Redox-sensitive polymeric nanoparticles for drug delivery

Article abstract of DOI:10.1039/c2cc31463k

Bioresponsive polymeric nanoparticles have been extensively pursued for the development of tumor-targeted drug delivery. A novel redox-sensitive biodegradable polymer with trimethyl-locked benzoquinone was synthesized for the preparation of paclitaxel-incorporated nanoparticles. The synthesized redox-sensitive nanoparticles released paclitaxel in response to chemically triggered reduction.

Full text of DOI:10.1039/c2cc31463k

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COMMUNICATION
Redox-sensitive polymeric nanoparticles for drug deliveryw
Hanjoung Cho,a Jungeun Bae,a Vivek K. Garripelli,a Joel M. Anderson,b Ho-Wook Junb and
Seongbong Jo*a
Received 27th February 2012, Accepted 24th April 2012
DOI: 10.1039/c2cc31463k
Bioresponsive polymeric nanoparticles have been extensively pursued
for the development of tumor-targeted drug delivery. A novel redox-
sensitive biodegradable polymer with ‘‘trimethyl-locked’’ benzoqui-
none was synthesized for the preparation of paclitaxel-incorporated
nanoparticles. The synthesized redox-sensitive nanoparticles released
paclitaxel in response to chemically triggered reduction.
prodrug, tested on T47D cells, the active drug resulted in
cytotoxicity after the reductive activation by oxidoreductases at a
low oxygen concentration.10a Even redox-triggered liposomes that
electrochemically release chemicals have been designed with the
TMBQ chemistry.11e Chemical reduction-induced shedding of
TMBQ from the liposome surface resulted in the structural change
of the supramolecular assemblies, which later destabilized the
redox-sensitive liposomes to electrochemically release a hydrophilic
fluorescent dye, calcein.11e However, the TMBQ-based redox-
chemistry has never been applied for a polymeric drug delivery
system thus far. The approach to use TMBQ-based redox-sensitive
polymeric nanoparticles is advantageous over the TMBQ-based
prodrugs or liposomes to target the redox stress in solid tumors.
Polymeric nanoparticles can be easily modified with the ligands
that selectively interact with the molecules expressed on cancer
cells to achieve active targeting. In addition, the nanoparticles
may be more flexible than the above-mentioned liposome for the
formulation of hydrophobic cancer drugs since they may disrupt
exquisitely balanced non-covalent interactions of TMBQ at the
liposome surface. Furthermore, the nanoparticles can easily
achieve prolonged circulation in blood and preferred accumula-
tion in tumor by poly(ethylene glycol) (PEG) coupling.12
Multifunctional polymeric nanoparticles have been widely adopted
for cancer-targeted drug delivery with their potential therapeutic
benefit to minimize the dose-limiting systemic toxicity by
preferential accumulation in tumors via the enhanced permeability
and retention (EPR) effect.1 These types of nanoparticles have
frequently been modified to respond to the signals stemming from
tumor microenvironments so that they can release incorporated
drugs selectively in tumor sites.2 Thus far, various bioresponsive
materials that are sensitive to external stimuli, such as pH,3
temperature,4 enzymes,5 and light,6 have been intensively
considered for targeted drug delivery to cancer.
Since redox changes associated with tumor hypoxia have been
identified as a viable biomarker for tumor progression and cancer
drug resistance, reductive enzymes overexpressed in tumor
microenvironments, such as 12- to 18-fold overexpression of
DT-diaphorase (EC 1.6.99.2) in lung cancer,7 provide an
important strategy for selective tumor targeting. Various bioreduc-
tive prodrugs have been reported and some of them are currently
under clinical trials.8 However, the prodrug approach to target
tumors may be complicated with ubiquitous expression of various
reductive enzymes in normal cells.9
In this article, we describe a proof-of-concept novel redox-
sensitive polymer containing amino groups protected by TMBQ.
