Well-defined star-shaped polyglutamates with improved pharmacokinetic profiles as excellent candidates for biomedical applications

Article abstract of DOI:10.1021/acs.molpharmaceut.5b00358

There is a need to develop new and innovative polymer carriers to be used as drug delivery systems and/or imaging agents owing to the fact that there is no universal polymeric system that can be used in the treatment of all diseases. Additionally, limitat

Full text of DOI:10.1021/acs.molpharmaceut.5b00358

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Article
WELL-DEFINED STAR-SHAPED POLYGLUTAMATES
WITH IMPROVED PHARMACOKINETIC PROFILES AS
EXCELLENT CANDIDATES FOR BIOMEDICAL APPLICATIONS
Aroa Duro-Castano, Richard M. England, David Razola, Eduardo
Romero, Marta Oteo-Vives, Miguel Angel Morcillo, and Maria J. Vicent
Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00358 • Publication Date (Web): 10 Sep 2015
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WELLꢀDEFINED STARꢀSHAPED
POLYGLUTAMATES WITH IMPROVED
PHARMACOKINETIC PROFILES AS
EXCELLENT CANDIDATES FOR BIOMEDICAL
APPLICATIONS
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Aroa Duro-Castano,1 Richard M. England,1 David Razola,2 Eduardo Romero,2 Marta Oteo-
Vives,2 Miguel Angel Morcillo,2 María J. Vicent1*
1. Polymer Therapeutics Lab., Centro de Investigación Príncipe Felipe (CIPF), Av. Eduardo
Primo Yúfera 3, Valencia 46012, Spain.
2. Biomedical Applications of Radioisotopes and Pharmacokinetics Unit, CIEMAT, Av.
Complutense 40, Madrid 28040, Spain
KEYWORDS ((Ringꢀopening polymerization, polypeptides, starꢀshaped polymers,
polyglutamates, NCA polymerization, polymer therapeutics, drug delivery))
ABSTRACT
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There is a need to develop new and innovative polymer carriers to be used as drug delivery
systems and/or imaging agents owing to the fact that there is no universal polymeric system that
can be used in the treatment of all diseases. Additionally, limitations with existing systems such
as lack of biodegradability and biocompatibility inevitably lead to side effects and poor patient
compliance. New polymer therapeutics based on amino acids are excellent candidates for drug
delivery, as they do not suffer from these limitations. This article reports on a simple yet
powerful methodology for the synthesis of 3ꢀarm starꢀshaped polyglutamic acid with wellꢀ
defined structures, precise molecular weights (MW) and low polydispersity (Đ = <1.3). These
were synthesized by ring opening polymerization (ROP) of Nꢀcarboxyanhydrides (NCA) in a
divergent method from novel multifunctional initiators. Herein, their exhaustive physicochemical
characterization is presented. Furthermore, preliminary in vitro evaluation in selected cell
models, and exhaustive in vivo biodistribution and pharmacokinetics highlighted the advantages
of these branched systems when compared with their linear counterparts in terms of cell uptake
enhancement and prolonged plasma halfꢀlife.
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1. Introduction
There has been a considerable effort devoted to the development of new and more
versatile polymeric architectures with specific and predictable properties to be used as targeted
drug delivery systems.1 Such desirables features in these materials include; adjustable molecular
weights (higher MW to enhance passive targeting by the Enhanced Permeability and Retention
(EPR) effect),2ꢀ4 predictable structure and conformation in solution, lower heterogeneity, and
greater possibility for multivalency. Nevertheless, the design and synthesis of new polymeric
constructs of relevant MW, together with their physicochemical characterization, conformational
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studies, and especially their potential for biological applications still remain to be fully exploited
in this area. To this aim, polypeptideꢀbased architectures can be considered suitable aspirants.
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The ring opening polymerization (ROP) of αꢀaminoꢀNꢀcarboxyanhydrides (NCAs)5ꢀ7
can be considered an excellent method to produce a wide variety of polypeptide architectures
including homopolymers, copolymers, block copolymers, and branched systems that do not exist
naturally, on multigram scale with relative ease. Since the first NCA synthesis by Leuch and
coworkers8ꢀ10 many methodologies have been described with the aim of improving and
overcoming the inherent limitations of this polymerization technique, as reviewed in the
following excellent literature.5ꢀ7 Among them, the use of high vacuum techniques (HVT),11
amine hydrochloride salts,12 heavy metal catalysts,13 hexamethyldisilazanes (HMDS)14 as well as
by means of optimization of reaction conditions (pressure, temperature etc.)15 can be considered.
Nevertheless, all methods have their own limitations. Previously in our group we have reported
the use of primary amine tetrafluoroborate (BF4) salts as an improvement over the use of
hydrochloride salts. This potent alternative is based on the nonꢀnucleophilicity of the BF4 salts
allowing the synthesis of wellꢀdefined polyglutamates (amongst other polypeptides), on
multigram scale and with low polydispersity (Đ<1.3), adjustable MW, controlled chain end
functionality and adequate stereoselectivity, ideal features for biomedical applications.16,17
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Star polypeptides are branched polymers, which consist of various linear chains linked
to a central core. There are two main synthetic strategies described: the coreꢀfirst approach (or
multifunctional initiators or divergent approach) and the armꢀfirst approach (or the use of
multifunctional linking agents, or convergent approach). The coreꢀfirst approach is based on the
use of a multifunctional initiator that simultaneously initiates the polymerization of several arms
while in the arm first approach the living arms are linked to a multifunctional agent. Besides
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those technologies, there is a recent classification including another strategy based on the
reaction of living macroinitiators (MI) (or macromonomers) with a multifunctional crossꢀlinker
to provide the soꢀcalled core crossꢀlinked star (CCS) polymers.
