Biotin-decorated all-HPMA polymeric micelles for paclitaxel delivery

Article abstract of DOI:10.1016/j.jconrel.2020.09.013

To avoid poly(ethylene glycol)-related issues of nanomedicines such as accelerated blood clearance, fully N-2-hydroxypropyl methacrylamide (HPMAm)-based polymeric micelles decorated with biotin for drug delivery were designed. To this end, a biotin-functionalized chain transfer agent (CTA), 4-cyano-4-[(dodecylsulfanylthiocarbonyl)-sulfanyl]pentanoic acid (biotin-CDTPA), was synthesized for reversible addition–fragmentation chain-transfer (RAFT) polymerization. Amphiphilic poly(N-2-hydroxypropyl methacrylamide)-block-poly(N-2-benzoyloxypropyl methacrylamide) (p(HPMAm)-b-p(HPMAm-Bz)) with molecular weights ranging from 8 to 24 kDa were synthesized using CDTPA or biotin-CDTPA as CTA and 2,2′-azobis(2-methylpropionitrile) as initiator. The copolymers self-assembled in aqueous media into micelles with sizes of 40–90 nm which positively correlated to the chain length of the hydrophobic block in the polymers, whereas the critical micelle concentrations decreased with increasing hydrophobic block length. The polymer with a molecular weight of 22.1 kDa was used to prepare paclitaxel-loaded micelles which had sizes between 61 and 70 nm, and a maximum loading capacity of around 10 wt%. A549 lung cancer cells overexpressing the biotin receptor, internalized the biotin-decorated micelles more efficiently than non-targeted micelles, while very low internalization of both types of micelles by HEK293 human embryonic kidney cells lacking the biotin receptor was observed. As a consequence, the paclitaxel-loaded micelles with biotin decoration exhibited stronger cytotoxicity in A549 cells than non-targeted micelles. Overall, a synthetic pathway to obtain actively targeted poly(ethylene glycol)-free micelles fully based on a poly(HPMAm) backbone was established. These polymeric micelles are promising systems for the delivery of hydrophobic anticancer drugs.

Full text of DOI:10.1016/j.jconrel.2020.09.013

Journal of Controlled Release xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Journal of Controlled Release
journal homepage: www.elsevier.com/locate/jconrel
Biotin-decorated all-HPMA polymeric micelles for paclitaxel delivery
a
a
a
b
b
Yan Wang , Mies J. van Steenbergen , Nataliia Beztsinna , Yang Shi , Twan Lammers ,
a
a,⁎
Cornelus F. van Nostrum , Wim E. Hennink
a
b
Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences, Utrecht University, Universiteitsweg 99, 3508 TB Utrecht, the Netherlands
Department of Nanomedicine and Theranostics, Institute for Experimental Molecular Imaging, RWTH Aachen University Clinic, Forckenbecktrasse 55, 52074 Aachen,
Germany
A R T I C L E I N F O
A B S T R A C T
Keywords:
To avoid poly(ethylene glycol)-related issues of nanomedicines such as accelerated blood clearance, fully N-2-
hydroxypropyl methacrylamide (HPMAm)-based polymeric micelles decorated with biotin for drug delivery
were designed. To this end, a biotin-functionalized chain transfer agent (CTA), 4-cyano-4-[(dodecylsulfa-
nylthiocarbonyl)-sulfanyl]pentanoic acid (biotin-CDTPA), was synthesized for reversible addition–fragmenta-
tion chain-transfer (RAFT) polymerization. Amphiphilic poly(N-2-hydroxypropyl methacrylamide)-block-poly(N-
Polymeric micelles
N-2-Hydroxypropyl methacrylamide
Biotin
Drug targeting
Nanomedicines
2
2
-benzoyloxypropyl methacrylamide) (p(HPMAm)-b-p(HPMAm-Bz)) with molecular weights ranging from 8 to
4 kDa were synthesized using CDTPA or biotin-CDTPA as CTA and 2,2′-azobis(2-methylpropionitrile) as in-
itiator. The copolymers self-assembled in aqueous media into micelles with sizes of 40–90 nm which positively
correlated to the chain length of the hydrophobic block in the polymers, whereas the critical micelle con-
centrations decreased with increasing hydrophobic block length. The polymer with a molecular weight of
2
2.1 kDa was used to prepare paclitaxel-loaded micelles which had sizes between 61 and 70 nm, and a maximum
loading capacity of around 10 wt%. A549 lung cancer cells overexpressing the biotin receptor, internalized the
biotin-decorated micelles more efficiently than non-targeted micelles, while very low internalization of both
types of micelles by HEK293 human embryonic kidney cells lacking the biotin receptor was observed. As a
consequence, the paclitaxel-loaded micelles with biotin decoration exhibited stronger cytotoxicity in A549 cells
than non-targeted micelles. Overall, a synthetic pathway to obtain actively targeted poly(ethylene glycol)-free
micelles fully based on a poly(HPMAm) backbone was established. These polymeric micelles are promising
systems for the delivery of hydrophobic anticancer drugs.
1
. Introduction
During the last decades, drug delivery systems have been ex-
(EPR) effect [10–12]. Besides passive targeting, micelles can be func-
tionalized with ligands for active targeting to increase the efficacy of
anti-cancer drugs while reducing their unwanted localization in healthy
tissues by means of receptor-mediated endocytosis [13–15]. Various
ligands, including antibody (fragments), peptides, aptamers and small
molecules (such as folate) have been employed for the design of tar-
geted nanomedicines [16–19].
tensively investigated for cancer chemotherapy, and particularly, sub-
stantial attention has been devoted to polymeric micelles [1–5]. Poly-
meric micelles are nanosized colloidal particles with a core-shell
structure, which spontaneously self-assemble from amphiphilic copo-
lymers above a certain concentration (CMC, critical micelle con-
centration) in aqueous solution. The outer hydrophilic shell ensures
colloidal stability of polymeric micelles and provides long circulation in
the blood circulation, whereas the hydrophobic core is highly suitable
for accommodating and solubilizing hydrophobic drugs [6–9]. Due to
their small size (< 100 nm) and long circulation kinetics, polymeric
micelles passively accumulate in cancerous or inflamed tissues by a
phenomenon referred to as the enhanced permeability and retention
Biotin, as a water-soluble vitamin, is essential for normal cellular
functions, growth and development [20,21], and the sodium-dependent
multivitamin transporter (SMVT) has been proved to be its main
transporter [22], which is overexpressed in many cancer cells such as
lung cancer cells (A549 and M109) [20,23]. In 2004 Russell-Jones et al.
reported that a biotin-targeted doxorubicin-poly(N-2-benzoyloxypropyl
methacrylamide) (pHPMAm) conjugate caused significant killing en-
hancement in colon carcinoma xenografts. Importantly, the same
⁎
Corresponding author.
E-mail addresses: y.wang3@uu.nl (Y. Wang), M.J.vanSteenbergen@uu.nl (M.J. van Steenbergen), nataliia.beztsinna@gmail.com (N. Beztsinna),
yshi@ukaachen.de (Y. Shi), tlammers@ukaachen.de (T. Lammers), C.F.vanNostrum@uu.nl (C.F. van Nostrum), W.E.Hennink@uu.nl (W.E. Hennink).
https://doi.org/10.1016/j.jconrel.2020.09.013
Received 21 June 2020; Received in revised form 21 August 2020; Accepted 7 September 2020
0168-3659/ © 2020 Elsevier B.V. All rights reserved.
Please cite this article as: Yan Wang, et al., Journal of Controlled Release, https://doi.org/10.1016/j.jconrel.2020.09.013

Y. Wang, et al.
Journal of Controlled Release xxx (xxxx) xxx–xxx
Scheme 1. Synthesis of p(HPMAm)-b-p(HPMAm-Bz) block copolymer with or without a biotin terminus. (A) Synthesis of biotin-functionalized chain transfer agent,
biotin-CDTPA. (B) Synthesis of macro chain trasnfer agent, p(HPMAm). (C) Synthesis of block copolymer p(HPMAm)-b-p(HPMAm-Bz).
2

