Synthesis and Characterization of Dendrimer-Based Polysarcosine Star Polymers: Well-Defined, Versatile Platforms Designed for Drug-Delivery Applications

Article abstract of DOI:10.1021/acs.biomac.0c00768

This paper describes the synthesis of star polymers designed for future drug-delivery applications. A generation-5 lysine dendrimer was used as a macroinitiator for the ring-opening polymerization of the sarcosine N-carboxyanhydride monomer to produce 32-arm star polymers with narrow molar mass distributions and desirable hydrodynamic size control. Fluorescent dye-labeled polymers were dosed in mice to measure plasma pharmacokinetics. Long circulation times were observed, representing ideal properties for biophysical targeting of tumors. In vivo efficacy of one of these star polymers conjugated to the therapeutic molecule SN-38 was evaluated in mice bearing SW620 xenografted tumors to demonstrate high antitumor activity and low body weight loss compared to the SN-38 prodrug irinotecan and this shows the potential of these delivery systems. As a further build, we demonstrated that these star polymers can be easily chain-end-functionalized with useful chemical moieties, giving opportunities for future receptor-targeting strategies. Finally, we describe the synthetic advantages of these star polymers that make them attractive from a pharmaceutical manufacturing perspective and report characterization of the polymers with a variety of techniques.

Full text of DOI:10.1021/acs.biomac.0c00768

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Article
Synthesis and characterization of dendrimer-based
polysarcosine star polymers: well-defined, versatile
platforms designed for drug delivery applications
Richard M. England, Jennifer Hare, Anders Gunnarsson, Jeremy Stephen Parker, and Marianne B. Ashford
Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.0c00768 • Publication Date (Web): 16 Jul 2020
Downloaded from pubs.acs.org on July 17, 2020
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Synthesis and characterization of dendrimer-based
polysarcosine star polymers: well-defined, versatile
platforms designed for drug delivery applications
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Richard M. England, ‡,† * Jennifer I. Moss, Anders Gunnarsson, Jeremy S. Parker,
§
¥
†
and Marianne B. Ashford.‡
‡Advanced Drug Delivery, Pharmaceutical Sciences, R&D, AstraZeneca, Macclesfield,
UK.
†Early Chemical Development, Pharmaceutical Sciences, R&D, AstraZeneca,
Macclesfield, UK.
§Early TDE, Oncology R&D, AstraZeneca, Cambridge, UK,
¥Discovery Sciences, Pharmaceutical Sciences, R&D, AstraZeneca, Gothenburg,
Sweden.
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KEYWORDS, dendrimer, star polymer, drug delivery, nanomedicine, Polymer
Therapeutics, polysarcosine, N-carboxyanhydride, stealth polymer, lysine, branched
polymer, ring opening polymerization, linker chemistry, SN-38.
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ABSTRACT
This paper describes the synthesis of star polymers designed for future drug delivery
applications. A generation-5 lysine dendrimer was used as a macroinitiator for the ring
opening polymerization of sarcosine N-carboxyanhydride monomer to produce 32-arm
star polymers with narrow molar mass distributions and desirable hydrodynamic size
control. Fluorescent dye labeled polymers were dosed in mice to measure plasma
pharmacokinetics. Long circulation times were observed, representing ideal properties
for biophysical targeting of tumors. In vivo efficacy of one of these star polymers
conjugated to the therapeutic molecule SN-38 was evaluated in mice bearing SW620
xenografted tumors to demonstrate high anti-tumor activity and low body weight loss
compared to the SN-38 prodrug, irinotecan, and show the potential of these delivery
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systems. As a further build, we demonstrated that these star polymers can be easily
chain-end functionalized with useful chemical moieties, giving opportunities for future
receptor targeting strategies. Finally, we describe the synthetic advantages of these star
polymers that make them attractive from a pharmaceutical manufacturing perspective
and report characterization of the polymers with a variety of techniques.
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INTRODUCTION
When designing novel polymer-based drug delivery systems for translation into
the clinic, there are many considerations to take into account before and during
development.1-6 From a platform perspective, important features for polymer
carriers include: synthetic reproducibility, low toxicity/immunogenicity, a suitable
clearance mechanism/biodegradability and capacity for high drug loading. Other
considerations should include ease of manufacture, formulation, analytical
characterization as well as low cost of goods. For drug delivery to tumors by
biophysical targeting, the approach is typically to develop technologies with long
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residence times in circulation to enable better tumor uptake, and to limit release
of an active drug in the circulation so to minimize non-target organ accumulation.
