Liposomal bortezomib nanoparticles via boronic ester prodrug formulation for improved therapeutic efficacy in vivo

Article abstract of DOI:10.1021/jm500352v

In this study, we describe the development of liposomal bortezomib nanoparticles, which was accomplished by synthesizing bortezomib prodrugs with reversible boronic ester bonds and then incorporating the resulting prodrugs into the nanoparticles via surface conjugation. Initially, several prodrug candidates were screened based upon boronic ester stability using isobutylboronic acid as a model boronic acid compound. The two most stable candidates were then selected to create surface conjugated bortezomib prodrugs on the liposomes. Our strategy yielded stable liposomal bortezomib nanoparticles with a narrow size range of 100 nm and with high reproducibility. These liposomal bortezomib nanoparticles demonstrated significant proteasome inhibition and cytotoxicity against multiple myeloma cell lines in vitro and remarkable tumor growth inhibition with reduced systemic toxicity compared to free bortezomib in vivo. Taken together, this study demonstrates the incorporation of bortezomib into liposomal nanoparticles via reversible boronic ester bond formation to enhance the therapeutic index for improved patient outcome.

Full text of DOI:10.1021/jm500352v

Article
pubs.acs.org/jmc
Liposomal Bortezomib Nanoparticles via Boronic Ester Prodrug
Formulation for Improved Therapeutic Efficacy in Vivo
Jonathan D. Ashley,† Jared F. Stefanick,† Valerie A. Schroeder,‡,§ Mark A. Suckow,‡,§ Tanyel Kiziltepe,*,†,∥
and Basar Bilgicer*,†,∥,⊥
†Department of Chemical and Biomolecular Engineering, University of Notre Dame, Notre Dame, 182 Fitzpatrick Hall, Indiana
46556, United States
‡Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana 46556, United States
§Freimann Life Science Center, University of Notre Dame, Notre Dame, Indiana 46556, United States
∥Advanced Diagnostics and Therapeutics, University of Notre Dame, Notre Dame, Indiana 46556, United States
⊥Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
S
* Supporting Information
ABSTRACT: In this study, we describe the development of
liposomal bortezomib nanoparticles, which was accomplished
by synthesizing bortezomib prodrugs with reversible boronic
ester bonds and then incorporating the resulting prodrugs into
the nanoparticles via surface conjugation. Initially, several
prodrug candidates were screened based upon boronic ester
stability using isobutylboronic acid as a model boronic acid
compound. The two most stable candidates were then selected
to create surface conjugated bortezomib prodrugs on the
liposomes. Our strategy yielded stable liposomal bortezomib
nanoparticles with a narrow size range of 100 nm and with
high reproducibility. These liposomal bortezomib nano-
particles demonstrated significant proteasome inhibition and cytotoxicity against multiple myeloma cell lines in vitro and
remarkable tumor growth inhibition with reduced systemic toxicity compared to free bortezomib in vivo. Taken together, this
study demonstrates the incorporation of bortezomib into liposomal nanoparticles via reversible boronic ester bond formation to
enhance the therapeutic index for improved patient outcome.
proteasome activity by binding with high affinity to the catalytic
sites of the 20S proteasome, particularly the chymotrypsin site,
physically blocking the enzymatic activity and preventing
proteolytic cleavage.12 Although bortezomib has proven to be
an effective treatment for several cancers, the dose limiting side
effects, particularly peripheral neuropathy and thrombocytope-
nia, have inhibited it from reaching its true therapeutic potential
in a broader patient population.13−16
In recent years, nanoparticle-based drug delivery systems
have gained remarkable interest as they have greatly improved
the efficacy of traditional therapeutics while decreasing the
associated systemic toxicities. Nanoparticles with a diameter of
20−200 nm can selectively target and preferentially home at the
tumor site via the enhanced permeability and retention (EPR)
effect, a phenomenon that arises from the angiogenic blood
vessels present in the tumor microenvironment which is not
found in healthy endothelia.17,18 Numerous studies have shown
that nanoparticle-based therapies selectively deliver therapeutics
to tumor cells which results in the decreased systemic toxicity
INTRODUCTION
■
To develop more effective treatments for cancer, studies have
focused on the identification of key pathways and proteins
responsible for the survival and progression of malignant cells.
One target that has gained much attention over the past decade
is the proteasome.1−5 Proteasomes are intracellular proteins
responsible for the degradation of damaged or misfolded
proteins as well as the systematic degradation of regulatory
proteins associated with cell cycle progression, cell growth, cell
survival, gene expression, and stress response.2,4,6−8 Disruption
of this pathway increases the accumulation of pro-apoptotic
proteins, cyclins, and cyclin-dependent kinase inhibitors while
decreasing NF-κB activity within tumor cells, ultimately
resulting in cell cycle arrest and apoptosis.4,8−10 Because
malignant cells exhibit a greater sensitivity to disruptions in
proteasome activity when compared to healthy cells,
proteasomes can be used as a viable therapeutic target for the
treatment of cancers.5,9,11 Bortezomib, a dipeptide boronic acid
analogue, was the first proteasome inhibitor approved by the
FDA for the treatment of cancers, specifically multiple myeloma
(MM) and mantle cell lymphoma, and remains one of the most
potent proteasome inhibitors available. Bortezomib inhibits
Received: March 5, 2014
© XXXX American Chemical Society
A
dx.doi.org/10.1021/jm500352v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
associated with the off-target activity of the therapeutic
agent.19−22 Therefore, nanoparticles present ideal drug delivery
vehicles for therapeutics such as bortezomib in an attempt to
reduce the associated dose limiting side effects while enhancing
its efficacy to improve the overall patient outcome.
Moreover, the reversibility of this conjugation strategy ensures
that the therapeutic is not chemically altered and that free
bortezomib is released upon hydrolysis and maintains its
activity.
To create a bortezomib prodrug with favorable release
kinetics, the stability of the different boronic esters needs to be
evaluated. Using bortezomib to evaluate the stability of the
various boronic esters formed when conjugated to different
linkage molecules is not a practical approach, primarily due to
the large quantities of bortezomib required for each synthesis
and the associated prohibitive cost. Instead, in previous studies,
phenylboronic acid (PBA) was frequently used as a model
compound to investigate boronic ester hydrolysis.33−35 In these
studies, PBA was conjugated to various diol-like functionalities
to evaluate the stability and pH sensitivity of different PBA
boronic ester bonds.33−35 It is known that PBA, which is an aryl
boronic acid, forms more stable boronic esters than alkyl
boronic acids such as bortezomib at physiological pH due to
the lack of an electron withdrawing group adjacent to the
boronic acid.29,36 Therefore, while the studies performed with
PBA have provided insight into conjugation strategies and
hydrolysis trends associated with boronic acid containing
molecules, the information gained from these studies cannot
be directly applied to bortezomib due to the differences in
chemical properties. Instead, isobutylboronic acid (IBBA),
which is an alkyl boronic acid with a similar chemical structure
around the boronic acid as bortezomib, provides a better model
compound to evaluate different boronic ester stabilities for
nanoparticle-based bortezomib drug delivery (Figure 2a). Thus,
we used IBBA as a model boronic acid to identify a linkage
molecule that forms a boronic ester conjugate with optimal
stability for drug delivery applications.
To investigate the stabilities of various boronic esters using
IBBA as a model compound, several different compounds
containing different carboxyl, hydroxyl, and amine function-
alities were selected as linkage molecules to form boronic esters
with IBBA (Figure 2b; 1−6). Iminodiacetic acid (1) was shown
to form very stable boronic esters in several studies and can be
easily modified for nanoparticle functionalization.37,38 Thus, it
was selected as a candidate for this application. One possible
caveat of 1 is that it may form boronic esters that are too stable,
which would not release bortezomib from the nanoparticle and
render the formulation ineffective. In that case, the carboxyl
groups in 1 can be replaced with hydroxyl groups to reduce the
stability of the bond while maintaining the dative bond between
the boron and nitrogen atoms. Thus, linkage molecules 2 and 3
were also chosen as candidates to identify the optimal rate of
hydrolysis for drug delivery. Given that the carboxyl groups
significantly contribute to the stability of boronic esters, other
molecules containing carboxyl groups were also selected for
evaluation. Methyl salicylic acid (4), a derivative of salicylic
acid, contains a carboxyl that is adjacent to a hydroxyl group on
a phenyl ring, which provides a structure that could help
stabilize the boronic ester. In addition, salicylic acid is the
precursor to salicylhydroxamic acid which was shown to form
boronic esters with PBA in a pH sensitive manner.39,40
Therefore, methyl salicylhydroxamic acid (5) was also selected
for this study. Lastly, to evaluate the effect of dative bond
formation in stability, citric acid (6) was selected as a candidate
because it can adopt similar boronic ester configurations to 1
without the presence of the dative bond to reduce the stability
and facilitate drug release.