Y. Wang, et al.
Journal of Controlled Release xxx (xxxx) xxx–xxx
outcomes were not observed when either vitamin B12 or folate were
used as targeting agents [23,24]. Therefore, biotin has emerged as a
remarkable active targeting ligand for nanocarriers [25–29]. Biotin has
a valeric acid tail, through which it can be conjugated to other mole-
cules to achieve biotinylation (Scheme 1A) [30]. Biotin can be deco-
rated on the surface of nanocarriers either through pre-conjugation of
biotin to the polymers before their assembly into nanocarriers [31–33],
or by attaching biotin onto the surface of nanocarriers after formation
tetrahydrofuran (THF, HPLC grade), dimethylacetamide (DMAc, pep-
tide synthesis grade), 2-propanol, diethyl ether, and acetone were
purchased from Biosolve Ltd. (Valkenswaard, the Netherlands) and
used as received, unless indicated otherwise. 2,2′-Azobis(2-methylpro-
pionitrile) (AIBN) was obtained from Sigma-Aldrich (Zwijndrecht, the
Netherlands), recrystallized from ethanol and stored at −20 °C.
Phosphate buffered saline (PBS, pH 7.4, containing 11.9 mM phos-
phate, 137 mM sodium chloride, and 2.7 mM potassium chloride) was
purchased from Fisher Scientific (Geel, Belgium). Paclitaxel (PTX) was
ordered from LC Laboratories (MA, USA). N-2-Hydroxypropyl metha-
crylamide (HPMAm) was synthesized and characterized as described in
a previous publication [50]. N-2-Benzoyloxypropyl methacrylamide
(HPMAm-Bz) was synthesized and characterized as previously reported
[43]. PEG standards for gel permeation chromatography (GPC) analysis
were supplied by Agilent (Santa Clara, USA). Cyanine 3 (Cy3) amine
was supplied by Lumiprobe GmbH (Hannover, Germany). Spectra/Por
dialysis membrane (MW 6–8 kDa), DNA stain Hoechst 33430, and re-
duced serum medium Opti-MEM were obtained from Thermo Fisher
Scientific (Landsmeer, the Netherlands), while 0.45 μm RC membrane
filters were ordered from Phenomenex (Utrecht, the Netherlands). 3-
(4,5-Dimethylthiazol-2-yl)-5-(3-carboxy-methoxyphenyl)-2-(4-sulfo-
phenyl)-2H-tetrazolium (MTS) reagent was purchased from Benelux
B.V. (Leiden, the Netherlands).
(
post-conjugation) [34,35].
Poly(ethylene glycol) (PEG) remains the ‘gold standard’ as hydro-
philic shell-forming block in polymeric micelles, due to its high water-
solubility and stealth properties that avoids adsorption of opsonins onto
the surface of nanocarriers, which in turn reduces unspecific uptake of
these particles by mononuclear phagocyte system (MPS) [36,37]. Sev-
eral formulations based on PEGylated micelles for anti-cancer therapy
are currently under evaluation in clinical trials [8,38,39]. However, in
contrary to the general assumption that PEGylated substances lack of
immunogenicity, it has been shown that repeated injections of PEGy-
lated nanoparticles can induce a phenomenon termed the accelerated
blood clearance (ABC) effect, which is mediated by anti-PEG im-
munoglobulin M antibodies (IgM) [40,41]. Therefore, the search for
alternative polymers that do not result in an immune response but
maintain the long circulation is required. p(HPMAm) is a promising
alternative for PEG, besides good hydrophilicity and biocompatibility,
because of its multifunctionality, which enables modification with ei-
ther hydrophobic moieties to serve as a micellar core, or conjugated
with multiple drugs and bioactive ligands for targeted delivery [42–47].
Importantly, Szoka et al. reported that the ABC phenomenon was not
observed upon repeated administration of p(HPMAm) modified lipo-
somes due to the lack of formation of anti-p(HPMAm) IgM antibodies in
rats, whereas a pronounced IgM response was observed for animals
repeatedly treated with PEG-coated liposomes [48].
2.2. Synthesis of the biotin-functionalized RAFT chain transfer agent
(biotin-CDTPA)
2.2.1. Synthesis of (+)-biotinyl-N-hydroxysuccinimide (NHS-biotin)
Biotin was activated according to literature procedures [51,52]. In
brief, biotin (5.00 g, 20.5 mmol) and NHS (2.83 g, 24.6 mmol) were
dissolved in dry DMF (150 mL) at 60 °C. DCC (5.07 g, 24.6 mmol) was
added after cooling down to room temperature. The solution was stirred
overnight at room temperature. Next, the formed reaction product di-
cyclohexylurea (DCU) was filtered off and washed with DMF. The
combined filtrates were dropped into cold diethyl ether to precipitate
the crude product which was then refluxed in 2-propanol, filtered, and
Recently the synthesis of poly(ethylene glycol)-block-poly(N-2-ben-
zoyloxypropyl methacrylamide) (mPEG-b-p(HPMAm-Bz)) was reported
[
49]. These micelles showed high stability, drug loading and drug re-
tention as a result of π-π stacking interactions between the aromatic
groups of the drugs and the polymer chains. In the same study it was
shown that paclitaxel (PTX)-loaded mPEG-b-p(HPMAm-Bz) micelles
exhibited high therapeutic efficacy and complete tumor regression in
mice bearing human epidermoid and breast carcinoma xenografts.
Taken this in mind, we here set out to develop a biotin-decorated fully p
1
dried under vacuum to yield NHS-biotin (4.60 g, 66%). H NMR
(600 MHz, DMSO‑d ) δ (ppm): 6.43 (s, 1H, NHC(=O)), 6.37 (s, 1H,
6
NHC(=O)), 4.31 (ddt, 1H, J = 7.5, 5.1, 1.1 Hz, CHNHC(=O)), 4.15
(ddd, 1H, J = 7.7, 4.4, 1.8 Hz, CHNHC(=O)), 3.11 (ddd, 1H, J = 8.3,
(
(
HPMAm)-based anti-cancer drug delivery system. To this end, p
HPMAm)-b-p(HPMAm-Bz) block copolymers with and without biotin
6.3, 4.4 Hz, CHS), 2.83—2.86 (m, 1H, CH S), 2.78—2.83(m, 4H,
2
(O=)C(CH
2
)
2
C(=O)), 2.65—2.70 (m, 2H, CH
2
C(=O)), 2.59 (d, 1H,
CH C(=O)). ESI-MS m/
S 341.4.
modification were synthesized by reversible addition−fragmentation
chain-transfer (RAFT) polymerization and used for the formation of
biotinylated micelles. The cellular uptake and cytotoxicity of these
micelles loaded with PTX were evaluated using A549 human lung
cancer cells overexpressing the biotin receptor as well as in HEK293
kidney cells lacking this receptor.
J = 12.4 Hz, CH
2
S), 1.38—1.69 (m, 6H, (CH
2
)
3
3 5
O
2
+
z 363.0 (M + Na) , calculated for C14
H
19
N
2.2.2. Synthesis of tert-butyl(6-(5-((3aS,4S,6aR)-2-oxohexahydro-1H-
thieno[3,4-d]imidazol-4-yl)pentanamido)hexyl)carbamate (N-Boc-HDA-
biotin)
2
. Materials and methods
N-Boc-HDA-biotin was synthesized as described in previous pub-
lications [53,54]. Briefly, NHS-biotin (3.00 g, 8.8 mmol) and N-Boc-1,6-
hexanediamine (1.90 g, 8.8 mmol) were dissolved in anhydrous DMF
2.1. Materials
(
150 mL) and then TEA (1.22 mL, 12.0 mmol) was added. The reaction
D-(+)-Biotin was purchased from Santa Cruz Biotechnology, Inc.
mixture was stirred at room temperature overnight. The product was
(
Heidelberg, Germany). N-Hydroxysuccinimide (NHS), dicyclohex-
isolated by precipitation in cold diethyl ether, dried under vacuum to
1
ylcarbodiimide (DCC), N-Boc-1,6-hexanediamine, triethylamine (TEA),
trifluoroacetic acid (TFA), 4-cyano-4-[(dodecylsulfanylthiocarbonyl)-
sulfanyl]pentanoic acid (CDTPA), N-(3-dimethylaminopropyl)-N′-
ethylcarbodiimide hydrochloride (EDC), sodium bicarbonate
yield N-Boc-HDA-biotin (3.50 g, 90%). H NMR (600 MHz, DMSO‑d
6
) δ
(ppm): 7.72 (t, 1H, J = 5.6 Hz, NH), 6.75 (t, 1H, J = 5.8 Hz, NH), 6.41
(s, 1H, NH-biotin), 6.35 (s, 1H, NH-biotin), 4.30 (ddt, 1H, J = 7.6, 5.2,
1.1 Hz, CHNH), 4.12 (ddd, 1H, J = 7.7, 4.4, 1.9 Hz, CHNH), 3.09 (ddd,
(
(
(
NaHCO
3
), sodium chloride (NaCl), anhydrous sodium sulfate
1H, J = 8.7, 6.2, 4.4 Hz, SCHCH
2
), 3.00 (q, 2H, NCH ), 2.85—2.90 (m,
2
Na SO ),
2
4
4-(2-hydroxyethyl)piperazine-1-ethanesulfonic
acid
2H, NCH
2
), 2.82 (dd, 1H, J = 12.4, 5.1 Hz, SCHH), 2.57 (d, 1H,
HEPES), pyrene, and Dulbecco's modified Eagle's medium (DMEM)
J = 12.4 Hz, SCHH), 2.04 (t, 2H, J = 7.4 Hz, CH
2
C(=O)), 1.38—1.61
+
were obtained from Sigma-Aldrich (Zwijndrecht, the Netherlands) and
used without further purification. Dimethylformamide (DMF, peptide
synthesis grade), dichloromethane (DCM, peptide synthesis grade),
(m, 23H, 7CH
2
, C(CH
3
)
3
). ESI-MS m/z 443.2 (M + H) , 465.2
(M + Na)+, calculated for C21
H
O S 442.6.
38
N
4
4
3