To prolong systemic retention, the technology should avoid renal clearance by
having a hydrodynamic size above the threshold for filtration and minimize the
liver, spleen and lung accumulation by having properties to reduce scavenger cell
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uptake and resulting off-target toxicity. The latter is normally controlled by
addition of polymers and depends on polymer type, surface charge,
hydrophilicity, hydrodynamic size and protein binding. Recent work has shown
that nanoparticles of 20-30 nm with poly(ethylene glycol) (PEG), poly(sarcosine)
(PSar) or poly(N-hydroxypropylmethacrylamide) (pHPMA) as the hydrophilic
shells displayed negligible protein binding after incubation in human plasma and
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all showed prolonged blood circulation times when tested in mice. Whilst PEG
and pHPMA are well studied for in vivo drug delivery applications, PSar is now
quickly emerging as an interesting polymer and a suitable alternative for both
polymers. PSar is conveniently synthesized from the ring opening polymerization
of sarcosine N-carboxyanhydride (NCA) which can be initiated from nucleophilic
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groups typically under ambient conditions. This provides an advantage over PEG,
which is typically purchased from commercial sources for most applications thus
is more challenging to tailor structures or deliver copolymers with specific
numbers of repeat units for a specific purpose. Additionally, there is growing
evidence that anti-PEG antibodies can lead to accelerated clearance of
PEGylated drug delivery systems upon repeated dosing, thus reducing their
efficacy and increasing their accumulation in off-target clearance organs, leading
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to adverse effects. PSar is proving to be a versatile material and has been used
in many applications as a novel material, as comprehensively reviewed by Birke,
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Ling and Barz. Interest is growing in the use of PSar for in vivo applications
including: antibody and protein modification,12, 13 tumor imaging,14 peptide-based
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nanotubes, polyplexes, gold nanoparticles, liposomes, and micelles.
In
one of these reports, PSar modification of a therapeutic protein was shown to
improve efficacy when tested in tumor-bearing animals when compared to PEG.13
There are a small number of reports using amine-functional dendrimers as
macroinitiators for the polymerization of sarcosine NCA to give well-defined, star-
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shaped materials.21-23 This approach offers a facile route to high molar mass
material with functionality maintained on the polymer chain ends. However, these
reports described the use of poly(trimethyleneimine) and poly(amidoamine)
dendrimer cores, which retain cationic charge near the core. An overall cationic
charge on a nanoparticle can be detrimental to drug delivery systems, where
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prolonged blood circulation is required, because of strong interactions with cell
membranes.8,
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A further more recent example of a dendrimer used to
polymerize PSar encompassed a generation 2 poly(propyleneimine) dendrimer to
synthesize 8-arm star block copolymers of poly(benzyl-L-glutamate) and PSar.25
The resulting amphiphilic structures formed nanoaggregates in water and were
intended for crossing mucosal barriers for the delivery of therapeutics. This
general approach to synthesizing star polymers, whilst more costly or time
consuming in terms of synthetic steps of the dendrimer core, offers many benefits
in terms of precision of the end product. This seems to be true for another type of
star polymer involving the grafting-to approach of modifying dendrimer with
pHPMA, where well-defined drug delivery systems could be synthesized and
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applied.26-28 Furthermore, star polymers may provide advantages over linear
equivalents for drug delivery due to structural organization with increasing
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numbers of arms, and less overlapping at higher concentrations due to osmotic
repulsion. 29, 30
We previously reported on the synthesis of an L-lysine dendrimer modified with
31
different poly(2-alkyl-2-oxazoline)s, designed to improve cytocompatibility. One
of these systems, with a poly(2-methyl-2-oxazoline) modification was tested for in
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vivo efficacy to deliver SN-38, an anticancer drug. These experiments added to
growing evidence that poly(2-methyl-2-oxazoline) is another alternative to the
gold standard of drug delivery, PEG, that has also been extensively investigated
as a modifier for dendrimers.33-36 Indeed, docetaxel conjugated to a generation-5
lysine dendrimer modified with PEG, developed by StarPharma has progressed
into Phase 2 clinical trials (EudraCT Number: 2016-000877-19). Here, we were
interested to try a different “stealth” polymer to offer benefits in terms of the
practicality of synthesis while maintaining the capability previously described for
use as a flexible drug delivery system.32 PSar is attractive because it can be
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polymerized directly from the surface of the dendrimer. In this article we describe
a scalable method for the synthesis of 32-arm star polymers from a lysine
dendrimer “macroinitiator” with different PSar chain lengths. The potential of
these star polymers to be used as a biophysical targeted drug delivery systems
was investigated by comparing plasma pharmacokinetic data and evaluating the
anti-tumor efficacy of one star polymer conjugated to the clinically relevant drug
SN-38 via a hydrolytically cleavable linker. These star polymers offer numerous
advantages for drug delivery applications, including precise sites for drug
conjugation, facile control over chain length, control of hydrodynamic size and
chain end functionality, which can be used to introduce targeting ligands onto the
surface of the nanocarrier. This approach also offers a convenient purification by
precipitation and filtration making it attractive from a manufacturing perspective.
Finally, we examine a new characterization technique, mass photometry, as a
convenient technique to give rapid molar mass determination at nanomolar
concentrations, that we believe will find more frequent use in the area of polymer
therapeutics and nanomedicine.
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MATERIALS AND METHODS
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All materials were purchased from Sigma Aldrich unless stated. Sarcosine N-
carboxyanhydride was sourced from Wuxi AppTec and recrystallized from
EtOAc/Heptane prior to use. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium
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tetrafluoroborate (DMTMM.BF ) was synthesized as previously reported.