The polymer is intended to release lactone and unmask free
amino groups under a simulated redox stress with sodium
dithionite. Thus, its nanoparticle would be able to release
incorporated drugs upon increased polymer hydration and
subsequent nanoparticle swelling with the protonation of free
amino groups at physiological pH (Scheme 1).11 We primarily
focused to test the feasibility of novel TMBQ-based redox-
sensitive polymer nanoparticles as a bioresponsive drug delivery
system using a simple in vitro drug release study.
Trimethyl-locked benzoquinone (TMBQ) is known to be
chemically or enzymatically transformed into lactone via intra-
molecular cyclization triggered by two-electron reduction.10,11
Prodrugs and bio-imaging probes have been based on the
bioreductive cleavage of TMBQ by reductive enzymes and
suggested for solid tumor-selective drug delivery and imaging
based on redox changes occurring in tumor hypoxia.10 As
demonstrated with a bioreductive TMBQ-based aniline mustard
A redox-sensitive polymer was designed and synthesized
from a monomer containing TMBQ. Initially, benzoquinone
carboxylic acid (b,b,2,4,5-pentamethyl-3,6-dioxo-1,4-cyclo-
hexadiene-1-propanoic acid) activated with N-hydroxysuccimide
a Department of Pharmaceutics, School of Pharmacy,
The University of Mississippi, University, MS 38677, USA.
E-mail: seongjo@olemiss.edu; Fax: +1 662-915-1177;
Tel: +1 662-915-5166
b Department of Biomedical Engineering, University of Alabama at
Birmingham, Birmingham, AL 35294, USA
w Electronic supplementary information (ESI) available: Detailed
experimental procedures and characterization data. See DOI:
10.1039/c2cc31463k
Scheme 1 Chemical reduction of the redox-sensitive polymers based
on trimethyl-locked benzoquinone.
c
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was synthesized according to the previously reported procedure.11a
The activated compound was coupled with serinol (2-amino-1,3-
propanediol) to yield a redox-sensitive diol monomer (ESIw).
Serinol was selected for the polymer synthesis because of its
proven biocompatibility as a monomer for biodegradable
polyesters.13 A coupling reaction between TMBQ-succinimidyl
ester and serinol consequently yielded a diol with a pendant
TMBQ (compound 1). Synthesized serinol monomer was
Fig. 1 TEM image and size distribution of polymeric nanoparticles by a
1
successfully confirmed by H and 13C NMR spectroscopy and
dynamic light scattering method (DLS). Scale bar is 500 nm in length.
elemental analysis. The serinol monomer was polymerized by
the reaction described in Scheme 2. The isolated yield of the
synthesized redox-sensitive polyester was found to be 85% after
purification with precipitation in ether. The number-average
polymer molecular weight (Mn) and PDI were determined to be
9800 Da and 1.51, respectively, by gel permeation chromato-
graphy (GPC) with polystyrene standards.
nanoparticle swelling or dissolution at pH 7.4. Sodium dithionite-
induced reduction dramatically increased the hydrodynamic
diameter of the nanoparticles from 178 nm in a turbid solution
to 29.3 mm in a transparent solution, a size beyond the measurable
limit by the Zetasizer as shown in Fig. 2. The size changes might
have resulted from the protonation of free amine groups in serinol
of which pKa is around 9.15.
The proton NMR of polymer 2 in CDCl3 showed all the
characteristic peaks and splitting (ESIw), which indicated
successful polymer synthesis. The chemical shift of 4 protons,
which are located in adjacent carbonyls in adipoyl groups
appeared at d = 2.33 ppm indicating polyester backbone
formation. Additionally, the trimethyl group in benzoquinone
ring and dimethyl groups in the serinol monomer were shown
at d = 2.12, 1.95, and 1.41 ppm, respectively. The proton
peaks of the monomer were broadened after the polymeriza-
tion. It is worthwhile to note that this synthetic reaction is also
amenable to further PEG modification at the redox-sensitive
polymer chain ends by the treatment of adipoyl chloride to
secure terminal carboxyl groups that can be exploited for the
esterification with methoxy-capped PEG. Even cancer selective
cyclic RGD can be modified to the end carboxyl groups.