Y. Wang, et al.
Journal of Controlled Release xxx (xxxx) xxx–xxx
2
.2.3. Synthesis of N-(6-aminohexyl)-5-((3aS,4S,6aR)-2-oxohexahydro-
H-thieno[3,4-d]imidazole-4-yl)pentanamide trifluoroacetate (HDA-biotin)
The removal of the Boc protection group from N-Boc-HDA-biotin
2.3. Synthesis and characterization of p(HPMAm)-b-p(HPMAm-Bz) block
1
copolymers with or without biotin terminus
was performed following literature procedures [53,54]. In short, to a
2.3.1. Synthesis of poly[N-(2-hydroxypropyl) methacrylamide] macro
chain transfer agent (p(HPMAm) macroCTA) with or without biotin
terminus
solution of N-Boc-HDA-biotin (3.50 g, 7.9 mmol) in DCM (43 mL), TFA
(
8.5 mL, 111.0 mmol) was added. The mixture was stirred at room
temperature for 3 h. The solvent was evaporated under reduced pres-
sure to yield a yellowish oil. The crude product was subsequently
precipitated in an excess of cold diethyl ether. After repeatedly dissol-
ving in methanol and evaporation under vacuum to remove residual
The p(HPMAm) macroCTA with or without a biotin terminus was
synthesized using RAFT polymerization with CDTPA or biotin-CDTPA
as CTA and AIBN as initiator at 70 °C [57]. In detail, the reagents were
weighed in Schlenk tubes and subsequently dissolved in dry DMAc
(10 mL). The concentration of HPMAm was 300 mg/mL. To obtain p
(HPMAm) of different molecular weights, molar ratios of [HPMAm]/
[CDTPA]/[AIBN] of 180/5/1, 320/5/1 and 460/5/1 were applied. For
the polymerization of HPMAm with a biotin terminal end, the molar
ratio of [HPMAm]/[biotin-CDTPA]/[AIBN] was 460/5/1. The obtained
solutions were degassed by three cycles of freeze-vacuum-thaw, back-
filled with nitrogen, and the tubes were subsequently immersed in a
prewarmed oil bath at 70 °C. At different time points, samples were
TFA, the oil was dissolved in 10 mL water and lyophilized to yield HDA-
1
biotin (3.50 g, 97%). H NMR (600 MHz, DMSO‑d
6
) δ (ppm): 7.75 (t,
J = 5.6 Hz, 1H, NH), 7.40 (br, 3H, NH
3
), 6.43 (s, 1H, NH-biotin), 6.38
(
s, 1H, NH-biotin), 4.31 (dd, 1H, J = 7.7, 5.1 Hz, CHNH), 4.12 (ddd,
1
H, J = 7.2, 4.4, 1.8 Hz, CHNH), 3.09 (ddd, 1H, J = 8.6, 6.0, 4.4 Hz,
), 3.01 (q, 2H, J = 6.6 Hz, NCH ), 2.81 (dd, 1H, J = 12.4,
.1 Hz, SCHH), 2.74—2.78 (m, 2H, NH CH ), 2.57 (d, 1H, J = 12.5 Hz,
SCHCH
2
2
5
2
2
SCHH), 2.04 (t, 2H, J = 7.4 Hz, CH
2
C(=O)), 1.22—1.61 (m, 14H,
+
1
7
CH
2
). ESI-MS m/z 343.2 (M + H) , calculated for C16
H
30
4
N O
2
S
withdrawn and analyzed by H NMR and GPC. The reaction was carried
3
42.5.
out for 5–6 h. Next, the polymers were isolated by precipitation in
diethyl ether for three times (DMAc/diethyl ether = 1/49, v/v) and
dried overnight under vacuum to give the final products (entries 1–4 in
Table 1).
2
.2.4. Synthesis of 4-cyano-4-[(dodecylsulfanylthiocarbonyl)-sulfanyl]
pentanoic succinimide (NHS-CDTPA)
The synthesis of NHS-CDTPA was based on previously published
papers [55,56]. Shortly, to a solution of 4-cyano-4-[(dodecylsulfa-
nylthiocarbonyl)-sulfanyl]pentanoic acid (CDTPA) (5.00 g, 12.4 mmol)
in 50 mL dry DCM at 0 °C, NHS (1.80 g, 15.6 mmol) and EDC (3.02 g,
2.3.2. Synthesis of block copolymers of poly[N-(2-hydroxypropyl)
methacrylamide]-block-[N-(2-benzoyloxy-propyl) methacrylamide] (p
(HPMAm)-b-p(HPMAm-Bz)) with or without biotin terminus
The obtained p(HPMAm) macroCTAs with or without biotin ter-
minus were chain-extended with HPMAm-Bz under the same conditions
as for the synthesis of p(HPMAm). In detail, the reagents were weighed
in Schlenk tubes and subsequently dissolved in dry DMAc (7 mL). The
concentration of HPMAm-Bz was 300 mg/mL. For the synthesis of co-
polymers with different molecular weights of the hydrophilic and hy-
drophobic blocks, the molar ratios of [HPMAm-Bz]/[p(HPMAm)]/
[AIBN] were 250/5/1, 500/5/1 and 900/5/1, respectively. For the
synthesis of biotinylated copolymer, the molar ratio of [HPMAm-Bz]/
[biotinylated p(HPMAm)]/[AIBN] was 900/5/1. The solution was de-
gassed by three cycles of freeze-vacuum-thawing, backfilled with ni-
trogen, and then the tube was immersed in a prewarmed oil bath at
70 °C. At different time points, samples were withdrawn and analyzed
1
5.9 mmol) were added. The reaction mixture was stirred at 0 °C for 1 h
and then at room temperature for 9 h. Subsequently, the DCM solution
was washed with saturated NaHCO (aq.) and the resulting DCM phase
3
was dried over anhydrous Na
2
SO
4
. After 2 h, Na
2
SO was filtered off
4
and the solvent was removed under reduced pressure and further dried
1
under vacuum to yield NHS-CDTPA (5.30 g, 85%). H NMR (600 MHz,
chloroform‑d) δ (ppm): 3.33 (m, 2H, CH
2
CH S), 2.93 (ddd, J = 9.2, 6.3,
2
3
1
9
.9 Hz, 2H, CH
H, J = 14.4, 9.8, 6.5 Hz, CH
.8, 6.5 Hz, CH CH C(=O)), 1.88 (s, 3H, C(CH
2
C(=O)), 2.85 (s, 4H, (O=)C(CH
CH C(=O)), 2.53 (ddd, 1H, J = 14.4,
)), 1.70 (tt, 2H,
2
)
2
C(=O)), 2.66 (ddd,
2
2
2
2
3
J = 12.8, 3.9 Hz, CH
2
CH
2
S), 1.37—1.42 (m, 2H, CH
2
(CH
2
) S),
2
1
.23—1.31 (m, 16H, CH
3
(CH
2
)
8
CH
2
), 0.88 (t, J = 7.0 Hz, 3H,
+
1
CH
3
CH
2
CH
2
). ESI-MS m/z 559.2 (M
+
Na
+
2H
2
O)
,
541.2
by H NMR and GPC. The reaction was carried out for 18–20 h. The
+
(
M + Na + H
2
O) , calculated for C23
H
36
2
N O
4
S
3
500.7.
polymers were isolated by precipitation in diethyl ether for three times
(
DMAc/diethyl ether = 1/49, v/v) and dried overnight under vacuum
to give the final products (entries 5–14 in Table 1).
2
.2.5. Synthesis of biotin-functionalized chain transfer agent (biotin-
1
CDTPA)
2.3.3. Characterizations of the polymers by H NMR spectroscopy and GPC
1
Coupling of NHS-CDTPA with HDA-biotin afforded a RAFT chain
H NMR spectra were recorded using a Bruker 600 MHz spectro-
transfer agent, biotin-CDTPA. Briefly, to a solution of HDA-biotin
meter (Billerica, MA, USA). The polymers (5–10 mg) were dissolved in
(
(
658 mg, 1.4 mmol) in a mixture of 18 mL anhydrous DMF and DCM
600 μL DMSO‑d
6
. The DMSO‑d peak at 2.50 ppm was used as the re-
6
1:1, v/v), NHS-CDTPA (656 mg, 1.3 mmol) and TEA (450 μL,
ference line.
3
.3 mmol) were added. The reaction mixture was stirred at room
Chemical shifts of p(HPMAm) (Fig. S2B): 7.18 (b, C(=O)NHCH
2
),
temperature for 16 h. Next, DCM was evaporated under vacuum at
4.70 (s, CH(CH
3
)OH), 3.68 (s, NHCH
2
CH(CH
3
) OH), 2.9 (b, NHCH
2
CH),
4
0 °C, and subsequently the reaction mixture was dropped into a large
0.4—2.0 (b, the rest of protons are from the methyl and backbone CH
2
excess of reverse osmosis (RO) water to precipitate the crude product
which was then washed with 2-propanol, filtered and dried under va-
protons). Chemical shifts of the biotinylated p(HPMAm) (Fig. S3B): in
addition to protons from p(HPMAm), 7.89 (s, NH-linker), 7.74 (s, NH-
linker), 6.43 (NH-biotin), 6.37 (NH-biotin), 4.30 (CHNH), 4.14
(CHNH).
1
cuum to yield biotin-CDTPA (~900 mg, 94%). H NMR (600 MHz,
DMSO‑d ) δ (ppm): 7.96 (t, 1H, J = 5.6 Hz, NH), 7.73 (t, 1H,
6
J = 5.6 Hz, NH), 6.41 (s, 1H, NH-biotin), 6.35 (s, 1H, NH-biotin), 4.30
dd, 1H, J = 7.8, 5.1 Hz, CHNH), 4.12 (ddd, 1H, J = 7.6, 4.5, 1.8 Hz,
CHNH), 3.09 (ddd, 1H, J = 8.7, 6.0, 4.4 Hz, SCHCH ), 3.04—2.98 (m,
H, 2NHCH ), 2.82 (dd, 1H, J = 12.4, 5.1 Hz, SCHH), 2.58 (d, 1H,
J = 12.4 Hz, SCHH), 2.43—2.26 (m, 4H, 2CH C(=O)), 2.04 (t, 2H,
J = 7.4 Hz, CH CH S), 1.85 (s, 3H, C(CH )), 1.63—1.20 (m, 36H,
8CH ), 0.85 (t, 3H, J = 6.9 Hz, CH CH CH ). ESI-MS m/z 728.3
M + H) , 750.2 (M + Na) , calculated for C35 728.2.
The theoretical number average of molecular weight (Mn, theory) of p
(HPMAm) with or without biotin terminus was calculated using the
following Eq. (1):
(
2
4
2
Mn,theory = [monomer]/[CTA] × conversion × Mmonomer + MCTA
(1)
2
2
2
3
where the conversion of HPMAm was determined by comparing the
integration areas of resonances from the vinyl protons of HPMAm at
5.30 ppm and the methine protons of HPMAm at 3.68 ppm (Fig. S2A &
1
2
3
2
2
+
+
(
H
61
5
N O
S
3 4
3
A), and [monomer], [CTA], Mmonomer and MCTA are the initial
4

