Indium-based and iodine-based labeling of HPMA copolymer-epirubicin conjugates: Impact of structure on the in vivo fate

Article abstract of DOI:10.1016/j.jconrel.2016.06.004

Recently, we developed 2nd generation backbone degradable N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-drug conjugates which contain enzymatically cleavable sequences (GFLG) in both polymeric backbone and side-chains. This design allows using polymeric carriers with molecular weights above renal threshold without impairing their biocompatibility, thereby leading to significant improvement in therapeutic efficacy. For example, 2nd generation HPMA copolymer-epirubicin (EPI) conjugates (2P-EPI) demonstrated complete tumor regression in the treatment of mice bearing ovarian carcinoma. To obtain a better understanding of the in vivo fate of this system, we developed a dual-labeling strategy to simultaneously investigate the pharmacokinetics and biodistribution of the polymer carrier and drug EPI. First, we synthesized two different types of dual-radiolabeled conjugates, including 1) ¹¹¹In-2P-EPI-¹²⁵I (polymeric carrier 2P was radiolabeled with ¹¹¹In and drug EPI with ¹²⁵I), and 2) ¹²⁵I-2P-EPI-¹¹¹In (polymeric carrier 2P was radiolabeled with ¹²⁵I and drug EPI with ¹¹¹In). Then, we compared the pharmacokinetics and biodistribution of these two dual-labeled conjugates in female nude mice bearing A2780 human ovarian carcinoma. There was no significant difference in the blood circulation between polymeric carrier and payload; the carriers (¹¹¹In-2P and ¹²⁵I-2P) showed similar retention of radioactivity in both tumor and major organs except kidney. However, compared to ¹¹¹In-labeled payload EPI, ¹²⁵I-labeled EPI showed lower radioactivity in normal organs and tumor at 48 h and 144 h after intravenous administration of conjugates. This may be due to different drug release rates resulting from steric hindrance to the formation of enzyme-substrate complex as indicated by cleavage experiments with lysosomal enzymes (Tritosomes). A slower release rate of EPI(DTPA)¹¹¹In than EPI(Tyr)¹²⁵I was observed. It may be also due to in vivo catabolism and subsequent iodine loss as literature reported. Nevertheless, tumor-to-tissue uptake ratios of both radionuclides were comparable, indicating that drug-labeling strategy does not affect the tumor targeting ability of HPMA copolymer conjugates.

Full text of DOI:10.1016/j.jconrel.2016.06.004

Journal of Controlled Release 235 (2016) 306–318
Contents lists available at ScienceDirect
Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel
Indium-based and iodine-based labeling of HPMA copolymer–epirubicin
conjugates: Impact of structure on the in vivo fate
a
a
a
a
a,b,
Libin Zhang , Rui Zhang , Jiyuan Yang , Jiawei Wang , Jindřich Kopeček
⁎
a
Department of Pharmaceutics and Pharmaceutical Chemistry/CCCD, University of Utah, Salt Lake City, UT 84112, USA
Department of Bioengineering, University of Utah, Salt Lake City, UT 84112, USA
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 6 April 2016
Received in revised form 27 May 2016
Accepted 1 June 2016
Available online 4 June 2016
Recently, we developed 2nd generation backbone degradable N-(2-hydroxypropyl)methacrylamide (HPMA) co-
polymer-drug conjugates which contain enzymatically cleavable sequences (GFLG) in both polymeric backbone
and side-chains. This design allows using polymeric carriers with molecular weights above renal threshold with-
out impairing their biocompatibility, thereby leading to significant improvement in therapeutic efficacy. For ex-
ample, 2nd generation HPMA copolymer-epirubicin (EPI) conjugates (2P-EPI) demonstrated complete tumor
regression in the treatment of mice bearing ovarian carcinoma. To obtain a better understanding of the in vivo
fate of this system, we developed a dual-labeling strategy to simultaneously investigate the pharmacokinetics
and biodistribution of the polymer carrier and drug EPI. First, we synthesized two different types of dual-
Keywords:
N-(2-hydroxypropyl)methacrylamide (HPMA)
Epirubicin
Polymer-drug conjugates
Dual-radiolabeling
111
125
111
radiolabeled conjugates, including 1)
In-2P-EPI- I (polymeric carrier 2P was radiolabeled with
In and
1
25
125
111
125
drug EPI with I), and 2) I-2P-EPI- In (polymeric carrier 2P was radiolabeled with I and drug EPI with
1
11
Pharmacokinetics and biodistribution
In). Then, we compared the pharmacokinetics and biodistribution of these two dual-labeled conjugates in fe-
male nude mice bearing A2780 human ovarian carcinoma. There was no significant difference in the blood circu-
lation between polymeric carrier and payload; the carriers (1 In-2P and
11
125
I-2P) showed similar retention of
111
radioactivity in both tumor and major organs except kidney. However, compared to In-labeled payload EPI,
I-labeled EPI showed lower radioactivity in normal organs and tumor at 48 h and 144 h after intravenous ad-
1
25
ministration of conjugates. This may be due to different drug release rates resulting from steric hindrance to the
formation of enzyme-substrate complex as indicated by cleavage experiments with lysosomal enzymes
Tritosomes). A slower release rate of EPI(DTPA)1 In than EPI(Tyr) I was observed. It may be also due to in
11
125
(
vivo catabolism and subsequent iodine loss as literature reported. Nevertheless, tumor-to-tissue uptake ratios
of both radionuclides were comparable, indicating that drug-labeling strategy does not affect the tumor targeting
ability of HPMA copolymer conjugates.
©
2016 Elsevier B.V. All rights reserved.
1
. Introduction
However, the use of PEG can be problematic in some instances, with re-
cent results indicating PEG-containing therapeutics can elicit comple-
ment activation, rapid clearance after repeated injections, and may
suffer from peroxidation [15,16]. Moreover, the inability to effectively
functionalize the polyether backbone mitigates the utility of many
PEG drug delivery systems [17]. HPMA copolymers have comparable
biocompatibility and advantages over PEG on non-immunogenicity
and established bioconjugation strategies. Its favorable properties
have been validated by diverse preclinical and clinical studies [18–22].
Recently, higher molecular weight (Mw) biodegradable HPMA copoly-
mer-drug conjugates have been designed with enzymatically degrad-
able tetrapeptide sequences (GFLG) in both polymer backbone and
side chains to prolong plasma circulation and enhance tumor accumula-
tion while preserving biocompatibility [23–28].
Conjugation of anticancer drugs to water-soluble polymers offers a
possibility to improve their solubility, decrease adverse effects, modify
pharmacokinetics, favorably change their biodistribution, and improve
therapeutic efficacy by enhanced permeability and retention (EPR) ef-
fect [1–4]. Numerous synthetic polymers have been used such as poly(-
ethylene glycol) (PEG) [5,6], [N-(2-hydroxypropyl)methacrylamide]
(
HPMA) copolymers [7–11], poly(amino acids) [12], polyoxazoline
[13], poly(malic acid) [14], etc. Among the various polymeric carriers,
the most commonly used is PEG, which has been approved by FDA for
clinical application and is commercial available in a wide range of
molar masses, end-functionalities as well as different architectures.
The knowledge of the pharmacokinetics and biodistribution of mac-
romolecular therapeutics is a prerequisite for the understanding of the
mechanism of their action and ultimate translation into clinical use.
⁎
Corresponding author at: Center for Controlled Chemical Delivery (CCCD), 20 S 2030
E, BPRB 205B, University of Utah, Salt Lake City, UT 84112-9452, USA.
E-mail address: jindrich.kopecek@utah.edu (J. Kopeček).
http://dx.doi.org/10.1016/j.jconrel.2016.06.004
0168-3659/© 2016 Elsevier B.V. All rights reserved.