While many nanoparticle formulations exist, poly(ethylene
glycol) (PEG) coated liposomes are an attractive choice for use
as pharmaceutical nanocarriers due to their ability to evade
detection by the immune system, ease of preparation, high drug
loading capabilities, and biocompatibility.23−25 Various chemo-
therapeutics such as doxorubicin, cisplatin, irinotecan, and
others have previously been incorporated in liposomes,
demonstrating an improved therapeutic index relative to free
drug.22,26,27 In the liposomal drugs developed to date, the
therapeutic agents have been predominately loaded in the
aqueous core exhibiting high drug to lipid ratios.27 However,
due to the relatively poor water solubility of bortezomib,
selective isolation in the aqueous core of the liposomes could
significantly limit the drug loading of the particle. A viable
option for incorporating bortezomib into liposomes is the
direct conjugation of bortezomib to the surface of liposomes via
a reversible boronic ester linkage.
In this study, we describe the development of liposomal
bortezomib nanoparticles, which was accomplished by synthe-
sizing bortezomib prodrugs with reversible boronic ester bonds
and then incorporating the resulting prodrugs into liposomal
nanoparticles via surface conjugation. Our results demonstrated
that the liposomal bortezomib nanoparticles inhibited protea-
some activity, were cytotoxic to cancer cells in vitro, and
showed enhanced tumor growth inhibition with reduced
systemic toxicity in vivo. Taken together, this study
demonstrates the synthesis and incorporation of bortezomib
prodrugs into liposomal nanoparticles to produce first-
generation liposomal bortezomib nanoparticles which have
enhanced therapeutic index with the long-term goal of
improving patient outcome in cancers.
RESULTS
■
Synthesis and Evaluation of the Boronic Ester Bond
Stability. Bortezomib contains a boronic acid moiety, which
plays a major role in its ability to inhibit proteasome
activity.11,28 Boronic acids are known to form boronic esters
with alcohols, diols, and carboxylic acid containing molecules
(linkage molecules) through a reversible reaction, which can
yield an unmodified boronic acid upon hydrolysis (Figure
1).29,30 The stability of these boronic esters is known to be
Figure 1. Schematic of the formation of boronic esters.
dependent upon the chemical structures present around the
boronic ester bond.29,31 For example, an unhindered cyclic
boronic ester made from ethylene glycol is less stable and
therefore hydrolyzes more rapidly than a boronic ester made
from a diol with a bulky side group that sterically inhibits
hydrolysis such as pinanediol.30−33 Thus, liposomal bortezomib
nanoparticles can be prepared by functionalizing liposomes
with diols or a similarly reactive moiety, which can then be used
to conjugate bortezomib to the liposome surface via a reversible
boronic ester bond to provide controlled drug release.
B
dx.doi.org/10.1021/jm500352v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 2. Linkage molecules screened for the synthesis of bortezomib prodrugs with reversible boronic ester bonds. (a) Structures of bortezomib
(left) and isobutylboronic acid (right). (b) Linkage molecules (1−6) were coupled to IBBA to form the boronic ester conjugates, 1a−6a, and were
evaluated for the relative boronic ester stability. (c) Linkage molecules 1 and 4 were selected as promising candidates based on relative hydrolysis
rates and were modified with a C16 aliphatic chain to yield the lipophilic molecules 1b and 4b, respectively. 1b and 4b were then used to synthesize
the bortezomib prodrug conjugates 1c and 4c, respectively. The C16 aliphatic chain enabled facile insertion to the liposomes.
To evaluate the relative bond stability of the boronic esters,
the model boronic acid, IBBA, was conjugated to linkage
molecules 1−6 in toluene under refluxing conditions to
generate molecules 1a−6a (Figure 2b), respectively. Excess of
the respective linkage molecule was used to ensure complete
boronic ester formation. Post formation, each conjugate was
monitored via 11B NMR spectroscopy to determine hydrolysis
of the boronic ester in PBS by observing the chemical shift of
the boron atom as the boronic ester (∼12 ppm) hydrolyzed to
a boronic acid (∼32 ppm) (Supporting Information Figure S1).
The boronic ester formed with iminodiacetic acid (1a) had a
half-life of t1/2 = 190 min, which was significantly more stable
than the boronic esters 2a−6a. The exchange of one (2a) or
both (3a) of the carboxyl groups in 1a with a hydroxyl group
significantly decreased the stability of the boronic ester
conjugate, reducing the half-lives by ∼21- and ∼118-fold for
2a (t1/2 = 8.8 min) and 3a (t1/2 = 1.6 min), respectively, when
compared to 1a. The increased stability of 1a over its
derivatives (2a and 3a) can be attributed to the strong dative
bond that results from the increased acidity of the boron due to
the two carboxyl groups. Methyl salicylic acid (4) formed the
second most stable boronic ester (4a) with a half-life of t1/2
=
10 min. This could be attributed to the stabilizing effects of the
phenyl ring in addition to the carboxyl group. This effect was
C
dx.doi.org/10.1021/jm500352v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
less pronounced with 5a (t1/2 = 0.4 min), where the half-life
was almost 30 times shorter than 4a. Comparing the stability of
6a (t1/2 = 1.6 min) to that of 1a highlighted the significance of
the dative bond in the stability of the boronic ester as 6a lacks a
central nitrogen atom to form a dative bond. The half-lives for
hydrolysis for molecules 1a−6a ranged from 0.4 to 190 min,
and are summarized in Table 1.
Table 1. Half-Lives of Different Boronic Esters in PBS
molecule
t1/2 (min)
1a
2a
3a
4a
5a
6a
190 45
8.8 0.2
1.6 0.4
10
1
0.4 0.1
1.6 0.1
It is noteworthy that although IBBA is a suitable model
compound to evaluate the relative boronic ester bond stability
formed with the linkage molecules (1−6) and provides a means
to rank these molecules, the hydrolysis rates obtained with
IBBA cannot be used as an absolute measure for bortezomib.
The chemical differences between IBBA and bortezomib
including the structural differences adjacent to the boronic
acids between the boro-leucine in bortezomib and IBBA will
alter the hydrolysis rates for bortezomib. Furthermore,
bortezomib, having a phenylalanine and pyrazinoic acid moiety,
possesses a more complex structure than IBBA which could
impede the hydrolysis of the boronic ester, resulting in longer
hydrolysis half-lives than those found using IBBA. Therefore,
on the basis of the ranking information gained from the relative
IBBA conjugate stabilities, linkage molecules 1 and 4 were
selected as promising candidates to synthesize the bortezomib
prodrugs and for their incorporation into the nanoparticles.
Synthesis of the Bortezomib Prodrugs and Formation
of Liposomal Bortezomib Nanoparticles. To incorporate
bortezomib into the liposomes, linkage molecules 1 and 4 were
modified with a C16 aliphatic chain, yielding lipophilic
molecules 1b and 4b (Figure 2c). These molecules were
synthesized in such a way as not to alter the functional aspects
of the molecules such that boronic esters could still be formed
with bortezomib. The C16 tail enables the facile insertion into
the bilayer of the liposomes and has a similar chain length as
the other lipids used to form the liposome to maintain
nanoparticle stability. Once the aliphatic modifications were
made to the linkage molecules, the bortezomib prodrug
conjugates 1c and 4c (Figure 2c) were synthesized by
conjugating the lipophilic molecules 1b and 4b, respectively,
to bortezomib via boronic ester formation by reacting in
toluene under refluxing conditions for 2 h. For the formation of
bortezomib prodrug incorporated nanoparticles, liposomes
were first prepared without the prodrugs and extruded through
a 100 nm polycarbonate membrane. Then, the prodrugs 1c and
4c were each postinserted into the particles with >80%
efficiency (Supporting Information Figure S2) at the molar
ratios of 92.5:5:2.5 DSPC:mPEG2000:Prodrug, creating the
liposomal bortezomib nanoparticles NP[1c] and NP[4c],
respectively (Figure 3a). The liposomal bortezomib nano-
particles yielded an average diameter of ∼100 nm based on
dynamic light scattering analysis (Figure 3b), which suggested
that the incorporation of the prodrugs did not alter the size of
the liposomes when compared to the nondrug loaded
Figure 3. Preparation and characterization of liposomal bortezomib
nanoparticles. (a) Illustration of the preparation of liposomal
bortezomib nanoparticles. (b) Dynamic light scattering analysis
revealed an average size distribution of ∼100 nm for all nanoparticles.
liposomes. As an alternative approach, prodrug analogues of
1c and 4c without the aliphatic chains were also synthesized
and incorporated into liposomal bilayer. Their encapsulation
efficiencies, however, were very low with 3.8% and 11.8%,
respectively, providing the rationale for the aliphatic
modification for efficient drug loading.