Y. Wang, et al.
Journal of Controlled Release xxx (xxxx) xxx–xxx
Table 1
Characteristics of the polymers synthesized by RAFT as determined by 1H NMR, GPC and fluorometric analysis.
a
n, NMR (kDa)b
n, GPC (kDa)c PDIc CMC (μg/
En-try Polymers
Molar Ratio of M/
Monomer Conversion
Mn, theory (kDa)
M
M
d
CTA/I
(%)
mL)
1
2
3
4
5
6
7
8
9
1
1
1
1
1
p(HPMAm)3.0k
180:5:1
320:5:1
460:5:1
460:5:1
250:5:1
500:5:1
900:5:1
250:5:1
500:5:1
900:5:1
250:5:1
500:5:1
900:5:1
51
50
51
46
36
39
44
42
38
40
45
39
39
41
3.0
–
–
–
3.2
1.19
1.22
1.26
1.33
–
–
–
–
p(HPMAm)4.9k
4.9
5.0
p(HPMAm)7.1k
7.1
7.7
biotinylated p(HPMAm)6.8k
6.8
7.0
8.1
p(HPMAm)3.0k-b-p(HPMAm-Bz)5.4k
p(HPMAm)3.0k-b-p(HPMAm-Bz)9.9k
p(HPMAm)3.0k-b-p(HPMAm-Bz)15.6k
p(HPMAm)4.9k-b-p(HPMAm-Bz)6.4k
p(HPMAm)4.9k-b-p(HPMAm-Bz)11.3k
p(HPMAm)4.9k-b-p(HPMAm-Bz)16.8k
p(HPMAm)7.1k-b-p(HPMAm-Bz)4.9k
p(HPMAm)7.1k-b-p(HPMAm-Bz)9.1k
p(HPMAm)7.1k-b-p(HPMAm-Bz)15.0k
7.5
8.4
6.6
1.39 33
1.43 27
1.55 21
1.36 32
1.41 22
12.6
22.5
10.1
14.3
22.8
12.7
16.7
24.9
25.3
12.9
18.6
11.3
16.2
21.7
12.0
16.2
22.1
23.1
8.7
10.6
7.4
9.2
0
1
2
3
4
10.0
9.1
1.41
6
1.42 43
1.49 22
8.5
11.0
12.9
1.48
1.48
6
–
biotinylated p(HPMAm)6.8k-b-p(HPMAm- 900:5:1
Bz)16.3k
1
5
Cy3-labeled p(HPMAm)4.9k-b-p(HPMAm-
–
–
–
23.6
10.0
1.41
–
e
Bz)18.7k
a
b
c
d
e
M
n, theory calculated using Eqs. (1) and (3);
n, NMR calculated using Eqs. (2) and (4);
M
Determined by GPC analysis (DMF containing 10 mM LiCl as eluent, PEGs as standards), PDI, equal to Mw, GPC/Mn, GPC
;
Determined using pyrene fluorescence method;
Obtained by labeling of p(HPMAm)4.9k-b-p(HPMAm-Bz)16.8k (Section 2.4).
monomer and CTA concentrations, molecular weights of monomer
biotin terminus at 3.68 ppm (Fig. S4B & 5B), and Mmonomer and Mn,
macroCTA are molecular weights of monomer (MHPMAm-Bz = 247 g/mol)
and macroCTA (Mn, p(HPMAm) = 3000, 4900, and 7100 g/mol, Mn, bio-
tinylated p(HPMAm) = 6800 g/mol), respectively.
(MHPMAm = 143 g/mol) and CTA (MCDTPA = 403 g/mol, Mbiotin-
CDTPA = 727 g/mol), respectively.
1
The number average of molecular weight determined by H NMR
analysis (Mn, NMR) of biotinylated p(HPMAm) was calculated using the
following Eq. (2):
GPC was carried out to determine the number average molecular
weight (M
n
), weight average molecular weight (M ) and dispersity of
w
molecular weight (PDI, equal to M
w
/M ) using two serial PLgel 5 μm
n
Mn,NMR = degree of polymerization × Mmonomer + MCTA
(2)
MIXED-D columns and PEGs of narrow molecular weight distribution as
calibration standards [58]. Samples were prepared by dissolving ap-
proximately 10 mg of the polymer in 3 mL of DMF and samples of 50 μL
were injected onto the column. The eluent was DMF containing 10 mM
LiCl, the elution rate was 1 mL/min, the temperature was 65 °C, and
detection was done using a refractive index (RI) detector and ultraviolet
(UV) detector at the wavelength of 254 and 309 nm.
where the degree of polymerization of biotinylated p(HPMAm) was
determined by comparing the integration areas of resonances from the
methine protons of biotin-CDTPA at 4.14 ppm and the methine protons
of biotinylated p(HPMAm) at 3.68 ppm (Fig. S3B), and Mmonomer and
M
CTA are molecular weights of monomer (MHPMAm = 143 g/mol) and
CTA (Mbiotin-CDTPA = 727 g/mol), respectively.
Chemical shifts of p(HPMAm)-b-p(HPMAm-Bz) with or without
biotin terminus (Fig. S4B & 5B): in addition to protons from p(HPMAm)
with or without biotin terminus, 7.93 (s, aromatic CH), 7.34—7.65 (b,
2
.4. Synthesis and characterizations of cyanine3 (Cy3)-labeled p
(
HPMAm)-b-p(HPMAm-Bz) block copolymer
aromatic CH), 5.00 (s, NHCH
CH )O(O=C)).
2
CH(CH
3
)O(O=C)), 3.15 (b, NHCH CH
2
(
3
The terminal carboxylic acid group of the p(HPMAm) block of p
Mn, theory of p(HPMAm)-b-p(HPMAm-Bz) with or without biotin
(
(
HPMAm)-b-p(HPMAm-Bz) was first activated by NHS/EDC, yielding p
HPMAm)-b-p(HPMAm-Bz) NHS ester, followed by its reaction with the
terminus was calculated using the following Eq. (3):
Mn,theory = [monomer]/[macroCTA] × conversion × Mmonomer
primary amine groups of Cy3 [59]. In detail, p(HPMAm)-b-p(HPMAm-
Bz) (100 mg, thus 0.0046 mmol COOH end groups, entry 10 in Table 1)
was dissolved in 1 mL dry DMAc and subsequently EDC (1.3 mg,
0.0069 mmol) and NHS (0.8 mg, 0.0069 mmol) dissolved in 1 mL DCM
were added. The reaction mixture was stirred at room temperature for
1 h followed by addition of Cy3 amine (3.5 mg, 0.0056 mmol) dissolved
in 0.5 mL dry DCM also containing TEA (8.0 μL, 0.058 mmol), and the
resulting reaction mixture was stirred at room temperature overnight
and subsequently transferred into a Spectra/Por dialysis membrane
with a molecular weight cutoff of 6–8 kDa and sealed. Dialysis was
carried out for 48 h against water/THF (1:1 v/v) which was changed
every 12 h [49]. The final product (entry 15 in Table 1) was collected
+
Mn,macroCTA
(3)
where the conversion of HPMAm-Bz was determined by comparing the
integration areas of resonances from the methine protons of p(HPMAm-
Bz) at 5.00 ppm and the vinyl protons of HPMAm-Bz at 5.60 ppm (Fig.
S4A & 5A), and [monomer], [macroCTA], Mmonomer and Mn, macroCTA
are the initial monomer and macroCTA concentrations, molecular
weights of monomer (MHPMAm-Bz = 247 g/mol) and macroCTA (Mn, p
(
(
HPMAm)
=
3000, 4900, and 7100 g/mol,
M
n, biotinylated
p
HPMAm) = 6800 g/mol), respectively.
M
n, NMR of p(HPMAm)-b-p(HPMAm-Bz) with or without biotin ter-
1
minus was calculated using the following Eq. (4):
after lyophilization and characterized by H NMR and GPC coupled
with a UV detector (detection wavelength of 550 nm) as described in
Section 2.3.3. The labeling efficiency was determined by Jasco FP8300
Spectrofluorometer (Easton, MD, USA) with a calibration curve of Cy3
amine in DMF at the concentration of 0.0625, 0.125, 0.25, 0.5 and
1 μg/mL. Fluorescence emission spectra of Cy3 from 500 to 800 nm
were recorded at room temperature with an excitation wavelength at
Mn,NMR = degree of polymerization × Mmonomer + Mn,macroCTA
(4)
where the degree of polymerization of p(HPMAm)-b-(pHPMAm-Bz)
with or without biotin terminus was determined by comparing the in-
tegration areas of resonances from the methine protons of p(HPMAm-
Bz) at 5.00 ppm and the methine protons of p(HPMAm) with or without
5

