C.; Ohtomi, M.; Ishihara, K.; Akiyoshi,
K. Novel Biodegradable Polyphosphate Cross-Linker for Making
Biocompatible Hydrogel. Biomacromolecules 2004, 5, 1110−1115.
J
DOI: 10.1021/acs.biomac.8b00069
Biomacromolecules XXXX, XXX, XXX−XXX

Biomacromolecules
Article
(43) Yang, X.-Z.; Wang, Y.-C.; Tang, L.-Y.; Xia, H.; Wang, J.
Synthesis and characterization of amphiphilic block copolymer of
polyphosphoester and poly(L-lactic acid). J. Polym. Sci., Part A: Polym.
Chem. 2008, 46, 6425−6434.
(44) Zhang, S.; Wang, H.; Shen, Y.; Zhang, F.; Seetho, K.; Zou, J.;
Taylor, J.-S. A.; Dove, A. P.; Wooley, K. L. A Simple and Efficient
Synthesis of an Acid-Labile Polyphosphoramidate by Organobase-
Catalyzed Ring-Opening Polymerization and Transformation to
Polyphosphoester Ionomers by Acid Treatment. Macromolecules
2013, 46, 5141−5149.
(45) Elsabahy, M.; Zhang, S.; Zhang, F.; Deng, Z. J.; Lim, Y. H.;
Wang, H.; Parsamian, P.; Hammond, P. T.; Wooley, K. L. Surface
charges and shell crosslinks each play significant roles in mediating
degradation, biofouling, cytotoxicity and immunotoxicity for poly-
phosphoester-based nanoparticles. Sci. Rep. 2013, 3, 3313.
(46) Elsabahy, M.; Zhang, M.; Gan, S. M.; Waldron, K. C.; Leroux, J.
C. Synthesis and enzymatic stability of PEGylated oligonucleotide
duplexes and their self-assemblies with polyamidoamine dendrimers.
Soft Matter 2008, 4, 294−302.
(47) Elsabahy, M.; Shrestha, R.; Clark, C.; Taylor, S.; Leonard, J.;
Wooley, K. L. Multifunctional hierarchically assembled nanostructures
as complex stage-wise dual-delivery systems for coincidental yet
differential trafficking of siRNA and paclitaxel. Nano Lett. 2013, 13,
2172−2181.
(48) Gao, H.; Elsabahy, M.; Giger, E. V.; Li, D.; Prud’homme, R. E.;
Leroux, J. C. Aminated linear and star-shape poly(glycerol
methacrylate)s: synthesis and self-assembling properties. Biomacromo-
lecules 2010, 11, 889−895.
(49) Shrestha, R.; Elsabahy, M.; Luehmann, H.; Samarajeewa, S.;
Florez-Malaver, S.; Lee, N. S.; Welch, M. J.; Liu, Y.; Wooley, K. L.
Hierarchically assembled theranostic nanostructures for siRNA
delivery and imaging applications. J. Am. Chem. Soc. 2012, 134,
17362−17365.
K
DOI: 10.1021/acs.biomac.8b00069
Biomacromolecules XXXX, XXX, XXX−XXX