Combinatorially designed lipid-like nanoparticles for intracellular delivery of cytotoxic protein for cancer therapy

Article abstract of DOI:10.1002/anie.201311245

An efficient and safe method to deliver active proteins into the cytosol of targeted cells is highly desirable to advance protein-based therapeutics. A novel protein delivery platform has been created by combinatorial design of cationic lipid-like materials (termed lipidoids ), coupled with a reversible chemical protein engineering approach. Using ribonuclease A (RNase A) and saporin as two representative cytotoxic proteins, the combinatorial lipidoids efficiently deliver proteins into cancer cells and inhibit cell proliferation. A study of the structure-function relationship reveals that the electrostatic and hydrophobic interactions between the lipidoids and the protein play a vital role in the formation of protein-lipidoid nanocomplexes and intracellular delivery. A representative lipidoid (EC16-1) protein nanoparticle formulation inhibits cell proliferation in vitro and suppresses tumor growth in a murine breast cancer model.

Full text of DOI:10.1002/anie.201311245

Angewandte
Chemie
DOI: 10.1002/anie.201311245
Protein Delivery
Combinatorially Designed Lipid-like Nanoparticles for Intracellular
Delivery of Cytotoxic Protein for Cancer Therapy**
Ming Wang, Kyle Alberti, Shuo Sun, Carlos Luis Arellano, and Qiaobing Xu*
Abstract: An efficient and safe method to deliver active
proteins into the cytosol of targeted cells is highly desirable to
advance protein-based therapeutics. A novel protein delivery
platform has been created by combinatorial design of cationic
lipid-like materials (termed “lipidoids”), coupled with a rever-
sible chemical protein engineering approach. Using ribonu-
clease A (RNase A) and saporin as two representative cyto-
toxic proteins, the combinatorial lipidoids efficiently deliver
proteins into cancer cells and inhibit cell proliferation. A study
of the structure–function relationship reveals that the electro-
static and hydrophobic interactions between the lipidoids and
the protein play a vital role in the formation of protein–lipidoid
nanocomplexes and intracellular delivery. A representative
lipidoid (EC16-1) protein nanoparticle formulation inhibits
cell proliferation in vitro and suppresses tumor growth in
a murine breast cancer model.
nanoparticle drug delivery systems have offered alternative
approaches for spatially and temporally controlled protein
delivery. A number of synthetic nanomaterials, including
liposomes,[5] polymers,[6] and inorganic nanoparticles,[7] have
been designed for this purpose. These nanoparticules, how-
ever, are still of limited utility for protein therapy owing to the
low delivery efficiency and/or complicated nanoparticle
fabrication processes. Thus, a facile and convenient approach
to develop novel nanomaterials for efficient intracellular
protein delivery has yet to be developed.
We report herein a novel and efficient protein delivery
platform that uses combinatorially designed cationic lipid-
based nanoparticles combined with a reversible protein
modification approach. Pioneered by Anderson, Langer,
et al.,[8] the combinatorial library strategy has recently been
used to generate cationic lipid-like materials (termed “lip-
idoids”) for siRNA delivery. We have further extended this
class of materials for use in DNA and mRNA delivery.[9] We
hypothesize that lipidoids can be used as a novel protein
delivery platform, as the charge–charge and hydrophobic
interactions between lipidoids and proteins can load proteins
into lipidoid nanoparticles. In turn, the hydrophobic nature of
lipidoid nanoparticles allows easy protein transport through
the cell membrane. In an attempt to strengthen the charge–
charge binding of proteins and lipidoids, we modified the
lysine residues of proteins with cis-aconitic anhydride in this
investigation. The conjugation reaction between the amine
groups of lysine and cis-aconitic anhydride converts the
positively charged lysines into negatively charged carboxylate
groups, thus increasing the negative charge density of protein
and its binding with cationic lipidoids. Moreover, the cis-
aconitic anhydride modification is reversible in the slightly
acidic intracellular environment (for example, the pH of
endosome and lysosome is in the range of 5–6),[10] leading to
the restoration of the biological activity of the modified
proteins.
