Effect of molecular weight of PGG-paclitaxel conjugates on in vitro and in vivo efficacy

Article abstract of DOI:10.1016/j.jconrel.2012.04.010

Polymeric prodrugs are one of the most promising chemotherapeutic agent delivery approaches, displaying unique drug release profiles, serum stability, formulation flexibility, and reduced drug resistance. One of the most important aspects of a polymeric prodrug, albeit a less-extensively studied one, is the polymer's molecular weight, which affects particle formation, drug release and PK/PD profiles, drug stability, and cell uptake; these factors in turn affect the prodrug's maximum tolerated dose and anticancer efficacy. Poly(l-γ-glutamylglutamine) (PGG) is a linear polymer designed to improve the therapeutic index of attached drugs. In this study we selected poly(l-γ-glutamylglutamine)-paclitaxel (PGG-PTX), as a model system for the methodical investigation into the effects of the poly(l-γ- glutamylglutamine) backbone molecular weight on its pharmacological performance. The polymeric prodrug was characterized by NMR, DLS and GPC-MALS, and its anticancer activity in vitro and in vivo was assessed. Herein we present data which provide valuable insight into improving anticancer polymer-based prodrug design and development.

Full text of DOI:10.1016/j.jconrel.2012.04.010

Journal of Controlled Release 161 (2012) 124–131
Contents lists available at SciVerse ScienceDirect
Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel
Effect of molecular weight of PGG–paclitaxel conjugates on
in vitro and in vivo efficacy
a
a
c
b
b
b
b
Danbo Yang , Xiaoqing Liu , Xinguo Jiang , Yun Liu , Wenbin Ying , Hai Wang , Hao Bai ,
b
b
b
b
b
Wendy D. Taylor , Yuwei Wang , Jean-Pierre Clamme , Erick Co , Padmanabh Chivukula ,
Kwok Yin Tsang , Yi Jin , Lei Yu
Institute for Advanced Interdisciplinary Research, East China Normal University, 3663 North Zhongshan Road, Shanghai, 200062, PR China
Biomedical Group, Nitto Denko Technical Corporation, 501 Via Del Monte, Oceanside, CA, 92058, USA
School of Pharmacy, Fudan University, Shanghai, 201203, PR China
b
b
a,b,
⁎
a
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Polymeric prodrugs are one of the most promising chemotherapeutic agent delivery approaches, displaying
unique drug release profiles, serum stability, formulation flexibility, and reduced drug resistance. One of
the most important aspects of a polymeric prodrug, albeit a less-extensively studied one, is the polymer's mo-
lecular weight, which affects particle formation, drug release and PK/PD profiles, drug stability, and cell up-
take; these factors in turn affect the prodrug's maximum tolerated dose and anticancer efficacy. Poly(L-γ-
glutamylglutamine) (PGG) is a linear polymer designed to improve the therapeutic index of attached
drugs. In this study we selected poly(L-γ-glutamylglutamine)–paclitaxel (PGG–PTX), as a model system for
the methodical investigation into the effects of the poly(L-γ-glutamylglutamine) backbone molecular weight
on its pharmacological performance. The polymeric prodrug was characterized by NMR, DLS and GPC–MALS,
and its anticancer activity in vitro and in vivo was assessed. Herein we present data which provide valuable
insight into improving anticancer polymer-based prodrug design and development.
Received 3 January 2012
Accepted 1 April 2012
Available online 12 April 2012
Keywords:
Polymers
PGG copolymer
Subcellular trafficking
Drug release
Drug delivery
Physicochemical characteristics
©
2012 Elsevier B.V. All rights reserved.
1
. Introduction
aqueous solution either through encapsulation using either various
micellar techniques or direct covalent conjugation, both of which sig-
nificantly improve chemotherapeutic agent compliance. Alteration of
the polymer–drug conjugate can also significantly increase the
amount of the chemotherapeutic agents delivered. Furthermore, the
polymer–drug conjugate can self-assemble into nanoparticles with
either individual polymer–drug conjugate or multiple conjugates to
form higher ordered structures through hydrophobic interactions.
The increase in “drug” size can prevent the conjugate from diffusing
from blood circulation into normal tissues and organs, but more easily
“leak” or permeate into the tumor tissue via the often disordered
tumor vasculature. Likewise, the increased “drug” size also signifi-
cantly prolongs the residence time of the polymeric prodrug in
blood circulation, which provides more opportunity for delivery of
the chemotherapeutic agent to tumor tissue [14,15].
It has long been recognized that molecular weight of a polymer
plays a significant role in its PK/PD properties after intravenous ad-
ministration [14]. Generally, higher molecular weight of a polymer
material results in longer blood circulation time, enhanced tumor ac-
cumulation, and reduced renal clearance. There is, however, no uni-
versal chemotherapeutic agent carrier identified because no two
cancers have identical structures or microenvironments, suggesting
the need for meticulous design of a polymer carrier to attain antican-
cer efficacy.
The most prominent advantage of utilizing a nanoparticle delivery
system is the enhanced permeability and retention (EPR) effect [1],
which directs the accumulation of therapeutic agents in tumor tissue
due to its unique morphology [2]. Nanoparticle prodrugs are one of
the most promising cancer agents for the delivery of chemotherapeu-
tics [3], exhibiting controlled drug release, favorable PK/PD profile,
improved serum stability, solubility enhancement of poorly soluble
drugs, the ability to overcome multi-drug resistance [4–8], and the re-
duction of non-specific toxicity [9].