3-
4
(Maleimido)propionic acid N-hydroxysuccinimide ester was purchased from
FluoroChem Ltd. Acetic acid N-hydroxysuccinimide ester was purchased from Apollo
Scientific Ltd. Bovine Serum Albumin MALDI-TOF-MS calibration kit was purchased
from AB SCIEX Ltd. NativeMark protein ladder was purchased from ThermoFisher.
Irinotecan hydrochloride was purchased from Carbosynth Ltd.
Instrumentation
Nuclear Magnetic Resonance (NMR). Spectra were recorded on either a Bruker
Ultrashield AVIII 500 MHz fitted with a QCI cryoprobe or a Bruker Ultrashield AVIII 400
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MHz spectrometer fitted with a BBFO probe. Both instruments were operated with
Topspin3.5pl5 software. Spectra were integrated using MestReNova version 9.0.
Size Exclusion Chromatography Triple Detection Array (SEC-TDA). Molar masses were
determined using a Malvern Panalytical OMNISEC system using a TSKgel Alpha 4000
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(300 x 7.8 mm, 10 µm) and a TSKgel Alpha 2500 (300 x 7.8 mm, 7 µm) in series. The
eluent used was 50 mM sodium phosphate + 100 mM sodium nitrate (pH 6.8). The
system was calibrated with a single Malvern Panalytical PEO 24 kDa narrow standard
and absolute molar masses determined using the refractive index and light scattering
signals. OMNISEC software version 11.0 was used.
Dynamic light scattering (DLS). A Malvern Zetasizer® Nano ZS instrument with back
scattering detector (173°, 633 nm laser wavelength) was used. The dispersant RI and
viscosity were assumed to be that of water (n = 1.59 and η = 0.888 mPa·s). The sample
RI was 1.59 and the temperature was set at 25°C. The hydrodynamic diameter (DH)
was reported as the intensity-weighted average after a minimum of twelve
measurements per sample and in triplicate and was calculated by the software.
Samples were made to concentrations of 5 mg/ml in PBS at pH 7.4 and were filtered
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using a 0.2 μm syringe filter prior to measurement. Data was obtained using Malvern
Zetasizer software version 7.12.
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Matrix Assisted Laser Desorption/Ionization Time of Flight Mass Spectrometry (MALDI-
TM
TOF-MS). Data were recorded using a Bruker UltrafleXtreme . Samples were
prepared at 5 mg/mL in methanol. A matrix solution of super-DHB (commercially
available 9:1 mixture of 2,5-Dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic
acid) was prepared at 40 mg/mL in THF. The matrix (1 µL) was applied to an MTP 384
polished steel target plate followed by sample (1 µL). Neighboring spots were spotted
with Bovine Serum Albumin calibrant in Sinapinic acid as per supplier’s protocol.
Spectra were recorded using Bruker flexControl software version 3.4 and analyzed
using Bruker flexAnalysis software version 3.4. In-built method LP_30-210 kDa was
used and the mass range extended for the largest star polymer analyzed.
Taylor Dispersion Analysis was performed on a Malvern Viscosizer TD fitted with a
o
coated capillary and 214 nm wavelength filter. Measurements were performed at 25 C
and buffer viscosity set at 0.8905 cP. Samples were made up at 10 mg/ml in phosphate
buffered saline and the calibrant, caffeine was made up at 1 mg/ml in Milli-Q water. The
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pressures applied were 1500 mbar pressure for the calibration and 140 mbar for
samples. Data were recorded in Malvern Viscosizer TD software version 2.20.
Mass photometry was performed on a Refeyn One mass photometry instrument
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(Refeyn Ltd, UK). All samples were dissolved in HBS-N buffer (10mM HEPES, pH=7.4,
150mM NaCl), diluted to 50 and 100 nM and analyzed using 60 sec acquisition time.
Resulting histograms were fitted to Gaussian distributions using DiscoverMP (Refeyn
Ltd, UK ) to extract peak contrast. Contrast-to-mass conversion was achieved by
calibration using NativeMark protein ladder. Three protein species (with specified
masses) were fitted to corresponding Gaussians to extract a linear relation between
mass and contrast (Supporting Figure S1).
In vivo work
All animal studies were conducted in accordance with UK Home Office legislation, the
Animal Scientific Procedures Act 1986, and the AstraZeneca Global Bioethics policy. All
experimental work is outlined in project licences 70/8894 and P0EC1FFDF, which have
gone through the AstraZeneca Ethical Review Process. Female BALB/c
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(
BALB/cOlaHsd) or athymic nude mice were purchased from Envigo, UK. All mice
weighed more than 18 g at the time of the first procedure.
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Plasma pharmacokinetics
To determine the plasma pharmacokinetic profile of the three different size star
polymers, naïve female BALB/c mice (n = 3 per size) were injected intravenously via the
lateral tail vein with a single dose of Cy5-labeled, carboxyl functional SD100, SD56 or
SD28 at 50 mg/kg. The star polymers were dissolved in physiological saline and were
administered in an injection volume of 0.05 mL/10 g mouse weight. After dosing, serial
live bleeds were collected at various time points (5 min, 2 h, 6 h, 24 h, 48 h, 72 h and
144 h post-dose) for determination of plasma levels of the Cy5-labeled star polymer.