Y. Wang, et al.
Journal of Controlled Release xxx (xxxx) xxx–xxx
5
50 nm. The excitation and emission band slits were 2.5 nm.
and DMF was subsequently removed by dialysis followed by filtration
through 0.45 μm RC membrane filter.
2
.5. Preparation and characterizations of empty (Cy3-labeled) and PTX-
loaded p(HPMAm)-b-p(HPMAm-Bz) micelles with or without biotin
modification
2.5.2. P(HPMAm)-b-p(HPMAm-Bz) polymer concentration determination
The polymer concentrations in the micellar dispersions were de-
termined by GPC analysis (Waters Alliance System) after 10-fold dilu-
tion with DMF to destabilize the micelles. The elution was isocratic with
a mobile phase of DMF containing 10 mM LiCl. Two serial PLgel 5 μm
MIXED-D columns were used. Total run time was 30 min with a flow
rate of 1 mL/min, the injection volume was 50 μL, the column tem-
perature was 65 °C, and detection was done using a refractive index (RI)
detector and ultraviolet (UV) detector at the wavelength of 254 and
309 nm. The polymer concentrations in the different samples were
calculated using a calibration curve of samples of polymer dissolved in
DMF at concentrations between 0.1 and 2 mg/mL. The micelle recovery
yield was calculated by the following formula (7):
2
.5.1. Preparation of empty (Cy3-labeled) and PTX-loaded p(HPMAm)-b-p
(
HPMAm-Bz) micelles
Empty p(HPMAm)-b-p(HPMAm-Bz) micelles (with or without biotin
modification) were prepared by solvent extraction method. In detail,
1
mL DMF solution of 18.0 mg of nonbiotinylated p(HPMAm)-b-p
HPMAm-Bz) copolymer (entry 13 in Table 1) and 2.0 mg of biotiny-
lated p(HPMAm)-b-p(HPMAm-Bz) copolymer (entry 14 in Table 1), or
0.0 mg nonbiotinylated p(HPMAm)-b-p(HPMAm-Bz) copolymer only
(
2
(
entry 13 in Table 1) was pipetted into 1 mL Milli-Q water while stirring
for 1 min. To remove DMF, the solution was transferred into a Spectra/
Por dialysis membrane with a molecular weight cutoff of 6–8 kDa and
sealed. Dialysis was carried out for 24 h against Milli-Q water which
was changed at 2nd, 5th and 13th h. Next, the micellar dispersion was
filtered through 0.45 μm RC membrane filter. The size of the empty p
amountofobtainedpolymer
amountofpolymerusedforprepearation
Recovery yield =
× 100%
(7)
2
.5.3. In vitro stability of p(HPMAm)-b-p(HPMAm-Bz) micelles
(
HPMAm)-b-p(HPMAm-Bz) micelles was determined by dynamic light
The in vitro colloidal stability of empty micelles, empty p(HPMAm)-
scattering (DLS) after 10-fold dilution in water at 25 °C using a Zetasizer
Nano S at a fixed angle of 173° (Malvern Instruments Ltd., Malvern,
UK). The Z-average diameter (Zave) and polydispersity index (PDI) were
calculated by the Zetasizer software v.7.13. The zeta-potential of the
empty p(HPMAm)-b-p(HPMAm-Bz) micelles was measured after 10-
fold dilution in 10 mM HEPES buffer pH 7.4 using a Zetasizer Nano Z
b-p(HPMAm-Bz) (entry 13 in Table 1) micelles (with or without biotin
decoration), prepared as described in Section 2.5.1 and diluted 10-fold
with PBS pH 7.4 (final polymer concentration was 0.9 and 0.6 mg/mL
for biotinylated and nonbiotinylated micelles), was investigated. The
samples were incubated at 37 °C for 48 h. Small sample aliquots were
analyzed by DLS.
(
Malvern Instruments Ltd., Malvern, UK). The residual DMF content of
1
the micellar dispersion was measured by H NMR as follows. Two
hundred μL micelle dispersion was mixed with 400 μL D
2
O, and no any
2.5.4. Effect of the hydrophobic/hydrophilic block molecular weight ratio of
p(HPMAm)-b-p(HPMAm-Bz) on micelle size
peak corresponding to DMF was detected (detection limit ⁓50 ppm).
PTX-loaded micelles were prepared similarly as for empty micelles.
In detail, 1 mL DMF in which the polymers (20 mg/mL, entries 13 and/
or 14 in Table 1) and PTX (concentrations ranging from 1 to 25 mg/mL)
were dissolved was rapidly pipetted into 1 mL Milli-Q water while
stirring for 1 min, and DMF was subsequently removed by dialysis
followed by filtration through 0.45 μm RC membrane filter to remove
non-encapsulated PTX. To determine the drug content of PTX-loaded
micelles, the micellar dispersions were diluted 10-fold with THF (HPLC
grade) to destabilize the micelles, and the dissolved PTX was subse-
quently quantified by HPLC analysis (Waters Alliance System). The
elution was isocratic with a mobile phase of THF/water = 55:45 (v/v)
containing 0.1% formic acid. The total run time was 8 min with a flow
rate of 1 mL/min. A Sunfire C18 column (5 μm, 4.6 × 150 mm) was
used and the detection wavelength was 227 nm. The injection volume
was 10 μL and the PTX concentration in the different samples was
calculated using a calibration curve of PTX standards prepared in THF
in a concentration range of 6–100 μg/mL. The encapsulation efficiency
The effect of hydrophobic/hydrophilic block molecular weight ratio
of p(HPMAm)-b-p(HPMAm-Bz) on the size of the formed micelles was
investigated using the small library of copolymers synthesized as de-
scribed in Section 2.3 (entries 5–13 in Table 1). The different diblock
copolymers (20 mg) were dissolved in 1 mL DMF and the obtained
solutions were rapidly pipetted into 1 mL Milli-Q water while stirring
for 1 min. DMF was removed by dialysis, and the obtained micellar
dispersions were subsequently filtered through 0.45 μm RC membrane
filter. The size of micelles was determined by DLS.
2.6. Critical micelle concentration (CMC) determination
The CMCs of the different p(HPMAm)-b-p(HPMAm-Bz) block co-
polymers were determined using pyrene fluorescence method as de-
scribed previously [60,61]. Briefly, the p(HPMAm)-b-p(HPMAm-Bz)
micelles were prepared by the solvent extraction method as described
in Section 2.5.1, and the p(HPMAm)-b-p(HPMAm-Bz) polymer con-
centration was determined as described in Section 2.5.2. Subsequently,
the micelle dispersions were diluted with distilled water to obtain
(
EE) and loading capacity (LC) were calculated by the following for-
mulas:
−5
concentrations that ranged from 1.0 × 10
to 1 mg/mL. Next, 50 μL
amountofloaded PTX
amountof PTX usedforloading
EE =
× 100%
of pyrene dissolved in acetone (0.18 mM) was added to 500 μL of the
polymer dispersion. The dispersions were incubated at room tempera-
ture overnight to evaporate acetone. Fluorescence excitation spectra of
pyrene from 300 to 360 nm were recorded at 37 °C while the intensity
of the emitted light at 390 nm was recorded. The excitation and
emission band slits were 10 and 2.5 nm, respectively. The intensity
ratio of I338/I333 was plotted against the polymer concentration to de-
termine CMC.
(5)
(6)
amountofloaded PTX
LC =
× 100%
amountofloaded PTX + amountofobtainedpolymer
To study cellular uptake, empty Cy3-labeled p(HPMAm)-b-p
(
HPMAm-Bz) micelles (with or without biotin modification) were pre-
pared similarly as for empty micelles without Cy3 label. Briefly, 1 mL
DMF solution of 17.4 mg of nonbiotinylated p(HPMAm)-b-p(HPMAm-
Bz) copolymer (entry 13 in Table 1), 2.0 mg of biotinylated p(HPMAm)-
b-p(HPMAm-Bz) copolymer (entry 14 in Table 1) and 0.6 mg Cy3-la-
beled p(HPMAm)-b-p(HPMAm-Bz) (entry 15 in Table 1), or 19.4 mg
nonbiotinylated p(HPMAm)-b-p(HPMAm-Bz) (entry 13 in Table 1) and
2.7. Cell culture
Human adenocarcinoma alveolar based lung cancer cell line (A549)
and human embryonic kidney cell line (HEK293) were obtained from
the American Type Culture Collection (ATCC, Manassas, Virginia, USA).
A549 cells are biotin receptor-positive whereas HEK293 cells are biotin
0
.6 mg Cy3-labeled p(HPMAm)-b-p(HPMAm-Bz) (entry 15 in Table 1)
was rapidly pipetted into 1 mL Milli-Q water while stirring for 1 min,
6