Prodrug design comprises an area of drug research that is associat-
ed with the reduction of non-specific toxicity. A prodrug is a biologi-
cally inactive derivative of a parent drug molecule that usually
requires chemical or enzymatic transformation in vivo to release the
active agent; the primary advantage of the prodrug is the improved
toxicity profile over its parent molecule [10–13]. Polymers can confer
enhanced solubility of hydrophobic chemotherapeutic agents in
⁎
Corresponding author at: Nitto Denko Technical Corporation, 501 Via Del Monte,
Oceanside, CA 92058, USA. Tel.: +1 760 435 7026; fax: +1 760 435 7050.
E-mail address: yu_lei@gg.nitto.co.jp (L. Yu).
0168-3659/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
doi:10.1016/j.jconrel.2012.04.010

D. Yang et al. / Journal of Controlled Release 161 (2012) 124–131
Table 1
125
Recently we reported the synthesis, characterization, and
PTX loading and impurity profile of PGG–PTX. Levels of free PTX and DMAP in PGG–PTX
samples were determined by SEC–HPLC (Agilent 1200, Shodex OHpak SB-804-HQ col-
umn) equipped with a dual-wavelength detector (254 nm and 228 nm).
bio-evaluation of a linear polymeric drug nano-carrier, poly(L-
γ-glutamylglutamine)–paclitaxel conjugate (PGG–PTX) [16], as
well as its preclinical efficacy studies. PGG–PTX was designed with
the addition of a hydrophilic glutamic acid layer onto poly(L-gluta-
mic acid) followed by the conjugation of a hydrophobic drug, namely
paclitaxel; the resulting conjugate was found to self-assemble spon-
taneously into a nanoparticle and possessed enhanced water solubil-
ity compared to its precursor. Previous studies have demonstrated a
significant reduction in systemic toxicity, coupled with a favorable
drug-tumor accumulation profile and superior performance to
Abraxane® (Albumin-bound paclitaxel nanoparticle) in several ani-
mal models [17–19].
Polymer
conjugate
PTX feeding
ratio (%)
PTX contenta
(%)
Free paclitaxel
(%)
Free DMAP
(%)
PGG–PTX12.5K-35%
PGG–PTX25K-35%
PGG–PTX37.5K-35%
PGG–PTX50K-35%
35
35
35
35
32.1
34.2
33.1
38.3
2.17
1.06
0.62
0.97
0.1
0.05
ND
0.09
a
Calculated by 1H NMR.
The physical properties of a given polymeric prodrug, including
water solubility, polymer backbone degradation rate, size, shape,
surface charge, inertness of the carrier materials, as well as the sta-
bility of the particle in vivo, significantly contribute to the overall
pharmacologic performance in the body. In present studies, we
have found that PGG polymer molecular weight has great bearing
2. Materials and methods
2.1. Materials
Paclitaxel (PTX) was obtained from Shanghai Junjie Biotech-
nology Co., Ltd. (Shanghai, China). N-(3-dimethylaminopropyl)-
N′-ethylcarbodiimide (EDC) and H-Glu(OtBu)-OtBu·HCl were
purchased from EMD Chemicals Inc. (Darmstadt, Germany). 1-
Hydroxybenzotriazole (HOBt) was purchased from Spectrum Labora-
tory Products, Inc. (New Brunswick, US). N,N′-dimethylaminopyridine
was supplied from Sigma-Aldrich, Inc. (United States). All other chemi-
cals and reagents were commercially available and directly used.
Human NCI-H460 carcinoma cell lines were obtained from ATCC.
In vitro assay reagents were purchased from Gibco (USA), Promega
on the nanoparticle's physical property and thus modulates IC50
,
drug release, cell uptake, MTD, and ultimately, tumor growth inhibi-
tion. To our knowledge, there has not been documentation or a sys-
tematic investigation into the effects of the anionic polymer
molecular weight on drug release profiles and in vitro and in vivo
pharmacology of drug conjugates. Data from these studies provide
valuable insight for improving polymer–prodrug design and
development.
(
USA), and Sigma (USA).
2.2. Synthesis and characterization of PGG–PTX
PGG–PTX was synthesized as shown in Fig. 1 [16]. PGG–PTX was pre-
pared by chemical synthesis in six steps starting from commercially-
available γ-benzyl-L-glutamate. Five intermediate compounds were
isolated and characterized before use in the subsequent step. The final
active pharmaceutical ingredient, PGG–PTX, was obtained by dialysis
with a tangential flow filtration system followed by lyophilization to
afford a white solid.
PGG–PTX was characterized by NMR spectroscopy (Joel Delta
4
00 MHz spectrometer), dynamic light scattering using a Malvern
Zetasizer Nano-ZS (Worcestershire, United Kingdom), and gel perme-
ation chromatography with a Wyatt GPC–MALS system (Santa Barbara,
California) (Tables 1 and 2).
Levels of free PTX and 4-dimethylaminopyridine (DMAP) in PGG–
PTX samples were determined by SEC–HPLC (Agilent 1200, Shodex
OHpak SB-804-HQ column) equipped with a dual-wavelength detec-
tor (228 nm and 254 nm). DMAP was detected at 254 nm and the
2
method was linear in the range between 0.0008 and 0.2 μg (r =
0
.9999, n=8). PTX was detected simultaneously at 228 nm and the
2
linear range for detection was between 0.075 and 10 μg (r =0.9999,
n=10).
All GPC–MALS analysis was performed with an Agilent 1200 HPLC
equipped with Wyatt Dawn Heleos-II MALS and Wyatt Optilab T-rEX
IR detectors using Shodex OHpak SB-804 HQ column eluting
Table 2
Characterization of polymer conjugates.
Sample #
Molecular weight (kDa) and PDI
PG PGG
Particle
size
PGG–PTX
(
nm)
PGG–PTX12.5K-35%
PGG–PTX25K-35%
PGG–PTX37.5K-35%
PGG–PTX50K-35%
3.16 (1.46)
6.11 (1.37)
12.30 (1.13)
19.70 (1.32)
45.00 (1.60)
81.00 (1.51)
13
17
19
22
8.00 (1.25)
13.44 (1.22)
22.20 (1.31)
14.67 (1.50)
27.33 (1.44)
50.60 (1.28)
Fig. 1. PGG–PTX synthetic scheme.