As a proof-of-concept for developing cationic lipid-based
nanoparticles for protein delivery, we designed and synthe-
sized a library of lipidoids through the ring-opening reaction
of 1, 2-epoxyhexadecane and aliphatic amines with diversified
chemical structures (Figure 1). Using RNase A and saporin,
two representative cytotoxic proteins, along with the cis-
aconitic anhydride modified versions (RNase A-Aco and
saporin-Aco), we demonstrate that the lipidoid nanoparticles
can deliver protein into cancer cells and inhibit cell prolifer-
ation, for potential applications such as cancer therapy.
RNase A can cleave intracellular RNA and induce cytotoxic
effects when taken up by cells,[11] while saporin irreversibly
inhibits protein synthesis in eukaryotic cells by rending the
28S subunit of ribosomes.[12] RNase A and saporin have both
P
rotein therapy has been considered as the safest and most
direct approach to manipulate cell function and treat human
disease since the early 1980s, when insulin began to be used as
the first human recombinant protein therapeutic.[1] A major-
ity of protein pharmaceuticals (for example, cytokines,
growth factors, and monoclonal antibodies) elicit their
biological activity by targeting cell surface ligands or extra-
cellular domains.[2] Nevertheless, advancements in molecular
biology have suggested that proteins that target intracellular
biological activity could be potent therapeutics.[3] The deliv-
ery of proteins safely and efficiently through the cell
membrane to reach their intracellular targets remains a chal-
lenge for the success of protein therapy.[3b] As such, the
development of methods for intracellular protein delivery is
needed. Over the past few decades, the most thoroughly
studied protein delivery approach has been fusing target
protein cargos with protein transduction domains (PTD) or
membrane transport signals. The delivery efficiency of PTD-
protein fusions vary with protein type[4] and lack the
capability to target a specific tissue or organ. More recently,
[*] Dr. M. Wang, K. Alberti, S. Sun, C. L. Arellano, Prof. Dr. Q. B. Xu
Department of Biomedical Engineering, Tufts University
4 Colby Street, Medford, MA (USA)
E-mail: qiaobing.xu@tufts.edu
[**] We thank Prof. Gary Sahagian and Dr. Min Fang at Tufts University,
School of Medicine for providing 4T1-12B cells and developing the
breast cancer mice model. This research was supported by Tufts
University. Q.B.X. also acknowledges the Tufts FRAC award and
Charlton Award from Tufts University School of Medicine and Pew
Scholar for Biomedical Sciences program from Pew Charitable
Trusts. K.A. acknowledges the IGERT fellowship from NSF.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201311245.
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Figure 2. Evaluation of lipidoid-facilitated protein delivery on B16F10
cell line via a cytotoxicity assay. Black: lipidoid controls (4 mgmLÀ1);
green: RNase A (3.3 mgmLÀ1); yellow: RNase A-Aco(3.3 mgmLÀ1); blue:
saporin(0.17 mgmLÀ1); red: Saporin-Aco(0.17 mgmLÀ1). Cytotoxicity
was determined by MTT assay. Data are presented as mean ÆSD
(n=4).
Figure 1. a) Route of synthesis for lipidoids. b) The chemical structures
of the library of amines used for lipidoid synthesis (The lipidoids are
named EC16, for 1, 2-epoxyhexadecane, followed by the amine
number).
toxicity and inhibits cell proliferation,[11,13b] the protein
delivery efficiency by various lipidoid-based nanoparticles
was compared by measuring the viability of differently
treated cells. As shown in Figure 2, all lipidoids displayed
low carrier cytotoxicity, with cell viabilities greater than 90%
following exposure to each of the lipidoids at a concentration
of 4 mgmLÀ1. Similarly, we observed no detectable toxicity to
B16F10 cells following exposure to the four proteins
(3.3 mgmLÀ1 for RNase A and RNase A-Aco, 0.17 mgmLÀ1
for saporin and saporin-Aco). This can be attributed to the
naked protein lacking an efficient mode of entry to cells.