Redox-sensitive polymeric nanoparticles were prepared from
the synthesized polymer by an emulsion method.14 Tween 80 was
used as a surfactant to form emulsion instead of poly(vinyl
alcohol) partially because of a low polymer molecular weight of
the redox polymer. The final polymeric nanoparticles resulted in a
yellowish fluffy powder. Particle size determined by dynamic light
scattering (DLS) using a Zetasizer (Malvern Instruments, UK)
revealed successful nanoparticle preparation with a Z-averaged
hydrodynamic diameter of 180 nm (PDI = 0.138) (Fig. 1) which
would allow tumor site accumulation by EPR.1 A TEM image
of the nanoparticles showed that the morphology of the nano-
particles was spherical with the particle size qualitatively compar-
able to that determined by DLS (Fig. 1), indicating that the
nanoparticle preparation via an emulsion was reliable.
A hydrophobic anticancer drug, paclitaxel, was loaded into the
redox-sensitive polymeric nanoparticles of which the particle size
was determined to be 267 nm (PDI = 0.21). The slight size
increase from 180 nm to 267 nm upon paclitaxel incorporation
might be attributed to the hydrophobicity of paclitaxel.15 As
reported with amphiphilic polymer micelles, hydrophobic inter-
action between paclitaxel and redox-sensitive polymer could
expand the particles.15 However, the morphology of drug-loaded
nanoparticles observed by TEM was similar to that of blank
nanoparticles and spherical (ESIw). The paclitaxel loading
efficiency in the nanoparticles was determined to be 77.9%
when a drug to polymer ratio of 1 : 10 was used.
Redox-triggered drug release from the nanoparticles was
tested by an in vitro release study using a medium containing
sodium dithionite. At a 200 molar excess of sodium dithionite
to the molar amount of benzoquinone, the release of paclitaxel
and lactone was dramatically increased over 36 h as shown in
Fig. 3. The amount of sodium dithionite used in the experiment
might exceed the redox stress occurring in tumors. The primary
purpose of using an excess amount of sodium dithionite was to
test the feasibility of the nanoparticles as a novel redox-sensitive
drug delivery system. Further investigation with DT-diaphorase
and NADPH mimicking tumor microenvironments would
better evaluate the TMBQ-based polymer nanoparticles.
The inclusion of sodium salicylate at a concentration of 80 mM
maintained sink conditions during the release study. It has
been known that sodium salicylate is able to increase paclitaxel
The effect of redox stress on the nanoparticles was tested
with a chemical reductant, sodium dithionite (Na2S2O4). It
was expected that sodium dithionite would reduce TMBQ to
leave free amine groups on the polymer, which would result in
physico-chemical changes in nanoparticles that would induce
Scheme 2 Synthesis of redox-sensitive polymer with adipoyl chloride
Fig. 2 Change in nanoparticle size in the presence of sodium dithionite
under basic conditions.
determined by DLS.
c
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in response to a simulated redox stress, these nanoparticles
would be useful for targeted drug delivery to solid tumors.
We acknowledge that funding for this work came from the
Department of Defense W81XWH-10-1-0414 and National
Science Foundation EPS-0903787 (S.J.). We also acknowledge
the HRSA grant off which the Malvern Zetasizer was purchased.
Individual fellowship support was provided by the Ruth L.
Kirschstein National Research Service Award Individual Fellow-
ship (F31DE021286) from the National Institute of Dental &
Craniofacial Research (J.M.A.)