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Various polypeptideꢀbased star polymers have been synthesized over the years. For
example, Klok et al.18 used perylene derivatives with four primary amine groups as initiators to
lead 4ꢀarm PBLG and PZLL and Inoue et al.19ꢀ20 used hexafunctional initiators for the synthesis
of 6ꢀarm PBLG star polymers both taking profit of the NCA polymerization techniques. Other
examples are provided from the work of Aliferis et al.21 who used 2ꢀ(aminomethyl)ꢀ2ꢀmethylꢀ
1,3ꢀpropanediamine as a trifunctional initiator for the synthesis of P(BLLꢀbꢀBLG)3 3ꢀarm starꢀ
block coꢀpolypeptides; or the studies of Karatzas et al.22 in the synthesis of 4ꢀarm (PEOꢀbꢀ
PBLG) hybrid star block coꢀpolymers using 4ꢀarm PEO stars endꢀfunctionalized with primary
amines as initiators for the polymerization of BLGꢀNCA among others. More recent examples
include the use of dendritic cores based on poly(ethylene imine) (PEI),23 poly(propylene imine)
(PPI)24ꢀ26 or poly(amidoamine) (PAMAM)27 as initiators. All these examples are based on the
use of the amine initiated NCA approach with its inherent limitations that could lead to star
polymers with either low molecular weight or with very broad molecular weight distributions.
The use of HVT from Hadjichristidis group can be seen as a suitable solution,21ꢀ22 however, the
requirement of complex equipment is always a disadvantage in order to develop a versatile
synthetic procedure.
Despite the growing interest in the development of hybrid and peptideꢀbased star
polymers as prospective advanced materials for biological applications, only recently, they have
been explored as drug delivery systems. For instance, Sulistio et al.28ꢀ30 synthesized peptideꢀ
based CCS polymers composed of PLL arms emerging from a poly(Lꢀcysteine) (PLC) able to
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entrap hydrophobic drugs, such as the antiꢀcancer drug pirarubicin. Xing et al.31 prepared CCS
polymers using MeOPEG1900ꢀNH2 as a macroinitiator for ROP cysteine and benzyl glutamate
NCA derivatives which resulted in the formation of nanogels able to entrap the hydrophobic drug
indomethacin. Also, the PPI initiated multiꢀarm stars from Byrne et al. were efficiently loaded
with rhodamine B.25
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Taken into account all the stated above, herein, we pursued the development of novel
starꢀshaped architectures for their use as polymeric carriers by means of a simple NCA
polymerization methodology. Our approach does not require of neither complex and expensive
equipment nor low temperatures, but is capable to yield starꢀshaped polypeptides with low
polydispersities in a relatively short time and in a variety of MW including high molecular mass.
As the applicability of these novel constructs will be first demonstrated as nanocarriers, their in
vitro validation in selected cell models was also performed, in terms of biodegradability, cell
viability and cellular uptake. Furthermore, their in vivo biodistribution profile and
pharmacokinetics in comparison with their linear polymeric counterpart is also presented.
2. Experimental Section
Materials
All chemicals were reagent grade, obtained from Aldrich and used without further
purification, unless indicated otherwise. HꢀLꢀGlu(OBzl)ꢀOH, was obtained from Iris Biotech.
Phenazinemethosulfate (PMS) were supplied by Sigma (Sp). Dulbecco’s Modified Eagle’s
Medium (DMEM), Leibovitz, Phosphate buffer saline (PBS), Foetal bovine serum (FBS) and
Trypsin,
were
provided
from
Gibco.
(3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ5ꢀ
(3ꢀ
carboxymethoxyphenyl)ꢀ2ꢀ(4ꢀsulfophenyl)ꢀ2Hꢀtetrazolium) (MTS) was supplied by Promega
(Sp). DO3AtBu was purchased from Chematec. 111In was obtained from Covidien Spain, S.L. All
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solvents were of analytical grade and were dried and freshly distilled. Deuterated chloroformꢀd1,
DMSOꢀd6, DMFꢀd7 and D2O were purchased from Deutero GmbH. Preparative SEC was
performed using Sephadex Gꢀ25 superfine from GE as well as PD MiniTrap Gꢀ10 ™ columns
containing 2.1 mL of Sephadex™ Gꢀ10. Ultrafiltration was performed in a Millipore
ultrafiltration device fitted with a 1, 3, or 10 kDa molecular weight cut off (MWCO) regenerated
cellulose membrane (Vivaspin ®)
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Characterization techniques
NMR spectroscopy.1H and 13CꢀNMR spectra were recorded on a Bruker AC 300 at room
temperature and at a frequency of 300 and 75 MHz respectively and analyzed using the
MestreNova 6.2 software.
Dimethylformamide (DMF) gel permeation chromatography (GPC). For SEC measurements in
DMF containing 1g/L of lithium bromide as an additive, an Agilent 1100 series system was used
with a flow rate of 1 mL/min at 70 °C as an integrated instrument, including three HEMAꢀbased
columns (105/103/102 Å porosity) from MZꢀAnalysentechnik GmbH, Viscoteck TDATM 302
triple detector 87 with UV detection coupled, and Calibration was achieved with wellꢀdefined
poly(methyl methacrylate) (PMMA)/DMF standards, provided by Polymer Standards Service
(PSS)/Mainz Germany.
Circular Dichroism (CD). CD Spectroscopy was performed with a Jꢀ815 CD Spectrometer
(JASCO Corporation) using a Peltierthermostated cell holder (PTCꢀ423, JASCO Corporation)
with a recirculating cooler (JULABO F250, JASCO Corporation). A nitrogen flow (~2.7 L/min)
was lead through the spectrometer and controlled with a nitrogen flow monitor (Afriso Euroꢀ
Index).The samples were dissolved in HFIP for protected and PBS pH=7.4 for deprotected
samples and diluted to a concentration of 0.1 mg/mL. Samples were measured repeatedly (n=3)
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in a quartz cuvette with d= 0.1 cm at 20 °C for HFIP samples and 37 ºC for PBS dissolved
samples. Obtained molar ellipticities were plotted as mean residue ellipticity.
Dynamic Light Scattering (DLS). DLS measurements were performed at 25 °C using a Malvern
ZetasizerNanoZS instrument, equipped with a 532 nm laser at a fixed scattering angle of 173°.
Polymer conjugate solutions (0.2 mg/mL) were prepared in PBS pH= 7.4. The solutions were
sonicated for 10 min and filtered through a 0.45 ꢁm cellulose membrane filter. Size distribution
by volume was measured (diameter, nm) for each conjugate per triplicate with n>3
measurements.