L. Zhang et al. / Journal of Controlled Release 235 (2016) 306–318
307
The fate of HPMA copolymers after administration to animals has been
intensively studied. Initially, drug concentration in various organs was
determined by direct extraction of drug from lyophilized tissue samples
followed by HPLC analysis or fluorescence assay [29,30]. This approach
needs large groups of animals and tedious work; it was replaced by
radiolabeling strategy, which has high sensitivity and was widely ap-
plied in preclinical studies and clinical investigations [31–33]. Among
the distribution and tumor accumulation of polymer carrier and a cleav-
able model drug [44]. Recently we designed dual-isotope-labeled 2nd
generation HPMA copolymer-drug model conjugate, whose HPMA copol-
ymer backbone was labeled with 1 I, whereas In-DTPA complex was
bound at GFLG side-chain termini and served as the drug model [26].
We have reported the pharmacokinetics and therapeutic efficacy of
2nd generation diblock HPMA copolymer-epirubicin (EPI) conjugates
(2P-EPI) in the treatment of experimental ovarian cancer [28]. Notably,
treatment with 2P-EPI resulted in complete tumor remission and long-
term inhibition of tumorigenesis (N100 days), whereas the tumor re-
currence was observed in mice treated with the 1st generation HPMA
copolymer-EPI conjugate (P-EPI, with Mw b 50 kDa). To demonstrate
the different pharmacologies between these two generations of conju-
gates, we designed and synthesized a series of dual radiolabeled
25
111
124 125
131
different radionuclides single-step iodination ( I,
I and
I) and
two-step radiometal labeling (90Y,
111
In and
177
Lu, etc.) are often per-
formed. Iodination has been frequently used due to low cost and simple
radiochemistry. For example, it was reported that radioiodination of
drugs (daunomycin and doxorubicin) was achieved by mixing drug
(
or conjugate) solution with iodide in an iodogen reaction vial under
ambient condition for a few minutes [19,34]. In the majority cases, how-
ever, tyrosine moiety was typically incorporated into polymer carrier
via copolymerization followed by iodine labeling [35–41]. In these
cases, the radioactive signals were correlated to the polymer carrier
rather than drug. To examine the circulation and accumulation of real
HPMA copolymer-EPI conjugates in which 1 I and
25
111
In were used to
label polymer carrier and drug (EPI), respectively, or vice versa. This
paper is devoted to the study of pharmacokinetics and biodistribution
of 2nd generation HPMA copolymer-EPI conjugates in nude mice. In
1
4
125
drug molecules, C-labeled drug might be an option. Nevertheless,
such isotope-labeled drugs are expensive; the synthesis of conjugates
will be complicated because 14C has a long half-life and will cause
large level of irradiation. Recently, dual-labeling strategies have been
developed in which one probe aims to track the polymer carrier, while
the other one monitor the fate of drug (modified or model drug) [26,
one approach, we labeled the polymer carrier with
I and used
DTPA-1 In to modify the EPI structure. In the second design, we used
11
111
125
DTPA- In to label the polymer carrier and
I for drug modification.
The issues we addressed are: A) How does modification of the conjugate
structure influence its fate? Is there a difference between the two label-
ing designs? B) How does the modification of drug structure impact the
formation of the enzyme-substrate complex, rate of enzymatic drug re-
lease and the biodistribution of the drug? C) How does the data obtain-
ed differ from the behavior of the unlabeled conjugate that would be
used as the macromolecular therapeutics?
4
2–43]. For example, dual-fluorescent conjugates were studied using
Fluorescence resonance energy transfer (FRET) as a tool to track chain
scission of the conjugates and drug release from the carrier [28], or
using noninvasive multispectral optical imaging to real time monitor
2
. Experimental section
2
.1. Abbreviations
APMA
Boc-GFLG-OMe
N-(3-aminopropyl)methacrylamide hydrochloride
methyl9-benzyl-2,2-dimethyl-4,7,10,13-tetraoxo-12-propyl-3-oxa-5,8,11,14-tetraazahexadecan-16-oate
Boc-GFLG-NH
CTA
2
tert-butyl(14-amino-4-benzyl-7-isobutyl-2,5,8,11-tetraoxo-3,6,9,12-tetraazatetradecyl)carbamate
chain transfer agent (4-cyanopentanoic acid dithiobenzoate)
CTA-GFLG-CTA
10-benzyl-2,25-dicyano-13-isobutyl-5,8,11,14,17,22-hexaoxo-6,9,12,15,18,21-hexaazahexacosane-2,25-diyl dibenzodithioate
DCC
DMAP
EPI
N,N′-dicyclohexylcarbodiimide
4-(dimethylamino) pyridine
epirubicin
Fmoc-Abu(N
HPMA
3
)-OH
(S)-2-(Fmoc-amino)-4-azidobutanoic acid
N-(2-hydroxypropyl)methacrylamide
HATU
HOBt
1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxidhexafluorophosphate
1-hydroxybenzotriazole
1
1
1
11
11
25
In-P-EPI-125
111
125
I
frist generation HPMA copolymer-EPI conjugate with
second generation HPMA copolymer-EPI conjugate with
In-DTPA labeled polymer backbone and
In-DTPA labeled polymer backbone and
I-Tyr labeled EPI
I-Tyr labeled EPI
125
111
125
In-2P-EPI-
I
I-2P-EPI-111In
second generation HPMA copolymer-EPI conjugate with 125I-Tyr labeled polymer backbone and 111In-DTPA labeled EPI
N-methacryloyltyrosinamide
MA-Tyr-NH
2
MA-GFLG-OH
N-methacryloylglycylphenylalanylleucylglycine
MA-GFLG-Abu(N
3
)-OH N-(2-((4-azido-1-(((2S,3R,4S,6R)-3-hydroxy-2-methyl-6-((3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)
tetrahydro-2H-pyran-4-yl)amino)-1-oxobutan-2-yl)amino)-2-oxoethyl)-2-(2-(2-methacrylamidoacetamido)-3-phenylpropanamido)-4-methylpentanamide
)-EPI N-(2-((4-azido-1-(((2S,3R,4S,6R)-3-hydroxy-2-methyl-6-((3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)
oxy)tetrahydro-2H-pyran-4-yl)amino)-1-oxobutan-2-yl)amino)-2-oxoethyl)-2-(2-(2-methacrylamidoacetamido)-3-phenylpropanamido)-4-methylpentanamide
MA-GFLG-Abu(N
3
MA-GFLG-Abu(Tyr)-EPI 2-(4-(1-(12-benzyl-3-(((2S,3R,4S,6R)-3-hydroxy-2-methyl-6-((3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)
oxy)tetrahydro-2H-pyran-4-yl)carbamoyl)-9-isobutyl-18-methyl-5,8,11,14,17-pentaoxo-4,7,10,13,16-pentaazanonadec-18-en-1-yl)-1H-1,2,3-triazol-5-yl)butanamido)-
3-(4-hydroxyphenyl)propanoic acid
MA-GFLG-NHBoc
MA-GG-EPI
tert-butyl(11-benzyl-8-isobutyl-17-methyl-4,7,10,13,16-pentaoxo-3,6,9,12,15-pentaazaoctadec-17-en-1-yl)carbamate
N-(2-((2-(((2S,3R,4S,6R)-3-hydroxy-2-methyl-6-((3,5,12-trihydroxy-3-(2-hydroxyacetyl)-10-methoxy-6,11-dioxo-1,2,3,4,6,11-hexahydrotetracen-1-yl)oxy)tetrahydro-
2H-pyran-4-yl)amino)-2-oxoethyl)amino)-2-oxoethyl)methacrylamide
NH
2
-GFLG-NH
2
2-(2-(2-aminoacetamido)-3-phenylpropanamido)-N-(2-((2-aminoethyl)amino)-2-oxoethyl)-4-methylpentanamide
2,2′-((1-((2-((2-carboxyethyl)(carboxymethyl)amino)ethyl)(carboxymethyl)amino)-3-(4-thiocyanatophenyl)propan-2-yl)azanediyl)diacetic acid
first generation HPMA and MA-GFLG-EPI copolymer conjugate
p-SCN-Bn-DTPA
P-EPI
P-EPI-Tyr
first generation HPMA and MA-GFLG-Abu(Tyr)-EPI copolymer conjugate
P-EPI-N
P-DTPA-EPI(Tyr)
3
first generation HPMA, APMA and MA-GFLG-Abu(N
first generation HPMA copolymer-EPI conjugate with DTPA pendent on polymer backbone and tyrosine moiety attachment with EPI
second generation HPMA, APMA and MA-GFLG-Abu(N )-EPI copolymer conjugate
second generation HPMA copolymer-EPI conjugate with DTPA pendent on polymer back bone and tyrosine moiety attachment with EPI
3
)-EPI copolymer conjugate
2
P-EPI-N
P-DTPA-EPI(Tyr)
3
3
2
(continued on next page)