Briefly, 20 µL of blood was collected from the tail vein using capillary tubes. Blood was
diluted with 80 µL of PBS and centrifuged at 19,000 xg for 4 min at 4°C. The
supernatant was collected and stored at -80°C until analysis. For analysis, a calibration
curve was prepared using serial dilutions of the dosing solutions in a 96-well plate. 40
µL samples from the live bleeds were plated as well. Fluorescent measurements were
made using a CLARIOstar plate reader using the Cy5 pre-set. The measurement
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parameters were: excitation = 610-30 nm; emission = 675-50 nm; dichroic filter = auto
637.5; gain = obtained by enhanced dynamic range; number of flashes = 20.
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Anti-tumor efficacy in SW620 xenograft model in mice
The anti-tumor efficacy of SD100-SN38 was evaluated in the SW620 xenograft model
in female nude mice. Mice were implanted subcutaneously in the left flank with 1 x 106
cells in 50% Matrigel. Tumor growth was monitored twice weekly using intersecting
2
calliper measurements; tumor volume was calculated using 3.14*length*width /6000,
where length is the longer dimension. Body weights were recorded at the time of tumor
measurement, and daily if weight loss exceeded 8%. Mice were randomized based on
tumor volume to ensure an equal volume distribution across groups; the average tumor
3
volume at the start of dosing was ~0.240 cm . Mice (n = 7 mice/group) were dosed once
weekly for 3 weeks with saline, irinotecan at 50 mg/kg or SD100-SN38 at 4 mg
SN38/kg. Saline and SD100-SN38 were administered intravenously via the lateral tail
vein; irinotecan was administered intraperitoneally. At 24 h after the final dose, 3 mice
per group were euthanized via Schedule 1 method with secondary confirmation, and
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tissues were excised, weighed and processed to determine the SN38 concentration in
liver, spleen and tumor.
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Analysis of SN-38 concentration in tumor, liver and spleen
Tissues were weighed and put into FastPrep Lysing Matrix A Tubes (MP Biomedicals
UK). Three times the volume of water per tissue weight was added to the tissue for
homogenization. The samples were homogenized using a FastPrep-24™ machine (MP
Biomedicals UK) at 6 m/s for 45 seconds. The cycle was repeated twice to gain
complete homogenization. Control tissue from undosed animals was homogenized as
above and spiked with an 11 point standard calibration curve (1-10000 nM SN-38)
prepared in DMSO. Tissue homogenates (25 µL) were precipitated with acetonitrile (100
µl) containing internal standard, followed by centrifugation at 4500 rpm for 10 minutes.
The supernatant (50 µl) was then diluted in 300 µl water and analyzed via UPLC-
MS/MS.
Synthetic procedures
Poly(L-Lysine) dendrimers. The synthetic procedures can be found in the supporting
information.
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SN-38 ester linker synthesis has been reported previously.32 Release rate data and
methodology can be found in the supporting information.
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Synthesis of 4-(2-furylmethylamino)-4-oxo-butanoic acid. Succinic anhydride (1.7 g, 17
mmol) was added to a 250 mL round bottom flask and partially dissolved in
dichloromethane (DCM) (25 mL) with stirring (400 RPM). Furfurylamine (1 mL, 11
mmol) was added drop-wise in 0.1 mL portions every 10 min. Almost immediately a
thick precipitate formed that was stirred for 1 h following full addition of the amine. The
precipitate was diluted with further DCM (50 mL) and filtered and then washed on the
filter pad with additional DCM (2 x 15 mL). The white solid was dried overnight under
o
1
vacuum at 40 C to yield the title compound. Yield = 1.03 g, 47 %. H NMR (d -DMSO,
6
5
00 MHz): δ= 2.34 (t, COCH ), δ= 2.34 (t, CH CO H), δ= 4.23 (d, NH-CH ), δ= 6.37 (dd,
2
2
2
2
13
Fur-H), δ= 6.62 (dd, Fur-H), δ= 7.55 (d, Fur-H), δ= 8.29 (-NH-), δ= 12.05 (t, CO H). C
2
NMR (d -DMSO, 500 MHz): δ= 28.99 (COCH ), δ= 29.81 (COOH ), δ= 35.46 (NHCH ),
6
2
2
2
δ= 106.63 (Fur-CH), δ= 110.38 (Fur-CH), δ= 141.96 (Fur-CH), δ= 152.37 (Fur-C-CH2),
δ= 170.83 (NH-CO), δ= 173.78 (CO H).
2
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G5-PLL[P(Sar) ] [boc] (SD28Boc). Sarcosine NCA (4.5 g, 39 mmol) was weighed
28 32
32
out into an oven-dried 100 mL Schlenk tube and fitted with a septum and purged with
nitrogen for 10 minutes. Anhydrous DMF (20 mL) was added via syringe and stirred
until the solid was fully dissolved. A solution of G5-PLL[NH ] [boc] (512 mg, 0.044
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mmol) dissolved in anhydrous DMF (2 mL) was added via syringe in one rapid addition.
The reaction was stirred overnight at room temperature with nitrogen flow.