Liposomal Bortezomib Nanoparticles Inhibit Protea-
some Activity of MM Cells and Induce Apoptosis. Once
the nanoparticles were formed, proteasome inhibition assays
were performed to evaluate the potency of the liposomal
bortezomib nanoparticles, NP[1c] and NP[4c], to arrest
proteasome activity. MM.1S and NCI-H929 MM cell lines
were incubated with 25 nM bortezomib equivalent concen-
trations of NP[1c], NP[4c], and free bortezomib for 1, 4, and 8
h, before assessing proteasome activity (Figure 4a). At 1 h,
NP[1c] had increased proteasome activity compared to free
bortezomib and NP[4c]. However, at 4 and 8 h free
bortezomib, NP[1c], and NP[4c] all effectively inhibited
proteasome activity. The kinetic delay observed with NP[1c]
in inhibition of proteasome activity can be attributed to the
boronic ester bond stability of the 1c bortezomib prodrug, and
is in accordance with the expected differences based on
hydrolysis rates obtained with the IBBA model molecule (Table
1).
After assessing proteasome activity, we evaluated if the
proteasome inhibition by the liposomal bortezomib nano-
particles induced apoptosis by observing the early apoptotic
marker, annexin V. MM.1S and NCI-H929 cells were incubated
with 12.5 nM of equivalent bortezomib concentrations of
NP[1c], NP[4c], and free bortezomib for 12 h (Figure 4b).
Both nanoparticles NP[1c], and NP[4c] induced apoptosis
similar to free bortezomib. Taken together, these results
demonstrated that the liposomal nanoparticles NP[1c] and
NP[4c] inhibited proteasome activity and induced apoptosis in
MM cells similar to free bortezomib.
Liposomal Bortezomib Nanoparticles Are Cytotoxic
to MM Cells. Next, we evaluated the cytotoxicity of liposomal
bortezomib nanoparticles against MM cells. For this, MM.1S
and NCI-H929 cells were incubated with NP[1c], NP[4c], or
free bortezomib for 48 h, and cell viability was assayed by Cell
D
dx.doi.org/10.1021/jm500352v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
were remarkably cytotoxicity against MM cells. Importantly,
although NP[1c] and NP[4c] demonstrated similar cytotox-
icity to free bortezomib in vitro, they have the potential to
further enhance efficacy in vivo due to preferential accumu-
lation in the tumor site via the EPR effect as has been
previously observed for other therapeutics.22,24 In addition, the
esterases and acidic conditions present in the tumor micro-
environment could further enhance the antitumor efficacy of
NP[1c] and NP[4c] by facilitating the drug release from the
particles at the tumor site.
Liposomal Nanoparticles Are Taken up by MM Cell
Lines. To evaluate if liposomal nanoparticles were taken up by
MM cells, fluorescein labeled liposomes were prepared and
incubated with MM.1S and NCI-H929 cells for 24 h. The
intracellular endocytic vesicles were stained with LyosTracker
Red, the cells were fixed with 4% paraformaldehyde, and the
nuclei were stained with Hoechst dye, respectively. Confocal
images were collected along the z-axis. Our results demon-
strated that the liposomes were efficiently internalized by MM
cells (Figure 5). Moreover, the fluorescein signal colocalized
with the LysoTracker Red signal indicating that the liposomes
resided within endocytic vesicles. Control experiments
performed in the absence of liposomes did not exhibit any
fluorescein fluorescence.
Liposomal Bortezomib Nanoparticles Inhibit Tumor
Growth and Reduce Systemic Toxicity in Vivo. An
important property associated with nanoparticles is the
preferential accumulation in the tumor due to the EPR effect,
which reduces nonspecific toxicities associated with the free
drug. Thus, the cytotoxicity observed in vitro with the
liposomal bortezomib nanoparticles suggests the potential for
improved efficacy in vivo. To evaluate the in vivo therapeutic
potential of the liposomal bortezomib nanoparticles, CB-17
SCID mice were injected subcutaneously with NCI-H929 cells.
When the tumors reached a volume of 100 mm3, mice were
randomized into treatment groups and injected with PBS, free
bortezomib, NP[1c], or NP[4c] at a dose of 1 mg/kg
bortezomib equivalent concentration on days 1, 4, 8, and 11.
Mice were analyzed for tumor growth inhibition and systemic
toxic effects. Our results indicated that both bortezomib
nanoparticles NP[1c] and NP[4c] were very efficacious in
tumor growth inhibition (Figure 6a). In addition, both NP[1c]
and NP[4c] nanoparticles significantly improved the systemic
toxicity profiles. Both nanoparticles resulted in only <10% loss
in body mass during the 2 week study period, whereas the free
bortezomib group demonstrated >20% weight loss and
moribundity on day 7 and was consequently sacrificed due to
clinical decline (Figure 6b). This improved overall systemic
toxicity profile of the liposomal nanoparticles when compared
to the free drug is most likely due to the EPR effect and the
selective release at the tumor site. In a separate experiment, we
injected tumor bearing mice with 1 mg/kg bortezomib
equivalents of NP[1c], NP[4c], or free bortezomib on days 1
and 4 and sacrificed on day 5 for ex vivo analyses. Ex vivo
proteasome inhibition studies demonstrated that both NP[1c]
and NP[4c] demonstrated significant inhibition of proteasome
activity of the tumor (Figure 6c) and induced apoptosis via
caspase-3 activation (Figure 6d).
Figure 4. Liposomal bortezomib nanoparticles inhibit proteasome
activity and induce apoptosis in MM cells. (a) The effect of NP[1c]
and NP[4c] on proteasome inhibition was analyzed by using MM.1S
and NCI-H929 MM cells. Cells were cultured in the presence of the
25 nM bortezomib equivalent concentrations of NP[1c], NP[4c], and
free bortezomib for 1, 4, and 8 h. Proteasome activity was assessed
using 20S Proteasome Activity Assay Kit. (b) Apoptosis was assessed
by flow cytometry following Annexin-V staining at 12 h. (c)
Cytotoxicity of NP[1c], NP[4c],and free bortezomib was assessed at
48 by using Cell Counting Kit-8. All data represents means ( sd) of
triplicate cultures.
Counting Kit-8 reagent (Figure 4c). For MM.1S and NCI-
H929 cell lines, free bortezomib had IC50 values of ∼17 and 20
nM, respectively. The liposomal nanoparticles demonstrated
slightly reduced cytotoxic effects to both MM.1S and NCI-
H929 cells compared to free bortezomib, with NP[1c] having
IC50 values of 25 and 45 nM, respectively, while NP[4c] had
IC50 values of ∼20 and 37 nM, respectively. The minor
differences in the IC50 values between free bortezomib and the
nanoparticles could be attributed to the kinetics of bortezomib
release from the nanoparticles. In addition, the differences of
cellular uptake pathways can also contribute to the observed
differences. While free drugs enter the cells via passive diffusion,
nanoparticles are taken up by endocytosis, which can result in
kinetic delays in drug uptake. Taken together, these results
demonstrated that the liposomal bortezomib nanoparticles
Given the relatively faster hydrolysis rate observed for linkage
molecule 4 used to generate NP[4c] (Table 1), it is surprising
but not unexpected that NP[4c] also demonstrated improved
systemic toxicity. It is noteworthy that serum albumin has been
shown to stabilize ester bonds in different therapeutics in
E
dx.doi.org/10.1021/jm500352v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 5. Cellular uptake of fluorescein labeled liposomes in MM.1S and NCI-H929 MM cell lines. MM.1S and NCI-H929 cells were incubated with
fluorescein labeled liposomal nanoparticles for 24 h. Nuclei were labeled with Hoechst dye, and endosomes were labeled with LysoTracker Red.