308
L. Zhang et al. / Journal of Controlled Release 235 (2016) 306–318
(continued)
2
2
P-Tyr-EPI-N
P-Tyr-EPI(DTPA)
3
2 3
second generation HPMA, MA-Tyr-NH and MA-GFLG-Abu(N )-EPI copolymer conjugate
second generation HPMA copolymer-EPI conjugate with tyrosine moiety pendent on polymer backbone and DTPA attachment with EPI
reversible addition-fragmentation chain transfer
tris(benzyltriazolylmethyl)amine
RAFT
TBTA
TFA
trifluoroacetic acid
Hexynoic-Tyr
V-65
2-(hex-5-ynamido)-3-(4-hydroxyphenyl)propanoic acid
2,2′-azobis(2,4-dimethylvaleronitrile)
V-501
VA-044
4,4-azobis (4-cyanopentanoic acid)
2,2′-azobis[2-(2-imidazolin-2-yl) propane]dihydrochloride
2
.2. Materials
Common reagents were purchased from Sigma-Aldrich and used as received unless otherwise specified. 2,2′-Azobis(2,4-dimethylvaleronitrile)
V-65), 2,2′-azobis[2-(2-imidazolin-2-yl) propane]dihydrochloride (VA-044) were from Wako; 4,4-azobis (4-cyanopentanoic acid) (V-501) was
from Fisher Scientific. N,N′-Dicyclohexylcarbodiimide (DCC), Fmoc-Abu(N )-OH (S)-2-(Fmoc-amino)-4-azidobutanoic acid and 4-(dimethylamino)
(
3
pyridine (DMAP) were obtained from AAPPTEC. Iodine-125 [1 I] was obtained from Perkin-Elmer. InCl
25
111
was from Intermountain Radiopharmacy.
3
1
-Hydroxybenzotriazole (HOBt) and N-Boc-ethylenediamine were purchased from AnaSpec. p-SCN-Bn-DTPA was purchased from Macrocyclics. Re-
versible addition-fragmentation chain transfer (RAFT) agent, 4-cyanopentanoic acid dithiobenzoate (chain transfer agent, CTA), was synthesized ac-
cording to the literature [45].
2
2
.3. Methods
.3.1. Synthesis of monomers
N-(2-Hydroxypropyl)methacrylamide (HPMA) was synthesized by acylating 1-aminopropan-2-ol with methacryloyl chloride in acetonitrile, as
previously described [46]. Melting point: 69–71 °C; N-methacryloyltyrosinamide (MA-Tyr-NH ) [47], N-methacryloylglycylphenylalanylleucylglycine
2
(MA-GFLG-OH), Boc-GFLG-OMe [48] and MA-GG-EPI [49] were synthesized as previously described.
2
.3.1.1. Synthesis of Hexynoic-Tyr. Fmoc-Tyr(Bu)-OH (230 mg, 0.5 eq.) and DIPEA (313 μL, 4 eq.) were dissolved in 9 mL dry dichloromethane (DCM).
The solution was added to the resin and shook for 2 h at room temperature (r.t.). The solution was drained and washed with 5 × DCM/MeOH/DIPEA
17/2/1, 9 mL), 3 × DCM and 3 × DMF. Kaiser Test showed a negative result. Piperidine/DMF (1/4, 9 mL) was added and shook for 5 min, a positive
Kaiser Test result showed by dark blue solution and beads. Hex-5-ynoic acid (140 mg, 132 μL, 2.5 eq.), DIPEA (391 μL, 5 eq.) and HATU (456 mg,
.4 eq.) in 5 mL DMF were added and shook for about 2 h. The solution was drained and washed with 3 × DMF, 3 × DCM and 3 × MeOH. TFA/
(
2
1
DCM (3/7, 9 mL) was added and shook for 2 h. After filtration and removing the solvent, 97 mg of product was obtained (35%) as yellow oil. H
NMR (300 MHz, CD OD) δ 1.67–1.74 (m, 2H), 2.09–2.11 (m, 2H), 2.2 (t, 1H, J = 2.1), 2.26–2.29 (m, 2H), 2.83 (dd, 1H, J = 10.5, 6.9), 3.11 (dd, 1H,
J = 10.5, 6.9), 4.59 (dd, 1H, J = 6.9, 3.6), 6.68–6.70 (m, 2H), 7.03 (dd, 2H, J = 4.3, 1.5).
3
2
.3.1.2. Synthesis of MA-GLFG-Abu(N
was added to the resin and shook for 2 h at r.t. The solution was drained and the resin was washed with 5 × DCM/MeOH/DIPEA (17/2/1, 6 mL),
× DCM and 3 × DMF. Kaiser Test was negative. The solvent was drained and piperidine/DMF (1/4, 2.5 mL) was added. After shaking for 5 min, a
3 3
)-OH. Fmoc-Abu(N )-OH (73 mg, 0.5 eq.) and DIPEA (139 μL, 4 eq.) were dissolved in 4 mL dry DCM. The solution
3
positive Kaiser Test was obtained. MA-GFLG-OH (230 mg, 2.5 eq.), DIPEA (208 μL, 5 eq.) and HBTU (182 mg, 2.4 eq.) in 2 mL DMF were added
and shook for 2 h. The solution was drained and washed with 3 × DMF, 3 × DCM, 3 × MeOH. TFE/DCM (3/7, 4 mL) was added and shook for 2 h.
After filtration and removing the solvent, 95 mg of product was obtained by precipitation in ethyl ether (81%) as white powder.
2
(
.3.1.3. Synthesis of MA-GFLG-Abu(N
19 mg, 0.05 mmol) were dissolved in 400 μL DMF. 2 min later, EPI (27 mg, 0.05 mmol) was added and the solution was stirred at r.t. for 1 h. Ethyl
acetate (100 mL) was added and the solution was washed with NaHCO (0.1 M, 50 mL), HCl (0.1 M, 50 mL) and NaCl (sat., 50 mL) three times, re-
spectively. The organic layer was dried with anhydrous MgSO and the solvent was removed under vacuum. The residue was precipitated into ethyl
ether to yield 30 mg of product as red powder (yield 54%). The structure and purity of the product were confirmed by MALDI-TOF-MS and HPLC
3 2 3
)-EPI. Under N atmosphere, MA-GFLG-Abu(N )-OH (30 mg, 0.05 mmol), DIPEA (23 μL, 0.13 mmol) and HATU
3
4
(
3
Agilent ZORBAX, 5 μm, 300SB-C18 column 4.6 × 250 mm, using flow rate 1.0 mL/min and gradient elution from 2% to 90% of buffer B within
+
+
2
0 min. Buffer A: DI H O with 0.1% TFA, Buffer B: acetonitrile with 0.1% TFA) analysis. MS (MALDI-TOF) m/z: 1134.46 [M + Na] , 1150.43 [M + K] .
2
.3.1.4. Synthesis of MA-GFLG-Abu(Tyr)-EPI. MA-GFLG-Abu(N )-EPI (36 mg, 32 μmol) was dissolved in 600 μL DMF and purged with N for 20 min.
3 2
Hexynoic-Tyr (40 mg, 145 μmol), CuBr (4.5 mg, 32 μmol) and tris(benzyltriazolylmethyl)amine (TBTA) (15 mg, 32 μmol) were dissolved in
00 μL DMF (purged with N for 20 min) and added into the MA-GFLG-Abu(N )-EPI solution. The mixture was stirred at r.t. and purged with N
for 20 min. Ascorbic acid (100 mg/mL × 54 μL, in H O purged with N for 20 min) was added and stirred at room temperature overnight. After work-
ing up, added 150 mL of ethyl acetate and washed with HCl (2 × 50 mL, 0.5 M). The aqueous phase turned red after washing with NaHCO (sat.,
0 mL). The pH value was adjusted to about 1 with HCl (1 M) and the aqueous layer was extracted with ethyl acetate (3 × 50 mL). The organic
4
2
3
2
2
2
3
5
4
phase was combined and dried with anhydrous MgSO . The solvent was removed by rotary evaporation below 35 °C, the residue was precipitated
into ethyl ether to yield 19 mg of product as red powder (yield 42%). The structure and purity of the product were confirmed by MALDI-TOF-MS
+
+
and HPLC analysis. MS (MALDI-TOF) m/z: 1409.60 [M + Na] , 1425.59 [M + K] .
2
.3.2. Synthesis of diarm chain transfer agent CTA-GFLG-CTA (Fig. S3)
Boc-GFLG-OMe (505 mg, 1 mmol) was dissolved in CH OH (5 mL), NH
working up, the solvent and part of NH (CH NH were removed by rotary evaporation below 35 °C; then 150 mL ethyl acetate were added. After
3
2 2 2 2
(CH ) NH (647 μL, 10 mmol) was added and stirred at r.t. for 3 h. After
2
)
2 2
2

