Synthetic enzyme inhibitor: a novel targeting ligand for nanotherapeutic drug delivery inhibiting tumor growth without systemic toxicity.
ABSTRACT Unresolved problems associated with ligand-targeting of liposomal nanoparticles (NPs) to solid tumors include variable target receptor expression due to genetic heterogeneity and insufficient target specificity, leading to systemic toxicities. This study addresses these issues by developing a novel ligand-targeting strategy for liposomal NPs using RR-11a, a synthetic enzyme inhibitor of Legumain, an asparaginyl endopeptidase. Cell-surface expression of Legumain is driven by hypoxic stress, a hallmark of solid tumors. Legumain-targeted RR-11a-coupled NPs revealed high ligand-receptor affinity, enhanced solid-tumor penetration and uptake by tumor cells. Treatment of tumor-bearing mice with RR-11a-coupled NPs encapsulating doxorubicin resulted in improved tumor selectivity and drug sensitivity, leading to complete inhibition of tumor growth. These antitumor effects were achieved while eliminating systemic drug toxicity. Therefore, synthetic enzyme inhibitors, such as RR-11a, represent a new class of compounds that can be used for highly specific ligand-targeting of NPs to solid tumors. FROM THE CLINICAL EDITOR: This study addresses the problems associated with ligand-targeting of liposomal nanoparticles to solid tumors with variable target receptor expression. A novel and efficacious targeting strategy has been developed towards a synthetic enzyme inhibitor of Legumain. The authors demonstrate successful tumor growth inhibiting effect while eliminating systemic drug toxicity in an animal model using this strategy.
- SourceAvailable from: Richard D Kennedy[Show abstract] [Hide abstract]
ABSTRACT: TBX2 is an oncogenic transcription factor known to drive breast cancer proliferation. We have identified the cysteine protease inhibitor Cystatin 6 (CST6) as a consistently repressed TBX2 target gene, co-repressed through a mechanism involving Early Growth Response 1 (EGR1). Exogenous expression of CST6 in TBX2-expressing breast cancer cells resulted in significant apoptosis whilst non-tumorigenic breast cells remained unaffected. CST6 is an important tumor suppressor in multiple tissues, acting as a dual protease inhibitor of both papain-like cathepsins and asparaginyl endopeptidases (AEPs) such as Legumain (LGMN). Mutation of the CST6 LGMN-inhibitory domain completely abrogated its ability to induce apoptosis in TBX2-expressing breast cancer cells, whilst mutation of the cathepsin-inhibitory domain or treatment with a pan-cathepsin inhibitor had no effect, suggesting that LGMN is the key oncogenic driver enzyme. LGMN activity assays confirmed the observed growth inhibitory effects were consistent with CST6 inhibition of LGMN. Knockdown of LGMN and the only other known AEP enzyme (GPI8) by siRNA confirmed that LGMN was the enzyme responsible for maintaining breast cancer proliferation. CST6 did not require secretion or glycosylation to elicit its cell killing effects, suggesting an intracellular mode of action. Finally, we show that TBX2 and CST6 displayed reciprocal expression in a cohort of primary breast cancers with increased TBX2 expression associating with increased metastases. We have also noted that tumors with altered TBX2/CST6 expression show poor overall survival. This novel TBX2-CST6-LGMN signaling pathway, therefore, represents an exciting opportunity for the development of novel therapies to target TBX2 driven breast cancers.Oncotarget 02/2014; · 6.63 Impact Factor
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ABSTRACT: Specific targeting and cellular internalization are key properties for carriers of antitumor therapeutic agents. Here, we develop a drug carrier through the attachment of substrate of endoprotease legumain, alanine-alanine-asparagine (AAN), to cell-penetrating peptides (TAT, trans-activating factor). The addition of the AAN moiety to the fourth lysine in the TAT creates a branched peptide moiety, which leads to a decrease in the transmembrane transport capacity of TAT by 72.65%. Legumain efficiently catalyses the release of TAT-liposome from the AAN-TAT-liposome and thereby recovers the penetrating capacity of TAT. Doxorubicin carried by the AAN-TAT-liposome led to an increase in the tumoricidal effect of doxorubicin and a reduction in its systemic adverse effects in comparison with doxorubicin carried by a control delivery system. Thus, the specific targeting and high efficiency of this delivery platform offers a novel approach to limit the toxicity of anticancer agents as well as increasing their efficacy in cancer therapy.Nature Communications 06/2014; 5:4280. · 10.74 Impact Factor