Notes and references
1 H. Maeda, J. Wu, T. Sawa, Y. Matsumura and K. Hori,
J. Controlled Release, 2000, 65, 271–284.
2 (a) S. Sengupta, D. Eavarone, I. Capila, G. Zhao, N. Watson,
T. Kiziltepe and R. Sasisekharan, Nature, 2005, 436, 568–572; (b) W.
Wang and C. Alexander, Angew. Chem., Int. Ed., 2008, 47, 7804–7806.
3 (a) A. P. Griset, J. Walpole, R. Liu, A. Gaffey, Y. L. Colson and
M. W. Grinstaff, J. Am. Chem. Soc., 2009, 131, 2469–2471;
(b) N. Murthy, M. Xu, S. Schuck, J. Kunisawa, N. Shastri and
J. M. Frechet, Proc. Natl. Acad. Sci. U. S. A., 2003, 100,
4995–5000; (c) J. K. Kim, V. K. Garripelli, U. H. Jeong,
J. S. Park, M. A. Repka and S. Jo, Int. J. Pharm., 2010, 401, 79–86.
4 (a) J. E. Chung, M. Yokoyama, M. Yamato, T. Aoyagi, Y. Sakurai
and T. Okano, J. Controlled Release, 1999, 62, 115–127;
(b) S. Q. Liu, Y. W. Tong and Y. Y. Yang, Biomaterials, 2005,
26, 5064–5074; (c) K. Na, K. H. Lee, D. H. Lee and Y. H. Bae, Eur.
J. Pharm. Sci., 2006, 27, 115–122.
5 F. M. Veronese, O. Schiavon, G. Pasut, R. Mendichi,
L. Andersson, A. Tsirk, J. Ford, G. Wu, S. Kneller, J. Davies
and R. Duncan, Bioconjugate Chem., 2005, 16, 775–784.
6 (a) N. Fomina, C. McFearin, M. Sermsakdi, O. Edigin and
A. Almutairi, J. Am. Chem. Soc., 2010, 132, 9540–9542;
(b) A. P. Goodwin, J. L. Mynar, Y. Ma, G. R. Fleming and
J. M. Frechet, J. Am. Chem. Soc., 2005, 127, 9952–9953.
7 S. Y. Sharp, L. R. Kelland, M. R. Valenti, L. A. Brunton,
S. Hobbs and P. Workman, Mol. Pharmacol., 2000, 58, 1146–1155.
8 (a) C. P. Guise, M. R. Abbattista, R. S. Singleton, S. D. Holford,
J. Connolly, G. U. Dachs, S. B. Fox, R. Pollock, J. Harvey, P. Guilford,
F. Donate, W. R. Wilson and A. V. Patterson, Cancer Res., 2010, 70,
1573–1584; (b) M. R. Albertella, P. M. Loadman, P. H. Jones,
R. M. Phillips, R. Rampling, N. Burnet, C. Alcock, A. Anthoney,
E. Vjaters, C. R. Dunk, P. A. Harris, A. Wong, A. S. Lalani and
C. J. Twelves, Clin. Cancer Res., 2008, 14, 1096–1104.
Fig. 3 In vitro release profiles of (a) paclitaxel and (b) lactone in the
presence of sodium dithionite (’) and without sodium dithionite (m)
in PBS. Triggered paclitaxel release was tested with an alternating
addition of sodium dithionite (c). Significant increase (*) in cumulative
paclitaxel release (P o 0.05) was noted during the release study. Data
represent the mean Æ SD (n = 3).
solubility in aqueous solution without affecting physical properties
of paclitaxel.16 As shown in Fig. 3, about 52% of encapsulated
paclitaxel was released within 3 h in the presence of sodium
dithionite while 13% of paclitaxel was released over 12 h in the
absence of the reducing agent. An achieved cumulative paclitaxel
release over 36 h was 79% in the presence of the reducing agent.
However, only 17% of incorporated paclitaxel was released from
the redox-sensitive nanoparticles without the chemical reductant.