L. Zhang et al. / Journal of Controlled Release 235 (2016) 306–318
309
washing with H
yield 361 mg of product as white solid (Boc-GFLG-NH
Boc-GFLG-NH (390 mg, 0.73 mmol) was dissolved in DCM (10 mL), 4 mL TFA was added and stirred at r.t. for 4 h and at 4 °C overnight. After re-
moving the solvent, the product was dried under reduced pressure to yield NH -GFLG-NH ∙2TFA as white solid (315 mg, 98%). CTA (217 mg,
.78 mmol), DCC (167 mg, 0.8 mmol) and HOBt (105 mg, 0.78 mmol) were dissolved in DMF/acetonitrile (2 mL/10 mL). The solution was stirred for
0 min at r.t. NH -GFLG-NH ∙2TFA (173 mg, 0.26 mmol) was dissolved in 5 mL acetonitrile with 104 μL DIPEA (0.6 mmol). After 30 min, the solution
-GFLG-NH ) was added to the CTA solution and stirred at room temperature overnight. After working up, filtered and removed solvent,
00 mL ethyl acetate was added and washed with HCl (0.5 M, 3 × 100 mL), NaHCO (sat., 3 × 100 mL) and NaCl (sat. 1 × 100 mL). The organic phase was
, the solvent was removed by rotary evaporation below 35 °C. The residue was purified by silica gel column chromatog-
raphy (acetone: petroleum ether = 1: 5–5: 1) to yield CTA-GFLG-CTA as pink solid (260 mg, 50%). The purity and structure of CTA-GFLG-CTA were con-
2 4
O (3 × 50 mL) and NaCl (sat. 3 × 50 mL), the organic phase was dried with MgSO , the solvent was removed by rotary evaporation to
2
, 68%).
2
2
2
0
3
2
2
(
containing NH
2
2
6
3
combined and dried by MgSO
4
1
firmed by HPLC, NMR and MALDI-TOF-MS analysis (Fig. S9). H NMR (400 MHz, CD
3
OD) δ 0.71–0.79 (m, 3H), 0.88–0.92 (m, 3H), 1.43–1.48 (m, 1H),
1
.50–1.68 (m, 1H), 1.93 (s, 6H), 2.36–2.43 (m, 2H), 2.51–2.59(m, 6H), 2.93–3.03 (m, 2H), 3.16–3.23 (m, 1H), 3.70–3.91 (m, 4H), 4.07–4.20 (m, 1H),
+
+
4.45–4.86 (m, 1H), 7.22–7.26 (m, 5H), 7.44–7.44 (m, 4H), 7.59 (s, 2H), 7.91–7,92 (m, 4H). MS (MALDI-TOF) m/z: 957.35 [M + H] , 979.33 [M + Na] .
2
2
.3.3. Synthesis of HPMA copolymer-EPI conjugates
.3.3.1. Synthesis of first generation HPMA copolymer-EPI conjugate P-DTPA-EPI(Tyr)
2
3 3
.3.3.1.1. Synthesis of P-EPI-N . An ampoule containing HPMA (92 mg, 0.69 mmol, 92%), APMA (5.1 mg, 0.027 mmol, 4%) and MA-GFLG-Abu(N )-
EPI (30 mg, 0.027 mmol, 4%) were attached to the Schlenk-line. After three vacuum-nitrogen cycles to remove oxygen, 150 μL degassed MeOH was
added and stirred at r.t. CTA (2.3 mg/mL × 150 μL, 0.000622 mmol, in degassed MeOH) and VA-044 (1 mg/mL × 145 μL, 0.000201 mmol, in degassed
H O) were added via syringe under magnetic stirring and bubbled with N for 10 min in ice bath. The ampoule was sealed, and polymerization was
2 2
performed at 40 °C for 22 h. The copolymer was obtained by precipitation into acetone/ethyl ether (3:1) and purified by redissolving in methanol and
precipitation in acetone/ethyl ether (3:1) two more times. The copolymer was isolated as red powder and dried under vacuum. Yield: 24 mg (19%).
The average molecular weight (Mw) and the polydispersity (PDI) of the conjugates were determined by size-exclusion chromatography (SEC) on
an AKTA FPLC system equipped with a UV detector (GE Healthcare), mini DAWN TREOS, and Optilab rEX (refractive index) detector (Wyatt Technol-
ogy) using a Superose 6 HR10/30 column with sodium acetate buffer containing 30% (vol/vol) acetonitrile (pH 6.5) as mobile phase. HPMA homo-
polymer fractions were used as molecular weight standards.
The red powder (24 mg) was further reacted with V-65 (8.4 mg, 0.034 mmol, over 40-times excess with respect to the polymer end groups) in
0
.3 mL MeOH at 55 °C for 2 h, purified by precipitation into acetone/ethyl ether (3:1) twice, resulting in intermediate P-EPI-N
alyzed by ninhydrin assay [50] to determine amino content. The EPI content of copolymers was determined spectrophotometrically with free EPI
standard working curve. ([NH ]: 216 nmol/mg polymer; [EPI]: 10.5 wt%).
.3.3.1.2. Attachment of tyrosine moiety and DTPA chelator. The polymer precursor (P-EPI-N
for 20 min. Hexynoic-Tyr (3 eq.), CuBr (1 eq.) and TBTA (1 eq.) were dissolved in 200 μL DMF (purged with N
polymer precursor solution. The mixture was stirred at room temperature and purged with N for 20 min. Ascorbic acid (10 eq., in H
with N for 20 min) was added and the mixture was stirred at room temperature overnight. After purification by ultrafiltration (10,000 Da cut-off,
Millipore) with DI H O three times and freeze drying, the product was obtained as red powder (16 mg, 71%). The red powder (15 mg) was dissolved
in 400 μL NaHCO buffer (0.2 M, containing 10 mM EDTA, pH 8.5) and mixed with p-SCN-Bn-DTPA (10 mg) in 200 μL DMSO. After stirring at room
temperature overnight, the sample was applied to a pre-equilibrated PD-10 Sephadex G25 column (GE Healthcare) with DI H O for primary purifi-
cation. The fraction of 2.5–4.5 mL was collected and further purified by ultrafiltration (10,000 Da cut-off) with NaHCO buffer three times and DI H
three times. The final product in DI H O was freeze dried to remove solvent (13 mg, P-DTPA-EPI(Tyr), 87%). The DTPA content was determined using
spectrophotometric method [51]. The content of DTPA in P-DTPA-EPI(Tyr) was calculated as 140 nmol/mg polymer. The conversion was 64%.
3
. The polymer was an-
2
2
3
, 23 mg) was dissolved in 200 μL DMF and purged with
for 20 min) and added into the
O purged
N
2
2
2
2
2
2
3
2
3
2
O
2
2
.3.3.2. Synthesis of second generation HPMA copolymer-EPI conjugate 2P-DTPA-EPI(Tyr)
3
.3.3.2.1. Synthesis of 2P-EPI-N . An ampoule containing HPMA (80 mg, 0.56 mmol, 93%), APMA (3.4 mg, 0.02 mmol, 4%) and MA-GFLG-Abu(N )-
2
3
EPI (20 mg, 0.018 mmol, 3%) was attached to the Schlenk-line. After three vacuum-nitrogen cycles to remove oxygen, 136 μL degassed MeOH was
added and the reaction mixture stirred at r.t. CTA-GFLG-CTA (10 mg/mL × 38 μL, 0.0004 mmol, in degassed MeOH) and VA-044 (2 mg/
mL × 36 μL, 0.000223 mmol, in degassed H O) were added via syringe under magnetic stirring and bubbled with N for 10 min in ice bath. The am-
2 2
poule was sealed, and polymerization was performed at 40 °C for 44 h. The copolymer was obtained by precipitation into acetone/ethyl ether (3:1)
and purified by redissolving in methanol and precipitation in acetone/ethyl ether (3:1) two more times. The copolymer was isolated as red powder
and dried under vacuum. Yield: 35 mg (34%). The red powder (35 mg) was further reacted with V-65 (5.3 mg, 0.034 mmol, over 40-times excess with
respect to the polymer end groups) in 0.4 mL MeOH at 55 °C for 2 h, purified by precipitation into acetone/ether (3:1) twice, resulting in intermediate
2
P-EPI-N
mg polymer).
.3.3.2.2. Attachment of tyrosine moiety and DTPA chelator. The synthetic method was the same as in Section 2.3.3.1.2. Obtained 15 mg of final prod-
uct (yield 87%). The content of DTPA in 2P-DTPA-EPI(Tyr) was determined as 83 nmol/mg polymer.
3 3 2
. EPI and amino group content were determined by the same methods as P-EPI-N (see Section 2.3.3.1.1). ([EPI]: 9.7 wt%; [NH ]: 320 nmol/
2
2
.3.3.3. Synthesis of second generation conjugate 2P-Tyr-EPI-DTPA
.3.3.3.1. Synthesis of 2P-Tyr-EPI-N . An ampoule containing HPMA (80 mg, 0.56 mmol, 94.8%), MA-Tyr-NH
GFLG-Abu(N )-EPI (20 mg, 0.018 mmol, 3%) was attached to the Schlenk-line. After three vacuum-nitrogen cycles to remove oxygen, 46 μL degassed
MeOH was added and the mixture was stirred at r.t. CTA-GFLG-CTA (3 mg/mL × 128 μL, 0.4 μmol, in degassed MeOH) and VA-044 (2 mg/mL × 36 μL,
.223 μmol, in de-gassed H O) were added via syringe under magnetic stirring and bubbled with N for 10 min in ice bath. The ampoule was sealed,
2
3
2
(3 mg, 0.013 mmol, 2.2%) and MA-
3
0
2
2
and polymerization was performed at 40 °C for 44 h. The copolymer was obtained by precipitation into acetone/ethyl ether (3:1) and purified by
redissolving in methanol and precipitation in acetone/ethyl ether (3:1) two more times. The copolymer was isolated as red powder and dried
under vacuum. Yield: 40 mg (39%). The red powder (40 mg) was further reacted with V-65 (5.3 mg, 0.034 mmol, over 40-times excess with respect
to the polymer end groups) in 0.4 mL MeOH at 55 °C for 2 h, purified by precipitation into acetone/ethyl ether (3:1) twice, resulting in 2P-Tyr-EPI-N
3
.
The EPI content was 5.5 wt%.