Methoxyacetyl chloride (0.261 mL, 2.79 mmol) was added via Eppendorf pipette
followed by pyridine (0.226 mL, 2.79 mmol) and the reaction mixture stirred for 1 h at
room temperature. The solution was added to a rapidly stirring mixture of ethyl acetate
(360 mL) and ethanol (40 mL) to form a precipitate. The precipitate was vacuum filtered
and dried for 30 minutes on the filter pad under nitrogen. The solid was re-dissolved in
methanol (10 mL) and added drop-wise to a mixture of ethyl acetate (280 mL) and
o
ethanol (20 mL) and the solid obtained by vacuum filtration and after drying at 40 C in a
1
vacuum oven for 24 h. Yield = 3.08 g, 91.5 %. H NMR (500 MHz, D O) 1.04 – 1.98
2
(374H, br), 2.61 – 3.22 (2814H, br), 3.27 – 3.41 (94H, br), 3.87 – 4.55 (1896H, br).
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G5-PLL[P(Sar) ] [boc]
(SD56Boc).
Method
as
for
compound
G5-
56 32
32
PLL[P(Sar) ] [boc] (SD28). Reagent quantities: Sarcosine NCA (4.5 g, 39 mmol),
28 32
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G5-PLL[NH ] [boc] (256 mg, 0.022 mmol), methoxyacetyl chloride (120 µL, 1.284
2
32
32
1
mmol), pyridine (104 µL, 1.284 mmol). Yield = 2.94 g, 95.7%. H NMR (500 MHz, D O)
2
1.04 – 1.98 (374H, br), 2.61 – 3.22 (5891H, br), 3.27 – 3.41 (102H, br), 3.87 – 4.55
(3945H, br).
G5-PLL[P(Sar) ] [boc]
(SD100Boc).
Method
as
for
compound
G5-
100 32
32
PLL[P(Sar) ] [boc] (SD28). Reagent quantities: Sarcosine NCA (3.13 g, 27.2 mmol),
28 32
32
G5-PLL[NH ] [boc] (100 mg, 0.0085 mmol), methoxyacetyl chloride (38 µL, 0.411
2
32
32
1
mmol), pyridine (33 µL, 0.411 mmol). Yield = 1.96 g, 95.5%. H NMR (500 MHz, D O)
2
1.04 – 1.98 (374H, br), 2.61 – 3.22 (10728H, br), 3.27 – 3.41 (94H, br), 3.87 – 4.55
(7123H, br).
G5-PLL[P(Sar) ] [NH .TFA] (SD28). SD28Boc (3.5 g, 0.045 mmol) was suspended
28 32
2
32
in DCM (18 mL) under nitrogen flow. TFA (9 mL) was added dropwise over 5 minutes
and the reaction was stirred for 2 h at room temperature. The reaction mixture was
added to rapidly stirring TBME (400 mL) to form a precipitate. The solid was collected
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by vacuum filtration and dried for 1 h on the filter pad under nitrogen flow. The solid was
o
dried in a vacuum oven at 40 C overnight and then freeze-dried for 48 h from Milli-Q
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water (30 mL) to give the title compound. Yield = 2.84 g, 0.037 mmol). H NMR (400
MHz, D O) 1.04 – 1.98 (374H, br), 2.61 – 3.22 (2814H, br), 3.27 – 3.41 (94H, br), 3.87 –
2
4.55 (1896H, br).
G5-PLL[P(Sar) ] [NH .TFA] (SD56). The method was followed as for SD28 starting
56 32
2
32
1
from 2.8 g of SD56Boc. Yield = 2.5 g, 91.0 %. H NMR (500 MHz, D O) 1.04 – 1.98
2
(374H, br), 2.61 – 3.22 (5891H, br), 3.27 – 3.41 (102H, br), 3.87 – 4.55 (3945H, br).
G5-PLL[P(Sar) ] [NH .TFA] (SD100). The method was followed as for SD28 starting
100 32
2
32
1
from 1.9 g of SD100Boc. Yield = 1.75 g, 85.1 %. H NMR (400 MHz, D O) 1.04 – 1.98
2
(374H, br), 2.61 – 3.22 (10728H, br), 3.27 – 3.41 (94H, br), 3.87 – 4.55 (7123H, br).
Reaction time course and end group modifications:
G5-PLL[P(Sar) -NH ] [boc] (SD56Boc-NH ). Sarcosine NCA (1.47 g, 12.8 mmol)
56
2 32
32
2
was added to an oven-dried Schlenk tube fitted with a septum. The solid was purged
with nitrogen and anhydrous DMF (8 mL) was added via syringe. A solution of G5-
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PLL[NH ] [boc] (85 mg, 0.007 mmol) dissolved in anhydrous DMF (0.5 mL) was
2 32
32
added in one rapid addition via syringe. Aliquots of the reaction mixture (100 µL) were
0
1
2
3
4
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9
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taken every 30 minutes for 3.5 h and added to ethyl acetate (12 mL) in 15 mL Falcon
tubes to form a precipitate. The precipitate was agitated and centrifuged at 2800 xg for
o
5
minutes. The obtained solids were dried under vacuum at 40 C for 24 h. The
remaining reaction was stirred overnight and added to a rapidly stirring ethyl acetate
(100 mL) to form a precipitate. The solid was collected by vacuum filtration and dried
o
overnight in a vacuum oven at 40 C. Yield = 0.95 g, 96 %. The samples taken at
1
different timepoints were evaluated by MALDI-TOF-MS and Mass Photometry. H NMR
(500 MHz, D O) 0.89 – 1.84 (594H, br), 2.29 – 2.46 (102H, br), 2.63 – 3.27 (5479H, br),
2
3.37 – 3.44 (19H, br), 3.67 – 3.72 (55H, br), 3.89 – 4.60 (3584H, br).