Lactone release from the paclitaxel-loaded nanoparticles was also
consistent with the paclitaxel release. Cumulative lactone release
lasted for 12 h to reach 66.8% while no lactone was released
without reduction (Fig. 3b). Therefore, lactone release upon
nanoparticle reduction indicates that property change occurred
in the redox-sensitive nanoparticles was indeed mediated by
the two-electron reduction of TMBQ moiety. Furthermore, the
consequent size change occurred to nanoparticles might be trans-
lated into increased paclitaxel release.
9 F. P. Guengerich and W. W. Johnson, Biochemistry, 1997, 36,
14741–14750.
We also challenged the nanoparticles with the reducing agent
after incubating them in PBS for 48 h. An addition of sodium
dithionite immediately increased drug release, which peaked within
24 h (Fig. 3c). The percentage release of paclitaxel within initial
48 h was only 20%. However, paclitaxel release increased to a
cumulative release of 90% within 24 h after the addition of sodium
dithionite. Taken together, the results indicated that paclitaxel
could be released from the TMBQ-based redox-sensitive polymer
nanoparticles in response to redox changes. Although a gap needs
to be filled with better simulation of the redox stress using tumor-
related reductive enzymes that are able to reduce TMBQ, the
TMBQ-based nanoparticles would be useful for targeting solid
tumors.
10 (a) M. Volpato, N. Abou-Zeid, R. W. Tanner, L. T. Glassbrook,
J. Taylor, I. Stratford, P. M. Loadman, M. Jaffar and R. M. Phillips,
Mol. Cancer. Ther., 2007, 6, 3122–3130; (b) S. T. Huang, Y. X. Peng
and K. L. Wang, Biosens. Bioelectron., 2008, 23, 1793–1798;
(c) S. T. Huang and Y. L. Lin, Org. Lett., 2006, 8, 265–268.
11 (a) A. Zheng, D. Shan and B. Wang, J. Org. Chem., 1999, 64,
156–161; (b) W. Ong and R. L. McCarley, Chem. Commun., 2005,
4699–4701; (c) C. Yan, W. Matsuda, D. R. Pepperberg,
S. C. Zimmerman and D. E. Leckband, J. Colloid Interface Sci.,
2006, 296, 165–177; (d) W. Ong and R. L. McCarley, Macromo-
lecules, 2006, 39, 7295–7301; (e) W. Ong, Y. Yang, A. C. Cruciano
and R. L. McCarley, J. Am. Chem. Soc., 2008, 130, 14739–14744.
12 V. P. Torchilin, Pharm. Res., 2007, 24, 1–16.
13 J. Rickerby, R. Prabhakar, A. Patel, J. Knowles and S. Brocchini,
J. Controlled Release, 2005, 101, 21–34.
14 J. Jung, I. H. Lee, E. Lee, J. Park and S. Jon, Biomacromolecules,
2007, 8, 3401–3407.
15 J.-K. Kim, V. K. Garripelli, U.-H. Jeong, J.-S. Park, M. A. Repka
and S. Jo, Int. J. Pharm., 2010, 401, 79–86.
16 (a) Y. W. Cho, L. Lee, S. C. Lee, K. M. Huh and K. Park,
J. Controlled Release, 2004, 97, 259–257; (b) K. M. Huh, H. S. Min,
S. C. Lee, H. J. Lee, S. Kim and K. Park, J. Controlled Release,
2008, 126, 122–129.
Novel redox-sensitive polymeric nanoparticles were prepared
from a synthesized monomer containing TMBQ as a redox
sensitive group. A hydrophobic cancer drug, paclitaxel, was
incorporated into the polymeric nanoparticles and released by
sodium dithionite-mediated reduction in a triggered manner. Since
the polymeric nanoparticles are able to release incorporated drugs
c
This journal is The Royal Society of Chemistry 2012
Chem. Commun., 2012, 48, 6043–6045 6045