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L. Zhang et al. / Journal of Controlled Release 235 (2016) 306–318
2
.3.3.3.2. Attachment of DTPA chelator. 2P-Tyr-EPI-N
were dissolved in 400 μL DMF and purged with N for 20 min. The mixture was stirred at room temperature and purged with N
acid (100 mg/mL × 12 μL, in H O which was purged with N for 20 min) was added and stirred at room temperature overnight. After working up, the
reaction mixture was purified by ultrafiltration (30,000 Da cut-off) with DI H O three times. The remnant was precipitated in 14 mL acetone/ethyl
ether (4:1) twice. 24 mg of red powder was obtained (73%). Amino content was determined as 75 nmol/mg polymer. Red powder (20 mg) was dis-
solved in 0.8 mL of NaHCO (0.2 M) containing 10 mM EDTA (pH 8.5), then mixed with p-SCN-Bn-DTPA (10 mg) in 200 μL of DMSO. After stirring at
room temperature overnight, the sample was applied to a pre-equilibrated PD-10 Sephadex G25 column (GE Healthcare) with DI H O for primary
purification. The fraction of 2.5–4.5 mL was collected and further purified by ultrafiltration (30,000 Da cut-off) with NaHCO buffer three times
and DI H O three times. The final sample in DI H O was freeze dried to remove solvent (16 mg, 80%). The content of DTPA was determined spectro-
photometrically (16 nmol/mg) [51]. The conversion was 22%.
3
(30 mg, 1 eq.), propargylamine (1.2 mg, 3 eq.), CuBr (1.0 mg, 1 eq.) and TBTA (3.0 mg, 1 eq.)
2
2
for 20 min. Ascorbic
2
2
2
3
2
3
2
2
2
.3.3.4. Synthesis of conjugates P-EPI and P-EPI(Tyr) for Tritosomes cleavage kinetic study. An ampoule containing 50 mg (or 73 mg) of HPMA and 10 mg
of MA-GFLG-EPI (or 7 mg MA-GFLG-Abu(Tyr)-EPI) was attached to the Schlenk-line. After three vacuum-nitrogen cycles to remove oxygen, 100 μL
or 120 μL) degassed MeOH was added and stirred at r.t. CTA, 4 mg/mL × 50 μL (or 2 mg/mL × 70 μL) in degassed MeOH and 2 mg/mL × 39 μL (or
mg/mL × 27 μL) of VA-044 in degassed MeOH were added via syringe under magnetic stirring and bubbled with N for 10 min in ice bath. The am-
(
2
2
poule was sealed, and polymerization was performed at 44 °C for 22 h. The copolymer was obtained by precipitation into acetone/ethyl ether (3:1)
and purified by redissolving in methanol and precipitation in acetone/ethyl ether (3:1) two more times. The copolymer was isolated as red powder
and dried under vacuum. Yield: 47 mg of P-EPI (57%) and 26 mg of P-EPI(Tyr) (46%). After end-modification with 40-times excess of V-65, the EPI
content of copolymers was determined spectrophotometrically as 86 nmol/mg polymer for P-EPI and 120 nmol/mg polymer for P-EPI(Tyr).
2
.3.4. Radiolabeling
125
I labeling of tyrosine moiety and 111In labeling of DTPA were conducted immediately before use by the method as previously described [26]. For
125
single labeling of I, HPMA copolymer-EPI conjugates, containing tyrosine amide in the side chains or pendant on the backbones, were reacted with
Na I (Perkin-Elmer) at r.t. in 0.01 M phosphate buffer containing chloramine-T for 30 min. After purification with ultrafiltration (10,000 Da cut-off
125
125
tube for first generation conjugate and 30,000 Da cut-off for second generation conjugates), the copolymer with I labeling in 0.1 M sodium acetate
solution (pH 5.2) was mixed with an aqueous solution of 1 InCl
11
at r.t. for 30 min and then purified by ultrafiltration (10,000 Da cut-off for first gen-
3
eration conjugate and 30,000 Da cut-off for second generation conjugates) to produce dual radiolabeled conjugates.
2
2
.3.5. Tritosomes cleavage of P-EPI, P-EPI(Tyr)
.3.5.1. Tritosomes preparation and activity toward Bz-Phe-Val-Arg-NAp substrate. Rat liver Tritosomes were prepared according to the method of
Trouet [52]. Briefly, 20% Triton WR-1339 in NaCl (0.15 M) solution was intraperitoneally injected into rats (1 mL per 100 g of rat weight). After
days, the rats were sacrificed, liver isolated and placed into 0.25 M sucrose solution at 4 °C. The liver was diced into little pieces and pushed through
4
a strainer. The mixture was homogenized gently and centrifuged at 2400 rpm for 10 min at 4 °C. The supernatants were collected and the pellet was
washed with 0.25 M sucrose solution and centrifuged at 2400 rpm for 10 min at 4 °C. The combined supernatants were centrifuged at 22,700 rpm for
1
0 min at 4 °C. The supernatant and the pink fluffy layer were removed. The pellets were centrifuged at 22,700 rpm for 10 min at 4 °C and the su-
pernatant and pink fluffy layer were removed again. The pellets and 45% sucrose solution were thoroughly mixed gently. A 34.5% w/w sucrose solu-
tion was added on the top of the 45% sucrose/pellet mixture and 14.3% w/w sucrose solution was added on the top of 34.5% sucrose solution. The
mixture was centrifuged at 24,000 rpm for 2 h at 4 °C. The lysosomal fraction (Tritosomes) was the layer between the 34.5% and 14.3% sucrose
solutions.
2
.3.5.2. Tritosomes activity toward Bz-Phe-Val-Arg-NAp substrate. Tritosomes (0.1 mL) were mixed with 0.84 mL sodium phosphate buffer (213 mM
sodium phosphate, 44 mM citrate, pH 5.5), 0.02 mL glutathione (0.25 M) in sodium phosphate buffer and 0.02 mL 10% Triton-X-100 in sodium phos-
phate buffer. The mixture was incubated at 37 °C for 5 min. The substrate (Bz-Phe-Val-Arg-NAp) solution (0.00102 g in 0.138 mL DMSO) was added
and incubated at 37 °C for 15 min. The absorption of cleaved p-nitroaniline was measured at 410 nm. Within 10 min, 4.7% of p-nitroaniline was re-
leased from Bz-Phe-Val-Arg-NAp.
2
.3.5.3. Tritosomes cleavage of P-EPI, P-EPI(Tyr). Tritosomes (0.48 mL) were mixed with 0.40 mL buffer B-GSH (27 mM citrate, 20 mM sodium phos-
phate, 2 mM EDTA, 5 mM glutathione, pH 4.6) and 0.02 mL 10% Triton-X-100 (0.1 g/mL in B-GSH buffer). After incubation for 5 min at 37 °C, conju-
gate (35 mg/mL × 0.1 mL of P-EPI or 25 mg/mL × 0.1 mL of P-EPI(Tyr) in H O) was added into the mixture and incubated at 37 °C. At predetermined
time intervals samples (0.1 mL) were withdrawn, added 0.05 mL of iodoacetic acid sodium (1 mg/mL in H O, inhibitor) and 0.05 mL of daunomycin
0.1 mg/mL in H O, internal standard for P-EPI and P-EPI(Tyr)). After centrifugation for 5 min (13,500 rpm), 80 μL of supernatant was analyzed by
HPLC. Gradient elution: 20% B to 35% B in 25 min, 35% B to 60% B in 15 min, 60% B to 90% B in 15 min for P-EPI and P-EPI(Tyr). Time intervals:
2
2
(
2
1
min, 1, 2, 4, 6, 8, 23, and 48 h.
2
.3.6. Cleavage of 2P-DTPA-EPI(Tyr) and 2P-Tyr-EPI(DTPA) by Tritosomes
Tritosomes (200 μL) were buffer changed with phosphate buffer-A (200 mM, 0.2% Triton-X-100, EDTA 2 mM, pH 5.5) and mixed with 5 μL of glu-
tathione (100 mM). After incubation at 37 °C for 5 min, 2P-DTPA-EPI(Tyr) or 2P-Tyr-EPI(DTPA), 50 mg/mL × 10 μL in H
2
O) was added and incubated
at 37 °C. After 45 h, to 200 μL of reaction mixture was added 20 μL of iodoacetic acid sodium (2 mg/mL in H O, inhibitor) and daunomycin (2 mg/
2
mL × 4 μL in MeOH; internal standard for 2P-DTPA-EPI(Tyr) and 2P-Tyr-EPI(DTPA)). The mixture was centrifuged for 5 min (13,500 rpm). The su-
pernatant was analyzed by HPLC. Tritosomes cleavage products were collected and determined by MALDI-TOF-MS. The Tritosomes cleavage products
+
+
of 2P-DTPA-EPI(Tyr) include [G-EPI-Tyr + Na] ,1024.39 and [EPI-Tyr + Na] , 967.37. The Tritosomes cleavage products of 2P-Tyr-EPI(DTPA) in-
+
+
+
clude [LG-EPI-DTPA + Ca + 2 K] , 1549.39, [G-EPI-DTPA + Ca + 3 K] , 1492.28 and [EPI-DTPA + Zn + 2 K + 2Na] , 1447.22.