126
D. Yang et al. / Journal of Controlled Release 161 (2012) 124–131
isocratically at flow rate 0.7 mL/min dn/dc values for PG-Na (0.180)
and PGG (0.145) were measured offline using the slope of a plot of re-
fractive index against concentration with an Optilab T-rEX (Wyatt)
refractive index detector. Particle size for PGG–PTX samples were
measured with a Malvern Zetasizer Nano-ZS at 2 mg/mL in pH 6.5
PBS-45% MeOH buffer.
2.5. Synthesis of PG-Na
Concomitant deprotection of PG-ester and MW control toward
PG-Na was performed as described previously in the literature [20].
A 5 L three-necked, round-bottomed flask equipped with a mechani-
cal stirrer was fitted with thermometer and drying tube (Drierite).
Dichloroacetic acid (4 L, 40 mL per gram of PG-ester) was added
and maintained at 28 °C in an oil bath. PG-ester (100.0 g, 556 mmol,
Unless otherwise stated, all reactions were carried out under an
inert atmosphere, using commercially available anhydrous solvents.
1
.0 eq.) was added portionwise and stirred overnight, while main-
taining internal temperature at 28 °C. 33% HBr in acetic acid solution
(350 mL, 2000 mmol, 4.37 eq.) was added in one portion and the
mixture was stirred for a period of time in order to obtain a specific
desired molecular weight of product PG-Na (5 h for 20 kDa PG). Hex-
ane (2 L) prechilled to −20 °C was added in one portion upon which
PG-acid precipitated as a white solid. After 10 min additional stirring,
the reaction was removed from the oil bath and allowed to stand for
30 min while equilibrating to room temperature. The supernatant
was removed by cannula and acetone (1 L) was added to the resulting
sediment. Following vigorous stirring for 10 min, stirring was stopped
and the PG-acid was allowed to settle for 30 min. The supernatant
was removed by decanting and the resulting slurry was isolated by
filter paper (#50 Whatman). The precipitate was washed with ace-
tone (2×500 mL) and air-dried for 2 min. The resulting solid was
2
.3. Synthesis of NCA-benzylester
γ-Benzyl-L-glutamate (100 g, 422 mmol, 1.0 eq) and THF (1000 mL)
were added to an oven-dried 2 L round-bottomed flask equipped with a
Teflon magnetic stirbar. The suspension was stirred for 5 min while
bubbling with argon. Triphosgene (48.0 g, 160.2 mmol, 0.38 eq.) was
added and the viscous mixture was heated at 50 °C for 4 h during
which the mixture became clear and homogeneous. The reaction mix-
ture was poured into rapidly stirring n-hexane (2.4 L) whereupon the
product precipitated as fine, white crystalline needles. The suspension
was allowed to stir for 30 min and then is stored at −20 °C for 24 h.
The precipitate was collected by filtration and washed thoroughly
with n-hexane to remove any residual triphosgene. The solid was
dried under vacuum and then dissolved in anhydrous ethyl acetate
added to a 1 L Erlenmeyer flask containing 1N aqueous NaHCO
3
(
1 L). With stirring, n-hexane (~1 L) was added upon which precipitate
(250 mL) while maintaining pH at ~7.0–8.0. The resulting solution
was stirred overnight, diluted with water to a volume of 3 L, and
was processed by tangential flow filtration (1 kDa molecular weight
cutoff regenerated cellulose membrane). The solution was lyophilized
to give PG-Na as a white crystalline solid (64 g, 93%). 1H NMR
began to form. The mixture was stored at −20 °C overnight. The solid
was collected by filtration and dried under vacuum to yield 5-benzyl
ester glutamic acid-N-carboxyanhydride as white crystals (83 g, 75%).
1
3
H NMR (400 MHz, CDCl ): δ 7.35 (m, 5H), 6.29 (s, 1H), 5.13 (s, 2H),
4
.36 (t, 1H), 2.59 (t, 2H), 2.25 (m, 1H), 2.11 (m, 1H) ppm.
2
(400 MHz, D O): δ 4.29–4.26 (t, 1H), 2.23 (m, 2H), 1.90 (m, 2H)
ppm. GPC-MALS: Zimm Model was used for Mw determination;
Mw, 23730 (0.86%), Mw/Mn, 1.30; 84.4% recovery.