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ABSTRACT: MicroRNAs (miRs) are small single-stranded RNA molecules, which function as key negative regulators of post-transcriptional modulation in almost all biological processes. Abnormal expression of microRNAs has been observed in various types of cancer including breast cancer. Great efforts have been made to identify an association between microRNA expression profiles and breast cancer, and to understand the functional role and molecular mechanism of aberrant-expressed microRNAs. As research progressed, 'oncogenic microRNAs' and 'tumor suppressive microRNAs' became a focus of interest. The potential of candidate microRNAs from both intercellular (tissue) and extracellular (serum) sources for clinical diagnosis and prognosis was revealed, and treatments involving microRNA achieved some amazing curative effects in cancer disease models. In this review, advances from the most recent studies of microRNAs in one of the most common cancers, breast cancer, are highlighted, especially the functions of specifically selected microRNAs. We also assess the potential value of these microRNAs as diagnostic and prognostic markers, and discuss the possible development of microRNA-based therapies.Journal of Zhejiang University SCIENCE B 01/2015; 16(1):18-31. · 1.29 Impact Factor
Synthetic enzyme inhibitor: a novel targeting ligand for nanotherapeutic
drug delivery inhibiting tumor growth without systemic toxicity
Debbie Liao, PhDa,1, Ze Liu, BSa,b,1, Wolfgang Wrasidlo, PhDc, Tingmei Chen, PhDa,
Yunping Luo, MD, PhDa, Rong Xiang, MD, PhDa,b, Ralph A. Reisfeld, PhDa,⁎
aDepartment of Immunology and Microbial Science, The Scripps Research Institute, La Jolla, California, USA
bDepartment of Immunology, Nankai University School of Medicine, Tianjin, China
cMoores Cancer Center, University of California, San Diego, California, USA
Received 14 September 2010; accepted 3 March 2011
Unresolved problems associated with ligand-targeting of liposomal nanoparticles (NPs) to solid tumors include variable target receptor
expression due to genetic heterogeneity and insufficient target specificity, leading to systemic toxicities. This study addresses these issues by
developing a novel ligand-targeting strategy for liposomal NPs using RR-11a, a synthetic enzyme inhibitor of Legumain, an asparaginyl
NPs revealed high ligand-receptor affinity, enhanced solid-tumor penetration and uptake by tumor cells. Treatment of tumor-bearing mice with
RR-11a-coupled NPs encapsulating doxorubicin resulted in improved tumor selectivity and drug sensitivity, leading to complete inhibition of
tumor growth. These antitumor effects were achieved while eliminating systemic drug toxicity. Therefore, synthetic enzyme inhibitors, such as
RR-11a, represent a new class of compounds that can be used for highly specific ligand-targeting of NPs to solid tumors.
From the Clinical Editor: This study addresses the problems associated with ligand-targeting of liposomal nanoparticles to solid tumors with
variable target receptor expression. A novel and efficacious targeting strategy has been developed towards a synthetic enzyme inhibitor of
Legumain. The authors demonstrate successful tumor growth inhibiting effect while eliminating systemic drug toxicity in an animal model
using this strategy.
© 2011 Published by Elsevier Inc.
Key words: Ligand-targeting; Liposome; Legumain; Cancer
Ligand targeting represents a major advancement in the
evolution of nanoparticle (NP)-mediated drug delivery, with the
aim of reducing systemic drug toxicity while maintaining a
biologically optimal drug dose delivery to target cells.1Initial
prolonged their circulatory life and reduced accumulation in the
such NPs has shown dose-limiting toxicity in the clinic due to
persistent nonspecific accumulation,3,4thus prompting the
transition to specific ligand targeting. Despite promising proof
of concept in solid tumors, unresolved problems associated with
tumor cells due to genetic heterogeneity and insufficient target
specificity resulting in systemic toxicity.5These limitations
enhanced specificity and targeting ability capable of resolving
these problems. To address these limitations, we developed a
Nanomedicine: Nanotechnology, Biology, and Medicine
7 (2011) 665–673
This study was supported by National Cancer Institute grant 5 R01
CA134364-01A1 (to R.A.R) and Merck Serono (to R.A.R.). D. Liao was
supported by the National Heart, Lung, and Blood Institute (T32HL007195)
training grant; the content of this study is solely the responsibility of the
authors and does not necessarily represent the official views of the National
Heart, Lung, and Blood Institute or the National Institutes of Health.