G5-PLL[P(Sar) -DBCO/Acetyl amide] [boc] (SD56Boc-DBCO/Ac). G5-PLL[P(Sar) -
56
32
32
56
NH ] [boc] (400 mg, 2.89 µmol) was dissolved in anhydrous DMF (3 mL). DIPEA (48
2
32
32
µL, 0.28 mmol) was added and the solution stirred briefly. A solution of
Dibenzocyclooctyne-N-hydroxysuccinimidyl ester (DBCO-NHS) (10 mg, 24.9 µmol) in
DMF (0.5 mL) was added and the reaction mixture stirred for 1 h at RT. Acetic acid N-
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hydroxysuccinimide ester (20 mg, 127 µmol) was added as a solid and the reaction
stirred for 1 h at RT. The reaction mixture was added dropwise to a stirred mixture of
ethyl acetate (150 mL) and acetonitrile (50 mL) to form a precipitate. The precipitate
was stirred for 15 minutes and then collected by vacuum filtration and washed with ethyl
acetate (20 mL). The solid was dried for 30 minutes on the filter pad under nitrogen and
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
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3
4
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5
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1
2
3
4
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9
0
1
2
3
4
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8
9
0
1
2
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4
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8
9
0
then dissolved in Milli-Q water, filtered (0.45 µm PVDF) and freeze-dried to obtain the
1
title compound. Yield = 345 mg, 84 %. H NMR (400 MHz, D O) 0.89 – 1.82 (644H, br),
2
1.93 – 1.97 (17H, br), 2.06 – 2.12 (43H, br), 2.47 – 3.30 (5505H, br), 3.75 – 4.60
(3584H, br), 5.00 – 5.10 (25H, br), 7.12 – 7.79 (103H, br).
G5-PLL[P(Sar) -Maleimide] [boc]
(SD56Boc-Mal). G5-PLL[P(Sar) -NH ] [boc]
56
32
32
56
2 32
32
(100 mg, 2.89 µmol) was dissolved in anhydrous DMF (2 mL). DIPEA (8 µL, 0.28 mmol)
was added and the solution stirred briefly. A solution of N-succinimidyl 3-
maleimidopropionate (Maleimide-NHS) (13 mg, 46.4 µmol) in DMF (0.5 mL) was added
and the reaction mixture stirred for 2 h at RT. The reaction mixture was added to a
rapidly stirring mixture of ethyl acetate (100 mL) and acetonitrile (100 mL) to form a
precipitate. The suspension was stirred for 15 minutes and then filtered and freeze-dried
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1
1
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
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4
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4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
as for SD56Boc-DBCO/Ac above. Yield = 95 mg, 91.8 %. H NMR (500 MHz, D O) 0.77
2
–
2.06 (624H, br), 2.56 – 3.38 (5693H, br), 3.61 – 3.75 (85H, br), 3.80 – 3.89 (38H, br),
.89 – 4.58 (3584H, br), 6.67 – 6.87 (79H, br).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
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0
3
G5-PLL[P(Sar) -Biotin] [boc] (SD56Boc-Biotin). G5-PLL[P(Sar) -NH ] [boc] (100
56
32
32
56
2 32
32
mg, 2.89 µmol) was dissolved in anhydrous N,N’-dimethylformamide (2 mL). DIPEA (8
µL, 0.28 mmol) was added and the solution stirred briefly. A solution of Biotin-NHS ester
(
16 mg, 46.9 µmol) in DMF (0.5 mL) was added and the reaction mixture stirred for 3 h
at RT. The reaction mixture was added to a rapidly stirring mixture of ethyl acetate (100
mL) and acetonitrile (100 mL) to form a precipitate. The suspension was stirred for 15
minutes and then filtered and freeze-dried as for SD56Boc-DBCO/Ac above. Yield = 95
1
mg, 90.3 %. H NMR (500 MHz, D O) 0.96 – 1.92 (714H, br), 2.30 – 2.38 (17H, br), 2.47
2
– 2.58 (34H, br), 2.75 – 2.82 (72H, br), 2.83 – 3.30 (5497H, br), 3.31 – 3.42 (45H, br),
3.99 – 4.47 (3584H, br), 4.49 – 4.68 (57H, br).
G5-PLL[P(Sar) -furfuryl] [boc]
(SD56Boc-furfuryl). G5-PLL[P(Sar) -NH ] [boc]
56
32
32
56
2 32
32
(
100 mg, 2.89 µmol) was dissolved in anhydrous N,N’-dimethylformamide (2 mL).