Images were taken with a Nikon A1R confocal microscope with a 40× oil lens. Image acquisition was performed by Nikon Elements Ar software.
serum.41 This effect likely also plays a role in the boronic ester
bond stability. This suggests that despite the relatively rapid
hydrolysis when compared to 1c, 4c is stabilized long enough
to reach the tumor before releasing active drug. Taken together,
these results demonstrate improved efficacy and decreased
systemic toxicity for liposomal bortezomib nanoparticles
NP[1c] and NP[4c] when compared to free bortezomib.
controlled release application, IBBA was used only as a model
compound to rank the stabilities of the different boronic esters
and the absolute hydrolysis rates using bortezomib will likely be
slower due to its more complex structure. Thus, we chose both
1 and 4 as promising candidates for the synthesis of bortezomib
prodrugs for further evaluation. For nanoparticle preparation,
aliphatically modified linkage molecules 1b and 4b were
conjugated to bortezomib to create the boronic ester prodrugs
which were then incorporated into nanoparticles to generate
NP[1c] and NP[4c].
DISCUSSION AND CONCLUSION
■
In this study, we describe the synthesis, characterization, and
incorporation of bortezomib prodrug conjugates into liposomes
for optimal drug delivery of bortezomib to tumors and
improved therapeutic efficacy. The boronic acid moiety of
bortezomib plays a major role in the proteasome inhibitory
properties of the therapeutic by forming a complex with the
threonine residue in the chymotrypsin-like site of the 20S
proteasome. Boronic acids are known to form boronic esters
with diols or diol-like moieties which can be used to sequester
therapeutics containing boronic acids through the formation of
reversible boronic ester bonds.29,30 However, boronic esters are
susceptible to hydrolysis and their instability poses a significant
challenge in the synthesis of such prodrugs, particularly with
boronic esters involving alkyl boronic acids such as
bortezomib.29,31 Therefore, in our approach, by using IBBA
as a model compound, we first performed a thorough screen of
several different linkage molecules with various functional
groups to determine promising candidates that would yield
boronic ester bonds suitable for the synthesis of bortezomib
prodrugs based upon their relative stabilities. Among these,
linkage molecules 1 and 4 yielded the most stable bond with
IBBA with t1/2 of 190 and 10 min, respectively. It is noteworthy
that while the hydrolysis rate of 4 appears relatively fast for a
In our approach, we used PEGylated liposomes to prepare
bortezomib loaded nanoparticles because of the significant
advantages they provide including biocompatibility, particle size
control, high drug loading capacities, and facile incorporation of
different functionalities.23−25 In addition, liposomes have been
previously used in other therapeutic applications with
remarkable clinical success.27 In previous clinical applications
of liposomal drug delivery systems, therapeutics were
commonly encapsulated within the aqueous interior and
exhibited high drug-to-lipid ratios while maintaining particle
stability.27 In our design, given the hydrophobic nature of
bortezomib, we have loaded the bortezomib prodrugs onto the
liposomes via a surface conjugation strategy. Therefore, in our
liposomal nanoparticles, the drug loading of bortezomib
directly correlated to its surface density. While this approach
provided advantages such as controlled release and improved
solubility, it also posed a challenge for drug loading and particle
stability. Although high drug loading is desirable, the increased
bortezomib density could result in the reduction of nano-
particle stealth due to steric disruptions in PEG coating. To
prevent this problem, we used 5 mol % PEG2000 in our
liposome formulations as was previously determined to provide
F
dx.doi.org/10.1021/jm500352v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 6. In vivo characterization of liposomal bortezomib nanoparticles in a xenograft multiple myeloma model. Tumor bearing SCID mice were
injected, intravenously, on days 1, 4, 8, and 11, with 1 mg/kg bortezomib equivalent doses of NP[1c], NP[4c], and free bortemozib. (a) Tumor
growth inhibition was detected by caliper measurements. All of the mice in the free bortezomib group were sacrificed on day 7 due to high systemic
toxicity (>20% body mass). Data shown are means ( sd) of n = 6−8 mice per treatment group. (b) Percentage of body weight of the animals was
used as an indication of systemic toxicity. The free bortezomib group significantly lost body mass (>20%) and showed moribundity by day 7 and was
therefore sacrificed. Only <10% weight loss was observed with NP[1c] and NP[4c] during this study. (c) Ex vivo proteasome inhibition assay of
excised tumors. Four additional mice per group were injected on days 1 and 4 with a dose of 1 mg/kg bortezomib equivalent concentrations. Mice
were sacrificed 24 h after treatment, and tumors were analyzed for proteasome inhibition. (d) Ex vivo mechanistic analysis of the tumors for
apoptosis. Excised tumors were stained for activated caspase-3. Representative images of the tumor cross sections obtained using a Nikon Eclipse
TS100 microscope at ×20 magnification are shown.
optimal stealth and maintained the drug loading below 5 mol %
to avoid PEG crowding in order to maintain the stability of the
liposomes.42,43 Thus, a prodrug loading of 2.5 mol % was
selected for the preparation of liposomal bortezomib nano-
particles, which yielded stable liposomes with high reproduci-
bility and a narrow size range of 100 nm. Ongoing studies in
our lab are investigating methods to further increase the
nanoparticle drug loading without compromising particle
stability.
While this study focuses solely on the synthesis of
bortezomib prodrugs for nanoparticle incorporation, the
strategies developed within this report can also be applied to
other therapeutic or diagnostic molecules containing a boronic
acid moiety including serine protease inhibitors, boron neutron
capture therapeutic agents, and other proteasome inhibitors for
improved targeting and efficacy. Importantly, the strategies
described in this study for the incorporation of bortezomib into
nanoparticles are not limited to liposomes but can be applied to
other types of nanoparticles such as polymeric nanoparticles,
gold nanoparticles, dendrimers, or micelles. Hence, this
methodology establishes a platform that utilizes boronic ester
chemistry to facilitate the incorporation of boronic acid
containing molecules into various nanoparticle-based drug
delivery systems for improved diagnostic and therapeutic
outcomes.
In preclinical evaluation of the liposomal bortezomib
nanoparticles, both NP[1c] and NP[4c] demonstrated
significant proteasome inhibition, induced apoptosis, and
cytotoxicity in MM cells in vitro and dramatically reduced
the nonspecific toxicities associated with free bortezomib while
maintaining significant tumor growth inhibition in vivo. These
studies demonstrated that, despite being an effective chemo-
therapeutic, the therapeutic efficacy of bortezomib can be
further improved by utilizing the enhanced tumor accumulation
properties afforded by nanoparticle-based drug delivery systems
to reduce nonspecific toxicities. In an ongoing study in our lab,
we are developing quantitative analytical methods to accurately
determine bortezomib concentration in tissues in order to
perform a thorough in vivo biodistribution study with liposomal
bortezomib. In this study, we have shown that the improved
therapeutic index and reduced systemic toxicity profile
associated with the liposomal bortezomib nanoparticles
demonstrates their potential to overcome the adverse events
associated with bortezomib in the clinic such that a broader
patient population can benefit from this effective therapeutic.
Taken together, this study provides the preclinical rationale for
the clinical evaluation of liposomal bortezomib nanoparticles
for improved patient outcome in cancers.
G
dx.doi.org/10.1021/jm500352v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
vacuo and the intermediate was dissolved in 40 mL of carbon
tetrachloride. Then 2.225 g of N-bromosuccinimide (12.5 mmol) and
0.726 g of benzoyl peroxide (3 mmol) were added to the solution. The
solution was refluxed while stirring for 4 h. The brominated
intermediate was purified via flash chromatography using 2% methanol
in chloroform solution and concentrated by rotary evaporation to a
volume of 5 mL. To the concentrate solution, 2.4 g of hexadecylamine
(10 mmol) and 2.5 g of DIPEA (20 mmol) in 70 mL of MeOH were
added. The reaction proceeded under reflux for 24 h while stirring.
