A tumor microenvironment (TME)-responsive nanoplatform for systemic saporin delivery and effective breast cancer therapy

Article abstract of DOI:10.1039/d0cc07808e

Intracellular delivery of therapeutic proteins remains a challenge for the success of protein-mediated disease treatment. We herein develop a robust nanoplatform made with a TME-pH responsive Meo-PEG-b-PPMEMA polymer and a cationic lipid-like compoundG0-C14forin vivodelivery of cytotoxic saporin and breast cancer therapy. This nanoplatform could respond to a TME pH to rapidly release saporin/G0-C14complexes, which could significantly improve the uptake of cytosolic saporin by tumor cells and subsequent endosomal escape, thereby leading to an effective inhibition of tumor growth.

Full text of DOI:10.1039/d0cc07808e

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A tumor microenvironment (TME)-responsive
nanoplatform for systemic saporin delivery
and effective breast cancer therapy†
Cite this: Chem. Commun., 2021,
57, 2563
Received 30th November 2020,
Accepted 1st February 2021
Qian Shen,‡a Lei Xu,‡b Rong Li,*a Guang Wu,a Senlin Li,b Phei Er Saw,b
ab
Yusheng Zhou*ac and Xiaoding Xu
*
DOI: 10.1039/d0cc07808e
rsc.li/chemcomm
Intracellular delivery of therapeutic proteins remains a challenge
In the past few decades, nanoparticles (NPs) have emerged
for the success of protein-mediated disease treatment. We herein as an important tool for systemic drug delivery.7 Due to their
develop a robust nanoplatform made with a TME-pH responsive advantages such as long blood circulation and high tumor
Meo-PEG-b-PPMEMA polymer and a cationic lipid-like compound accumulation, various NPs made with cationic lipids, polymers,
G0–C14 for in vivo delivery of cytotoxic saporin and breast cancer or lipid/polymer hybrids have been developed for spatially and
therapy. This nanoplatform could respond to a TME pH to rapidly temporally controlled protein delivery.8–12 Nevertheless, the
release saporin/G0–C14 complexes, which could significantly in vivo therapeutic efficacy of proteins is still unsatisfactory
improve the uptake of cytosolic saporin by tumor cells and subsequent due to their weak uptake and/or slow release in the target cells.
endosomal escape, thereby leading to an effective inhibition of tumor Arising from the distinguishing tumor microenvironment
growth.
(TME) compared to normal tissues, TME-responsive NPs have
been recently designed and developed for effective cancer
Proteins play vital roles in various cellular processes (e.g., signaling therapy. These NPs can respond to a TME signal (e.g., acidic
transduction, metabolism, and gene regulation) and their dysfunc- pH and hypoxia)2 to change their physicochemical properties
tion is widely involved in the development and progression of such as size, surface charge, and hydrophilic–hydrophobic
various diseases, including cancer.1–3 Therefore, protein therapy balance, thereby inducing enhanced diffusion, cellular uptake,
has shown great potential for cancer therapy with the advantages of and/or intracellular cargo release.13–16 At present, TME-responsive
high selectivity, strong activity, and low toxicity.4 However, due to NPs have been successfully applied for the systemic delivery of
their intrinsically vulnerable structure and susceptibility to enzy- chemotherapeutic drugs to achieve a better anticancer effect.
matic degradation, most therapeutic proteins (e.g., enzymes, Nevertheless, few efforts have been made to develop TME-
growth factors, and cytokines) suffer from poor physicochemical/ responsive NPs for in vivo delivery of therapeutic proteins. We
biological stability and potential immunogenicity.5,6 In addition, have previously reported a polymeric NP platform made with TME
when therapeutic proteins function in the cytoplasm, their inter- pH-responsive PEGylated polymer, and demonstrated its feasibility
nalization and biological activity are significantly restricted by poor to improve in vivo small interference RNA (siRNA) delivery
membrane permeability and weak endosomal escape ability. There- efficacy.17 Since both siRNA and protein are biomacromolecules,
fore, the development of an effective protein delivery strategy is this TME pH-responsive polymer may be also applicable for
essential to improve therapeutic outcomes.
systemic protein delivery. However, the anticancer mechanisms
of siRNA and therapeutic protein are totally different, i.e., siRNA
silences protumoral gene expression but therapeutic protein
directly induces cell death; therefore, a technology platform still
needs to be rationally designed to achieve efficient protein delivery
in vivo and effective cancer therapy.