Lipidoid–protein complex treated cells, however, showed
distinct changes in cell viability, depending on the lipidoid and
protein type. No appreciable viability decrease was observed
for lipidoid/RNase A treated cells, indicating a low RNase A
delivery efficiency by all lipidoids in the library. This is most
likely because of the high intrinsic positive charge, and
hydrophilic nature of RNase A, which prevented the forma-
tion of stable nanocomplexes for membrane penetration. In
contrast, RNase A-Aco, saporin, and saporin-Aco can be
delivered using seven of the lipidoids in the library (EC16-1,
EC16-3, EC16-4, EC16-5, EC16-6, EC16-12, and EC16-14). A
notable example of a lipidoid with high protein delivery
efficiency is EC16-1, which facilitates the delivery of
RNase A-Aco, saporin, and saporin-Aco, and reduces
B16F10 cell viability down to 30%. It is also noteworthy
that EC16-1 delivered both chemically modified and non-
modified saporin with comparable efficiency. This indicates
that the charge–charge interaction is not the only driving
force facilitating lipidoid and protein binding, and that
hydrophobic interactions may also contribute to the complex-
ation between protein (saporin) and lipidoid. The significance
of hydrophobic interaction between lipidoid and protein in
facilitating protein delivery was further demonstrated by
delivering RNase A-Aco and saporin with lipidoids of varied
tail length.
been used in clinical trials in cancer patients that are
refractory to traditional chemotherapy.[13] In an in vivo
study, we demonstrate that the administration of a represen-
tative lipidoid/saporin nanoparticle formulation suppresses
tumor growth in a 4T1 murine breast cancer model by
accumulating saporin at tumor sites.
The library of lipidoids was synthesized through the ring-
opening reaction between 1,2-epoxyhexadecane and amine
under mild conditions according to the methods of previous
reports.[9a,b] The crude products were used directly for initial
identification of protein delivery materials, and subsequently
purified for detailed formulation studies. The lipidoids are
named EC16 followed by the amine number in the library
(Figure 1), where EC16 indicates 1,2-epoxyhexadecane. The
reversible chemical modifications of RNase A or saporin
were achieved by reacting proteins with excessive amounts of
cis-aconitic anhydride, followed by a dialysis purification
process. RNase A-Aco was selected for detailed study to
confirm the acid-labile nature and chemical reversibility of
the cis-aconitic anhydride modification of the proteins. The
treatment of RNase A-Aco with an acidic buffer solution
(NaOAc, pH 5.2) restored the protein back to RNase A, as
confirmed by SDS-PAGE analysis (Supporting Information,
Figure S1A). Moreover, the acid-treated RNase A-Aco sig-
nificantly enhances ribonuclease activity compared to that of
neutral PBS treated RNase A-Aco (Figure S1B). These
results suggest that protein modification using cis-aconitic
anhydride is acid-labile and chemically reversible. Such
a reversible protein modification approach has the potential
to boost electrostatic binding of protein with cationic lipidoids
while having minor effect on their intracellular function.
The capability of lipidoids to deliver protein was eval-
uated by co-culturing murine melanoma cancer cells
(B16F10) with lipidoid–protein complexes of RNase A,
saporin, RNase A-Aco, or saporin-Aco. As the successful
transduction of RNase A or saporin into cell induces cyto-
Two lipidoids with shorter hydrophobic tails, EC14-1 and
EC14-12, delivered RNase A-Aco and saporin and subse-
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quently inhibited cell proliferation less efficiently than EC16-
1 and EC16-12 (Figure S2). This suggests that lipidoids with
longer hydrophobic tails have higher protein delivery effi-
ciency. Moreover, these findings demonstrate the advantage
of a combinatorial approach for the discovery of novel
nanocarriers for protein delivery and the investigation of
structure–function relationships. The complexation between
EC16-1 and proteins was characterized by dynamic light
scattering (DLS) analysis. The complexation between neg-
atively charged RNase A-Aco or saporin-Aco and EC16-
1 increased the size and decreased the zeta-potential of the
EC16-1 particles, while the addition of unmodified RNase A
or saporin into the EC16-1 solution had only minor effect on
the size and surface charge of EC16-1 (Table S1). The
representative nanoparticle structures of EC16-1 and EC16-
1/RNase A-Aco complexes were further characterized by
TEM imaging (Figure S3). The dispersed EC16-1 solution
formed nanoparticles with a size of about 200 nm, while
typical lipsome-like structures were observed for the EC16-1/
RNase A-Aco complexs.