Additional support was received from the National Science Foundation of
China (NSFC) grant 30830096 and 973 program grant 2007CB914804
(to R.X.). This is TSRI manuscript number IMM20750.
D.Liao and Z.L. designed the study, performed experiments, interpreted
data and wrote the manuscript. W.W. and T.C. contributed to the formulation
of NPs. W.W. and R.X. contributed to experiments in Figure 1. Y.L.
contributed to experiments in Figure 2. R.A.R. contributed to the design of
the study. R.A.R. and W.W. provided editorial assistance.
The authors disclose a provisional patent application filed jointly by
Merck Serono and The Scripps Research Institute.
⁎Corresponding author: The Scripps Research Institute, La Jolla, CA
E-mail address: firstname.lastname@example.org (R.A. Reisfeld).
1These authors contributed equally to this work.
1549-9634/$ – see front matter © 2011 Published by Elsevier Inc.
Please cite this article as: D., Liao, et al, Synthetic enzyme inhibitor: a novel targeting ligand for nanotherapeutic drug delivery inhibiting tumor growth
without systemic toxicity. Nanomedicine: NBM 2011;7:665-673, doi:10.1016/j.nano.2011.03.001
novel ligand-targeting strategy forNPsusingRR-11a,a synthetic
enzyme inhibitor of Legumain.6
We hypothesized that Legumain, an asparaginyl endopepti-
dase, would be an ideal target because it is overexpressed by a
majority of human solid tumors, including breast, colon and
prostate tumors, and sparsely expressed in normal tissues.7-10
Our previous work demonstrated Legumain to be overexpressed
in vivo by both tumor cells and proliferating endothelial cells in
the tumor microenvironment (TME).11Additionally, we found
that Legumain overexpression on tumor cells occurred only in
response to environmental stress, such as serum starvation or in
vivo growth, and was not detectable in cells under typical culture
conditions.10One important finding was that Legumain
expression was absent in the normal tissues of corresponding
solid tumors.10,11These results suggested that Legumain
expression on tumor cells is governed by a stress response
induced by a function of the TME. Thus, we predicted that this
characteristic of Legumain expression would facilitate homing of
NPs to solid tumors and free our targeting strategy from
limitations arising from genetic heterogeneity.
We further hypothesized that ligand targeting of Legumain
could be achieved by coupling liposomal NPs to RR-11a, a
synthetic small molecule (451 M.W.) inhibitor of Legumain.6
RR-11a is from a class of aza-peptide Michael acceptor
Figure 1.Formulationandcharacterization ofLegumain-targeted liposomalNPs. (A) TUBOmurinebreast cancercellstreated with100 μM CoCl2for 24 hwere
analyzed by immunofluorescence using anti-Legumain antibody and Alexa Fluor 488 (green) conjugated secondary antibody. Scale bars, 100 μm. (B)
Quantification of Legumain binding sites on 4TO7 tumor cells by Scatchard Plot analysis. The mean Kds for control and CoCl2treated tumor cells were 1.107 ±
0.232 nM and 1.208 ± 0.107 nM and the number of Legumain binding sites were calculated to be 46,760 and 117,800 sites/cell, respectively. (C) The synthetic
enzyme inhibitor of Legumain, RR-11a, was conjugated to DOPE using triethylamine as a catalyst.
666 D. Liao et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 665–673
inhibitors that was designed with clan CD specific sequences
and is therefore highly specific for clan CD proteases, such as
Legumain, which it inhibits irreversibly at IC50values in the
nanomolar range (IC50= 31–55 nM).6,12,13Importantly, RR-
11a does not interact with other related cysteine proteases,
including caspases, clostripain or gingipain K, and is resistant
to cleavage by proteases,6,14thus increasing the stability of the
ligand in vivo.