After the 24 h, the solvent was removed via rotary evaporation and the
solids were dissolved in chloroform. The intermediate product was
purified via flash chromatography using 3% methanol in chloroform
solution. The solution was concentrated by rotary evaporation then
diluted into 400 mL of 0.5 M NaOH. The solution was boiled for 2 h.
A white precipitate formed and the product was filtered, washed, and
EXPERIMENTAL SECTION
■
Reagents. The membranes (0.1 μm), mini-extruder, and all lipid
components were purchased from Avanti Polar Lipids, Inc. (Alabaster,
AL). Bortezomib was purchased from GenDepot (Barker, TX). All
other chemicals, including molecules 1, 3, 4, and 6, were purchased
from Sigma-Aldrich and were reagent grade or better (St. Louis, MO).
1
Purity of titled compounds was determined via either HPLC or H
NMR and were tested to be >95% pure. High resolution mass
spectrometry (HRMS) analysis was performed on a Bruker Q-TOF
system.
Synthesis of 2-((2-Hydroxyethyl)amino)acetic Acid [2]. First,
2 mmol of ethanolamine and 2 mmol of diisopropylethylamine
(DIPEA) were mixed in 30 mL of methanol in a flame-dried 200 mL
round-bottom flask. Then 2 mmol of methyl bromoacetate was added
dropwise over a period of 1 min into the solution while stirring. The
reaction proceeded under reflux overnight while stirring. The solution
was cooled to room temperature, and the solvent was removed under
vacuum. The solids were dissolved in 1.3 mL of EtOH and diluted into
6 mL of 1 M NaOH. The solution was heated to reflux for 1 h. The
pH of the solution was then adjusted to 7 with 10 M HCl and
evaporated to dryness. 1H NMR (400 MHz, D2O) δ 4.24 (s, 1H), 4.00
(s, 1H), 3.87 (s, 2H), 3.81 (t, 2H), 3.13 (t, 2H). ESI HRMS for
C4H9NO3: calculated m/z 120.0655 [M + H]+; found 120.0691.
Synthesis of N,2-Dihydroxy-4-methylbenzamide [5]. First,
252.5 mg of 4-methylsalicylic acid (1.66 mmol) was dissolved with 20
mL of MeOH in a 50 mL round-bottom flask. Then 0.5 mL of 18 M
sulfuric acid was added while stirring. The reaction proceeded under
reflux for 24 h while stirring. The reaction was quenched by adding
100 mL of water to the solution. The intermediate was extracted with
ethyl ether (3 × 30 mL). The organic phases were combined and
washed with a saturated sodium bicarbonate solution (2 × 100 mL).
The ether was evaporated in vacuo, and the intermediate was dissolved
in 0.5 mL of THF. The intermediate solution was added dropwise to a
NaOH/NH2OH solution (6.72 mL of 1.64 M NH2OH in water was
added to 8.38 mL of 3 M NaOH) while stirring. The reaction was
allowed to proceed at room temperature for 24 h. After 24 h, the
reaction was cooled to 0 °C using an ice bath, and the pH was adjusted
to 5 with 10 M HCl. The solution was allowed to warm to room
temperature before extracting the product with ethyl acetate (3 × 15
mL). The organic layers were combined, and the solvent was
evaporated. 1H NMR (400 MHz, DMSO-d6) δ12.30 (s, 1H), 11.39 (s,
1H), 9.28 (s, 1H), 7.56 (d, J = 8.28 Hz, 1H), 6.71 (s, 1H), 6.67 (d, J =
7.89 Hz, 1H), 2.54 (s, 1H), 2.25 (s, 3H). ESI HRMS for C8H9NO3:
calculated m/z 168.0655 [M + H]+; found 168.0711.
Synthesis of 2,2′-(Hexadecylazanediyl)diacetic Acid [1b].
First, 1.81 g of hexadecylamine (7.5 mmol) was dissolved in 75 mL of
MeOH in a flame-dried 250 mL round-bottom flask. Then 3.92 mL of
DIPEA (30 mmol) and 2.29 g of methyl bromoacetate (15 mmol)
were added to the flask while stirring. The reaction proceeded under
reflux for 96 h while stirring. Next, the solvent was removed under
vacuum and the solids were dissolved in chloroform. The intermediate
product was purified via flash chromatography using 5% methanol in
chloroform solution. The solution was concentrated by rotary
evaporation then diluted into 400 mL of 0.5 M NaOH. The solution
was refluxed until it became clear (∼2 h). The solution was then
cooled to room temperature and the pH adjusted to 2 using 10 M HCl
causing a white precipitate to form. The product was filtered, washed,
and dried in vacuo overnight. 1H NMR (500 MHz, DMSO-d6) δ 3.41
(s, 4H), 2.62 (t, 2H), 1.23 (s, 28H), 0.85 (t, 3H). ESI HRMS for
C20H39NO4: calculated m/z 358.2952 [M + H]+; found 358.2899.
Synthesis of 4-((Hexadecylamino)methyl)-2-hydroxybenzoic
Acid [4b]. First, 1.521 g of methyl salicylic acid (10 mmol) was
dissolved in 50 mL of MeOH in a 200 mL round-bottom flask. Then 3
mL of sulfuric acid was added to the solution. The flask was connected
to a condenser (T = 5 °C) and heated to reflux for 24 h. The reaction
was quenched by adding 100 mL of water to the solution. The
intermediate was extracted with ethyl ether (3 × 50 mL). The organic
phases were combined and washed with a saturated sodium
bicarbonate solution (2 × 100 mL). The ether was evaporated in
1
dried in vacuo overnight. H NMR (400 MHz, CDCl3) δ 7.42−7.81
(m, 1H), 6.57−7.01 (m, 2H), 3.67−4.16 (m, 2H), 2.76−3.20 (m, 2H),
1.53−1.88 (m, 2H), 0.97−1.44 (m, 26H), 0.87 (t, J = 6.69 Hz, 3H).
ESI HRMS for C24H41NO3: calculated m/z 392.3159 [M + H]+; found
392.3116.
Synthesis of the Boronic Acid Conjugates [1a−6a, 1c, and
4c]. The linkage molecule and the boronic acid (IBBA or bortezomib)
at a molar ratio of 1:1 were placed in a flame-dried 25 mL flask with 7
mL of toluene. The solution was allowed to reflux for 2 h while stirring
before being removed from the heat. The solvent was then evaporated
1
in vacuo. Conjugation was verified using H NMR and 11B NMR
spectroscopy to observe the peak shift of the boron from ∼32 ppm
(boronic acid) to ∼9 ppm (boronic ester) via Bruker AVANCE III
HD 400 MHz spectrometer (Bruker, Billerica, MA).
2-Isobutyl-1,3,6,2-dioxazaborocane-4,8-dione (1a). 1H NMR
(400 MHz, DMSO-d6) δ 3.90−4.09 (m, 4H), 1.63−1.74 (m, 1H),
0.90 (d, J = 6.69 Hz, 6H), 0.45 (d, J = 7.08 Hz, 2H). ESI HRMS m/z
calculated for C8H14BNO4: calculated m/z 200.1090 [M + H]+, found
200.1077.
N-((S)-1-(((R)-1-(6-Hexadecyl-4,8-dioxo-1,3,6,2-dioxazaborocan-
2-yl)-3-methylbutyl)amino)-1-oxo-3-phenylpropan-2-yl)pyrazine-2-
carboxamide (1c). 1H NMR (500 MHz, DMSO-d6) δ 9.05−9.08 (m,
1H), 8.85−8.92 (m, 3H), 8.71−8.75 (m, 1H), 7.21−7.29 (m, 5H),
4.66−4.69 (m, 1H), 3.36 (s, 4H), 3.06−3.08 (m, 2H), 2.63 (t, 1H),
1.41−1.43 (m, 1H), 1.39−1.41 (m, 1H), 1.36−1.38 (m, 1H), 1.16−
1.32 (m, 28H), 0.82−0.82 (m, 0H), 0.84 (t, 6H), 0.81 (t, 3H). ESI
HRMS for C39H60BN5O6: calculated m/z 706.4735 [M + H]+; found
706.4709.
2-Isobutyl-1,3,6,2-dioxazaborocan-4-one (2a). 1H NMR (400
MHz, DMSO-d6) δ 3.86 (s, 1H), 3.61−3.69 (m, 2H), 3.55 (t, J = 5.31
Hz, 2H), 2.80 (t, 2H) δ 1.74 (m, 1H), 0.78 (d, 6H), 0.48 (d, 2H). ESI
HRMS for C8H16BNO3: calculated m/z 186.1298 [M + H]+; found
186.1300.