To investigate lipidoid facilitated protein delivery in
detail, RNase A-Aco, and saporin were selected along with
the lipidoid EC16-1 as representative proteins and lipidoid for
the remainder of the studies. Protein delivery conditions were
initially optimized by treating B16F10 cells with lipidoid–
protein complexes mixed at varied EC16-1 to protein ratio,
while the concentrations of RNase A-Aco and saporin that
the cells were exposed to was fixed at 3.3 mgmLÀ1 and
0.17 mgmLÀ1, respectively (Figure S4). Optimal results were
achieved at mass ratio of 2: 5 for EC16-1/RNase A-Aco, and
20:1 for EC16-1/saporin complex, and further increases in
lipidoid to protein ratio did not improve the delivery
efficiency. Furthermore, the cytotoxicity of EC16-1/
RNase A-Aco and EC16-1/saporin complexes against
B16F10 cells is dependent on protein dose. As shown in
Figure 3, when used as stand-alone agents, RNase A-Aco and
mined to be 64 nm and 5.3 nm respectively, which was greatly
improved compared to RNase A-Aco and saporin alone.
Whether lipidoids such as EC16-1 are able to deliver
proteins to a panel of cancer cell lines was also investigated.
An efficient protein delivery platform for cancer therapy
must be able to transfect various cell lines, as each consists of
different cell surface environments which may affect the
internalization of nanoparticles and the protein delivery. We
selected human breast cancer cells (MCF-7 and MDA-MB-
231), human liver hepatocellular carcinoma cells (HepG 2),
human prostate cancer cell lines (PC-3 and LNCaP), human
cervical carcinoma cells (HeLa), and murine breast cancer
cells (4T1) as target cell lines. These cells were treated with
EC16-1/RNase A-Aco or EC16-1/saporin complexes at pre-
viously optimized delivery conditions. Table 1 summarizes the
IC50 values that were determined following the treatments of
EC16-1/RNase A-Aco or EC16-1/saporin complexes to all of
the cancer cell lines. The delivery of EC16-1/RNase A-Aco or
EC16-1/saporin complexes resulted in significant cytotoxicity
in all cell lines, suggesting the general applicability of lipidoid
EC16-1 for protein delivery.
Table 1: IC50 values of RNase A-Aco and saporin delivered by EC16-
1 against various cancerous cell lines.
Cell line
Cancer type
RNaseA-Aco [nm]
Saporin [nm]
MCF-7
MDA-MB-231
B16F10
HepG 2
PC-3
LNCaP
HeLa
4T1
breast
breast
40.6
57
64
2.9
2.5
5.3
3.9
2.3
1.4
3.2
0.9
melanoma
liver
prostate
prostate
cervix
28
125
243
48
breast
412
Having demonstrated the high efficiency and generality of
EC16-1 for protein delivery, we next developed a general
protein delivery formulation that comprises protein
(RNase A-Aco or saporin), lipidoid EC16-1, 1,2-dioleoyl-sn-
glycero-3-phosphoethanolamine (DOPE), cholesterol, and
N-(palmitoyl)sphingosine[succinyl{methoxy(polyethylene
glycol)2000}] (C16-mPEG-ceramide) for in vivo protein
delivery. We further post-modified the lipidoid/protein for-
mulations with DSPE-PEG2000-biotin to target tumor cells
and tissues. Biotinylated polymers or nanoparticles can be
selectively taken up by cancer cells and accumulate in the
tumor tissue, improving therapeutic efficacy.[14] The EC16-1/
RNase A-Aco and EC16-1/saporin nanoparticle formulation
and also empty EC16-1 formulated nanoparticles are about
120 nm in size, as determined by dynamic light scattering
(DLS) analysis (Figure 4a). The typical nanoparticular struc-
ture of EC16-1/saporin formulation was visualized by TEM
(Figure 4b). Subsequent zeta-potential analysis of the nano-
particles revealed the positively charged nature of EC16-
1 (7.6 Æ 0.3 mV) and EC16-1/saporin formulations (5.6 Æ
0.2 mV), while EC16-1/RNase A-Aco formulation had a re-
duced zeta-potential of À13.5 Æ 1.4 mV (Figure 4a). The
surface charge measurements of EC16-1/protein nanoformu-
Figure 3. Protein concentration-dependent cytotoxicity of EC16-1 medi-
ated RNase A-Aco (a) and saporin (b) delivery on B16F10 cells (Protein
~
&
only: ; Lipidoid/protein nanoparticle: ). Data are presented as
mean ÆSD (n=4).