Y. Wang, et al.
Journal of Controlled Release xxx (xxxx) xxx–xxx
1
Fig. 1. Conversion as a function of reaction time for the RAFT polymerization of HPMAm and HPMAm-Bz (A). M
n
(based on GPC and H NMR analysis) and Mw, GPC
/
M
n, GPC (PDI) versus conversion of HPMAm and HPMAM-Bz (B, p(HPMAm), molar ratio of M/CTA/I was 320/5/1; C, p(HPMAm-Bz), molar ratio of M/CTA/I was
5
00/5/1; and D, p(HPMAm)-b-p(HPMAm-Bz), molar ratio of M/macroCTA/I was 900/5/1).
receptor-negative [20]. A549 and HEK293 cells were cultured in DMEM
supplemented with 10% of fetal bovine serum (FBS) at 37 °C in a hu-
bio-reduced by living cells into a colored formazan product that is so-
luble in tissue culture medium and that can be quantified by colori-
metric method [58,62]. A549 and HEK293 cells were seeded into 96-
midified atmosphere containing 5% of CO . Both cell lines were grown
2
in 75 cm2 cell culture flasks and passaged twice a week.
well plates at a density of 5 × 10 cells/well and incubated for 24 h at
3
3
7 °C in a humidified atmosphere containing 5% of CO . Stock solutions
2
2
.8. In vitro cellular uptake of empty Cy3-labeled p(HPMAm)-b-p
of empty micelles with biotin decoration (polymer concentration was
9 mg/mL), empty micelles without biotin decoration (polymer con-
centration was 6 mg/mL), PTX-loaded micelles with biotin decoration
(polymer concentration was 7 mg/mL) and PTX-loaded micelles
without biotin decoration (polymer concentration was 6 mg/mL) were
prepared as described in Section 2.5.1. PTX as Taxol formulation was
prepared by dissolving 12 mg of PTX in 1 mL ethanol followed by ad-
dition of 1 mL Cremophor EL and sonication for 30 min [43]. A Taxol
formulation without PTX was prepared by mixing Cremophor EL and
ethanol (1:1, v/v). PTX-loaded micellar and Taxol formulations were
diluted in cell culture media to yield concentrations of PTX that ranged
(
HPMAm-Bz) micelles
A549 and HEK293 cells were seeded into 96-well plates at a density
4
of 1 × 10 cells/well and incubated for 24 h at 37 °C in a humidified
atmosphere containing 5% of CO . Stock solutions of empty Cy3-la-
2
beled micelles with or without biotin decoration (polymer concentra-
tion was 6 mg/mL) were prepared as described in Section 2.5.1. The
cells were incubated with 100 μL of different formulations at 140 μg/
mL at 37 °C in a humidified atmosphere containing 5% of CO for 1, 4,
2
8
, and 24 h. Next, Hoechst 33430 was added to the wells 30 min before
−5
imaging with a final concentration of 10 nM. The media were replaced
with Opti-MEM and the plate was transferred into a Yokogawa CV7000
from 1.0 × 10 to 100 μg/mL. The empty micelles were diluted in cell
culture medium to yield polymer concentrations ranging from
−4
(
Tokyo, Japan) spinning disk microscope with a 40 × 1.2 NA water
1.7 × 10
to 1700 μg/mL, subsequently, Cremophor EL/ethanol so-
objective. To investigate whether cellular internalization of the bioti-
nylated micelles indeed occurs via biotin-receptor mediated en-
docytosis, A549 and HEK293 cells were pre-incubated with 2 mM free
biotin for 1 h. Subsequently, the cells were incubated with the Cy3-
labeled micelles with or without biotin decoration for 4 h, followed by
confocal imaging according to the above procedures.
lution was diluted in cell culture medium the same way as the Taxol
formulation. The cells were incubated with 100 μL of the different
formulations at 37 °C in a humidified atmosphere containing 5% of CO
2
for 48 h. Next, the media were replaced by 100 μL of fresh medium and
20 μL of MTS reagent followed by incubation for 2 h. The cell viability
was determined by measuring the absorbance at 492 nm using a Bio-
chrome EZ microplate reader (Jakarta Utara, Indonesia). IC50 values
were calculated as drug concentration that inhibits the cell growth by
2
.9. In vitro cytotoxicity of empty and PTX-loaded p(HPMAm)-b-p
5
0% after 48 h of cultivation.
(
HPMAm-Bz) micelles
The cell viability was evaluated after 48 h-exposure to the for-
mulations by using the MTS assay. The tetrazolium reagent MTS can be
7