The mechanism of Legumain inhibition by RR-11a involves a
nucleophilic attack by the catalytic cysteine residue on the
Michael Acceptor double bond at C2, forming a covalent bond
that irreversibly inhibits Legumain.15Based on this character-
istic, we predicted that binding of RR-11a-coupled liposomal
NPs to Legumain on the surface of tumor cells would result in
covalent attachment of the entire liposomal composite to the
receptor and result in subsequent irreversible internalization.
This proposed mechanism is supported by NMR studies16and is
biologically relevant because high binding affinity will improve
targeting. Thus we sought to demonstrate that the characteristics
of Legumain as (a) stress-induced tumor cell surface expression,
(b) high RR-11a binding specificity and (c) irreversible
internalization of RR-11a following covalent binding, would
enhance NP-mediated drug delivery to solid tumors in vivo.
Animals and cell lines
Female BALB/c mice were purchased from The Scripps
Research Institute Rodent Breeding Facility and housed in an
AAALAC accredited facility. All animal procedures were
performed in accordance with institutional guidelines pertaining
to the humane care of animals and approved by The Scripps
Research Institute Animal Care Committee. Furthermore, 4T1
and 4TO7 murine breast carcinoma cell lines were provided by
Suzanne Ostrand-Rosenberg (University of Maryland, College
Park, Maryland). In addition, CT26 murine colon carcinoma
cells were purchased from ATCC (Manassas, Virginia). TUBO
murine breast carcinoma cells were a gift from Jay A. Berzofsky
(National Cancer Institute, Bethesda, Maryland).
TUBO cells were incubated for 24 hours, with or without 100
μM cobalt chloride (CoCl2), a hypoxia-mimetic agent that
induces the DNA binding activity of hypoxia-inducible factor-1
alpha (HIF-1a), a key regulator of the cellular response to
hypoxia.17,18Cells were washed with PBS and fixed with 4%
paraformaldehyde. The unpermeabilized cells were blocked with
5% BSA for 30 minutes followed by overnight incubation with
sheep-anti-mouse Legumain antibody (R&D Systems, Minne-
apolis, Minnesota) and detected using an Alexa Fluor 488
donkey anti-sheep IgG secondary antibody (Life Technologies,
Carlsbad, California). For visualization of in vitro uptake of
rhodamine B and doxorubicin (DOX)-loaded NPs, cells were
imaged using a Zeiss Axiovert 100 TV inverted microscope
(Carl Zeiss MicroImaging, LCC, Thornwood, New York) (555
nm excitation wavelength/580 nm emission wavelength).
Figure 2. RR-11a conjugated NPs show increased uptake by tumor cells in
vitro and enhanced homing to solid tumors in vivo. Targeted (RR-11a+) and
Non-targeted (RR-11a-) NPs were labeled with the fluorescent dye
rhodamine B. (A) Murine breast (4T1/4TO7) and colon (CT26) carcinoma
cells were cultured for 24 h with 100 μM CoCl2, after which Targeted or
Non-targeted NPs were added to the cells. After the times indicated,
uninternalized NPs were removed and the tumor cells imaged by
fluorescence microscopy to quantify the percentage of rhodamine B-positive
cells. (n = 3 wells/group) Data represent means ± s.e.m. (B) Mice with
established 4T1 breast tumors were injected once with Non-targeted or
Targeted liposomes and sacrificed 24 h later. Organs were analyzed
immediately by fluorescence microscopy to visualize liposomal NPs (red).
(n = 2 mice/group) Scale bars, 100 μm.
667D. Liao et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 665–673
Binding studies and Scatchard plot analyses
Anti-mouse Legumain antibody (40 mg) (R&D Systems) was
incubated for 30 minutes with 0.5 mCi of
of lodo-Gen reagent (Pierce Chemical Co., Rockford, Illinois).
Non-incorporated125I was removed by PD-10 gel filtration (GE
105) were cultured with or without 100 μM CoCl2for 24 hours
and incubated with 14 nM of serially diluted
antibody for 2 hours at 4°C. Cells were washed with PBS
containing 1% BSA, and the amount of bound radiolabel was
determined with a g-scintillation counter. The corresponding
counts per minute (CPM) were used for Scatchard plot analysis
with Prism software (GraphPad, La Jolla, California) and used to
calculate number of Legumain binding sites.