2-Isobutyl-1,3,6,2-dioxazaborocane (3a). 1H NMR (400 MHz,
DMSO-d6) δ 6.44 (s, 1H), 3.62−3.72 (m, 2H), 3.55 (d, J = 3.54 Hz,
2H), 2.89−3.01 (m, 2H), 2.60−2.70 (m, 2H), 1.50−1.66 (m, 1H),
0.83 (d, J = 6.29 Hz, 6H), 0.18 (d, J = 6.69 Hz, 2H). ESI HRMS for
C8H18BNO2: calculated m/z 172.1503 [M + H]+; found 172.1500.
2-Isobutyl-7-methyl-4H-benzo[d][1,3,2]dioxaborinin-4-one (4a).
1H NMR (500 MHz, DMSO-d6) δ 7.59 (d, 1H), 6.64−6.71 (m,
2H), 2.26 (s, 3H), 1.57−1.71 (m, 1H), 0.85 (d, 6H), 0.38 (d, 4H). ESI
HRMS for C12H15BO3: calculated m/z 219.1187 [M + H]+; found
219.1179.
N-((S)-1-(((R)-1-(7-((Hexadecylamino)methyl)-4-oxo-4H-benzo-
[d][1,3,2]dioxaborinin-2-yl)-3-methylbutyl)amino)-1-oxo-3-phenyl-
propan-2-yl)pyrazine-2-carboxamide (4c). 1H NMR (400 MHz,
DMSO-d6) 9.09−9.14 (m, 1H), 8.86−8.93 (m, 2H), 8.71−8.78 (m,
2H), 7.73−7.87 (m, 5H), δ 7.19−7.32 (m, 1H), 6.84−7.10 (m, 2H),
5.00−5.14 (m, 2H), 4.06−4.21 (m, 2H), 3.12−3.18 (m, 2H), 2.85−
2.94 (m, 2H), 2.53−2.55 (m, 1H), 1.45−1.65 (m, 2H), 1.39 (none,
2H), 1.23 (s, 26H), 0.78−0.88 (m, 9H). ESI HRMS for C43H62BN5O5:
calculated m/z 740.4917 [M + H]+; found 740.4927.
3-Hydroxy-2-isobutyl-7-methyl-2,3-dihydro-4H-benzo[e][1,3,2]-
1
oxazaborinin-4-one (5a). H NMR (300 MHz, DMSO-d6) δ 11.38
(s, 1H), 7.56 (d, J = 8.01 Hz, 1H), 6.71 (s, 1H), 6.67 (d, 1H), 2.25 (s,
H
dx.doi.org/10.1021/jm500352v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
3H), 1.69−1.86 (m, 1H), 0.85 (d, J = 6.63 Hz, 6H), 0.53 (d, J = 7.46
Hz, 2H). ESI HRMS for C12H16BNO3: calculated m/z 234.1299 [M +
H]+; found 234.1335.
Gold Antifade (Molecular Probes). Cells were visualized with a Nikon
A1R confocal microscope using a 40× oil lens (Nikon Instruments,
Melville, NY). Image acquisition was performed by Nikon Elements Ar
software (Nikon).
4-(Carboxymethyl)-2-isobutyl-6-oxo-1,3,2-dioxaborinane-4-car-
1
boxylic Acid (6a). H NMR (400 MHz, DMSO-d6) δ 2.57−2.65 (m,
MM Xenograft Mouse Model. CB-17 SCID mice (Harlan
Laboratories, Indianapolis, IN) were irradiated with 150 rad and were
inoculated subcutaneously with 5 × 106 NCI-H929 cells. When
tumors reached a volume of 100 mm3, mice were distributed into four
groups of 6−8 mice and were treated intravenously with NP[1c],
NP[4c], free bortezomib, or vehicle (PBS), on days 1, 4, 8, and 11 at a
dose of 1 mg/kg bortezomib equivalents. Animals were monitored for
body weight and tumor volume. Tumor volume was measured via
calipers (volume = 0.5 × length × (width)2). Mice were treated
humanely and in accordance with the protocol approved by the
Institutional Animal Care and Use Committee (IACUC) at the
Freimann Life Science Center (Notre Dame, IN).
Ex Vivo Proteasome Inhibition and Apoptosis Assays. CB-17
SCID mice were irradiated with 150 rad and were inoculated
subcutaneously with 5 × 106 NCI-H929 cells. When tumors were
palpable, mice were distributed into four groups of 4 mice and were
treated intravenously with NP[1c], NP[4c], free bortezomib, or PBS,
on days 1 and 4 at a dose of 1 mg/kg bortezomib equivalents. The
mice were sacrificed on day five, and the tumors were excised and
divided in half. For detection of apoptosis, one-half was fixed in
formalin solution and immunohistochemical staining for caspase-3 was
performed as previously described.19 The other half was used for the
proteasome inhibition assay. Tumors were homogenized in 0.2 mL of
lysis buffer (100 mM Tris, pH 7.8, 150 mM NaCl, 1% Triton-X100)
per 50 mg tissue. The homogenate was centrifuged at 18400g for 10
min at 4 °C. The supernatant was removed and centrifuged again to
ensure complete removal of any precipitate. The protein concentration
of each sample was determined using Bradford assay, and samples
were diluted to a protein concentration of 15 μg/mL. 20S Proteasome
Activity Assay Kit (EMD Millipore) was used to measure proteasome
activity. Then 10 μL of the respective homogenate, along with 10 μL
of proteasome substrate (Suc-LLVY-AMC) and 80 μL of the assay
buffer (25 mM HEPES, pH 7.5, 0.5 mM EDTA, 0.05% NP-40, and
0.001% SDS (w/v)), was placed into a 96 well plate and incubated at
37 °C for 1 h. Proteasome activity was assessed via fluorescence
spectroscopy (ex 380 nm/em 460 nm).
4H), 1.58−1.72 (m, 1H), 0.84 (d, J = 6.69 Hz, 6H), 0.37 (d, J = 6.69
Hz, 2H). ESI HRMS for C10H15BO7: calculated m/z 257.0829 [M −
H]−; found 257.0821.
Hydrolysis of Boronic Esters. 0.2 mmol of the boronic ester
conjugates, 1a−6a, were dissolved in 0.4 mL of DMSO-d6 and divided
into two 0.2 mL vials. One solution was diluted with 0.2 mL of PBS,
while the other was diluted with 0.2 mL of DMSO-d6 as a control. 11
B
spectra were obtained at t = 0, 5, 10, and 30 min for each solution at
128 MHz using the Bruker AVANCE III HD 400 MHz spectrometer
equipped with a standard 5 mm broadband probe (Bruker, Billerica,
MA). The following experimental parameters were used: acquisition
time 1.42 s, relaxation delay 2.0 s, 256 scans. Hydrolysis of the boronic
ester was measured by observing the reduction of the peak at ∼12
ppm.
Liposome Preparation. Liposomes were prepared by dry film
hydration as described previously.44 Briefly, lipids were mixed at the
indicated ratio in chloroform, dried to form a thin film using nitrogen
gas, and then placed under vacuum overnight to remove residual
solvent. The lipid films were hydrated at 65 °C in PBS pH 7.4 with
gently agitated and extruded at 65 °C through a 0.1 μm polycarbonate
filter. Liposomes all adhered to the following formula 92.5:5:2.5
DSPC:mPEG2000:Prodrug.
Particle Sizing. Dynamic light scattering analysis was performed
using the 90Plus nanoparticle size analyzer (Brookhaven Instruments,
Holtsville, NY) as previously described.19
Cell Culture. MM.1S and NCI-H929 cell lines were obtained from
American Type Culture Collection (Rockville, MD). Both cell lines
were supplemented with 10% fetal bovine serum (FBS), 2 mM L-
glutamine (Gibco, Carlsbad, CA), 100 μg/mL penicillin, and 100 μg/
mL streptomycin (Gibco). NCI-H929 cells were further supplemented
with 55 μM 2-mercaptoethanol.