2
.4. Synthesis of PG-ester
2.6. Synthesis of PGG-ester
A 5 L, three-necked, round-bottomed flask equipped with a mag-
netic stirrer was placed in a water bath (22 °C), purged with dry nitro-
gen for 5 min and sealed with septa. Anhydrous dioxane (3500 mL) was
added to the flask. With stirring, γ-benzyl-N-carboxyl-L-glutamate
anhydride (200 g, 760 mmol, 1.0 eq.) was added in portions via a
funnel, followed by anhydrous dioxane (350 mL) to rinse any residual
solid on the funnel. The solution was stirred until homogeneous where-
upon dry nitrogen was bubbled through the solution for 15 min via
glass pipette. To this solution, a prepared mixture of NaOMe (3.47 mL,
To an argon-purged, 5000 mL pear-shaped flask equipped with
mechanical stirrer was added PG-Na (40.0 g, 265 mmol, 1.0 eq.) and
DMF (2000 mL). The solution was stirred at room temperature for
30 min. In the following sequence at 30 min intervals, H-Glu(OtBu)-
OtBu·HCl (157 g, 529 mmol, 2.0 eq.), 1-hydroxybenzotriazole hy-
drate (48.6 g, 318 mmol, 1.2 eq.), and N-(3-dimethylaminopropyl)-
N′-ethylcarbodiimide (152 g, 794 mmol, 3.0 eq.) were added to the
reaction flask. The reaction mixture was stirred under nitrogen atmo-
sphere for 36 h. In two equal and separate portions, half of the solu-
tion was poured slowly into a 10 L Nalgene vessel containing
mechanically stirred deionized water (5 L), upon which a white pre-
cipitate formed. After stirring for 10 min, the material was isolated
by vacuum filtration. The solids from each filtration were transferred
to a 10 L Nalgene vessel, suspended in deionized water (5 L), and stir-
red for 10 min by mechanical stirrer. The solids were again isolated by
vacuum filtration and washed with addition MilliQ water (2 L). The
material was lyophilized to afford PGG-ester as a white solid
1
5.2 mmol, 0.02 eq.) and MeOH (11.7 mL) in dioxane (152 mL) was
added in one portion. After stirring at 22 °C for 1 h, the mechanical stir-
rer was removed and the flask was capped. The reaction mixture was
allowed to stand at 22 °C for 72 h, resulting in a clear, colorless, viscous
solution. One quarter of the reaction mixture was poured slowly into a
5
L Nalgene vessel equipped with a mechanical stirrer containing etha-
nol (3 L). After stirring for 10 min at room temperature, the resulting
fibrous polymer was isolated by vacuum filtration (#50 Whatman
paper). These two steps were repeated three more times to process
the remainder of the reaction mixture. The residual material in the
flask was treated with additional ethanol (2×200 mL), filtered, and
combined with the previously collected solids. The combined solids
were transferred to a 5 L plastic beaker containing ethanol (4 L). The
mixture was stirred for 10 min via mechanical stirrer and then allowed
to settle for 1 h. The polymer was collected by vacuum filtration (#50
Whatman paper). The sequence of stirring the solid in ethanol followed
by vacuum filtration was repeated. The polymer was separated into
small pieces and placed on a non-reactive, absorbent material at room
temperature to facilitate drying overnight. The material was further
dried under vacuum at 40 °C to a constant weight to afford PG-ester
as a white fibrous solid (134 g, 82%). 1H NMR (400 MHz, TFA-d): δ
(96.6 g, 98.5%). 1H NMR (400 MHz, CDCl
3
): δ 8.81 (br, 1H), 8.40 (br,
1H), 4.48 (br, 1H), 4.13 (br, 1H), 2.41–2.10 (m, 4H), 2.00–1.30 (m,
22 H) ppm. GPC–MALS: Zimm Model was used for Mw determina-
tion; Mw, 43930; Mw/Mn, 1.31.
2.7. Synthesis of PGG
PGG-ester (95 g, 256 mmol, 1.0 eq) and trifluoroacetic acid (760 mL,
8 mL per gram of PGG-ester) were added to a nitrogen-purged,
2000 mL round-bottomed flask equipped with magnetic Teflon stirbar.
The reaction mixture was stirred at room temperature for 4 h. The TFA
was removed in vacuo at 40 °C to give a gray, glassy foam. The residue
was reconstituted in TFA (380 mL, 4 mL per gram of PGG-ester) and
stirred overnight at room temperature. The TFA was again removed in
7
1
.31–7.09 (m, 5H), 4.98 (m, 2H), 4.53 (m, 1H), 2.32 (m, 2H), 2.02 (m,
H), 1.81 (m, 1H)ppm.

D. Yang et al. / Journal of Controlled Release 161 (2012) 124–131
127
5
vacuo at 40 °C. The residue was dissolved in deionized water (2 L) and
was vacuum filtered (Whatman #50). The filtrate was diluted to 4 L
with deionized water and the pH and conductivity were measured.
The solution was dialyzed via Tangential Flow Filtration (3×10 kDa
molecular weight cutoff cassettes) until the permeate conductivity
was b0.1 mS/cm. The permeate solution was lyophilized to a constant
The cells were seeded in 6-well plates at a density of 3×10 cells per
well and incubated in medium for overnight (37 °C, 5% CO ). Then cells
2
were incubated with the fluorescence-labeled-PGG–PTX samples of
different molecular weights at 37 °C for 0.5, 1, and 2 h. Cells without
treatment were used as control. The concentration of drug used was
10 μg/mL. Meanwhile, the initial amount of fluorescence intensity in
each sample was measured and used for data normalization. After
incubation, medium was removed. Cells were washed twice with cold
0.2 M glycine buffer containing 0.15 M NaCl (pH 3.0) (30 s), followed
by cold PBS wash (30 s). After washing, 500 μL of trypsin–EDTA solution
weight with additional P
(
2
2
O
5
drying to afford a PGG as a white solid
50.5 g, 76%). H NMR (400 MHz, D O): δ 4.50 (br, 1H), 4.15 (br, 1H),
.4 (br, 4H), 2.2–1.9 (br, 4H) ppm. GPC–MALS: Zimm Model was used
1
2
for Mw determination; Mw, 38510; Mw/Mn, 1.23.
(
Gibco, USA) was added to each well, and the cells were detached
completely from the dishes by incubation at 37 °C for 15 min. Cells
were fixed with 2% paraformaldehyde and resuspended in 200 μL PBS
followed immediately by flow cytometry analysis. The amount of
2
.8. Synthesis of PGG–PTX
PGG (10.0 g, 0.063 mol/monomer-unit of polymer) was added
1
×104 cells was collected, and the mean fluorescence intensity was
into a 1000-mL glass flask equipped with a Teflon magnetic stir bar
and a septum under argon atmosphere. N,N-dimethylformamide
recorded for each sample using a FACS C6 flow cytometer and analyzed
using FlowJo software (Accuri).