Mice bearing 4T1 orthothopic tumors of approximately
500mm3were injected once intravenously with RR-11a+or
RR-11a-NPs labeled with rhodamine B. Alternatively, mice
were injected intravenously 3 times at 48-hour intervals with
RDZ-218, NP-DOX, free DOX, or saline. Twenty-four hours
after the final treatment, animals were sacrificed and spleen,
kidney, lungs, liver, heart and tumor were collected, frozen in
OCT compound (Fisher Scientific, Waltham, Massachusetts),
immediately sectioned and imaged by fluorescence microscopy
(555 nm excitation wavelength/580 nm emission wavelength).
Thoracic mammary fat pads of female BALB/c mice were
injected with 1 × 1054TO7 cells. Seven days later, mice received
5 IV injections, at 3-day intervals, of RDZ-218, NP-Dox, free
Dox (at 1 mg/kg DOX and NP-DOX) or saline. Mice were
sacrificed 24 hours after the final treatment and body and tumor
weights were determined and tissues subjected to histological
analysis. TUNEL (Promega, Madison, Wisconsin) immunohis-
tochemical staining was performed according to the manufac-
In this study 6- to 8-week-old age-matched female BALB/c
in size, were given 5 consecutive IV injections of free DOX,
NP-DOX, or RDZ-218 at 24-hour intervals. The dose of
DOX administered per animal for all groups was approximately
5 mg/kg (see Supplementary Methods). After the first injection,
survival of animals was monitored daily for 10 days.
The statistical significance of differential findings between
experimental groups and controls was determined by 2-tailed
Student's t test using Prism software (GraphPad, La Jolla,
California). Findings were regarded as significant if P b 0.05.
Hypoxia-induced expression of Legumain
Our previous work demonstrated that Legumain overexpres-
sion by tumor cells was induced by a function of the TME.10
TME,19we sought to determine whether hypoxia could induce
cell-surface expression of Legumain in tumor cells. To this end,
murine TUBO breast cancer cells were cultured with 100 μM
CoCl2, a hypoxia mimetic, and cell-surface expression of
Legumain visualized by immunofluorescence. As shown in
Figure 1, A, cell-surface expression of Legumain was markedly
induced by hypoxic stress in comparison with cells under normal
culture conditions. Furthermore, we quantified the number of
Legumain binding sites per tumor cell by binding studies with
125I-labeled anti-Legumain antibody and Scatchard Plot analysis
on untreated or CoCl2(100 μM) treated 4TO7 murine breast
cancer cells (Figure 1, B). The mean Kds of untreated and CoCl2
treated cells were 1.107 ± 0.232 nM and 1.208 ± 0.107 nM,
respectively. From these values, we calculated the number of
Legumain-binding sites under normal oxygen tension versus
hypoxia to be approximately 46,760 and 117,800 sites/cell,
respectively, representing a threefold increase in Legumain cell
surface expression in response to hypoxic stress.
In vitro uptake and in vivo biodistribution of RR-11a
To test whether Legumain targeting can improve tumor-cell
uptake of NPs, we coupled the synthetic Legumain inhibitor RR-
11a to the 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine
(DOPE) component of liposomal NPs by reaction with
triethylamine (TEA), as described in detail in Methods (Figure
1, C). Analysis of RR-11a coupled (Targeted) or native (Non-
targeted) NPs by dynamic light scattering (DLS) revealed a
uniform size distribution of 150 and 110 nm, respectively
(Supplementary Figure 1). Next, murine breast (4T1 and 4TO7)
Figure 3. Legumain-targeting enhances uptake of DOX-encapsulated NPs and improves drug delivery to solid tumors. DOX was loaded into Legumain-targeted
NPs conjugated with RR-11a to generate RDZ-218. DOX was also loaded into Non-targeted NPs (NP- DOX) as a control. (A) 4T1 and 4TO7 murine breast
cancer cells were cultured with CoCl2for 24 h and then incubated with either RDZ-218, NP- DOX or free DOX for the indicated times. Mean fluorescence
(B) The percentage of total DOX uptake was determined by comparing the MFI of RDZ-218, NP- DOX, and free DOX -treated tumor cells with that of serially
diluted DOX. (C) The relative percentage of dead 4T1 and 4TO7 cells 24 h following treatment with either RDZ-218, NP- DOX, free DOX, free RR-11a, or
empty Targeted liposomes (NP-RR-11a) was determined by analyzing the forward and side scatter plot of flow cytometry. Data are shown relative to untreated
cells (Control). (n = 3 wells/group) Data represent means ± s.e.m. ⁎P b 0.05, ⁎⁎P b 0.005. (D) Mice bearing 4TO7 orthotopic breast tumors of 500 mm3in size
were given 2 IV injections with either RDZ-218, NP-DOX or free DOX. Tissues were isolated 24 h later and immediately analyzed by fluorescence microscopy
to detect distribution of DOX (red). Tissue sections were stained with DAPI (blue) to visualize cell nuclei. (n = 2 mice/group) Scale bars, 100 μm.