L. Zhang et al. / Journal of Controlled Release 235 (2016) 306–318
311
2
.3.7. Cell culture
A2780 human ovarian carcinoma cells (ATCC) were maintained at 37 °C in a humidified atmosphere containing 5% CO
Gibco) supplemented with 10% FBS and a mixture of antibiotics (100 units/mL penicillin, 0.1 mg/mL streptomycin).
2
in RPMI-1640 medium
(
2
.3.8. Tumor model
All animal studies were carried out in accordance with the University of Utah Institutional Animal Care and Use Committee guidelines under ap-
6
proved protocols. A2780 human ovarian cancer cells (5 × 10 ) in 100 μL of PBS were subcutaneously inoculated in the right flank of 6- to 8-wk-old
syngeneic female nude mice (22–25 g; Charles River Laboratories).
2
.3.9. Pharmacokinetics and biodistribution study
For pharmacokinetic study, 6–8 week-old healthy female nude mice (22–25 g; Charles River Laboratories) (n = 5) were intravenously
injected with dual-radiolabeled HPMA copolymer-EPI conjugates (1 mg, 20 μCi per mouse). At predetermined intervals, blood samples
1
25
(
10 μL) were taken from the tail vein, and the radioactivity of each sample was measured with Gamma Counter (Packard). The
I activity
In activity was counted in a channel having windows set for 237–257 keV
In channel were 1.5%. Gross cpm values were corrected to compensate for
cross-counting. The blood pharmacokinetic parameters for the radiotracer were analyzed using a noncompartmental model with WinNonlin
1
11
was counted in a channel with windows set for 15–85 keV and
26]. Cross-counts in the 1 I channel were 5% and in the
25
111
[
5
.0.1 software (Pharsight).
For biodistribution study, 6–8 week-old female nude mice bearing s.c. A2780 tumors (22–25 g; Charles River Laboratories) received in-
travenous injection of dual-radiolabeled copolymer-EPI conjugates (1 mg, 20 μCi per mouse). At 48 h and 144 h after administration, the
mice were sacrificed. Various tissues (heart, liver, spleen, lung, stomach, intestine, muscle, bone, brain and tumor) were harvested, weighed,
and counted for radioactivity with Gamma Counter (Packard) with the aforementioned 111In/ I dual-isotope protocol. Uptake of the con-
jugate was calculated as the percentage of the injected dose per gram of tissue (%ID/g). Data are presented as mean ± standard deviation
125
(
n = 5).
2
.3.10. Statistics
Statistical analyses were done using a two-tailed unpaired Student's t-test, with p values of b0.01 indicating statistically significant
differences.
Scheme 1. Illustration of a general strategy to synthesis of dual-fluorophore labeled and/or dual-radiolabeled HPMA copolymer conjugates.

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L. Zhang et al. / Journal of Controlled Release 235 (2016) 306–318
Scheme 2. Synthesis of HPMA copolymer-EPI conjugates and dual-radiolabeling strategy. (A) Synthesis of 2nd-generation conjugate 2P-Tyr-EPI(DTPA) by RAFT copolymerization of MA-
GFLG-Abu(N
labeling with
3
)-EPI with HPMA and MA-Tyr-NH
In. The polymer precursor was consecutively labeled with
2
using a cleavable diarm RAFT agent CTA-GFLG-CTA. Propargylamine was coupled with N
3
group and chelator DTPA was attached for drug
1
11
125
111
I (polymer backbone) and
In (EPI). (B) Synthesis of 2nd-generation conjugate 2P-DTPA-EPI(Tyr) by
3
copolymerization of MA-GFLG-Abu(N )-EPI with HPMA and APMA. Tyrosine residue was incorporated via Cu(I) assisted alkyne-azide click reaction. DTPA was attached to polymer
125
111
backbone via pendent amino group modification with p-SCN-Bn-DTPA. The polymer precursor was then labeled with I and In, consecutively.
Table 1
Summary of dual labeled HPMA copolymer conjugates.
No
Polymer precursor
Dual-labeled conjugate
Mw kDa
Mw/Mn
EPI (wt%)
DTPA
Label isotode
Backbone
No./per chain
3.7
EPI
I
II
III
IV
V
P-DTPA-EPI(Tyr)
2P-DTPA-EPI(Tyr)
2P-Tyr-EPI(DTPA)
28.2
65.8
75.2
1.07
1.20
1.25
10.5
9.7
111In-P-EPI-125
111In-2P-EPI-125
125I-2P-EPI-111In
I
111In
111In
125I
125I
4.5
1
I
125I
5.5
VI
111In

L. Zhang et al. / Journal of Controlled Release 235 (2016) 306–318
313
Table 2
Model conjugates for enzyme cleavage.
Conjugate
P-EPI (Tyr)
Comonomer
Mw, kDa
50.7
Mw/Mn
1.09
EPI (wt%)
6.5
P-EPI
55.6
1.20
4.7
3
. Results and discussion
concurrently investigate two different molecular functions via multiple
imaging modalities. As depicted in Scheme 1, the key component in our
3
.1. Design and synthesis of dual-radiolabeled HPMA copolymer-EPI
design is an azide-containing drug monomer MA-GFLG-Abu(N
3
)-Drug.
conjugates
By copolymerization of MA-GFLG-Abu(N )-Drug with HPMA and
3
APMA, a heterobifunctional polymer precursor will be obtained. The
pendant amino groups can be used to incorporate a fluorescence dye
containing N-hydroxysuccinimide (NHS) ester reactive group to poly-
mer backbone. The second fluorophore will be introduced via azide-al-
kyne click reaction that has high specificity. The resulting dual-
fluorophore labeled conjugates could be used for optical imaging stud-
ies such as bioluminescence imaging or fluorescence tomography
(FMT). This approach can also be used to synthesize a dual-radiolabeled
polymer-drug conjugate for PET/CT imaging or pharmacokinetics and
biodistribution studies due to high sensitivity of radionuclides. For
HPMA-based polymer drug conjugates have been investigated for
decades; most of studies focused on the treatment of solid tumors. It
has been demonstrated in preclinical studies that polymer-drug conju-
gates have numerous advantages compared to current marketed
chemotherapeutical agents as a result of enhanced permeability and re-
tention (EPR) effect. To accelerate the translation process from bench to
bed, we propose a general approach to the synthesis of dual-labeled
polymer-drug conjugates; one radiolabel is bound to polymeric carrier
and the other is attached to drug (or model drug), in order to
Fig. 1. Tritosomes cleavage of P-EPI and P-EPI(Tyr). HPLC analysis of P-EPI (A) and P-EPI(Tyr) (B) at different time intervals (1 min, 1, 2, 4, 6, 8, 23, and 48 h). Daunomycin was used as
internal standard. MALDI-TOF-MS analysis of cleaved products from P-EPI(Tyr) (C, D).