(
500 mL) was added into the flask, and the solution was stirred at
For confocal image analyses: 24 h before imaging, 5×104 NCI-H460
cells were seeded in a LabTek 8 well chambered coverslides. 20 h before
imaging, cells were incubated with CellLight late endosome (Invitrogen,
Carlsbad, USA), or CellLight lysosome (Invitrogen, Carlsbad, USA). 1 h
before imaging, cells were washed twice with PBS, and incubated with
room temperature for an hour to allow complete dispersion. N-(3-
dimethylaminopropyl)-N′-ethylcarbodiimide (9.4 g, 0.049 mol) and
4
the mixture solution was stirred at room temperature for 15 min to
allow complete dispersion. PTX (5.4 g, 0.0063 mol) was added to
the mixture solution, and the reaction solution was stirred for
-dimethylaminopyridine (2.6 g) were added to the solution, and
0.16 mg/mL of Alexa Fluor 647-PGG-PTX. After incubation, cells were
washed twice with PBS, fixed 15 min in 2% paraformaldehyde. The para-
formaldehyde was then quenched with 50 mM Tris in PBS for 5 min. For
membrane and nucleus staining, cells were then incubated for 5 min
with Alexa 555-wheat germ agglutinin and Hoechst 33342 (Invitrogen,
Carlsbad, USA). The cells were washed again with PBS and covered with
a drop of SlowFade mounting media (Invitrogen, Carlsbad, USA) and
imaged with an Olympus Fluoview 1000 confocal microscope.
2
4–28 h until the PTX was no longer detected by thin layer chroma-
tography (100% ethyl acetate). The solution was poured slowly into
vigorously stirred 0.2 M aqueous hydrochloric acid solution (1.5 L),
during which a white precipitate formed. The precipitate was isolated
by centrifuging the solution for 10 min at 5000 rpm. The residue was
dissolved in 0.5 M sodium bicarbonate solution (1.5 L). The product,
PGG–PTX, was dialyzed against water for 24 h, including changing
water (4 L) four times (once every hour for 3 h and once overnight)
to remove impurities and free PTX. The solution was then filtered
through a 0.45 μm filter and lyophilized. PGG–PTX (14.7 g) was
obtained and characterized using GPC–MALS detector and SEC–
HPLC. The PTX content was determined to be 35.8% by ultraviolet–
visible methods. GPC–MALS: Zimm Model was used for Mw determi-
nation: Mw, 129,400 (0.9%); Mw/Mn: 1.87; 97.7% recovery.
2
2
.11. Toxicity dose-finding studies
.11.1. In vitro cytotoxicity test (MTS assay and cell viability)
Cell viability was evaluated using the MTS colorimetric assay based
on the reduction of a tetrazolium compound (MTS) to a colored forma-
zan product that was then measured at 490 nm; absorbance was direct-
ly proportional to the number of living cells in the culture. Cells (NCI-
3
H460) were seeded in 96-well plates at 2×10 cells/100 μL/well and
2
.9. Drug release experiments
allowed to adhere to the plate overnight. The next day the cells were
treated with free PTX or PGG–PTX at a concentration depending on its
potency. Dilutions were made using the assay medium (DMEM plus
A four-day drug release experiment was performed to compare
the paclitaxel release profile of various PGG–PTX conjugates in phos-
phate buffer system (PBS) containing 20% human plasma at 37 °C.
PGG–PTX samples were prepared in 1.5 mg/mL concentration and
then diluted with human plasma to give working solutions with a
final concentration of 1.2 mg/mL (20% human plasma by volume). A
series of 20 mL vials containing exactly 1.0 mL of each working solu-
tion was prepared with a repeat pipetter. These vials were continu-
ously agitated at 25 rpm in an incubator at 37 °C. In a given time
period, three vials of each drugs plus a control (containing 1.0 mL of
0
.5% FBS) from a stock solution of the drug in assay medium or DMSO
(
PTX, final DMSO concentration less than 0.08%) ethanol, respectively.
After incubation, the culture medium was aspirated and cells were
washed with PBS (pH 7.4); 100 μL of fresh culture medium without
drug and 20 μL of MTS were added to each well and the cells were incu-
bated for 2 h. The plates were read on a Microplate Reader (Synergy HT,
BIO-TEK). Cell survival was expressed as the percentage of viable cells in
treated samples relative to untreated control cells. All experiments
were repeated in duplicate. Two internal controls were set up for each
2
0% human plasma in PBS) were withdrawn from the incubator and
0
experiment: (1) an IC consisting of cells only; and (2) IC100 consisting
extracted with 2×2 mL of ethyl acetate. Analytical samples were fi-
nally prepared by evaporating the EtOAc extracts with a SpeedVac
and reconstituting the residues with 1 mL of methanol. The solutions
were passed through 0.2 μm syringe filters. Analysis of the drug re-
lease was performed by LC–MS (Agilent LC 1100, MS G1956B, Agilent
Eclipse XDB C18 5 μm, 150×4.6 mm column).
of medium only. Background absorbance due to the non-specific reac-
tion between test compounds and the MTS reagent was deducted
from exposed cell values.
2.11.2. Dose response curves of in vitro cytotoxicity data
Dose response curves were plotted for the test articles after cor-
rection by subtracting the background absorbance from the controls.
IC50 (50% inhibitory concentration) and values were extrapolated
from Sigmoidal dose response curve fitting (GraphPad Prism soft-
ware, San Diego, CA).
2
.10. Cell uptake studies
H460 cells, obtained from ATCC were maintained in RPMI 1640
medium supplemented with 10% fetal bovine serum (FBS) and
00 U/mL penicillin and 100 μg/mL streptomycin at 37 °C in an incu-
bator with 5% CO . All experiments were performed in the presence of
0% FBS.