668D. Liao et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 665–673
669D. Liao et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 665–673
and colon (CT26) carcinoma cells were subjected to CoCl2-
induced hypoxic stress and then incubated with Targeted or
Nontargeted NPs, labeled with the fluorescent dye rhodamine B,
for varying lengths of time. Analysis of these cells by
fluorescence microscopy after removal of unbound NPs revealed
markedly enhanced uptake of Targeted, in comparison with Non-
targeted, NPs by all three cell lines tested (Figure 2, A).
The efficacy of in vivo targeting was determined by IV
injection of Targeted or Non-targeted NPs into female BALB/c
mice bearing orthotopic 4T1 breast tumors of approximately 500
mm3size. Fluorescence microscopy of tumor and tissues from
these animals 24 hours later showed a marked increase in
Targeted NPs that had homed to primary tumors with decisively
reduced nonspecific accumulation in RES organs, including
liver, spleen and kidney, when compared with Non-targeted NPs
(Figure 2, B).
In vitro delivery of DOX-encapsulated NPs conjugated
The efficacy of RR-11a coupled liposomal NP-mediated
delivery of a drug payload to tumor cells in vitro was evaluated
with DOX, a chemotherapeutic drug commonly used to treat
breast cancer. DOX was loaded into Targeted (RDZ-218) or
Non-Targeted (NP-DOX) NPs via an ammonium phosphate
gradient as described in the Methods section above. In addition,
4T1 and 4TO7 murine breast tumor cells under CoCl2-induced
hypoxic stress were then incubated with either free DOX, NP-
DOX or RDZ-218 and analyzed by flow cytometry to quantify
the amount of internalized drug by measuring the mean
fluorescence intensity (MFI) of DOX, which exhibits red
fluorescence. As shown in Figure 3, A, RDZ-218-treated tumor
cells showed rapid drug uptake, and uptake of NP-DOX was
considerably less effective. Importantly, treatment with RDZ-
218 not only resulted in more rapid uptake over time in
comparison with NP-DOX but also increased the degree of drug
uptake by as much as 16-fold, with the rate of RDZ-218 uptake
being similar to that of free DOX (Figure 3, A). By comparing
the MFI values generated from cells treated with serially diluted
free DOX with that of encapsulated DOX, we determined that
close to 100% of RDZ-218 NPs were taken up by the tumor cells
within 4 hours (Figure 3, B). Significantly, the logarithmic
uptake of RDZ-218 that we observed was indicative of ligand-
receptor-mediated internalization, and was superior in compar-
ison with Non-targeted liposomal NPs.
In vitro bioactivity of RDZ-218
DOX bioactivity of RDZ-218 was determined in vitro with
flow cytometry by comparing the percentages of dead 4T1 or
4TO7 tumor cells 24 hours after treatment with either free DOX,
NP-DOX, free RR-11a or empty RR-11a-conjugated NPs (NP-
RR-11a). As depicted in Figure 3, C, tumor cells treated with
RDZ-218 showed a 3- to 15-fold increase in percentage of dead
cells in comparison with control or NP-DOX-treated cells.
Intriguingly, this cytotoxic effect of RDZ-218 was superior to
that of free DOX. An important finding was that no cytotoxic
effect was observed with free RR-11a or NP-RR-11a treated
cells, indicating that the enhanced cytotoxicity of RDZ-218 is
duesolely to increased uptake of DOX,and notdueto any effects
of Legumain inhibition in tumor cells by RR-11a alone.