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Protocols
Monomer Synthesis: Synthesis protocol followed as that reported in ConejosꢀSanchez et al.16ꢀ17
Detailed protocol can be found in Supporting Information (SI).
Initiator Synthesis: For clarity reasons, only the ethylꢀbased initiator will be included in the
manuscript. The others are included in the Supporting Information.
a) Synthesis of 1,3,5ꢀTriꢀtertꢀbutyl ((benzenetricarbonyltris(azanediyl)) tris(ethaneꢀ2,1ꢀ
diyl))tricarbamate. In a twoꢀneck round bottom flask fitted with a stirrer bar, and a N2 inlet and
outlet, 500 mg of 1,3,5ꢀbenzenetricarbonyl trichloride (1.88 mmol, 1 equivalent (eq.)) was
dissolved in 12 mL of anhydrous THF. N,N’,N’’ꢀdiisopropylethylendiamine (DIEA) (803.31 mg,
6.22 mmol, 3.3 eq.) was added to the reaction mixture followed by dropꢀwise addition of NꢀBocꢀ
ethylendiamine (1.34 g, 6.22 mmol, 3.3 eq.) over a period of 10 min. The reaction was then left
to proceed for 2 hours. After that time, the solvent was completely removed under vacuum. The
product was reꢀdissolved in chloroform and washed 3 times with deionized water (ddH2O), and 3
times with acidic water (pH~3). Finally, the organic phase was isolated under vacuum and the
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product was recrystallized 3 times from THF/Methanol/Hexane yielding a white crystalline solid.
The product was then dried under high vacuum and stored at ꢀ20 °C.
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Yield: 82 %. 1H NMR (300 MHz, DMSO) δ 8.68ꢀ8.65 (m, 3H), 8.41 (s, 3H), 6.92ꢀ6.88(m, 3H),
3.34ꢀ3.31 (m, 6H), 3.16ꢀ3.13 (m, 6H), 1.37 (s, 27H).13C NMR (75 MHz, CDCl3) δ 166.80
(C=O), 156.84 (C=O), 134.58 (CAr quaternary), 128.47 (CHAr), 79.57 (C quaternary), 40.93
(CH2), 40.43 (CH2), 28.45 (CH3).
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b) Synthesis of 1,3,5ꢀ(benzenetricarbonyltris(azanediyl))triethanamonium BF4 salt. In a round
bottom flask fitted with a stirrer bar and a stopper, 200 mg of 1,3,5ꢀTriꢀtertꢀbutyl
((benzenetricarbonyltris(azanediyl)) tris(ethaneꢀ2,1ꢀdiyl))tricarbamate (0.33 mmol, 1 eq.) was
dissolved in dichloromethane. Afterwards, 3.3 eq. of tetrafluoroboric acid diethyl ether complex,
HBF4.Et2O, (179 mg, 150 µL), was added to the solution leading to the instantaneous formation
of a white solid. The precipitate was filtered off and recrystallized three times from
THF/methanol/hexane. The product was then dried under high vacuum and stored at ꢀ20 °C.
Yield: 98 %. 1H NMR (300 MHz, D2O) δ 8.32 (s, 3H), 3.72ꢀ3.68 (m, 6H) 3.25ꢀ3.21 (m, 6H).13C
NMR (75 MHz, D2O) δ 169.45 (C=O), 134.38 (CAr quaternary), 129.36 (CAr), 39.23 (CH2),
37.52 (CH2).19FꢀNMR: ꢀ150.48 (BF4). MALDIꢀTOF: 337.1709 [M+1]
St-PBLG Polymer Synthesis: As for ConejosꢀSanchez et al. 16ꢀ17 with slight variations, briefly, γꢀ
Benzyl Lꢀglutamate Nꢀcarboxyanhydrides (0.5 g, 1.9 mmol) was added to a Schlenk tube fitted
with a stirrer bar, a stopper and purged with 3 cycles of vacuum/Ar, and dissolved in 5 mL of the
freshly purified solvent. The 3ꢀarm initiator was added and the mixture was left stirring at 4 °C
for 3 days under inert atmosphere. Finally, the reaction mixture was poured into a large excess of
1
cold diethyl ether leading to a white polymer after isolation. Yield: 70ꢀ90 %. HꢀNMR (300
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MHz, DMF, δ) 8.58 (s, xH), 7.42 (s, 5H), 5.19 (s, 2H), 4.21 (s, 1H), 2.81 (s, 2H), 2.45 (s,
2H).13CꢀNMR (300 MHz, DMF, δ) 175.94 (s), 172.26 (s), 162.77ꢀ162.18 (m), 161.98 (s), 136.76
(s), 128.87ꢀ127.75 (m), 66.05 (s), 57.13 (s), 35.41ꢀ34.17 (m), 32.48 (s), 30.84, 30.30 ꢀ29.04 (m),
27.28 (s), 25.99 (s). x: DP obtained/3 arms
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Deprotection: different methods were followed depending on the initiator used: acid conditions
were applied when ethyl and hexyl based initiators were used. On the other hand, basic
conditions were applied for DOOA (3,6ꢀdioxaꢀ8ꢀoctaneamine), and cystamine based initiators
synthesis. Briefly:
Deprotection of St-PBLG with HBr in trifluoroacetic acid (TFA): in a round bottom flask fitted
with a glass stopper and a stirrer bar, 50 mg of StꢀPBLG (0.23 mmol, 1 eq. glutamic acid unit,
GAU) were dissolved in TFA. Once dissolved, 2 eq. of HBr (48 % v/v) per carboxyl group were
added drop wise, and the mixture was stirred for five hours. *Note: For big scale deprotection of
StꢀPBLG, 16 hours were applied for full deprotection. Then, the solution was poured into a large
excess of cold diethyl ether leading to a white solid after isolation.
Deprotection of St-PBLG in basic conditions: in a round bottom flask, 40 mg of benzyl protected
StꢀPBLG (0.183 mmol, 1 eq. GAU) was dissolved in 16 mL of THF. The solution was cooled
down to 4 °C and 2 eq. of NaOH per carboxylic group of the polypeptide block (14.7 mg, 0.369
mmol) were added in 2 mL of ddH2O. The mixture was stirred for 16 hours. Finally, THF was
removed and the residue was diluted with ddH2O, concentrated and purified by ultrafiltration
(Vivaspin®, MWCO= 3000 Da) or by size exclusion columns (G25).