1
2.11.3. In vivo MTD studies
The maximum tolerated dose (MTD) for PGG–PTX was adminis-
tered intravenously or intraperitoneally in female nu/nu nude mice
2
1

128
D. Yang et al. / Journal of Controlled Release 161 (2012) 124–131
(
VitalRiver, Beijing, China). The studies were based on a single esca-
lysosomal release can impact negatively the amount of drug delivered
to the site of action. In addition to these factors, circulation residence
time, tumor tissue accumulation profile, prodrug stability, and the
drug release environment significantly impact the prodrug's antican-
cer efficacy and the general toxicity in vivo.
One of the most important factors that affect the cell uptake and
drug release profiles is the molecular weight of the polymeric back-
bone bearing the chemotherapeutic agent [24,25], which in turn in-
fluences pharmacologic performance and systemic toxicity after
intravenous administration [26]. In this study, we utilized our PGG
lating dose strategy. Three mice per group and three to four dose
levels were explored until the MTD was attained. PGG–PTX was pre-
pared on the day of administration by dissolving test articles in PBS;
toxicity was determined by daily monitoring of body weight and sur-
vival for two weeks following drug administration. The MTD was de-
termined to be the dose that induced ∼10% loss in body weight with
no lethality; extrapolation from Sigmoidal fitting curves were employed
where applicable. No notable behavior differences were noted in any
animals across all dosing groups.
(
poly(L-γ-glutamylglutamine)) polymer as a model system and syn-
2
.11.4. Evaluation of antitumor activity in human non-small lung cancer
thesized four different sizes by varying the molecular weight of PGG
(increasing 12.5 kDa increments). All variants were characterized
for molecular weight and particle size by GPC–MALS and DLS, respec-
tively (Table 2). The impact of PGG polymer weight on cell uptake
and drug release profiles while correlating the in vitro and in vivo ac-
tivity and systemic toxicities of PGG–PTX was investigated.
NCI-H460 xenograft models
NCI-H460 cells were maintained in RPMI-1640 supplemented
with 10% fetal bovine serum containing 100 U/mL penicillin and
1
00 μg/mL streptomycin. Cells were split 48 h before inoculation
into mice so that they were in log phase growth when harvested.
The prodrug linker that conjugates the chemotherapeutic agent to
the polymer backbone affects the drug release profile; a triggered re-
lease can be designed to take advantage of various environmental fac-
tors [27–31]. In the present study, the chemotherapeutic agent (PTX)
is attached to the PGG via an ester bond which can be cleaved by hy-
drolysis and/or esterases [32]. In addition to the prodrug linker, the
polymer molecular weight was also reported to affect the drug re-
lease profile [33,34]. Our studies, however, take advantage of our
ability to control the molecular weight of the penultimate PGG inter-
mediate, which enables the present studies and consequent analyses
on bioperformance parameters. To evaluate the net effects of hydroly-
sis and enzymatic cleavage, PGG–PTX drug release studies were stud-
ied at 37 °C in PBS solution containing 20% human plasma. As shown
in Fig. 2, no initial burst release was observed for the system with 20%
human plasma. In the absence of human plasma, less than 7% of the
paclitaxel was released from the conjugate in the first 24 and only
~12% at 96 h. These results indicate that PGG–PTX is relatively resis-
tant to plasma esterases and is unlikely to release substantial
amounts of free paclitaxel into circulation even with a prolonged res-
idence time. Drug release was controlled by hydrolytic cleavage of the
ester linkage between PGG and PTX. As a result, PGG is able to func-
tion as a stable reservoir in the bio-environment to deliver the anti-
cancer drug with a longer half-life and larger AUC0–340 h compared
with free paclitaxel [19].
2
.11.5. Efficacy studies and MTD measurement
NCI-H460 cells were suspended in serum-free RPMI 1640 medi-
um, and then injected subcutaneously. The size per inoculum site
was 4×10 cells for the NCI-H460 cells. NCI-H460 cells were injected
over each shoulder of 6- to 8-week-old female athymic nude (nu/nu)
mice (Charles River Laboratories, Wilmington, MA) (2 sites per
mouse). Perpendicular tumor diameters were measured with a cali-
per at two day intervals, and tumor volumes were calculated using
6
2
the formula (w ×l)/2, where “l” is the longest diameter of the
tumor and “w” is the width. Tumors were allowed to grow until
they reached an average volume of 100–300 mm3 [21]; the animals
then were randomly divided into 3 groups of 6–10 mice each. Both
PGG–PTX- and vehicle-treated animals were administrated through
intravenous injection. Mice were weighed daily and tumor volumes
were measured every other day until total tumor burden reached
1
were performed at the animal facility of East China Normal University
in accordance with institutional guidelines prescribed by the Institu-
tional Animal Care and Use Committee (IACUC).
3
.5 cm , at which time the mice were sacrificed. All in vivo studies
2
.11.6. Statistics
Tumor volume was graphed versus time and exponential growth
curves of the equation Y=Start∗exp(K∗t) were fit to each data set
by least squares nonlinear regression. The parameter “Start” was con-
strained to the mean initial tumor volume of 125 mm3 and the
growth rate parameter “K” was unconstrained. The growth rates of
the tumors were compared by the comparison of the parameter K be-
tween curve fits using an F-test. Volume data for each measurement
day were compared between treatment groups using a One-way
ANOVA with Bonferroni's t-test as pair-wise post hoc analysis.
For the conjugates with 35 wt.% drug loading but varying MW
(Fig. 2), release profiles for 12.5 kDa PGG–PTX were the fastest,
while those from 50 kDa PGG were slowest. PGG–PTX25K-35% and
PGG-PTX37.5K-35% behaved similarly with a medium release profile.