saporin show low cytotoxicities at all of the studied concen-
trations. EC16-1/RNase A-Aco and EC16-1/saporin com-
plexes prepared at optimized delivery conditions, however,
had significantly enhanced protein cytotoxicity and displayed
protein-concentration dependence. The half-maximal growth
inhibitory concentration (IC50) of EC16-1/RNase A-Aco and
EC16-1/saporin complexes against B16F10 cells was deter-
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FITC-RNase A-Aco nanoparticles at a lower temperature
(48C) have a significantly reduced nanoparticle uptake
compared to that at 378C (Figure S5). This is an indicative
of endocytosis process of EC16-1/FITC-RNase A-Aco nano-
particles, as endocytosis of nanoparticles is known as an
energy-dependent process. The endocytosized EC16-1/FITC-
RNase A-Aco nanoparticles efficiently escape from endo-
some/lysosome after entering cells. The CLSM imaging
studies by counterstaining endosome/lysosome reveals the
co-localization of EC16-1/FITC-RNase A-Aco nanoparticles
within endosome/lysosome after 4 h of incubation (white
arrows in Figure 5b). However, significant amounts of protein
had already escaped from the endosomal compartment, as
indicated by the green fluorescence throughout the cell.
The efficiency of EC16-1/protein formulations to inhibit
cell proliferation was further determined using B16F10 cells.
The treatment of cells with RNase A-Aco or saporin nano-
formulation inhibited cell proliferation in a protein-dose-
dependent manner (Figure S6). The potential cytotoxicity of
empty EC16-1 nanoparicles was excluded in Figure S7.
Meanwhile, B16F10 cells treated with varying concentrations
of RNase A-Aco or saporin formulations, without EC16-
1 caused only a minor decrease in cell viability, compared to
that of similar formulations containing EC16-1 (Figure S8).
This indicates the vital role that lipidoids play in the
formulation of protein nanoparticles for intracellular delivery.
The IC50 of RNase A-Aco and saporin nanoformulation
against B16F10 cells was determined to be 36.5 nm and
4.2 nm, respectively, with an improvement compared to that
of EC16-1/protein complexes without formulation processes
(Table 1).
Figure 4. Characterization of EC16-1/protein nanoformulations: a) size
and zeta-potential measurements; b) TEM images of EC16-1/saporin
nanoformulation.
lations further confirmed the different complexation modes
of lipidoids with RNase A-Aco and saporin. The charge–
charge interaction between RNase A-Aco and EC16-1 neu-
tralizes the positive charge of the lipidoid nanoparticles, while
saporin is encapsulated into the lipidoid primarily by hydro-
phobic interactions and thus has a minor effect on the surface
charge of EC16-1 nanoparticles.