Yields: 75ꢀ86 %. 1HꢀNMR (300 MHz, D2O, δ) 8.2 (s, xH), 4.31ꢀ4.26 (m, 1H), 2.38ꢀ2.14 (m, 2H)
2.10ꢀ1.80 (m, 2H) 2.10ꢀ1.80 (m, 2H). x: DP obtained/3 arms
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Note: depending on the initiator used, the corresponding signals of the Ethyl, Hexyl, or DOOA
were also present.
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Conjugation of Oregon Green Cadaverine to linear and star PGA: In a round two necked
bottom flask fitted with a stirrer bar and two septums, 29 mg of PGA (linear or star) (0.225 mmol
GAU, 1 eq.) was weighed and dissolved in 1.5 mL of dry DMF under N2 flow. Of
N,N’Diisopropylcarbodiimide (1.12 ꢁL) and DIC (0.85 mg, 0.00674 mmol, d= 0.806 g/mL, 0.03
eq.) were added and the reaction was left to proceed for 5 min at room temperature. Afterwards,
Hydroxybenzotriazole, HOBt (1 mg, 0.00674 mmol, 0.03 eq.) was added directly. The reaction
was then left to proceed for 10 min before Oregon Green Cadaverine (1 mg, 2.25·10ꢀ3 mmol,
0.01 eq.) was added. The pH was adjusted to 8 by adding ~100 ꢁL of DIEA. The mixture was
left stirring overnight protected from light. Finally, the solvent was removed under vacuo at
room temperature and the product was dissolved in 300 ꢁL of water and then adding ~50 ꢁL of
NaHCO3 1M. The solution was purified by Sephadex PD10 column eluting with distilled water.
The Oregon Green loading was calculated by fluorescence using a Victor2WallaceTM plate reader
with excitation filter of 490 and emission filter of 535. A calibration curve with Oregon green
was first performed. Yield: 95 %. OG loading: 0.8 mol glutamic acid unit.
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Conjugation of DO3AtBu-NH2 to St-PGA: In a twoꢀneck round bottom flask fitted with a stir bar
and two septums, 300 mg (of StꢀPGA (SE2), 110 GAU, 2.32 mmol GAU, 1 eq.) was dissolved in
20 mL of anhydrous DMF under nitrogen flow. Then, 53 ꢁL of DIC (88 mg, 0.70 mmol, 0.3 eq.)
were added and the reaction was left to proceed for 5 min at room temperature. Afterwards,
HOBt (94 mg, 0.70 mmol, 0.3 eq.) was added directly. The reaction was then left to proceed for
10 min before DO3AtBuꢀNH2 (282 mg, 0.46 mmol, 0.2 eq.) was added for 20 % modification.
The pH was adjusted to 8 by adding ~100 ꢁL of DIEA. The mixture was left stirring for 48 hours
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at room temperature and protected from light. Finally, the solvent was partially removed under
vacuo, precipitated into a large excess of cold acetone, filtered off and washed three times with
cold acetone. A pale yellow solid was obtained after drying. The percentage of modified GAU
was calculated as 20 % mol GAU, according to the tBu groups signal at 1.4 ppm in comparison
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with the alpha proton of PGA backbones in HꢀNMR spectra. Conjugation efficiency: 100 %.
Yield: 75 %.
Deprotection of DO3A tBu-NH2: The construct was dissolved in CH2Cl2/TFA (3/2, v/v) mixture
and left under vigorous stirring for 16 hours at room temperature. After that time, the solution
was precipitated by pouring into a large excess of cold diethyl ether. Pale yellow solid was
obtained after filtering, washing with diethyl ether and drying under vacuum. Complete
deprotection was achieved as confirmed by 1HꢀNMR. Yields: 90 %.
Labelling with 111In: StꢀPGAꢀDO3Aꢀ111In was prepared by dissolving 51.3 mg of StꢀPGAꢀDO3A
in deionized water to a final concentration of 10 mg/mL. Then, 0.25ꢀ 0.5 mL of this dissolution
was transferred into a microwave tube and the pH was adjusted to 3.5ꢀ4 by adding HEPES buffer
and HCl 2 M. Next 7ꢀ27 MBq of 111InCl3 in HCl 0.05 M was added and the reaction mixture
heated at 90 ºC for 5 min by using a laboratory microwave with monomodal radiation (Discover
Benchmate, CEM). After that, the reaction mixture was cooled down with nitrogen gas. The
reaction was stopped after 5 min at room temperature by the addition of 50 ꢁL of 50 mM
ethylenediaminetetraacetate acid (EDTA). StꢀPGAꢀDO3Aꢀ111In was purified from unreacted
111InꢀEDTA by exclusion molecular chromatography cartridge (Bio Gel Pꢀ6, BioRad) using
phosphate buffered saline (pH=7) as eluent, at flow rate 0.5 mL/min. The elution profile was
determined by fractionating, 0.77 mL per fraction, and measuring each with a dose calibrator
(VDC 405, Veenstra). Radiochemical yield (RY) was calculated as percentage of the activity in
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each fraction eluted from the molecular exclusion cartridge of the total activity purified and
corrected for the decay.