In vivo bioactivity of RDZ-218
To test the ability of RDZ-218 to deliver a bioactive payload
of DOX to solid tumors in vivo, we first tested drug delivery in
mice with established orthotopic 4TO7 breast tumors, of
approximately 500 mm3, by giving 2 IV injections of either
RDZ-218, NP-DOX, or free DOX at 48-hour intervals.
Microscopic analysis of tumors 24 hours after the last injection
revealed intense and widely spread DOX fluorescence in tumors
from RDZ-218 treated animals (Figure 3, D). In contrast,
fluorescence of DOX was strikingly reduced or appeared
punctate in tumors from mice treated with NP-DOX or free
DOX, respectively. Only RDZ-218 resulted in markedly reduced
accumulation of DOX in the liver and heart in comparison with
NP-DOX and free DOX, respectively.
Finally, we evaluated the therapeutic benefit of RDZ-218 in
mice with established 4TO7 breast tumors by giving these
animals 5 IVinjections at 3-day intervals of either RDZ-218,NP-
DOX, NP-RR-11a, free DOX, or saline. DOX was administered
at 1mg/kg for both encapsulated and free drug. Determination of
tumor size with microcalipers indicated that RDZ-218 treatment
alone essentially eliminated tumor growth, whereas all control
groups revealed only a delay in tumor growth in comparison with
animals treated with saline (Figure 4, A). Three weeks after
tumor-cell challenge, gross examination of primary tumors from
mice treated with RDZ-218 revealed only rudimentary tumor
nodules and control mice presented with large tumor masses of
approximately 500 mm3in size or greater (Figure 4, B).
Cumulatively, treatment with RDZ-218 resulted in an 8- to 12-
fold decrease in tumor weight when compared with controls
(Figure 4, C). Concordantly, TUNEL immunohistochemical
analysis of tumor sections from RDZ-218-treated mice showed
between a 9- and 35-fold increase in percentage of apoptotic cells
when compared to tumors from control animals (Figure 4, D).
Toxicity study with RDZ-218
One striking finding of our therapeutic study was that mice
treated with RDZ-218 did not lose body weight during the course
of the study, in contrast with mice treated with free DOX or NP-
DOX, which showed a decided loss in body weight (Figure 5).
Additionally, mice treated with NP-RR-11a, which did not
contain DOX, did not lose body weight, suggesting that
Legumain targeting by RDZ-218 markedly reduced DOX
toxicity in vivo. To confirm this observation, we performed a
toxicity study in tumor-bearing mice by daily IV injection of a
single dose of either free DOX, NP-DOX or RDZ-218, at
5 mg/kg DOX for all groups, over the course of 5 days. An
important finding was that the results summarized in Table 1
5 treatments. We found it striking that free DOX administered
at 5 mg/kg was lethal and all test animals in this group expired
immediately following the first treatment. Additionally, in the
Non-targeted NP-DOX-treated group, only 1 animal survived
670D. Liao et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 665–673
all 5 treatments, whereas lethal toxicity occurred after the
second and third treatment in 4 of 5 animals.