The cellular uptake and intracellular trafficking of EC16-
1/protein nanoparticles were studied by formulating FITC-
labeled RNase A-Aco (FITC-RNase A-Aco) with EC16-
1 and exposing to B16F10 cells. A confocal laser fluorescence
microscopy (CLSM) imaging study (Figure 5a) reveals that
Finally, in vivo protein delivery using EC16-1/saporin
nanoparticles as a representative protein formulation was
conducted to assess the potential of intracellular delivery of
EC16-1/protein nanoparticles for cancer therapy. First, the
accumulation of EC16-1/saporin nanoformulation at tumor
sites was investigated in a murine breast cancer model. Balb/c
mice brearing 4T1 tumors were intravenously injected with
EC16-1/saporin nanoparticles or free saporin (310 mgkgÀ1 of
saporin). The tumors were harvested 4 h post-injectection for
saporin analysis. Immunohistochemical studies (Figure 6a)
showed significant amounts of saporin accumulation at tumor
sites with EC16-1/saporin nanoformulation injection (as
indicated by the dark regions), while no protein accumulation
was observed for mice treated with free saporin. The in vivo
protein delivery ability of EC16-1/saporin nanoparticles and
saporin accumulation at tumor sites were also observed in
a B16F10 murine melanoma cancer model. As shown in
Figure S9, the intravenous injection of EC16-1/saporin nano-
particles into C57BL/6J mice bearing B16F10 melanoma
tumors resulted in similar accumulation of saporin at the
tumor site.
Figure 5. Confocal laser scanning microscopy (CLSM) images of
B16F10 cells incubated for 4 h with free FITC-RNase A-Aco (a) and
EC16-1/FITC-RNase A-Aco nanoformulations (b). Cells were counter-
stained with DAPI (for nuclei) and LysoTracker Red (for endosome/
lysosomes). Scale bar: 20 mm.
free FITC-RNase A-Aco had a low efficiency of entry of
protein. In contrast, significant cellular uptake and intra-
cellular accumulation of EC16-1/protein nanoparticles were
observed as green fluorescent pinpoints in the CLSM images
of cells treated with the EC16-1/FITC-RNase A-Aco nano-
formulation (Figure 5b). The different uptake of free protein
and EC16-1/FITC-RNase A-Aco nanoparticles was further
confirmed by a flow cytometry analysis (Figure S5). FITC-
RNase A-Aco treated cells had comparable mean fluores-
cence intensity to that of untreated cells, while the cells
exposed to EC16-1/FITC-RNase A-Aco nanoformulation
had significantly enhanced fluorescence intensities, suggest-
ing that the EC16-1 formulation is capable of delivering
proteins. Moreover, B16F10 cells incubated with EC16-1/
The accumulation of EC16-1/saporin nanoformulations at
the tumor sites could be ascribed to the enhanced perme-
ability and retention (EPR) effect of the leaky vascular
structure of the tumor tissue.[15] Having confirmed the
successful and efficient delivery of saporin into tumors,
a comparative tumor growth suppression study was per-
formed in the 4T1 murine breast cancer model. 4T1-tumor-
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(RNase A and saporin), we have found that the intrinsic
physical properties of the proteins (net negative charge and
hydrophobicity) determine the protein–lipidoid interactions
and affect the intracellular delivery efficiency. The lipidoid
that we selected for primary study, EC16-1, is able to deliver
cytotoxic RNase A-Aco and saporin to a panel of cancer cell
lines and inhibit cell proliferation. Furthermore, we devel-
oped several lipidoid/protein formulations that are efficient
for in vitro and in vivo protein delivery. EC16-1/saporin
nanoparticles were shown to accumulate at the tumor site
and they suppressed tumor growth in a murine breast cancer
model. Taken together, these results suggest that combinato-
rially developed lipidoids can be a highly efficient and
effective delivery platform for protein therapeutics. We
believe the results disclosed herein will help advance and
accelerate the clinical translation of protein pharmaceuticals
for cancer therapy.
Received: December 27, 2013
Published online: February 12, 2014
Figure 6. In vivo delivery of EC16-1/saporin nanoformulations into 4T1
breast tumor bearing mice: (a) immunohistochemistry (IHC) studies
of saporin accumulation at tumor sites. Saporin in an uncomplexed
form (left) and in EC16-1/saporin nanoparticles (right), administrated
by tail-vein injection. (b) EC16-1/saporin nanoparticle delivery sup-
presses tumor growth on a 4T1 breast cancer model. Tumor sizes
reported as mean ÆSEM.
Keywords: cancer therapy · lipidoids · nanoparticles ·
.
protein delivery
[1] D. V. Goeddel, D. G. Kleid, F. Bolivar, H. L. Heyneker, D. G.