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Degradations with Cathepsin B: StꢀPGA was degraded in presence of the lysosomal enzyme
cathepsin B as its linear counterpart. To test the profile and ratio of the degradation of the
polymers by cathepsin B, solutions of the polymers (3 mg/mL) were prepared. 3 mg exactly were
weighed and 700 µL of acetate buffer 20 mM, pH 6, 100 µL of EDTA 2mM, 100 µL of DTT
5mM were added. Finally, 6.25 units of Cathepsin B (100 µL of a solution of 25 units of
cathepsin B in 400 µL of acetate buffer pH 6 20 mM were added. Cathepsin B needs pH 6 to be
active, the DTT solution is added to activate Cathepsin B and the solution of EDTA was added to
complex the free cations (mainly Ca2+ that inactivates cathepsins). Once the solutions were
prepared, aliquots of 100 µL were picked at different time points (t= 0, 0.5, 1, 2, 4, 8, 24, 48 and
72 hours) after homogenization of the solutions. Meanwhile, the samples were kept at 37 ºC
under stirring. The aliquots taken were frozen and later analyzed by GPC. To evaluate the mass
of the conjugates, 100 ꢁL of 3 mg/mL conjugate solution in PBS was injected in the GPC using
two TSK Gel columns in series G2500 PWXL and G3000 PWXL with a Viscoteck TDATM 302
triple detector 87 with UV detection coupled. The mobile phase used was PBS 0.1 M, flow 1
mL/min
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Cell culture protocols: Human umbilical vein endothelial (HUVEC) cells were cultured in
Medium 200 supplemented with Low Serum Growth Supplement (LSGS). Human derived
neuroblastoma (SHSY5Y) cells were cultured in Dubelcco’s Modified Eagle Medium (DMEM)
supplemented with Lꢀglutamine and Foetal Bovine Serum (FBS). Cells were maintained at 37 °C
in an atmosphere of 5 % carbon dioxide and 95 % air. Medium was replaced every 2ꢀ3 days and
underwent passage once weekly when 80 % of cell confluence was reached.
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MTS assay for cell viability determination: Cells were seeded in sterile 96ꢀwell microtitre plates
at a cell density of 35000 cell/cm2 for SHSY5Y and 1260 cell/cm2 for HUVEC. Plates were
incubated for 24 hours and compounds (0.2 ꢁm filter sterilized) were then added to give a final
concentration of 0ꢀ3 mg/mL. After 72 hours of incubation, [3 ꢀ (4,5ꢀdimethylthiazolꢀ2ꢀyl) ꢀ 5 ꢀ
(3ꢀcarboxymethoxyphenyl) ꢀ 2 ꢀ (4ꢀsulfophenyl)2H ꢀ tetrazolium] (MTS) (10 µL of manufacturer
solution) was added to each well, and the cells were incubated for a further 2 hours.
Mitochondrial dehydrogenase enzymes of viable cells converted MTS tetrazolium into a colored
formazan product. The optical density of each well was measured at 490 nm. The plates were
read spectrophotometrically using a Victor2 WallaceTM plate reader. The absorbance values were
represented as the percentage of cell viability taken as 100 % cell viability of untreated control
cells.
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Cellular uptake by flow cytometry of OG-labeled polymers in SHSY5Y cells: SHSY5Y cells were
seeded in 6ꢀwell plates at a density of 35000 cell/cm2 (1 mL cell suspension per well) and
allowed to adhere for 24 hours. In the binding experiments conducted at 4 °C, the cells were preꢀ
incubated at this temperature for 30 min prior to start the experiment. For both experiments, 4 ºC,
and 37 ºC, the cathepsin B inhibitor CAꢀ074 (0.4 µL from a solution of 5µM to reach a final
concentration of 2 µM) was added 30 min before the addition of the conjugate. Then, 10 ꢁL of
OGꢀlabeled polymer (0.01 mg OG/mL) were added at different time points from 0 to 300 min
meanwhile the cells were incubated either at 37°C or 4°C for each experiment. Finally, the cells
were placed on iced in order to stop the energy dependent mechanisms and washed twice with
cold PBSꢀBSA 0.1 %. (PBS supplemented with Bovine Serum Albumin (BSA)). Then the cells
were suspended in 0.5 mL of cold PBS by the use of a scraper. Finally, the cell pellet was placed
in flow cytometer tubes. Cellꢀassociated fluorescence was then analyzed using a Becton
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Dickinson FACS Calibur cytometer (California, USA) equipped with an argon laser (488 nm)
and emission filter for 550 nm. Data collection involved 10,000 counts per sample, and the data
were analyzed using CELLQuestTM version 3.3 software. Data are expressed by plotting the cellꢀ
associated fluorescence, which is the result of the % of positive cells by multiplied by the mean
fluorescence. CAF= % positive cells*mean fluorescence/100. Also, the % positive cells is
shown. Cells incubated without polymer were used as control in order to account for the
background fluorescence (cell autofluorescence).
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Confocal fluorescence microscopy: Live-cell imaging. For liveꢀcell imaging, SHSY5Y cells were
seeded at a density of 35000 cell/cm2, on glass bottom culture dishes (1 cm2 Petri plate) and
allowed to seed for 24 hours at 37 ºC. The experiment was performed following a pulseꢀchase
mode at 37 ºC. First, the cathepsin B inhibitor CAꢀ074 (0.4 µL from a solution of 5 µM to reach
a final concentration of 2 µM) was added 30 min prior the addition of the StꢀPGAꢀOG. Then, 10
ꢁL of OGꢀlabeled polymer (0.01 mg OG/mL) were added and the cells were incubated for 2
hours at 37 °C (pulse). The medium was removed and the cells were washed twice with PBS. 1
mL of medium containing 2 µM of the CAꢀ074 inhibitor was then added, and the cells were
incubated for 4 hours at 37 ºC (chase). 30 min before washing the cells with PBSꢀBSA 0.1 %, the
nuclear marker Hoetch (1 µL from a solution 5 mM), and the lysosomal marker Lysotracker Red
(0.75 µL from a solution of 100 µM) were added in order to identify possible coꢀlocalizations
and therefore establish an endocytic pathway. Finally, the cells were washed with PBSꢀBSA 0.1
%. Then, the glass removed and placed on the microscope chamber with fresh media containing
2 µM of CAꢀ074 inhibitor. Samples were analyzed under the microscope and images were
captured after excitation of the fluorophore. Images were captured with an inverted DM IRE2
microscope equipped with a λꢀblue 60X oil immersion objective and handled with a TCS SP2
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system, equipped with an Acoustic Optical Beam Splitter (AOBS). Excitation was performed
with an argon laser ((OG 496 nm) and HeNe laser (Lysotracker Red 594 nm), and blue diode
(Hoetch 405 nm). Images were captured at an 8ꢀbit grey scale and processed with LCS software
(version 2.5.1347a, Leica Germany) containing multicolor, macro and 3D components. Control
cells that followed the same incubation time were also analyzed to establish the
autofluorescence, as well as cells treated only with Hoetch or Lysotracker Red.