DIPEA (8 µL, 0.28 mmol) was added and the solution stirred briefly. A premixed solution
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of 4-(2-furylmethylamino)-4-oxo-butanoic acid (7.0 mg, 35.5 µmol) and DMTMM.BF (16
4
mg, 46.33 µmol) in DMF (2 mL) was added and the reaction mixture stirred for 3 h at
RT. The reaction mixture was added to a rapidly stirring mixture of ethyl acetate (100
mL) and acetonitrile (100 mL) to form a precipitate. The suspension was stirred for 15
1
1
1
1
1
1
1
1
1
1
2
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2
2
2
2
2
2
2
2
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3
3
3
3
3
3
3
4
4
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5
5
5
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5
6
0
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9
0
minutes and then filtered and freeze-dried as for SD56Boc-DBCO/Ac above. Yield = 93
1
mg, 89.9 %. H NMR (500 MHz, D O) 0.79 – 1.96 (563H, br), 2.39 – 2.53 (77H, br), 2.56
2
–
3.44 (5498H, br), 3.80 – 4.59 (3584H, br), 6.17 – 6.45 (54H, br), 7.33 – 7.50 (23H, br).
G5-PLL[P(Sar) ] [Cy5] [COOH] (SD28-Cy5-COOH). G5-PLL[P(Sar) ] [NH .TFA]
28 32
2
30
28 32
2
32
(50 mg, 0.6 µmol) was dissolved in anhydrous DMF (2 mL) in a 10 mL round bottom
flask and stirred to dissolve. DIPEA (14 µL, 0.08 mmol) was added followed by a
solution of Cyanine 5 NHS ester (0.8 mg, 0.001 mmol) from a stock solution (4 mg/mL
in DMF). The reaction mixture was stirred for 1 h at RT. A solution of succinic anhydride
(4.1 mg, 0.041 mmol) from a stock of 40 mg/ml dissolved in DMF was added and the pH
adjusted with additional DIPEA to pH8-9 (5 µL added to 5µL of Milli-Q water and tested
on pH paper). The reaction mixture was stirred for 4 h at room temperature before being
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added drop-wise to rapidly stirring TBME (45 mL) to form a blue precipitate. The solid
was obtained by centrifuging at 3000 xg to obtain a blue pellet after decanting the
solvent to waste. The solid was dried overnight at RT under vacuum (50 mBar). The
solid was dissolved in Milli-Q water (0.5 mL) and the pH adjusted to 3-4 using 2 M HCl.
The solution was passed through a PierceTM Dextran desalting column (5 kDa MWCO)
and early blue fractions collected. The fractions were frozen and freeze-dried before
0
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8
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0
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4
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9
0
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9
0
collating pure fractions. The title compound was obtained as a dark blue solid. Yield =
1
42 mg, 80 %. H NMR (500 MHz, D O) 0.98 – 1.85 (364H, br), 3.23 – 3.60 (131H, br),
2
3.66 – 3.23 (2642H, br), 3.27 – 3.40 (91H, br), 3.86 – 4.55 (1792H, br).
G5-PLL[P(Sar) ] [Cy5] [COOH] (SD56-Cy5-COOH). The method for SD28-Cy5-
56 32
2
30
COOH was followed but using G5-PLL[P(Sar)56] [NH .TFA] (50 mg, 0.4 µmol),
32
2
32
DIPEA (8 µL, 0.046 mmol), Cyanine 5 NHS ester (0.43 mg, 0.7 µmol) from a stock
solution (4 mg/mL in DMF), succinic anhydride (2.3 mg, 0.023 mmol) (from stock
1
solution of 40 mg/mL in DMF. Yield = 41 mg, 82 %. H NMR (500 MHz, D O) 0.98 –
2
1.85 (329H, br), 3.23 – 3.60 (160H, br), 3.66 – 3.23 (5416H, br), 3.27 – 3.40 (100H, br),
3.86 – 4.55 (3584H, br).
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G5-PLL[P(Sar) ] [Cy5] [COOH] (SD100-Cy5-COOH). The method for SD28-Cy5-
100 32
2
30
COOH was followed but using G5-PLL[P(Sar)56] [NH .TFA] (50 mg, 0.4 µmol),
32
2
32
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
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8
9
0
1
2
3
4
5
6
7
8
9
0
DIPEA (8 µL, 0.046 mmol), Cyanine 5 NHS ester (0.43 mg, 0.7 µmol) from a stock
solution (4 mg/mL in DMF), succinic anhydride (2.3 mg, 0.023 mmol) (from stock
1
solution of 40 mg/mL in DMF. Yield = 46 mg, 92%. H NMR (500 MHz, D O) 0.98 – 1.85
2
(473H, br), 3.23 – 3.60 (276H, br), 3.66 – 3.23 (9876H, br), 3.27 – 3.40 (52H, br), 3.86 –
4.55 (6400H, br).