Cytotoxicity Assays. Cell Counting Kit-8 (Dojindo Molecular
Technologies, Rockville, MD) was used as previously described.19
In Vitro Proteasome Inhibition Assays. 20S Proteasome
Activity Assay Kit (EMD Millipore, Billerica, MA) was used to
measure proteasome activity. Briefly, 6 × 105 cells/well were plated in
a 6 well dish. NP[1c], NP[4c], or free bortezomib were added at 25
nM bortezomib equivalent concentrations to their respective wells and
incubated for 1, 4, or 8 h at 37 °C. Cells were washed twice with cold
PBS and lysed in 60 μL of lysis buffer (50 mM HEPES, 5 mM EDTA,
150 mM NaCl, 1% Triton X-100, pH 7.5) on ice for 30 min by
periodic vortexing. The lysate was centrifuged at 21130g for 15 min at
4 °C. Then 10 μL of the lysate, along with 10 μL of proteasome
substrate (Suc-LLVY-AMC) and 80 μL of the assay buffer (25 mM
HEPES, pH 7.5, 0.5 mM EDTA, 0.05% NP-40, and 0.001% SDS (w/
v)), was placed into a 96 well plate and incubated at 37 °C for 1 h.
Proteasome activity was assessed via fluorescence spectroscopy (ex
380 nm/em 460 nm).
ASSOCIATED CONTENT
■
S
* Supporting Information
Representative 11B-NMR spectra and HLPC chromatograms of
loading efficiency for the prodrugs. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Authors
*For T.K.: phone, 574-631-1603; E-mail, tkiziltepe@nd.edu.
*For B.B.: phone, 574-631-1429; E-mail, bbilgicer@nd.edu.
Flow Cytometry. Apoptotic cells were detected with Annexin-V
(FITC) antibody (BD Pharmigen, Sandiego, CA). Cells were analyzed
with Guava EasyCyte flow cytometer (EMD Millipore) as previously
described.19
Notes
The authors declare no competing financial interest.
Confocal Microscopy. Confocal experiments were performes as
previously described.45 Briefly, 1 × 105 cells/well were plated 24 h
prior to each experiment in a 24 well dish. Liposomes were added at
100 μM phospholipid concentration and incubated for 24 h at 37 °C.
1% DOPE-CF was added as a fluorescent marker to each liposomal
formulation. Cells were washed 3 times with PBS and incubated in 50
nM LysoTracker Red (Molecular Probes, Carlsbad, CA) in culture
media for 30 min at 37 °C to allow internalization. After 30 min, cells
were washed 3 times with PBS and spun onto slides, using a Cytospin
(Thermo Fisher Scientific, Waltham, MA) before being fixed with 4%
paraformaldehyde. Slides were rinsed with PBS then incubated with 2
μg/mL Hoechst dye (Sigma-Aldrich) in PBS for 15 min to stain the
nucleus. Coverslips were mounted on microscope slides with Prolong
ACKNOWLEDGMENTS
■
We thank the Notre Dame Integrated Imaging Facility for
confocal microscopy, the Center for Environmental Science
and Technology for the use of DLS, the Mass Spectrometry
and Proteomics Facility for the use of their mass spectrometers,
and the Magnetic Resonance Research Center for the use of the
Bruker 400 MHz spectrometer. We thank Deborah Donahue
and the W. M. Keck Center for the irradiation of the mice. We
thank Sarah Chapman for preparing the histology sections. This
work was supported by the Hollis Brownstein Research
Program Grant from Leukemia Research Foundation.
I
dx.doi.org/10.1021/jm500352v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
as risk factors to develop severe neuropathy in a mouse model. J.
Peripher. Nerv. Syst. 2011, 16, 199−212.
ABBREVIATIONS USED
■
DIPEA, diisopropylethylamine; DOPE-CF, 1,2-dioleoyl-sn-
glycero-3-phosphoethanolamine-N-(carboxyfluorescein);
DSPC, 1,2-distearoyl-sn-glycero-3-phosphocholine; EPR, en-
hanced permeability and retention; IBBA, isobutylboronic acid;
mPEG2000, 1,2-dipalmitoyl-sn-glycero-3-phosphoethanol-
amine-N-[methoxy(poly(ethylene glycol))-2000]; MM, multi-
ple myleoma; PBA, phenylboronic acid
(17) Maeda, H.; Wu, J.; Sawa, T.; Matsumura, Y.; Hori, K. Tumor
vascular permeability and the EPR effect in macromolecular
therapeutics: a review. J. Controlled Release 2000, 65, 271−284.
(18) Patricia Egusquiaguirre, S.; Igartua, M.; Maria Hernandez, R.;
Luis Pedraz, J. Nanoparticle delivery systems for cancer therapy:
advances in clinical and preclinical research. Clin. Transl. Oncol. 2012,
14, 83−93.
(19) Kiziltepe, T.; Ashley, J. D.; Stefanick, J. F.; Qi, Y. M.; Alves, N.
J.; Handlogten, M. W.; Suckow, M. A.; Navari, R. M.; Bilgicer, B.
Rationally engineered nanoparticles target multiple myeloma cells,
overcome cell-adhesion-mediated drug resistance, and show enhanced
efficacy in vivo. Blood Cancer J. 2012, 2, e64.
(20) Allen, T. M.; Cullis, P. R. Liposomal drug delivery systems: from
concept to clinical applications. Adv. Drug Delivery Rev. 2013, 65, 36−
48.
(21) Paraskar, A. S.; Soni, S.; Chin, K. T.; Chaudhuri, P.; Muto, K.
W.; Berkowitz, J.; Handlogten, M. W.; Alves, N. J.; Bilgicer, B.;
Dinulescu, D. M.; Mashelkar, R. A.; Sengupta, S. Harnessing
structure−activity relationship to engineer a cisplatin nanoparticle
for enhanced antitumor efficacy. Proc. Natl. Acad. Sci. U. S. A. 2010,
107, 12435−12440.
(22) Boulikas, T. Low toxicity and anticancer activity of a novel
liposomal cisplatin (Lipoplatin) in mouse xenografts. Oncol. Rep. 2004,
12, 3−12.
REFERENCES
■
(1) Genin, E.; Reboud-Ravaux, M.; Vidal, J. Proteasome inhibitors:
recent advances and new perspectives in medicinal chemistry. Curr.
Top. Med. Chem. 2010, 10, 232−256.
(2) Chauhan, D.; Hideshima, T.; Anderson, K. C. Proteasome
inhibition in multiple myeloma: therapeutic implication. Annu. Rev.
Pharmacol. Toxicol. 2005, 45, 465−476.
(3) Chauhan, D.; Hideshima, T.; Mitsiades, C.; Richardson, P.;
Anderson, K. Proteasome inhibitor therapy in multiple myeloma. Mol.
Cancer Ther. 2005, 4, 686−692.
(4) Almond, J. B.; Cohen, G. M. The proteasome: a novel target for
cancer chemotherapy. Leukemia 2002, 16, 433−443.
(5) Orlowski, R. Z.; Kuhn, D. J. Proteasome inhibitors in cancer
therapy: lessons from the first decade. Clin. Cancer Res. 2008, 14,
1649−1657.
(23) Gabizon, A.; Catane, R.; Uziely, B.; Kaufman, B.; Safra, T.;
Cohen, R.; Martin, F.; Huang, A.; Barenholz, Y. Prolonged circulation
time and enhanced accumulation in malignant exudates of doxorubicin
encapsulated in polyethylene-glycol coated liposomes. Cancer Res.
1994, 54, 987−992.
(6) Hideshima, T.; Mitsiades, C.; Akiyama, M.; Hayashi, T.;
Chauhan, D.; Richardson, P.; Schlossman, R.; Podar, K.; Munshi, N.
C.; Mitsiades, N.; Anderson, K. C. Molecular mechanisms mediating
antimyeloma activity of proteasome inhibitor PS-341. Blood 2003, 101,
1530−1534.
(7) Mitsiades, N.; Mitsiades, C. S.; Poulaki, V.; Chauhan, D.;
Fanourakis, G.; Gu, X. S.; Bailey, C.; Joseph, M.; Libermann, T. A.;
Treon, S. P.; Munshi, N. C.; Richardson, P. G.; Hideshima, T.;
Anderson, K. C. Molecular sequelae of proteasome inhibition in
human multiple myeloma cells. Proc. Natl. Acad. Sci. U. S. A. 2002, 99,
14374−14379.