In summary, we formulated a novel Legumain-targeted
liposomal NP capable of highly efficient drug payload delivery
to solid tumors in vivo. To our knowledge, we are first to
demonstrate successful ligand-targeting of solid tumors using a
synthetic small-molecule enzyme inhibitor. Legumain is a
stable target that is highly conserved among species.7
However, the efficiency of ligand-targeting depends in part
on abundance of the target receptor on the cell surface and is
maximized by its overexpression on target cells relative to
normal cells.5To this end, we identified hypoxia as a promoter
of Legumain cell-surface expression and showed that hypoxic
stress induced a threefold increase in the number of Legumain-
binding sites on the surface of tumor cells. Additionally, we
demonstrated that targeting of NPs via coupling to the
Legumain inhibitor RR-11a markedly enhanced NP uptake
by tumor cells under hypoxic stress. These observations are
biologically relevant because hypoxia is a universal hallmark
of the TME,19and thus should free our ligand-targeting
strategy from limits imposed by the genetic heterogeneity
commonly observed in solid tumors.20
The main therapeutic objective of ligand-targeting is to
eliminate undesirable systemic drug toxicities arising from
nonspecific accumulation of liposomal NPs in peripheral
tissues.21However, a further requirement for effective NP-
mediated drug delivery is not only their ability to carry an
optimal payload to the desired target cell, but also the effective
release of this payload.22To evaluate these parameters
critically, DOX, a chemotherapeutic drug commonly used to
treat breast cancer, was loaded into Targeted NPs via an
ammonium phosphate gradient, as previously described,23to
generate RDZ-218. We demonstrated that RDZ-218 increased
tumor sensitivity to low drug doses and inhibited breast tumor
growth in vivo. An important result is that we did not find
Figure 4. Therapeutic treatment with RDZ-218 inhibits primary breast tumor
growth in mice without toxicity. Female BALB/c mice were injected ortho-
topically with 2 × 1064TO7 tumor cells and 7 d later, given 5 IV injections at
3 d intervals with either RDZ-218, NP-DOX, free DOX, empty Targeted
was calculated using measurements obtained by microcalipers on each day of
treatment. Data represent means ± s.e.m. (B) Images of primary tumors were
from each group. (C) Primary tumor wet weight was also measured. Data
representmeans±s.e.m.⁎Pb 0.05.(D) TUNELstainingwasusedtovisualize
and quantify the percentage of apoptotic tumor cells in tumor sections. n = 5
fields/section. Data represent means ± s.e.m. ⁎⁎⁎P b 0.0005.
Figure 5. Mice treated with RDZ-218 maintain their body weight. Mice were
treated as describedin Figure 4. Initial bodyweightwassubtractedfrom body
weight at time of sacrifice (minus tumor weight) to determine change in body
weight. Data represent means ± s.e.m. Control groups were compared with
the RDZ-218 treated group, ⁎⁎P ≤ 0.0077, ⁎⁎⁎P b 0.001.
671D. Liao et al / Nanomedicine: Nanotechnology, Biology, and Medicine 7 (2011) 665–673
accumulation of Legumain-targeted NPs in the liver, which is
significant because this RES organ has been identified as a
major sink for native PEG-liposomes, which may result in
toxicity.1Additionally, we did not observe any accumulation of
RDZ-218 in the heart, which is significant because cardiotoxi-
city is a dose-limiting factor for DOX therapy.24A significant
finding was that RDZ-218 eliminated systemic toxicities
induced by lethal doses of DOX in mice. This finding does
not exclude the possibility that continuous and long-term
treatment with RDZ-218 may increase Legumain expression, in
response to cellular stress caused by the generation of reactive
oxygen species (ROS) derived from redox activation of DOX,25
in cells that are not exposed to lethal DOX doses. However, we
did not observe any incremental toxicity as a result of
continuous RDZ-218 treatment during our in vivo therapeutic
tumor study (5 injections given over the course of 15 days) or
acute toxicity study (5 injections given over the course of 5
days). These results confirm that improvements in tumor cell
killing and elimination of drug toxicity observed with RDZ-218
in our studies is a direct result of enhanced drug delivery
achieved by Legumain targeting of NPs.
We found it striking that these benefits were observed in
direct comparison with Non-targeted DOX-encapsulated PEG-
liposomal NPs, similar to the FDA-approved drug, Doxil. This
increase in drug sensitivity and decrease in toxicity observed
with RDZ-218 is clinically significant because it effectively
widens the therapeutic window for encapsulated DOX. We
predict that this improvement in nanomedicine can also be
achieved with other chemotherapeutic drugs when encapsulated
in Legumain-targeted NPs.
Collectively, our data indicate that Legumain-targeting via
coupling to the synthetic Legumain inhibitor RR-11a enhances
NP uptake by hypoxic tumor cells and effectively enhances
homing of drug-loaded NPs to solid tumors, and reduces non-
specific accumulation in the RES organs and heart. We
postulate that these phenomena, in conjunction with the high
binding affinity of RR-11a for Legumain, facilitate the specific
homing of RDZ-218 to solid tumors in vivo, thus eliminating
drug toxicity. Most important, this technology has the potential
to advance the current status of liposomal NP-mediated
chemotherapy in nanomedicine by reducing the biologically
optimal drug dose required for a significant antitumor effect
and eliminating toxicities, thereby significantly improving the
health and quality of life of cancer patients.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
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