G5-PLL[P(Sar) ] [SN-38] (SD100-SN38). G5-PLL[P(Sar) ] [NH .TFA] (200 mg,
100 32
32
100 32
2
32
0.8 µmol) was dissolved in anhydrous DMF. TEA (10.8 µL, 0.08 mmol) was added. In a
separate vial, 2-(4-(4-oxo-4-SN38 butyl)-1H-1,2,3-triazol-1-yl)acetic acid (19 mg, 0.032
mmol) and DMTMM.BF (18 mg, 0.040 mmol) were dissolved in anhydrous DMF (1 mL)
4
and then added slowly to the polymer containing solution. The reaction was stirred for 3
h at RT and then added drop-wise to rapidly stirring ethyl acetate (125 mL) to form a
precipitate, which was collected by filtration. The solid was re-dissolved in methanol (3
mL) and added to a rapidly stirring mixture of 50/50 ethyl acetate/tetrahydrofuran. This
process was repeated once more to give the title compound as a pale yellow solid. Yield
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
=
172 mg, 81 %. H NMR (400 MHz, d4-acetic acid) 0.65 – 0.96 (110H, br), 1.24 – 1.35
(
68H, br), 2.44 – 3.15 (9684H, br), 3.19 – 3.36 (168H, br), 3.46 – 4.87 (6400H, br), 5.06
0
1
2
3
4
5
6
7
8
9
0
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2
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8
9
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8
9
0
1
2
3
4
5
6
7
8
9
0
–
5.64 (143H. br), 6.98 – 8.34 (109H, br).
RESULTS AND DISCUSSION
Synthesis and characterization of polysarcosine star polymers. A generation-5 lysine
dendrimer (ε-boc protected) was used as a macroinitiator to polymerize sarcosine
N-carboxyanhydride to give 32-arm star polymers with different chain lengths
(Scheme 1). The polymerization was directed to the lysine α-amino groups with
the boc-protected ε-amine positioned for further functionalization e.g. with drug-
linker molecules after the polymerization to provide more precision for drug
loading and ultimately better reproducibility, which are key drivers for
pharmaceutical development. This controlled approach offered additional
benefits, as a pre-synthesized and characterized linear polymer with bespoke
end groups was not required, reducing reaction steps. The polymerization was
performed at room temperature with carbon dioxide as the by-product, which was
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removed as the reaction progressed by purging with nitrogen. This synthesis
gave high yields of material after a simple process of precipitation of the product
into an antisolvent (ethyl acetate/ethanol mixture), followed by filtration.
1
1
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O
O
O
N
Dendrimer macroinitiator
G5-PLL[α-NH ][ε-boc]
2
Functional N-terminus
Scheme 1. Polymerization of sarcosine N-carboxyanhydride from generation 5
lysine dendrimer macroinitiator to give a 32-arm star polymer with terminal amino
groups.
Previously, for the polyoxazoline-modified dendrimer, the excess linear
polyoxazoline had to be removed by a diafiltration step to yield the pure star
polymer before freeze-drying, adding to the technical challenge upon scale up.31
An interesting finding for the polysarcosine star polymers was the excellent
solubility in water, methanol, acetic acid, dimethylformamide and dimethyl
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sulfoxide but poor to no solubility in several organic solvents, including ethers,
ethyl acetate, tetrahydrofuran, acetonitrile and acetone. This allowed the removal
of excess reagents, reaction solvent and low molar mass linear polysarcosine
that may have formed in situ, during the precipitation into antisolvent. This
strategy could also be used to remove excess drug-linker from a drug conjugation
step, simplifying purification and making the process easier to scale. A useful
attribute of using the dendrimer core to polymerize Sar-NCA was that the chain
length, and therefore hydrodynamic size, could be tuned simply by altering the
feed ratios of dendrimer macroinitiator and monomer (Table 1).
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Table 1. Summary of characterization data for star polymers with degrees of
polymerization of 28, 56 and 100.
Description DPa Mn
(kDa)b
Ɖb
Mass
Mp
Inferred
Molar
DLS SEC Taylor
RH RH Dispersion
(
kDa)c
Mass
(nm) (nm) Analysis
(
kDa)d
R (nm)
H
SD28
29
78.9
1.08 74.8
79 ± 9
5.1
4.5
5.8
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SD56
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95
149.3 1.09 140.2
252.2 1.12 230.0
149 ± 16
7.1
7.2
8.3
8.0
9.3
SD100
228 ± 25 9.1
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a
1
b
c
d
Degree of Polymerization from HNMR. SEC-TDA. Peak Maximum molar mass from MALDI-TOF-MS. Molar
masses inferred from Mass Photometry.
PSar chain lengths of n= 28 and 56 were chosen based on molar mass
equivalents to linear PEG (2000 g/mol and 4000 g/mol), which are amongst the
most common PEGs used for drug delivery systems. A further nominal n= 100
sample was synthesized as the highest molar mass star polymer for
investigation. These samples were denoted SD28, SD56, SD100, signifying
PSar-modified dendrimer and the number of repeat units per chain. The amino
terminal chain ends were capped with methoxyacetyl chloride yielding charge
neutral, non-reactive methoxy terminal groups. The expected masses calculated
from the relative molar ratios of dendrimer core and monomer were 77 kDa, 141
kDa and 241 kDa. The resulting star polymers were characterized by SEC-TDA
(
Figure 1A) and determined to have number average molar masses of 79 kDa,
149 kDa and 252 kDa, which were in close agreement with the expected masses.
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