Yansura, R. Crea, T. Hirose, A. Kraszewski, K. Itakura, A. D.
Riggs, Proc. Natl. Acad. Sci. USA 1979, 76, 106 – 110.
[2] B. Leader, Q. J. Baca, D. E. Golan, Nat. Rev. Drug. Discovery
2008, 7, 21 – 39.
[3] a) G. Walsh, Nat. Biotechnol. 2010, 28, 917 – 924; b) Z. Gu, A.
Biswas, M. X. Zhao, Y. Tang, Chem. Soc. Rev. 2011, 40, 3638 –
3655.
bearing mice were divided into four groups, and each group of
mice received a different treatment (PBS, EC16-1, saporin, or
EC16-1/saporin) by intravenous tail vein injection every three
days for a total of three injections per mouse. Following each
treatment, the tumor growth was monitored by direct caliper
measurement. As shown in Figure 6b, EC16-1 and free
saporin treated groups had similar tumor volumes to that of
PBS control mice. EC16-1/saporin nanoparticle-treated mice,
however, had significantly reduced tumor volumes (by 80%)
compared to the control groups. Mouse body weight were also
monitored during the treatments (Figure S10) to verify that
administration did not result in significant toxicity. No
significant body weight changes during the treatments were
observed nor were a decrease in body condition. Further-
more, the immune response to EC16-1/saporin nanoparticle
injections was monitored by measuring the TNF-a and IFN-g
level in the blood samples of mice (Figure S11) to confirm
that reduction in tumor volume was not a result of immune
stimulation. No significant increase in TNF-a or IFN-g level
were observed in EC16-1/saporin-nanoparticle treated mice
compared to the levels in PBS or free saporin-treated mice.
The above results demonstrate that EC16-1/saporin nano-
particles can deliver protein in vivo and suppress tumor
growth. This suggests that lipidoids are a safe and efficient
protein delivery platform for the delivery of cytotoxic
proteins for cancer therapy.
[4] Z. B. Mi, J. Mai, X. L. Lu, P. D. Robbins, Mol. Ther. 2000, 2, 339 –
347.
[5] a) S. K. Kim, M. B. Foote, L. Huang, Biomaterials 2012, 33,
3959 – 3966; b) O. Zelphati, Y. Wang, S. Kitada, J. C. Reed, P. L.
Felgner, J. Corbeil, J. Biol. Chem. 2001, 276, 35103 – 35110.
[6] J. S. Lee, J. Feijen, J. Controlled Release 2012, 161, 473 – 483.
[7] P. Ghosh, X. Yang, R. Arvizo, Z.-J. Zhu, S. S. Agasti, Z. Mo,
V. M. Rotello, J. Am. Chem. Soc. 2010, 132, 2642 – 2645.
[8] a) A. Akinc, A. Zumbuehl, M. Goldberg, E. S. Leshchiner, V.
Busini, N. Hossain, S. A. Bacallado, D. N. Nguyen, J. Fuller, R.
Alvarez, A. Borodovsky, T. Borland, R. Constien, A. de Fou-
gerolles, J. R. Dorkin, K. N. Jayaprakash, M. Jayaraman, M.
John, V. Koteliansky, M. Manoharan, L. Nechev, J. Qin, T. Racie,
D. Raitcheva, K. G. Rajeev, D. W. Y. Sah, J. Soutschek, I.
Toudjarska, H. P. Vornlocher, T. S. Zimmermann, R. Langer,
D. G. Anderson, Nat. Biotechnol. 2008, 26, 561 – 569; b) Y.-H.
Huang, Y. Bao, W. Peng, M. Goldberg, K. Love, D. A. Bumcrot,
G. Cole, R. Langer, D. G. Anderson, J. A. Sawicki, Proc. Natl.
Acad. Sci. USA 2009, 106, 3426 – 3430; c) K. T. Love, K. P.
Mahon, C. G. Levins, K. A. Whitehead, W. Querbes, J. R.