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L. Zhang et al. / Journal of Controlled Release 235 (2016) 306–318
example, Bolton-Hunter reagent can be used to modify amino group to
tyrosine-like structure for backbone iodination, while copper-free ap-
proach could be applied for attachment of a chelator such as DTPA
followed by incorporation of a radiometal ion.
were summarized in Table 1 (I, III and V). They were radiolabeled by
125
111
I and
In consecutively, yielded dual-labeled products II, IV and
VI for pharmacokinetic and biodistribution studies.
In this study, we focused on dual-radiolabeling strategy. First gener-
ation polymer conjugate P-DTPA-EPI(Tyr) (Mw b 50 kDa, i.e. below the
renal threshold, see SI Fig. S6) and backbone degradable HPMA copoly-
mer-EPI conjugates 2P-DTPA-EPI(Tyr) (diblock with Mw N 50 kDa;
above the renal threshold) were synthesized to evaluate the impact of
molecular weight and degradability of the conjugate on its fate in the or-
ganism. We slightly modified the synthetic approach in Scheme 1: In-
stead of using Bolton-Hunter reagent, we attached p-SCN-Bn-DTPA to
the pendant amino group on the polymer backbone, while a tyrosine
3.2. Enzymatic cleavage study of HPMA copolymer-EPI conjugates
As a payload tracer, the cleavability of modified EPI is important for
interpretation of pharmacokinetic profile and biodistribution result. In
the ideal case, the radioisotope will be bound to the drug molecule
and cleaved together without change of drug release rate. If the label
and drug separate, then the radioactivity level will not be relevant to
the drug concentration. To examine whether the modified EPI keeps in-
tact when it is cleaved in the lysosomes, we synthesized model conju-
gate P-EPI(Tyr) and determined its stability in the presence of
lysosome enzyme. Unmodified P-EPI was synthesized and served as
control. Unlike the synthetic procedure described above (Scheme 2B),
in P-EPI(Tyr), the tyrosine moiety was first incorporated to the mono-
mer MA-GFLG-Abu(N3)-EPI via click reaction, then purified and poly-
merized to ensure there is no unmodified EPI left. The structure and
physicochemical characterization of two conjugates are listed in Table
2 and Fig. S10.
moiety was coupled with N
3
group via Cu(I) assisted alkyne-azide
click reaction. To further investigate how radiolabel type impacts prop-
1
25
erties of the conjugates, 2P-Tyr-EPI(DTPA) containing an
I labeling
site on the polymer backbone and an 1 In labeling site at the modified
EPI was synthesized. The overall synthetic process for 2nd generation
conjugates is described in Scheme 2, more detailed synthetic schemes
including monomers, RAFT agent and 1st generation conjugate are
listed in Supplementary Information. The conjugates to be evaluated
11
Fig. 2. Tritosomes cleavage of 2nd-generation HPMA copolymer-EPI conjugates. HPLC analysis of 2P-DTPA-EPI(Tyr) (A) and 2P-Tyr-EPI(DTPA) (B) following incubation with Tritosomes
for 45 h. MALDI-TOF-MS analysis of cleaved products from 2P-Tyr-EPI(DTPA) (C).

L. Zhang et al. / Journal of Controlled Release 235 (2016) 306–318
315
Table 3
within the lysosomal compartment [54]. In addition, the rate of enzy-
matically-catalyzed hydrolysis depends on the structure of the ligand
Structure of the conjugates and enzyme cleavage products.
(
drug) bound to the GFLG sequence. Since the GFLG spacer aligns into
S4-S1 subsites of cathepsin B, the different rate of drug release depends
on the fit of the drug (ligand) into subsites S1′ and S2′ [56]. The forma-
tion of G-EPI-Tyr indicates that part of the substrate bound to the active
site with phenylalanine in the (preferred [57]) P2 position. Probably,
based on the bulkiness of EPI-Tyr, the cleavage of G-EPI-Tyr by cathep-
sin B occurred first and other enzymes present in Tritosomes subse-
quently cleaved the bond between glycine and EPI. In addition, the
results indicated that the bond between the tyrosine moiety and EPI
was stable since only a very small amount of free EPI was detected.
The two different dual label 2nd generation conjugates, 2P-Tyr-
EPI(DTPA) and 2P-DTPA-EPI(Tyr), were also cleaved with Tritosomes.
2P-DTPA-EPI(Tyr) showed similar cleavage pattern to P-EPI(Tyr), main-
ly yielded EPI(Tyr) with little G-EPI(Tyr) and tiny free EPI. However, 2P-
Tyr-EPI(DTPA) resulted in more complicated cleavage products, as
shown in MALDI-TOF-MS (Fig. 2C). It revealed the presence of LG-
EPI(DTPA), G-EPI-DTPA and EPI-DTPA. This indicates the due to the
size of EPI-DTPA the substrate aligns to the cathepsin B active site
with GFLG in subsites S2–S2′. Other lysosomal enzymes participate in
the cleavage of LG-EPI(DTPA) to G-EPI(DTPA) and EPI(DTPA) (Table 3).
The results of enzymatic cleavage of the conjugates clearly indicate
the impact of the steric hindrance of the bulky payload on the S1′–P1′
and S2′–P2′ interactions. This needs to be taken into account when
interpreting biodistribution data.
For cleavage, we have chosen to use Tritosomes, a mixture of lyso-
somal enzymes isolated from liver of rats, to mimic in vivo enzyme en-
vironment. The conjugates containing 300 nmol equivalent EPI were
incubated with 0.48 mL Tritosomes at 37 °C for 48 h. Hydrolysis rate
was monitored using HPLC. The results are shown in Fig. 1.
When incubated with Tritosomes, HPLC showed that the wide peak
of conjugate P-EPI was decreased with incubation time, while free EPI
peak emerged. Eventually conjugate was not detectable, indicating EPI
was released from P-EPI gradually, and almost 100% release was
reached within 48 h. In contrast, a new unknown peak occurred in P-
EPI(Tyr) and increased slowly with incubation time (Fig. 1B). During
4
8 h incubation, a very small EPI peak also emerged. The fractions
were collected and determined using MALDI-TOF mass spectroscopy.
The result suggested that the cleavage mainly yielded two products, gly-
3.3. Pharmacokinetics and biodistribution study of dual-radiolabeled copol-
ymer-PEI conjugates
+
cine-EPI-Tyr ([G-EPI-Tyr + Na] , 1024.39) and EPI-Tyr ([EPI-
+
Tyr + Na] , 967.37) (Fig. 1C, D). The free drug EPI was only 2.5% com-
Dual-labeled products II, IV and VI (see Table 1) were administered
to nude mice via tail veins for pharmacokinetics and biodistribution
studies. The dual-radiolabel strategy allowed to simultaneously track
pared to the modified EPI.
These findings are agreement with our previous studies. It was well
established that the most important enzyme in the lysosomal compart-
ment (and in Tritosomes) in the cleavage of oligopeptide sequences at-
tached to HPMA copolymers is cathepsin B. Other enzymes participate
in the cleavage to a lesser extent [9,53–54]. Cathepsin B (EC 3.4.22.1)
is an important lysosomal enzyme active in the degradation of internal-
ized proteins and peptides. It is a cysteine protease and its active site ac-
1
25
111
the fate of the payload (EPI- I or EPI- In) and of the polymeric car-
riers (1 In-P/ In-2P or
11
111
125
I-2P). Fig. 3A shows blood radioactivity-
time profiles of 1st and 2nd generation conjugates in which polymer
1
11
125
backbone was labeled with In and drug with I. For each conjugate,
both 1 In and
11
125
I showed very similar pattern indicating the linker
GFLG between drug and polymeric carrier is stable in blood circulation
during transport. Moreover, it clearly demonstrated the superior prop-
2
commodates at least four amino acid residues toward the NH end of
erty of 2nd generation conjugate (1 In-2P-EPI- I) with higher molec-
11
125
the substrate (positions P4–P1; subsites S1–S4; nomenclature of
Schechter-Berger [55]) and two amino acid residues toward the COOH
end of the substrate (positions P1′ and P2′). The GFLG sequence was de-
signed to match the specificity of cathepsin B in the lysosomal compart-
ment [53] and is now widely used as an attachment/release moiety in
the design of lysosomotropic nanomedicines [9].
ular weight on enhanced plasma concentration and elongation of
1
11
125
circulation time as compared with
In-P-EPI- I. Consequently, the
111
125
111
tumor-uptake of
In-2P-EPI- I was 2–3-folds greater than
In-P-
EPI-1 I as illustrated in Fig. 3B.
25
This observation provides a direct proof that backbone degradable
diblock HPMA copolymer carriers can more effectively deliver drugs
to the tumor, resulting in the improvement of the treatment efficacy
Comparison of cleavage of polymer-drug conjugates containing the
GFLG spacer revealed that other enzymes participate in the cleavage
Fig. 3. Comparison of pharmacokinetic profiles and tumor-uptake of dual-labeled 1st generation conjugate 111In-P-EPI-125I and 2nd generation conjugate 111In-2P-EPI-125I in nu/nu mice.
1
11
125
(
4
A) Blood activity-time profiles of In-labeled polymer carriers and I labeled drug EPI in mice. (B) Radioactivity level in tumors for the mice bearing subcutaneous A2780 xenografts
111
125
8 h after intravenous injection of dual radiolabeled conjugates. Here In corresponds to polymer carrier (1st generation P- and 2nd generation 2P-) and I to drug EPI, respectively. The
data represent the mean radioactivity expressed as a percentage of the injected dose per gram of blood (n = 5). p b 0.001.