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Statistical Analysis. Values obtained from the experiments were analyzed using oneꢀway
ANOVA. In all cases, we considered differences to be significant when p*** < 0.001; p** <
0.01; p*< 0.05; ns: nonꢀsignificant.
Biodistribution and pharmacokinetics (PK): Biodistribution and pharmacokinetics of [111In]ꢀSt ꢀ
PGAꢀDO3A was evaluated by analysis of radioactivity. FVB/NJ adult mice (CIEMAT
Laboratory Animals Facility, Madrid, Spain) were housed on a 12ꢀhour light and 12ꢀhour dark
cycle. Free access to food and water was allowed at all times. All animal protocols were
approved by the Institutional Animal Care and Use Committee at CIEMAT (Madrid, Spain). For
the pharmacokinetics and biodistribution study, 30 mice (5 mice at each time point for each
compound, 23±1g of body weight) were anesthetized with 1.5 % isofluorane, and between 0.05ꢀ
2 MBq of [111In]ꢀSt ꢀPGAꢀDO3A (0.5ꢀ20 µg compound/g body weight) per mouse was injected
intravenously through the tail vein. At 0.5, 1, 2, 4, 8 and 24 hours following injection, blood
samples were obtained from mice by terminal bleeding via cardiac puncture following
isofluorane anaesthesia; organs (lungs, heart, spleen, kidneys, liver and brain) and tissues
(muscle, fat) were isolated, rinsed with normal saline, weighted, and radioactivity was measured
using the Cobra II autoꢀgamma counter. The blood samples were centrifuged (10 min, 3000 rpm)
and the supernatant of plasma was transferred to a new eppendorf tube for later analysis. The
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percentage injected dose per gram tissue (% ID/g) was calculated by comparison with standards
taken from the injected solution for each animal. The data were expressed as the mean ± standard
deviation.
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3. Results and Discussion
3ꢀarm star shaped polyglutamates have been synthesized throughout a facile and versatile
methodology based on the use of ammonium tetrafluoroborate (BF4) salts as initiators for NCA
polymerization16ꢀ17 to yield wellꢀdefined carriers with potential biological applications showing
tunable MW and low polydispersities (Scheme 1). Remarkably, this methodology allow us to
reach such described constructs in a high batch to batch reproducibility, within reasonable
reaction times and without the need of using complex or expensive equipment as for HVT, or the
incorporation of metal catalysts that, if not carefully removed, could compromise the desired bioꢀ
applications.
Scheme 1. a) Schematic representation of the NCA polymerization using starꢀshaped initiators.
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The control in the initiation step that this newly described methodology offers is key to
the synthesis of branched polymers with predictable molecular weights and low polydispersities.
By using a variety of multifunctional amine initiators in their BF4 salt form to ringꢀopen
polymerize the benzylꢀLꢀglutamate NCA results in a fast and homogeneous initiation and
efficient propagation to achieve wellꢀdefined architectures. To this aim we synthesized four
different multifunctional 3ꢀarm initiators, including one that contained disulfide bonds within the
arms. In the presence of a reducing agent (GSH or DTT) the star could then be disassembled into
the individual arms. This novel initiator for polypeptide synthesis enables the characterization of
the individual arms to ensure similar chain length resulting from a living polymerization and
efficient initiation. In all the cases, the synthesis of the initiator was carried out starting from the
multifunctional core 1,3,5ꢀbenzenetricarbonyl trichloride, to which, the monoꢀBoc protected
diamines, NꢀBocꢀethylendiamine NꢀBocꢀ1,6ꢀhexanediamine, 1ꢀ(tꢀButyloxycarbonylꢀamino)ꢀ3,6ꢀ
dioxaꢀ8ꢀoctaneamine (NꢀBocꢀDOOA), and NꢀBocꢀcystamine were efficiently coupled in a
simple and clean reaction. The monoꢀBoc protected 3ꢀarm initiator precursors were carefully
purified to avoid diꢀ or monoꢀfunctional cores that would lead to unwanted mixtures of 1ꢀ and 2ꢀ
arm star polymers. Once purified, the BF4 ammonium salt initiators were obtained simply by the
removal of the Boc groups using tetrafluoroboric acid.
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We have explored the versatility of the BF4 salts based methodology by polymerization
of OBzlꢀLꢀGlu NCA to yield 3ꢀarm star shaped polyglutamates with different molecular weights
ranging from 15000ꢀ50000 Da (Table 1, Figure 1) and polydispersities in the range of 1ꢀ1.3
independently of the initiator used. This appears to suggest that a large spacer between the
initiator core and the initiation site is not necessary, despite concerns over steric hindrance
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Table 1. Variety of initiators used in the polymerization processes and different DPs obtained for
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StꢀPBLGs, demonstrating the versatility of the technique.
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Mna
Mnb
(kDa) (kDa)
21.3 21.0 97 96
Star Initiator DPtheo
DPa DPb
Ð
SE1
SE2
SE3
SH1
SH2
SH3
SD1
SD2
SD3
SD4
100
150
250
75
1.26
Ethyl
based
24.0 27.6 110 126 1.22
50.3 51.5 229 235 1.09
16.4
ꢀ
75
ꢀ
1.25
Hexyl
based
150
250
75
23.9 23.6 109 108 1.23
51.5 52.7 235 240 1.17
15.7 16.9 72 77
1.13
100
150
200
22.2 24.1 101 110 1.23
33.2 31.1 152 142 1.10
40.4 41.6 185 190 1.12
DOOA
based
Cyst
SS1
200
43.1
ꢀ
196 ꢀ
1.22
based
a) Data obtained by SEC in DMF/LiBr (1 %) at 8 mg/mL. b) Data obtained by 1HꢀNMR from the
deprotected polymers by integrating the aromatic signal of the initiator. DP=degree of
polymerization, Ð =polydispersity
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Figure 1. a) Selected GPCs from starꢀshaped polyglutamates (StꢀPBLG) in DMF (1 % LiBr) at 8
mg/mL. b) Circular Dichroism (CD) of benzyl protected starꢀshaped polyglutamates at 20 ºC in
1,1,1,3,3,3ꢀhexafluoroisopropanol (HFIP) at 0.1 mg/mL.