3
2
1
0
0
0
0
3
. Results and discussion
Binding a drug to macromolecular scaffolds (polymer–drug conju-
gates) considerably alters its diffusibility, thereby significantly alter-
ing the biodistribution of the drug-conjugate. Intravascular half-life
for instance is greatly extended, particularly for polymer conjugates
larger than the glomerular filtration cutoff [22]. Drug concentrations
in tumor tissue are increased due to the EPR (enhanced permeability
and retention) effect as a result of high rates of leakage from the neo-
vasculature of tumor tissue [23]. Typically, cell uptake of synthetic
polymers has been shown to occur via endocytosis [24], whereby
higher molecular weight conjugates are trafficked to the lysosomal
compartment; consequently the chemotherapeutic agents are re-
leased from the polymer backbone and diffuse into cytoplasm or nu-
cleus to inhibit cell growth or kill the cell directly. Drug activity in this
case typically requires release of the drug from the carrier via labile
bonds. Factors such as low rates of cellular internalization and
PGG 50 kDa
PGG 37.5 kDa
PGG 25 kDa
PGG 12.5 kDa
0
24
48
72
96
Time (hours)
Fig. 2. Release of paclitaxel from PGG–PTX conjugate as function of time in 20% human
plasma in PBS at 37 °C; each point represents the mean of three with SEM vertical bars.
Error bars that are undetectable fall into the data points. There were significant differences
in percent PTX release between PGG at 50 kDa and all other groups using Bonferroni's post
hoc test to evaluate pairwise comparisons on all time points. There was no significant
difference in PTX release between PGG at 25 kDa compared to 37.5 kDa.

D. Yang et al. / Journal of Controlled Release 161 (2012) 124–131
129
This observation may be explained by the fact that nanoparticles de-
rived from higher MW material assemble to form tighter hydrophobic
cores and are relatively less accessible by enzymes or water.
Polymer–drug conjugates as macromolecules, characterized by
high molecular weight, cannot be internalized into cells by simple dif-
fusion; instead, these macromolecules enter cells through endocyto-
sis [24]. In order to investigate the effect of anionic polymer
molecular weight on PGG–PTX prodrug cell uptake profile, the four
PGG–PTX prodrug conjugates described above were labeled by
Alexa Fluor 647. Alexa Fluor 647 was chosen because of its robustness
and stability in acidic environments. Flow cytometry was used to
measure cell-associated fluorescence. The uptake model was evaluat-
ed in H460 cells with incubation times of 0.5, 1, and 2 h. Results sum-
marized in Fig. 3A show a correlation of MW with fluorescence
intensity. Cell uptake is more pronounced with conjugates based on
smaller PGG molecular weight, which is understandably a direct cor-
relation to the cell's ability to internalize smaller size conjugates more
easily. Additionally, we observed that cell uptake was essentially
inhibited at 4 °C, which suggests that the major uptake mechanism
of PGG–PTX occurred through endocytosis (data not shown). Further-
more, confocal microscopy images in Fig. 3B (control) and C (2 h)
show evidence of internalization of Alexa Fluor 647-PGG–PTX (red)
over time.
To track PGG–PTX (based on 12.5 kDa PGG with 35% PTX loading)
uptake into H460 cells, fluorescence microscopy-based cellular
uptake studies were performed. Fig. 4 shows that Alexa Fluor 647-
labeled PGG–PTX formulations colocalize with Rab7a-GFP and Lamp1-
GFP used as late endosome (CellLight™ late endosomes) and Lysosome
(
CellLight™ Lysosomes) markers, respectively. Both our FACS and im-
aging data confirm PGG–PTX uptake by endocytosis, consistent with re-
ports from previous studies.
Fig. 4. Confocal imaging results with PGG–PTX (based on 12.5 kDa PGG). Alexa Fluor
647-labeled PGG-PTX formulations colocalize with Rab7a-GFP and Lamp1-GFP used
as late endosome and lysosome markers, respectively. These results suggest that
PGG–PTX is taken up through endocytosis.
The cell uptake and drug release data indicate that PGG–PTX could
confer its cytotoxic effect through release of PTX in the lysosome, fol-
lowed by free drug diffusion into the cytoplasm. To compare overall
PGG–PTX and free PTX in vitro potencies, a human lung cancer
H460 cell line was employed in cytotoxicity assays. Cell-viability
without PGG–PTX co-incubation was used as a control. The mito-
chondrial activity of living cells was measured by an MTS assay. The
PGG–PTX conjugates tested displayed a typical Sigmoidal curve fitting
(
Fig. 5). After 72 h incubation of H460 cells, all conjugate sizes
revealed a dose-dependent cytotoxicity with EC50 values over a very
narrow concentration range. As shown in Fig. 5, an inversely propor-
tional trend of increasing carrier weight with potency was observed
between all groups treated. EC50 values of PGG–PTX derived from
1
2.5, 25, 37.5 and 50 kDa PGG were calculated to be 2.6, 4.9, 6.9,
and 7.1 nM, respectively. As mentioned in the experimental section,
dialysis was employed to remove free PTX from the conjugate; how-
ever, due to the labile nature of the PGG–PTX ester bond, it is likely
impossible to remove the free PTX from the conjugate entirely. Addi-
tionally, stronger intramolecular association or altered uptake profile
of higher molecular weight conjugates explain the variance in EC50 in
vitro; this effect should be compensated in vivo as higher molecular
Fig. 3. A. Cell uptake (Alexa Fluor 647 cell-associated fluorescence) as a function of in-
cubation time. Conjugates based on smaller PGG molecular weight display more pro-
nounced cell uptake as a function of incubation time. All values were normalized
with respect to PGG–PTX derived from 25 kDa PGG. In Fig. 3B and C, NCI-H460 cells
were incubated with Alexa Fluor 647-labeled PGG-PTX (10 μg/mL). Confocal microsco-
py images in 3B (control) and 3C (2 h) show that Alexa Fluor 647-PGG-PTX conjugates
(red) are internalized over time. The cell membrane is shown in green, while the nu-
cleus is shown in blue.