Y. Wang, et al.
Journal of Controlled Release xxx (xxxx) xxx–xxx
Fig. 2. GPC chromatograms of the synthesized block copolymers. (A–C) GPC chromatograms (RI detection) of the different p(HPMAm)-b-p(HPMAm-Bz) copolymers.
D) GPC chromatograms (RI and UV detection) of biotinylated p(HPMAm)-b-p(HPMAm-Bz).
(
Fig. 3. (A) Effect of the molecular weight of the p(HPMAm)-b-p(HPMAm-Bz) block copolymers on the Z-average size (black, red, and blue line) and PDI (purple line)
of the formed micelles. Data represent mean ± SD (n = 3). (B) Critical micelle concentration as a function of p(HPMAm)-b-p(HPMAm-Bz) polymer molecular
weight. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
2.10. Statistical analysis
3. Results and discussion
Statistical analysis was done by GraphPad Prism 8.3.0 software.
3.1. Synthesis of biotin-functionalized chain transfer agent, biotin-CDTPA
A biotin-functionalized RAFT chain transfer agent, namely 4-cyano-
Two-way analysis of variance (ANOVA) was used to determine the
statistical significance of cell viability data. A value of p < 0.05 was
considered significant.
4
[(dodecylsulfanylthiocarbonyl)-sulfanyl]pentanoic acid (biotin-
CDTPA), was synthesized as shown in Scheme 1A. Amine-
8

Y. Wang, et al.
Journal of Controlled Release xxx (xxxx) xxx–xxx
Fig. 4. (A) Encapsulation efficiency (red line) and loading capacity (blue line) of p(HPMAm)7.1k-b-p(HPMAm-Bz)15.0k micelles at different PTX feeds. Data represent
mean ± SD (n = 3). (B) The effect of PTX feed on the Z-average size (red line) and PDI (blue line) of p(HPMAm)7.1k-b-p(HPMAm-Bz)15.0k micelles. Data represent
mean ± SD (n = 3). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
functionalized biotin (HDA-biotin) and NHS-activated CDTPA were
synthesized as described previously [51–56], and obtained with the
yield of 97 and 85%, respectively. Subsequently, biotin-CDTPA was
obtained through NHS coupling of HDA-biotin with NHS-activated
To synthesize p(HPMAm)-b-p(HPMAm-Bz) diblock copolymers, the
different p(HPMAm) macroCTAs were chain-extended with HPMAm-Bz
under the same reaction conditions as for the synthesis of p(HPMAm).
We aimed to synthesize block copolymers with a molecular weight
below 45 kDa, which is the threshold of renal elimination for pHPMA
[71]. To obtain p(HPMAm)-b-p(HPMAm-Bz) of different molecular
weights, the molar ratios of HPMAm-Bz/p(HPMAm)/AIBN ([M]/
1
CDTPA with 94% yield. The H NMR spectrum (Fig. S1) and mass
spectrometric analysis (described in Section 2.2.5) demonstrate the
successful synthesis of the desired CTA.
[
CTA]/[I]) in the feed were 250/5/1, 500/5/1 and 900/5/1. For the
3.2. Synthesis of p(HPMAm)-b-p(HPMAm-Bz) block copolymers
synthesis of the biotinylated block copolymer, the feed molar ratio of
HPMAm-Bz/biotinylated p(HPMAm)/AIBN was 900/5/1. The RAFT
polymerization was terminated when the conversion was around 40%
with PDI of 1.36–1.55 (entries 5–14 in Table 1). The successful chain
The block copolymers p(HPMAm)-b-p(HPMAm-Bz) were synthe-
sized as illustrated in Scheme 1B & C. First, HPMAm was polymerized
by RAFT using either CDTPA or biotin-CDTPA as CTA and AIBN as
initiator, respectively (Scheme 1B). The obtained p(HPMAm) mac-
roCTA was subsequently extended with HPMAm-Bz (Scheme 1C). As
shown in Fig. 1A, fast polymerization rates up to 8 h were observed,
which slowed down after this time point likely due to loss of active
thiocarbonylthio chain ends affording ‘dead’ polymer chains that are
unable to participate in the RAFT process [63,64]. Similar pheono-
menon in RAFT polymerization at high conversions was also observed
in previous studies [57,65]. Fig. 1B–D show that the number average
1
extension was demonstrated by GPC and H NMR analysis. The ob-
tained block copolymers showed a decreased GPC retention time
compared to p(HPMAm), demonstrating an increased molecular weight
(Fig. 2A–D). The GPC traces of the synthesized block copolymers ex-
hibited slight tailing at low molecular weight and thus were relatively
asymmetric, probably due to the presence of p(HPMAm) chains with a
dead end. Such dead polymer chains formed during the macroCTA
synthesis cannot undergo chain extension, resulting in a small amount
of homopolymer in the final product (Fig. S7), which is inevitable when
block copolymers are synthesized via RAFT [72]. The repeating units of
molecular weight (M
conversion, which is typical for a controlled radical polymerization
66].
n
) increased linearly with HPMAm and HPMAm-Bz
1
both HPMAm and HPMAm-Bz in the H NMR spectra support the
[
composition of the formed block copolymers (Fig. S4B & 5B). The
We aimed to synthesize p(HPMAm) macroCTA with molecular
theoretical M 's of the block copolymers calculated from the HPMAm-
n
weights of 3, 5 and 7 kDa, respectively, to mimic PEG (2–5 kDa)
commonly used as a stealth polymer in polymeric micelles and
PEGylated liposomes [67–70]. To obtain p(HPMAm) of different mo-
lecular weights, the molar ratio of HPMAm/CDTPA/AIBN ([M]/[CTA]/
Bz conversion were close to the molecular weight values as determined
1
by H NMR analysis (entries 5–14 in Table 1), by comparing the in-
tegration areas of resonances from the methine protons of p(HPMAm-
Bz) at 5.00 ppm and the methine protons of p(HPMAm) at 3.68 ppm
1
[
I]) in the feed was varied (180/5/1, 320/5/1 and 460/5/1). For the
(Fig. S4B). A typical H NMR spectrum of the biotinylated p(HPMAm)-
synthesis of biotinylated p(HPMAm), the feed molar ratio of HPMAm/
biotin-CDTPA/AIBN was 460/5/1. The RAFT polymerization was ter-
minated when the conversion was around 50% to obtain p(HPMAm)
b-p(HPMAm-Bz) shows that in addition to the characteristic resonances
for p(HPMAm) and p(HPMAm-Bz) units, some small peaks ascribed to
biotin were found (Fig. S5B, chemical shifts as described in Section
2.3.3) providing evidence that the block copolymer p(HPMAm)-b-p
(HPMAm-Bz) indeed contains the biotin end group. Importantly, only
one peak was observed in the GPC chromatogram using UV detection
(wavelength 210 nm, Fig. 2D), which points to the free biotin con-
centration below detection.
with PDI of 1.19–1.33 (entries 1–4 in Table 1). The M 's of p(HPMAm)
n
measured by GPC were in accordance with the theoretical values cal-
culated from the conversion of HPMAm (entries 1–4 in Table 1), by
comparing the integration areas of resonances from the vinyl protons of
HPMAm at 5.30 ppm and the methine protons of HPMAm at 3.68 ppm
1
(
(
Fig. S2A). In a typical H NMR spectrum of the biotinylated p
HPMAm) (Fig. S3B), besides the characteristic peaks for p(HPMAm),
To obtain a fluorescently labeled block copolymer suitable for cel-
lular uptake studies, p(HPMAm)4.9k-b-p(HPMAm-Bz)16.8k with a term-
inal NHS ester group was synthesized and subsequently reacted with
the resonances attributed to the biotin unit could be clearly identified
(
chemical shifts as described in Section 2.3.3), which demonstrates that
Cy3 amine fluorophore (Scheme S1). The M
n
of the synthesized Cy3-
1
the biotin-terminated p(HPMAm) was successfully synthesized by RAFT
polymerization using a biotin-functionalized trithiocarbonate chain
transfer agent.
labeled p(HPMAm)-b-p(HPMAm-Bz) as determined by H NMR was
23.6 kDa, which is close to the number average of molecular weight of
the copolymer before Cy3 conjugation (Fig. S6A & B). The GPC
9