Dorkin, J. Qin, W. Cantley, L. L. Qin, T. Racie, M. Frank-
Kamenetsky, K. N. Yip, R. Alvarez, D. W. Sah, A. de Fouger-
olles, K. Fitzgerald, V. Koteliansky, A. Akinc, R. Langer, D. G.
Anderson, Proc. Natl. Acad. Sci. USA 2010, 107, 1864 – 1869;
d) D. N. Nguyen, K. P. Mahon, G. Chikh, P. Kim, H. Chung, A. P.
Vicari, K. T. Love, M. Goldberg, S. Chen, A. M. Krieg, J. Chen,
R. Langer, D. G. Anderson, Proc. Natl. Acad. Sci. USA 2012,
109, E797 – E803; e) C. A. Alabi, K. T. Love, G. Sahay, H. Yin,
K. M. Luly, R. Langer, D. G. Anderson, Proc. Natl. Acad. Sci.
USA 2013, 110, 12881 – 12886.
In summary, we have reported that our combinatorially
designed cationic lipidoid nanoparticles function as a novel
protein delivery platform. We synthesized a library of
lipidoids through the mild ring-opening reactions of 1,2-
epoxyhexadecane with primary or secondary aliphatic
amines. Using two representative cytotoxic proteins
Angew. Chem. Int. Ed. 2014, 53, 2893 –2898
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org 2897

.
Angewandte
Communications
[9] a) M. Wang, S. Sun, K. A. Alberti, Q. Xu, ACS Synth. Biol. 2012,
1, 403 – 407; b) S. Sun, M. Wang, S. A. Knupp, Y. Soto-Feliciano,
X. Hu, D. L. Kaplan, R. Langer, D. G. Anderson, Q. B. Xu,
Bioconjugate Chem. 2012, 23, 135 – 140; c) S. Sun, M. Wang,
K. A. Alberti, A. Choy, Q. B. Xu, Nanomed-Nanotechnol. 2013,
9, 849 – 854.
[10] a) Y. Lee, T. Ishii, H. Cabral, H. J. Kim, J. H. Seo, N. Nishiyama,
H. Oshima, K. Osada, K. Kataoka, Angew. Chem. 2009, 121,
5413 – 5416; Angew. Chem. Int. Ed. 2009, 48, 5309 – 5312; b) Y.
Lee, T. Ishii, H. J. Kim, N. Nishiyama, Y. Hayakawa, K. Itaka, K.
Kataoka, Angew. Chem. 2010, 122, 2606 – 2609; Angew. Chem.
Int. Ed. 2010, 49, 2552 – 2555; c) K. Maier, E. Wagner, J. Am.
Chem. Soc. 2012, 134, 10169 – 10173.
[12] J. G. Beitz, P. Davol, J. W. Clark, J. Kato, M. Medina, A. R.
Frackelton, D. A. Lappi, A. Baird, P. Calabresi, Cancer Res.
1992, 52, 227 – 230.
[13] a) M. S. Iordanov, O. P. Ryabinina, J. Wong, T. H. Dinh, D. L.
Newton, S. M. Rybak, B. E. Magun, Cancer Res. 2000, 60, 1983 –
1994; b) J. G. Beitz, P. Davol, J. W. Clark, J. Kato, M. Medina,
A. R. Frackelton, D. A. Lappi, A. Baird, P. Calabresi, Cancer
Res. 1992, 52, 227 – 230; c) L. Polito, M. Bortolotti, M. Pedrazzi,
A. Bolognesi, Toxins 2011, 3, 697 – 720.
[14] a) A. Taheri, R. Dinarvand, F. S. Nouri, M. R. Khorramizadeh,
A. T. Borougeni, P. Mansoori, F. Atyabi, Int. J. Nanomed. 2011,
6, 1863 – 1874; b) J. Su, F. Chen, V. L. Cryns, P. B. Messersmith, J.
Am. Chem. Soc. 2011, 133, 11850 – 11853.
[11] R. T. Raines, Chem. Rev. 1998, 98, 1045 – 1065.
[15] Y. Matsumura, H. Maeda, Cancer Res. 1986, 46, 6387 – 6392.
2898 www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 2893 –2898