130
D. Yang et al. / Journal of Controlled Release 161 (2012) 124–131
2
2
1
1
0
0
.5
.0
.5
.0
.5
.0
A 3
000
Vehicle (PBS)
5
0 kDa PGG
1
2
3
2.5 kDa PGG
5 kDa PGG
37.5 kDa PGG
2
1
5 kDa PGG
7.5 kDa PGG
2.5 kDa PGG
2000
50 kDa PGG
PTX
1000
0
1
0-4
10-2
100
102
0
5
10
15
20
Concentration (PTX Eq., M)
Days post Administration
Fig. 5. Cytotoxicity of free PTX and PGG–PTX in H460 cells. H460 cells were treated
with increasing concentrations of either free PTX or PGG–PTX for 72 h and cell viability
was assessed by an MTS assay. Data points represent mean absorption at 490 nm, error
bars were standard error of the means, and lines are best fit exponential growth curve
by least squares nonlinear regression. The EC50 values were extrapolated from the Sig-
moidal dose response curve fitting and were plotted vs. PGG molecular weight.
B 3
000
Vehicle (PBS)
0 mg/kg PTX
6
50 kDa PGG
2000
weight conjugates are expected to have a longer intravascular half-
life.
The molecular weight of the carrier and its corresponding conjugate–
efficacy relationship for PGG–PTX was also explored in mice bearing
NCI-H460 lung cancer xenografts in a single-dose regimen. To evaluate
their relative potency, the maximum tolerated doses were determined
first in female nu/nu mice. PGG–PTX was dissolved into pH 7.4 PBS on
the day of dosing, administered by a bolus injection (10 mL/kg) through
mouse tail vein. The maximum tolerated dose (MTD) was defined as the
dose that produced 10% body weight loss within two weeks. As shown
in Table 3, no significant difference in MTD was observed for 35% PTX
loading with regard to carrier size.
1000
0
0
5
10
15
20
Days post Administration
Fig. 6. A. Tumor growth inhibition of PGG–PTX conjugates as a function of PGG molec-
ular weight in mice bearing NCI-H460 tumors. Data points represent mean tumor vol-
ume and error bars are expressed as standard error of the mean. Dosing was chosen
according to MTD values specified in Table 3. PBS vehicle control (hollow circle),
Finally, the structure–activity relationship for carrier molecular
weight was compared in an animal xenograft model. NCI-H460 cells
were inoculated in female nude mice subcutaneously (SC) to gener-
ate two inoculations per mouse. Tumor dimensions (length and
width) were monitored and their volumes were calculated using
1
2.5 kDa PGG (filled diamond), 25 kDa PGG (filled triangle), 37.5 kDa PGG (filled
circle), 50 kDa PGG (filled square). Best fit exponential growth curve was conducted
by least squares nonlinear regression. B. NCI-H460 tumor growth inhibition of standa-
lone PTX vs PGG-PTX derived from 50 kDa PGG. Standalone PTX was prepared as a
cremophor–ethanol formulation. Data points represent mean tumor volume and
error bars are expressed as standard error of the mean. MTD was determined to be the
amount inducing 10% body weight loss without lethality. PBS vehicle control (hollow
circle), 60 mg/kg PTX (hollow square), 50 kDa PGG (filled square).
2
the formula of tumor volume=w ×l/2 where “w” is the smaller di-
mension and “l” is the larger dimension. Drug treatment was initiated
3
once the average tumor size reached 120 mm ; mice were assigned
into various groups to eliminate statistical differences in tumor vol-
umes across the treatment groups. Body weights were recorded
daily for the first 14 days and twice weekly thereafter. Fig. 6A displays
a trend in tumor growth inhibition of PGG–PTX conjugates based on
PGG molecular weights increasing from 12.5 kDa to 50 kDa. To assess
the statistical significance of this difference, tumor volumes were
monitored during the course of the experiment and the data were
graphed with respect to time and exponential growth curves by the
equation Y=Start∗exp(K∗t); curves were fit to each data set by
least squares nonlinear regression. The tumor growth rates (doubling
time) were compared by the comparison of the parameter K between
curve fits using an F-test. Experiment controls behaved as expected
and tumor growth rates were significantly lower with PGG–PTX at
higher molecular weights. For comparative purposes, we included
previously unpublished data in Fig. 6B showing the tumor inhibition
of standalone PTX (60 mg/kg) and PGG–PTX derived from 50 kDa
PGG. These data, in combination with 6A, clearly illustrate the im-
proved tumor inhibition of PGG–PTX conjugates vs PTX. In all cases,
dosing was determined to be at MTD, defined by 10% body weight
loss over two weeks with no lethality. Additionally, Fig. 7 plots
15
1
0
5
0
Table 3
EC50 and MTD of PGG-PTX conjugates. EC50 of free PTX was 1.4 nM.
Sample
PG MW
%
PTX
Dosing
route
EC50
(nM)
MTD
(PTX eq., mg/kg)
0
10
20
30
40
50
(
kDa)
PGG Molecular Weight (kDa)
PGG–PTX12.5K-35%
PGG–PTX25K-35%
PGG–PTX37.5K-35%
PGG–PTX50K-35%
5
35%
35%
35%
35%
IV
IV
IV
IV
2.6
4.9
6.9
7.1
381
393
317
350
10
15
20
Fig. 7. Tumor doubling time as a function PGG molecular weight. Dosing was chosen
according to the MTD determined in Table 3. 0 kDa corresponds to the PBS vehicle
control.

D. Yang et al. / Journal of Controlled Release 161 (2012) 124–131
131
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