The polymers were characterized using a number of physicoꢀchemical analytical
techniques, always yielding the desired molecular weight by Gel Permeation Chromatography
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(GPC) and proton Nuclear Magnetic Resonance HꢀNMR. The data obtained from GPC and Hꢀ
NMR regarding DP was always consistent. Wellꢀdefined polymers were obtained even up to 50
kDa (Table 1). This is a highly significant fact and a major benefit offered by the technology we
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are describing herein, since most star polypeptides described in the literature have not achieved
high MW with narrow polydispersities.25ꢀ34 CD spectra from the benzyl protected samples in
HFIP showed the typical alpha helix conformation from PBLGs (Figure 1b).
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All data obtained so far indicates an excellent control over the polymerization using
these initiators, however, to fully demonstrate this fact, we endeavored to prove that the arms on
the stars were of equal molecular weight and therefore with a low polydispersity.
To do this we employed the aboveꢀmentioned initiator bearing disulfide bonds on the
arms that was very efficiently reduced by using DTT, thus releasing the individual arms allowing
their monitoring by SEC. Remarkably, we were able to almost perfectly disassemble the star
polymers to yield linear chains of 1/3 MW of the original star and with low polydispersities
(<1.25) (Figure S1). This evidence, in addition to the expected MW of the stars from GPC and
1HꢀNMR fully demonstrate that the star polymers synthesized have very similar length arms
resulting from fast and efficient initiation and controlled propagation. Knowing that this
synthesis of polyglutamateꢀbased stars leads to homogenous material makes them much more
favorable candidates to be used as drug delivery carriers or imaging probes.
In order to have a full understanding on the quality of the star polymers synthesized, the
analysis of the deprotected polymers needed to be also performed. The benzyl protecting groups
were removed using conventional methods already reported for linear polymers.16ꢀ17 However, it
was important to choose the correct deprotection approach depending on the initiator used. For
example, to maintain the integrity of the DOOA core, a procedure using sodium hydroxide with
previously optimized conditions capable of preserving the polyglutamate stereochemistry whilst
16ꢀ17
at the same time completely removing the benzyl protecting groups was used.
Standard
HBr/TFA acidic conditions were used in all other cases. The complete removal of the benzyl
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protecting groups was certified by HꢀNMR (7.4 ppm and 5.3 ppm) and the remaining aromatic
signal observed, therefore, were assigned to the benzylic core of the star at 8.2 ppm (3 protons).
Integration of this signal against those of glutamic acid provided the average number of units per
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chain and consequently the MW. In all cases, the MW obtained by HꢀNMR was in agreement
with that predicted by GPC for the benzyl protected precursor.
After deprotection, the expected random coil conformation of the PGA chains was
observed in all samples when analyzed using Circular Dichroism (CD) (Figure 2). Finally, DLS
(Dynamic Light Scattering) at 0.2 mg/mL showed particle sizes between 4ꢀ6 nm in
hydrodynamic radius for all deprotected StꢀPGA, comparable to those obtained for their linear
counterparts of similar MW (also in the range of 4ꢀ6 nm radius) (Figure 2).
To be sure that the enzymeꢀdependent biodegradability of the polyglutamateꢀbased stars
had not been compromised by the architecture, all polymers were incubated in presence of the
lysosomal enzyme cathepsin B. The degradation rate or MW loss at different time points was
monitored by GPC as described in the Experimental Section. As expected, the star polymers
were found to be degraded by the lysosomal enzyme with similar kinetics independently of the
MW and initiator used for the polymerization (Figure 3).
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Figure 2. a) HꢀNMR of StꢀPGAs of different molecular weights in D2O. The red square is
surrounding the benzyl core signals. b) CD of StꢀPGAs in PBS at 37 ºC at 1 mg/mL showing
typical random coil conformation of PGA chains. c) Example of particle size obtained by DLS
from StꢀPGAs at 0.2 mg/mL in PBS 7.4 at 25 ºC.
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Figure 3. In vitro evaluation of the newly synthesized StꢀPGA (benzyl deprotected) carriers. a)
Example of degradation profile by cathepsin B of SE3 polymer monitored by GPC in PBS at 3
mg/mL and at different time points. b) Toxicity assay against SHSY5Y cell line of different Stꢀ
PGAs measure by MTS assay at 72 hours postꢀtreatments.
Another key feature for the validation of the newly synthesized 3ꢀarm star shaped
polymers as potential drug delivery carriers or imaging probes is their toxicity in cell cultures. To
this respect, 72 hours MTS assays were performed against SHSY5Y human derived
neuroblastoma cell line as well as in HUVEC human umbilical vein endothelial cells. Polymers
were found to be nonꢀtoxic up to 3 mg/mL (Figure 3, and Figure S2).
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For macromolecular therapeutics and nanoꢀsized drug delivery systems, fluorescent
labeling is commonly applied to allow intracellular trafficking studies, conjugate cellꢀspecific
localization and/or in vivo fate and PK. Probes such as the fluorophore Oregon Green (OG) have
been extensively reviewed for cellular studies to determine cell uptake and binding.32ꢀ34 To this
aim, the conjugated probe must fulfill some requirements such as high stability of the probe itself
as well as stability of the linkage point to ensure adequate carrier monitoring. On the other hand,
the minimal probe loading is desirable in order to avoid data misinterpretation due to changes in
the polymer conformation resulting from changes in charge and solubility. Thus, a minimal
amount of OG (0.8 % mol) was successfully conjugated to the starꢀshaped polyglutamates using
diisopropylcarbodimide (DIC) / Hydroxybenzotriazole (HOBt) as carboxylic acid activators
(Scheme S1).
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Flow cytometry (cell uptake and binding) together with liveꢀcell confocal microscopy
analysis (subcellular fate and pathway) in SHSY5Y human derived neuroblastoma cell line, were
used to study cellular trafficking of the OGꢀlabeled starꢀshaped polymers (Figure 4). Flow
cytometry experiments were carried out at different temperatures, 37 °C (to measure the total
uptake) and 4 °C (to measure cell binding) in order to determine the presence of energy
dependent or nonꢀdependent internalization mechanisms, such as endocytosis or diffusion,
respectively. It is worth mentioning that all experiments were done in presence of the cathepsin
B inhibitor CAꢀ047 in order to avoid the degradation of the polyglutamic acid chains along the
incubation periods.