Y. Wang, et al.
Journal of Controlled Release xxx (xxxx) xxx–xxx
(
caption on next page)
10

Y. Wang, et al.
Journal of Controlled Release xxx (xxxx) xxx–xxx
Fig. 5. Internalization of the fluorescently labeled targeted and non-targeted p(HPMAm)-b-p(HPMAm-Bz) micelles. Laser confocal scanning microscopy images of
A549 cells (A) and HEK293 cells (B) after incubation with Cy3-labeled p(HPMAm)-b-p(HPMAm-Bz) micelles with or without biotin decoration (140 μg/mL) for 1, 4,
8
, and 24 h. Cell nuclei are stained with Hoechst 33430 in blue while the micelles are visualized in red by Cy3. (C) The corresponding mean fluorescence intensity
calculated from the confocal images. Scale bar = 50 μm, data represent mean ± SD (n = 5 imaging fields), * p < 0.05. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)
chromatogram of Cy3-labeled p(HPMAm)4.9k-b-p(HPMAm-Bz)18.7k
3.5. PTX loading capacity
(
Fig. S8A) shows that the chromatograms as recorded by RI and UV
detection demonstrate identical retention time, which confirms that
Cy3 was conjugated to the block copolymer. Importantly, the absence
of UV–Vis signal in the Cy3-labeled p(HPMAm)4.9k-b-p(HPMAm-
Bz)18.7k sample at 18.5 min confirms that no free Cy3 amine was pre-
sent. After the coupling reaction, on average, the number of Cy3 units
was 0.85 per polymer chain as demonstrated by fluorometric analysis
PTX is an antineoplastic drug and used in the treatment of various
cancers [80], which is very hydrophobic and its aqueous solubility
is < 0.3 μg/mL [81]. By loading this drug in p(HPMAm)7.1k-b-p
(HPMAm-Bz)15.0k micelles, its solubility increased to 0.5 mg/mL (Table
S2). As shown in Fig. 4A, the loading capacity of p(HPMAm)7.1k-b-p
(HPMAm-Bz)15.0k micelles increased from 1 to 9 wt% when increasing
PTX feed from 1 to 15 wt%. The loading capacity did not further in-
crease at a 20 wt% feed of PTX, which can be attributed to the effect of
the preparation methods and the solvent used in this method [77]. Our
results are in accordance with previous reports showing that the in-
troduction of benzoyl group in these micelles yielded good incorpora-
tion of hydrophobic molecules due to the π–π stacking interaction be-
tween the aromatic groups of PTX and benzoyl groups of the polymer
[43,49,82]. The PTX-loaded p(HPMAm)7.1k-b-p(HPMAm-Bz)15.0k mi-
celles, dependent on the PTX content, had a slightly larger size than that
of empty micelles, ranging from 61 to 70 nm with a PDI < 0.12
(Fig. 4B).
(
Fig. S8B).
3.3. Preparation of empty p(HPMAm)-b-p(HPMAm-Bz) micelles
Polymeric micelles based on the different p(HPMAm)-b-p(HPMAm-
Bz) copolymers (entries 5–13 in Table 1) were prepared by rapid ad-
dition of DMF solution containing the block copolymer to the same
1
volume of water under stirring and followed by dialysis. H NMR
analysis showed that the residual DMF concentration was < 150 ppm,
which is below the 880 ppm limit according to International Commu-
nity of Harmonization (ICH) guideline for residual solvents for human
use [73]. The copolymers with molecular weights ranging from 8 to
3.6. Cellular uptake of Cy3-labeled p(HPMAm)-b-p(HPMAm-Bz) micelles
2
4 kDa resulted in the formation of micelles with tailored sizes from 40
to 90 nm (PDI < 0.12). As shown in Fig. 3A, for block copolymers with
a fixed molecular weight of the hydrophilic p(HPMAm) block (3.0, 4.9
and 7.1 kDa), the size of the micelles increased with increasing mole-
cular weight of hydrophobic block. This is in agreement with previous
findings reported by our group, where the micelle size and aggregation
number (Nagg) decreased with decreasing molecular weight of the hy-
drophobic block of the block copolymer mPEG-b-p(HPMAm-Bz) [74].
Further, the size of empty p(HPMAm)7.1k-b-p(HPMAm-Bz)15.0k micelles
Previous studies have shown that 10–20 mol% ligand (i.e., biotin
and folate) density on the surface of polymeric nanoparticles was op-
timal for efficient cellular uptake [83,84]. We therefore fixed the biotin
density at 10 mol% at the surface of the micelles. A549 (biotin receptor-
positive) and HEK293 (biotin receptor-negative) cells were incubated
with Cy3-labeled p(HPMAm)-b-p(HPMAm-Bz) micelles with or without
biotin decoration for 1, 4, 8, and 24 h and stained with Hoechst 33430
before imaging. In non-cancerous HEK293 cells which do not express
the biotin receptor, very low internalization of both biotinylated and
nonbiotinylated micelles was observed (Fig. 5B & C). However, it is
clear that the fluorescence intensity of biotinylated p(HPMAm)-b-p
(
with or without biotin decoration) in PBS (pH 7.4) did not change
upon incubation for 48 h at 37 °C (63 ± 5 and 62 ± 3 nm for bio-
tinylated and nonbiotinylated micelles), which demonstrates good
colloidal stability.
(
HPMAm-Bz) micelles was significantly higher as compared to the
nonbiotinylated micelles in biotin receptor-expressing A549 cells
(
(
(
Fig. 5A & C), confirming the enhanced internalization of biotinylated p
HPMAm)-b-p(HPMAm-Bz) micelles. Besides, the uptake of
3.4. CMC of p(HPMAm)-b-p(HPMAm-Bz)
p
HPMAm)-b-p(HPMAm-Bz) micelles in these cells was time-dependent.
The critical micelle concentration (CMC) of the micelles based on
In the presence of free biotin, biotinylated micelles showed significantly
lower uptake by A549 cells whereas the uptake of nonbiotinylated
micelles was not affected (Fig. S10). These observations validate that
the biotinylated micelles are taken up via biotin/SMVT receptor-
mediated endocytosis.
the library of p(HPMAm)-b-p(HPMAm-Bz) block copolymers (entries
5
–13 in Table 1) was determined using pyrene as a hydrophobic
fluorescence probe. In the excitation spectra of pyrene, a red shift oc-
curs as a result of pyrene partitioning from water to the hydrophobic
core of polymeric micelles [75,76]. When p(HPMAm)-b-p(HPMAm-Bz)
concentrations were below the CMC, a maximum peak of pyrene was
observed at 333 nm, whereas this peak shifted to 338 nm when the
polymer concentrations were above CMC (Fig. S9A). The I338/I333
fluorescence ratio can thus be used to determine the CMC of polymeric
micelles (Fig. S9B). The CMCs of p(HPMAm)7.1k-b-p(HPMAm-Bz)15.0k, p
3
.7. Cytotoxicity of empty and PTX-loaded p(HPMAm)-b-p(HPMAm-Bz)
micelles
A549 and HEK293 cell lines were used to assess the cytocompat-
ibility of formed micellar formulations with or without biotin mod-
ification and to determine the cytotoxicity of PTX-loaded micelles as
compared to Taxol formulations. The results indicate that empty p
(
HPMAm)7.1k-b-p(HPMAm-Bz)9.1k and p(HPMAm)7.1k-b-p(HPMAm-
Bz)4.9k were 6, 22 and 43 μg/mL, respectively (Table 1). It is clear that
for block copolymers with a fixed molecular weight of the hydrophilic
block (7.1 kDa), the CMCs of the polymers decreased with increasing
hydrophobic/hydrophilic ratio and thus with increasing molecular
weight of the hydrophobic block. The same trend was also observed for
the block polymers with fixed hydrophilic block of p(HPMAm)3.0k and p
(
HPMAm)7.1k-b-p(HPMAm-Bz)15.0k micelles with or without biotin
modification were non-toxic at the polymer concentrations up to
.7 mg/mL as compared to Cremophor EL/ethanol solution which
showed substantial cytotoxicity above a concentration of 1.7 μg/mL
1
(
Fig. 6A & B). PTX-loaded micelles with or without biotin decoration
(
HPMAm)4.9k (Table 1 and Fig. 3B), which is in line with previous
exhibited comparable cytotoxicity for both cell lines at low PTX con-
centrations (up to 0.1 μg/mL) (Fig. 6C & D). This can be explained by
the fact that the polymer concentration was below its CMC and thus
publications [6,74,76–79].
11

Y. Wang, et al.
Journal of Controlled Release xxx (xxxx) xxx–xxx
Fig. 6. Cytotoxicity of the targeted and non-targeted p(HPMAm)-b-p(HPMAm-Bz) micelles. Cell viability of A549 (A) and HEK293 cells (B) exposed to Cremophor
EL/ethanol mixture and p(HPMAm)-b-p(HPMAm-Bz) micelles with or without biotin decoration. Cell viability of A549 (C) and HEK293 cells (D) exposed to Taxol
and PTX-loaded p(HPMAm)-b-p(HPMAm-Bz) micelles with or without biotin decoration. Data represent mean ± SD (n = 3), * p < 0.05.
12

Y. Wang, et al.
Journal of Controlled Release xxx (xxxx) xxx–xxx
Table 2
Appendix A. Supplementary data
IC50 values in A549 and HEK293 cells after 48 h-treatment by PTX formula-
tions.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jconrel.2020.09.013.
IC50 (μg/mL)a
Cell line
Taxol
PTX-loaded micelles
without biotin
decoration
PTX-loaded micelles with
biotin decoration
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