(8) Hideshima, T.; Richardson, P.; Chauhan, D.; Palombella, V.;
Elliott, P.; Adams, J.; Anderson, K. The proteasome inhibitor PS-341
inhibits growth, induces apoptosis, and overcomes drug resistance in
human multiple myeloma cells. Cancer Res. 2001, 61, 3071−3076.
(9) Rajkumar, S.; Richardson, P.; Hideshima, T.; Anderson, K.
Proteasome inhibition as a novel therapeutic target in human cancer. J.
Clin. Oncol. 2005, 23, 630−639.
(24) Allen, T. M.; Cheng, W. W. K.; Hare, J. I.; Laginha, K. M.
Pharmacokinetics and pharmacodynamics of lipidic nano-particles in
cancer. Anti-Cancer Agents Med. Chem. 2006, 6, 513−523.
(25) Allen, T. Liposomal drug formulationsrationale for develop-
ment and what we can expect for the future. Drugs 1998, 56, 747−756.
(26) Barenholz, Y. DoxilThe first FDA-approved nano-drug:
lessons learned. J. Controlled Release 2012, 160, 117−134.
(27) Chang, H.; Yeh, M. Clinical development of liposome-based
drugs: formulation, characterization, and therapeutic efficacy. Int. J.
Nanomed. 2012, 7, 49−60.
(28) Trippier, P. C.; McGuigan, C. Boronic acids in medicinal
chemistry: anticancer, antibacterial and antiviral applications. Med-
ChemComm 2010, 1, 183−198.
(29) Hall, D. G. Structure, properties, and preparation of boronic
acid derivatives overview of their reactions and applications, in boronic
acids: preparation and applications in organic synthesis and medicine.
In Boronic Acids Preparations and Applications in Organic Synthesis and
Medicine; Hall, D. G., Ed.; Wiley-VCH Verlag GmbH & Co. KGaA:
Weinheim, 2006; pp 1−99.
(30) Roy, C. D.; Brown, H. C. Stability of boronic estersstructural
effects on the relative rates of transesterification of 2-(phenyl)-1,3,2-
dioxaborolane. J. Organomet. Chem. 2007, 692, 784−790.
(31) Roy, C. D.; Brown, H. C. A comparative study of the relative
stability of representative chiral and achiral boronic esters employing
transesterification. Monatsh. Chem. 2007, 138, 879−887.
(32) Roush, W. R.; Walts, A. E.; Hoong, L. K. Diastereoselective and
enantioselective aldehyde addition-reactions of 2-allyl-1,3,2-dioxabor-
olane-4,5-dicarboxylic esters, a useful class of tartrate ester modified
allylboronates. J. Am. Chem. Soc. 1985, 107, 8186−8190.
(33) Bernardini, R.; Oliva, A.; Paganelli, A.; Menta, E.; Grugni, M.;
De Munari, S.; Goldoni, L. Stability of boronic esters to hydrolysis: a
comparative study. Chem. Lett. 2009, 38, 750−751.
(10) Mitsiades, N.; Mitsiades, C.; Poulaki, V.; Chauhan, D.;
Fanourakis, G.; Gu, X.; Bailey, C.; Joseph, M.; Libermann, T.;
Treon, S.; Munshi, N.; Richardson, P.; Hideshima, T.; Anderson, K.
Molecular sequelae of proteasome inhibition in human multiple
myeloma cells. Proc. Natl. Acad. Sci. U. S. A. 2002, 99, 14374−14379.
(11) Richardson, P. G.; Hideshima, T.; Anderson, K. C. Bortezomib
(PS-341): a novel, first-in-class proteasome inhibitor for the treatment
of multiple myeloma and other cancers. Cancer Control 2003, 10, 361−
369.
(12) Dick, L. R.; Fleming, P. E. Building on bortezomib: second-
generation proteasome inhibitors as anti-cancer therapy. Drug
Discovery Today 2010, 15, 243−249.
(13) Arastu-Kapur, S.; Anderl, J. L.; Kraus, M.; Parlati, F.; Shenk, K.
D.; Lee, S. J.; Muchamuel, T.; Bennett, M. K.; Driessen, C.; Ball, A. J.;
Kirk, C. J. Nonproteasomal targets of the proteasome inhibitors
bortezomib and carfilzomib: a link to clinical adverse events. Clin.
Cancer Res. 2011, 17, 2734−2743.
(14) Chen, D.; Frezza, M.; Schmitt, S.; Kanwar, J.; Dou, Q. P.
Bortezomib as the first proteasome inhibitor anticancer drug: current
status and future perspectives. Curr. Cancer Drug Targets 2011, 11,
239−253.
(15) Curran, M. P.; McKeage, K. Bortezomib a review of its use in
patients with multiple myeloma. Drugs 2009, 69, 859−888.
(16) Bruna, J.; Ale, A.; Velasco, R.; Jaramillo, J.; Navarro, X.; Udina,
E. Evaluation of pre-existing neuropathy and bortezomib retreatment
(34) Yan, J.; Springsteen, G.; Deeter, S.; Skeuse, C.; Wang, B. H. The
relationship among pKa, pH, and binding constants in the interactions
between boronic acids and diolsit is not as simple as it appears.
Tetrahedron 2004, 60, 11205−11209.
(35) Aznar, E.; Dolores Marcos, M.; Martinez-Manez, R.; Sancenon,
F.; Soto, J.; Amoros, P.; Guillem, C. pH- and photo-switched release of
J
dx.doi.org/10.1021/jm500352v | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
guest molecules from mesoporous silica supports. J. Am. Chem. Soc.
2009, 131, 6833−6843.
(36) Springsteen, G.; Wang, B. H. A detailed examination of boronic
acid-diol complexation. Tetrahedron 2002, 58, 5291−5300.
(37) Gillis, E. P.; Burke, M. D. A simple and modular strategy for
small molecule synthesis: iterative Suzuki−Miyaura coupling of B-
protected haloboronic acid building blocks. J. Am. Chem. Soc. 2007,
129, 6716−+.
(38) Knapp, D. M.; Gillis, E. P.; Burke, M. D. A general solution for
unstable boronic acids: slow-release cross-coupling from air-stable
MIDA boronates. J. Am. Chem. Soc. 2009, 131, 6961−+.
(39) Stolowitz, M. L.; Ahlem, C.; Hughes, K. A.; Kaiser, R. J.; Kesicki,
E. A.; Li, G. S.; Lund, K. P.; Torkelson, S. M.; Wiley, J. P.
Phenylboronic acid−salicylhydroxamic acid biconjugates. 1. A novel
boronic acid complex for protein immobilization. Bioconjugate Chem.
2001, 12, 229−239.
(40) Wiley, J. P.; Hughes, K. A.; Kaiser, R. J.; Kesicki, E. A.; Lund, K.
P.; Stolowitz, M. L. Phenylboronic acid−salicylhydroxamic acid
bioconjugates. 2. Polyvalent immobilization of protein ligands for
affinity chromatography. Bioconjugate Chem. 2001, 12, 240−250.
(41) Greige-Gerges, H.; Diab, Y.; Farah, J.; Magdalou, J.; Haddad, C.;
Ouaini, N. Ferutinin stability in human plasma and interaction with
human serum albumin. Biopharm. Drug Dispos. 2008, 29, 83−89.
(42) Tirosh, O.; Barenholz, Y.; Katzhendler, J.; Priev, A. Hydration of
polyethylene glycol-grafted liposomes. Biophys. J. 1998, 74, 1371−
1379.
(43) Barenholz, Y. Liposome application: problems and prospects.
Curr. Opin. Colloid Interface Sci. 2001, 6, 66−77.
(44) Stefanick, J. F.; Ashley, J. D.; Kiziltepe, T.; Bilgicer, B. A
systematic analysis of peptide linker length and liposomal polyethylene
glycol coating on cellular uptake of peptide-targeted liposomes. ACS
Nano 2013, 7 (4), 2935−2947.
(45) Stefanick, J. F.; Ashley, J. D.; Bilgicer, B. Enhanced cellular
uptake of peptide-targeted nanoparticles through increased peptide
hydrophilicity and optimized ethylene glycol peptide-linker length.
ACS Nano 2013, 7 (9), 8115−8127.
K
dx.doi.org/10.1021/jm500352v | J. Med. Chem. XXXX, XXX, XXX−XXX