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Figure 4. In vitro evaluation of the newly synthesized carriers. a) Uptake study by flow
cytometry of a starꢀshaped fluorescently labeled PGA (SE3 deprotected) in SHSY5Y cell line.
Experiment at 4 ºC excludes the energy dependent mechanisms. The different among the uptake
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profile at 4 and 37 degrees (soꢀcalled “Energyꢀdependent” uptake) is also represented. CAF: Cell
associated fluorescence= % positive cells*mean fluorescence/100. The CAF represented
corresponds to the difference between CAF obtained with treated cells and CAF from untreated
control cells. b) Confocal image of the uptake at 2 hours postꢀtreatment of a starꢀshaped PGA in
SHSY5Y cell line following a pulseꢀchase experiment, with coꢀlocalization histogram. Coꢀ
localization with the lysosomal marker Lysotracker Red was observed.
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Liveꢀcell confocal imaging allows visualizing trafficking between multiple
compartments within individual living cells over time, avoiding any possible artifacts derived
from fixation protocols.34 Results from both techniques clearly showed an energyꢀdependent
mechanism of internalization due to the absence of uptake at 4 ºC as observed by flow
cytometry. This was further confirmed with the confocal microscopy studies at 2 hours postꢀ
treatment with an OG labeled polymer in SHSY5Y cells following a pulseꢀchase experiment,
where coꢀlocalization in the lysosomes was observed upon the use of lysosomal marker
Lysotracker Red (Figure 4b).
Interestingly, the StꢀPGAꢀOG showed a significant increase in cell uptake at 5 hours
when compared with linearꢀPGAꢀOG conjugate of similar MW (Figure 5). This might be
attributed to the inherent properties assigned to the starꢀshaped polymers. As general basis, star
polymers have a more compact structure, presumably with globular shape, and have large
surface areas, increased concentrations of functional end groups for polymers with equal
molecular weight, and unique rheological properties which make them optimal platforms for
drug delivery and imaging among other biological applications.35ꢀ36
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Figure 5. Uptake study of StꢀPGA (SE3, deprotected) in comparison with linear PGA of similar
MW (around 250 GAU). a) CAF of both polymers over time. b) CAF of both polymers at 5 hour
time point showing significant differences when statistics was performed using oneꢀway
ANOVA. p*< 0.05. c) % of positive (+) cells to Oregon Green (OG) fluorescence, comparison of
both polymers at 5 hour time point showing as expected statistical differences. p*< 0.05. d) % of
positive cells representation comparing both polymers together with the control used (cell
autofluorescence).
To further validate the synthesized nanocarriers, in vivo biodistribution as well as
pharmacokinetic profiles (PK) were obtained by radioactivity measurements. For that purpose, a
gamma emitting radionuclides 111In was introduced into the starꢀshaped PGAs through
complexation chemistry. In order to accomplish a stable complexation of the metal radioisotope,
the incorporation of bifunctional chelating agents into the polymer backbone is required. The
most commonly used chelating agents for 111In are based on polyamine carboxylic acids such as
diethylene triamine pentaacetic acid (DTPA), 1,4,7,10ꢀtetraazacyclododecaneꢀ1,4,7,10ꢀ
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tetraacetic acid (DOTA), or 1,4,7ꢀtriazacyclododecaneꢀ1,4,7ꢀtetraacetic acid (NOTA). For the
biodistribution of radiolabeled PGA based architectures, DOTA derivative chelating agent with a
free amine group suitable for conjugation (DO3AꢀNH2) was selected. It is wellꢀknown that,
DO3AꢀNH2 forms stable complexes with several M2+ and M3+ ions such as 68Ga or 111In.
Therefore, 20 % mol GAU of DO3AꢀtBuꢀNH2 was effectively conjugated via amide bond to a Stꢀ
PGA110 (Ð 1.23) following the strategy depicted in the Scheme S2.
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Conjugation efficiency was almost quantitative (since 10 % mol GAU was pursued)
with a reasonable mass yield of 75 %. The percentage of modification was calculated according
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to HꢀNMR analysis by comparing the corresponding integral of the CH alpha of PGA (4.24
ppm) with the 27 protons of the tBu groups at 1.40 ppm. The tBu groups were then easily
deprotected using the mixture CH2Cl2/TFA (3/2, v/v). Finally, the StꢀPGAꢀDO3A polymer was
labelled with 111In as described previously. Radiochemical yields were ≥ 85 % for StꢀPGAꢀ
DO3Aꢀ111In in all synthesis. In the exclusion molecular purification step (Figure 3S), the first 5
fractions were discarded and the next 3 fractions, containing the maximum specific activity of
StꢀPGAꢀDO3Aꢀ111In, were collected and used for further experiments.
Animal experiments to test the biodistribution and PK profile were then carried out with
i.v. injected doses between 37 KBq and 2.5 MBq of 111Inꢀlabelled polymers (1ꢀ20 µg/g body
weight) and monitored up to 24 hours (4ꢀ5 mice were sacrificed per time point 0.5, 1, 2, 4, 8 and
24 hours). Blood and organs were extracted and radioactivity was measured ex vivo in the
gamma counter.
According to the results obtained from the biodistribution, where the higher percentage
of injected dose (ID) corresponded to the kidneys, it can be concluded that these new polypeptide
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architectures follow renal excretion profiles with no specific accumulation in any organ (Figure
6).
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Figure 6.1 StꢀDO3Aꢀ111In biodistribution. Data expressed as normalized % ID per gram of tissue
at different time points.
The biodistribution profile obtained for StꢀPGA was then compared with the one obtained
for its linear counterpart of similar MW (100 GAU, Đ: 1.20).37 The biodistribution of linear PGA
was previously performed using 68Ga radioisotope, therefore, only short times (up to 3 hours)
could be recorded due to radionuclide decay (about 68 min for 68Ga). If short time points (0.5 h,
1 h and 2 h) of the % ID/g tissue of PGAꢀDO3Aꢀ68Ga and StꢀDO3Aꢀ111In are compared, a general
greater accumulation in all the organs of star shaped polymer is observed, in comparison with the
one found in the linear PGA construct as shown in Figure 7.
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