316
L. Zhang et al. / Journal of Controlled Release 235 (2016) 306–318
Fig. 4. Effect of radiolabeling strategy on pharmacokinetic profiles of the polymer carrier (A) and payload (EPI) (B) of backbone degradable diblock HPMA copolymer-EPI conjugates in
tumor-bearing nu/nu mice. 2P-DTPA-EPI(Tyr) with average Mw 66 kDa and 2P-Tyr-EPI(DTPA) with Mw 75 kDa were used. Data obtained using the radioactivity count method are
plotted as percentage of injected dose per gram of tissue (%ID/g). All data are expressed as mean ± standard deviation (n = 5).
and decrease of non-specific adverse effect, in agreement with our pre-
vious report [28].
125I (125I-2P, Fig. 5A & B). Two phenomena may contribute to this obser-
vation: First, the incorporation of chelator DTPA for 1 In labeling in-
duces negative charges to the polymer carrier; it has been observed
that this enhances the accumulation in the cortex and renal tubules
11
Fig. 4 shows the blood radioactivity-time profiles of different isotope-
labeled polymer carriers (A) and payload (modified EPI) (B). Conjugate IV
111
has similar Mw as conjugate VI. Their backbones were labeled with In
after intravenous administration in mice [32,58,59]. Second, it is more
1
25
125
111
and I, respectively, and side chain drug EPI was labeled with
I and
likely due to
In strong kidney accumulation and slow clearance as
111
111
In; similar pharmacokinetics for polymer carrier and for payload was
noted by others [60–62]. Other than In-2P in kidney, at 48 h after in-
travenous injection of 2nd generation conjugates, the highest concen-
tration of conjugates (both payload EPI and polymer carrier) was still
in blood stream, indicating the advantage of high molecular weight con-
jugates. Until 144 h, the concentrations of polymer carrier and payload
EPI(DTPA)1 In in tumor were clearly higher than the levels in the nor-
observed. Combined with results from Fig. 3, this suggests that for
water-soluble polymer-drug conjugates, the molecular weight plays a de-
cisive role on its circulation time and body clearance, while side-chain
modification has a minor impact. This result also suggests that the modi-
fication of EPI structure does not influence the PK of the conjugate.
Pharmacokinetic parameters are summarized in Table 4. Both 2nd
11
mal organs except liver, and spleen, demonstrating the targeting ability
1
11
125
125
111
125
generation conjugates,
In-2P-EPI- I and
I-2P-EPI- In, showed
of polymer conjugates to tumor (Fig. 5A & B). However, EPI(Tyr)
I
showed lower uptake than EPI(DTPA)1 In in major organs and tumor
11
similar half-life, which was much longer than that of the 1st generation
1
11
125
conjugate
In-P-EPI- I. For example, the terminal half-lives of pay-
at both 48 h and 144 h (Fig. 5A & B); at same time points payload
load EPI(Tyr)125I and EPI(DTPA) In in 2nd generation conjugates
111
EPI(DTPA) In showed similar uptake as polymeric carrier
111
125
I-2P in
were 29.79 h and 28.40 h, respectively; they are very close to the termi-
major organs and tumor. There are several factors that may contribute
to this result: different drug release rates resulting from steric hindrance
of EPI modification, and lower retention of radioiodine. It has been re-
ported that after endocytosis and proteolytic degradation in lysosomes,
the iodine-labeled conjugates generate iodotyrosine, which is rapidly
excreted from cells and shows poor retention in normal organs and tu-
mors [63,64]. In addition, radioiodide could be liberated from
iodotyrosine by dehalogenase enzymes and rapidly sequestered by thy-
roid [32]. Future in vivo experiments using D-amino acids could evaluate
the importance of this pathway [65–67].
We compared the tumor-to-tissue uptake ratios of payload and
polymeric carriers and found that the tumor-to-tissue uptake ratios of
both radionuclides were similar (Fig. S15). The tumor-to-tissue uptake
ratios of 2nd generation conjugates were higher than that of 1st gener-
ation conjugate for both payloads and polymeric carriers.
nal half-lives of polymeric carriers 1 In-2P (31.52 h) and
11
125
I-2P
31.11 h). However, terminal half-life of payload EPI(Tyr) I in 1st gen-
125
(
111
eration conjugates and the polymeric carrier
In-P were 9.63 h and
9
.20 h, respectively. The total area under the blood concentration versus
1
11
125
time curve (AUC) of In-2P-EPI- I (1135% injected dose per mL (ID/
mL) for 1 I and 1474% ID/mL for In) and of I-2P-EPI- In (1604%
25
111
125
111
111
125
ID/mL for In and 1628% ID/mL for I) were significantly higher than
that of 1 In-P-EPI- I (261% ID/mL for
11
125
125
I and 324% ID/mL for
111
In)
In-
In-
111
(
P b 0.001) (Table 4). Comparing with 1st generation conjugate
1
25
111
P-EPI- I, the increased exposure of 2nd generation conjugates
1
25
125
111
2
P-EPI- I and
I-2P-EPI- In resulted mainly from a significantly
slower mean systemic clearance (CL) (P b 0.001).
We also analyzed biodistribution of payload and carrier in female
nude mice bearing A2780 human ovarian carcinoma. A high and lasting
radioactivity level of polymer carrier labeled with 1 In ( In-2P) in
11
111
Taking together with the results we previously obtained [26] and
other literatures [19,68,69], pharmacokinetics and biodistribution
kidney was observed, in contrast to the polymer carrier labeled with
Table 4
Comparison of pharmacokinetic parameters of dual-labeled (125I- and 111In-based) 1st generation conjugate and 2nd generation conjugates in mice.
1
st generation
111In-P-EPI-125
EPI(Tyr)-125
0.57 ± 0.07
2nd generation
111In-2P-EPI-125
Conjugate
I
I
125I-2P-EPI-111In
EPI(DTPA)111In
Component
I
P(DTPA)111In
EPI(Tyr)125
I
2P(DTPA)111In
2P(Tyr)125
I
T
T
1/2, α (h)
1/2, β (h)
0.50 ± 0.08
9.20 ± 1.68
324.25 ± 36.98
0.30 ± 0.03
12.46 ± 2.25
3.84 ± 0.29
1.51 ± 0.20
29.79 ± 1.62
1135.00 ± 43.81
0.08 ± 0.003
41.85 ± 2.21
3.68 ± 0.08
1.58 ± 0.19
31.52 ± 2.01
1473.97 ± 65.83
0.06 ± 0.003
43.95 ± 2.73
2.98 ± 0.08
0.49 ± 0.13
28.40 ± 2.03
1604.36 ± 95.44
0.06 ± 0.003
40.71 ± 2.89
2.53 ± 0.05
0.47 ± 0.16
31.11 ± 2.19
1628.32 ± 97.28
0.06 ± 0.004
44.69 ± 13.15
2.74 ± 0.06
9.63 ± 11.47
260.72 ± 24.64
0.38 ± 0.03
AUC (% ID h/mL)
CL (mL/h)
MRT (h)
13.01 ± 1.96
4.99 ± 0.31
Vss (mL)
T1/2,α = initial half-life; T1/2,β = terminal half-life; AUC = total area under the blood concentration versus time curve; %ID = percentage of injected dose; CL = total body clearance; MRT
=
mean residence time; Vss = steady-state volume of distribution. Data are presented as mean ± standard deviation (n = 5).

L. Zhang et al. / Journal of Controlled Release 235 (2016) 306–318
317
Fig. 5. Effect of radiolabeling strategy on biodistribution of the polymer carrier and payload (EPI) of backbone degradable diblock HPMA copolymer-EPI conjugates in tumor-bearing mice
at 48 h (A) and 144 h (B) after intravenous administration. Data obtained using the radioactivity count method are plotted as percentage of injected dose per gram of tissue (%ID/g). All data
are expressed as mean ± standard deviation (n = 5).
results indicated: 1) GFLG linker between payload and polymeric carrier
remained stable in the bloodstream during transport. 2) After internal-
ization via endocytosis and localization in the lysosomes, the payload
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