To achieve this goal, we herein report a robust NP platform,
which is composed of our previously reported TME-responsive
PEGylated polymer17 and an amphiphilic cationic lipid-like
compound (alkyl-modified polyamidoamine dendrimer, denoted
as G0–C14),18 for systemic protein delivery and effective cancer
therapy (Fig. 1). After encapsulation of the therapeutic protein,
a Institute of Pharmacy & Pharmacology and the Second Affiliated Hospital,
University of South China, Hengyang 421001, P. R. China.
E-mail: l7979r@163.com, yszhou08@126.com
b Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene
Regulation, Medical Research Center, Sun Yat-sen Memorial Hospital, Sun Yat-sen
University, Guangzhou 510120, P. R. China
c The Affiliated Nanhua Hospital, University of South China, Hengyang 421001,
P. R. China. E-mail: xuxiaod5@mail.sysu.edu.cn
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0cc07808e
‡ These authors contributed equally to this work.
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Fig. 2 (A and B) Morphology (A) and size distribution (B) of the NP50
platform in aqueous solution at pH 7.4. (C) Morphology of the NP50
platform in aqueous solution at pH 6.8. (D) Cumulative saporin release
from the NP50 platform in aqueous solution at pH 7.4 or 6.8.
Fig. 1 Schematic illustration of the TME pH-responsive NP platform for
systemic saporin delivery and breast cancer therapy. After encapsulation of
saporin and then intravenous administration (a), the NPs could extravasate
from the leaky tumor vasculature and accumulate in the tumor tissues
(b). In response to TME pH, the NPs rapidly disassemble to release saporin/
G0–C14 complexes (c), which could be internalized by tumor cells (e) and
then escape from endosomes to cytoplasm (f). With the cytosolic release
of saporin (g), tumor growth is inhibited via saporin-mediated blocking of
the protein synthesis function of ribosome (h).
aqueous saporin solution with a dimethylformamide (DMF)
solution containing Meo-PEG-b-PPMEMA and G0–C14 followed
by adding to rapidly stirred deionized water, well-defined NPs
with a spherical morphology can be formed. In this self-
the resulting NP platform shows the following features for cytosolic assembly system, the negatively charged saporin could form
protein delivery: (i) the hydrophilic PEG outer shell prolongs blood complexes with the cationic lipid-like G0–C14 in the DMF
circulation19 and thereby enhances tumor accumulation via solution via electrostatic interaction with the hydrophobic tails of
the enhanced permeability and retention (EPR) effect;20 (ii) TME G0–C14 positioned on the surface of the complexes.11 When adding
pH-triggered protonation of the hydrophobic poly(2-(pentamethyle- these complexes to deionized water, they could be encapsulated
neimino)ethyl methacrylate) (PPMEMA) segment leads to the rapid into the hydrophobic cores of the self-assembled Meo-PEG-b-
NP dissociation and exposure of protein/G0–C14 complexes that PPMEMA polymer via hydrophobic interaction with the PPMEMA
could enhance protein uptake; (iii) the cationic characteristic of segment.17 We varied the feed composition to adjust the physico-
G0–C14 could induce endosomal swelling through the ‘‘proton chemical properties of the saporin-loaded NPs. As the amount of
sponge’’ effect21 and thus improve endosomal escape of the inter- G0–C14 increases, the resulting NPs (denoted as NP30, NP50, NP75,
nalized protein; and (iv) facile synthesis of the PEGylated polymer and NP100) show increased saporin encapsulation efficiency
and robust NP formulation enables the scale-up of this NP platform. from B30.7% to 87.6% and particle size from B80 to 150 nm
In this work, we chose the cytotoxic protein saporin and system- (Fig. S4, ESI†). One possible reason is that increasing G0–C14
atically evaluated the TME-pH responsive NPs for saporin delivery amount induces stronger electrostatic interaction with saporin,
and its anticancer efficacy. Saporin is a type I ribosome-inactivating leading to the encapsulation of more saporin into the NPs with
protein (RIP) that blocks the protein synthesis function of ribosome larger size.
and induces apoptosis.22 Due to the lack of a receptor, it is difficult
Having successfully constructed the saporin-loaded NPs, we
for saporin to enter cells to implement its biological function. Our next examined their TME pH response using NP50 platform as
results show that systemic saporin delivery with the TME pH- an example. As shown in Fig. 2C, when incubating the NPs in
responsive NPs could efficiently improve saporin uptake by tumor aqueous solution at pH 6.8, the spherical NPs (Fig. 2A) trans-
cells and significantly inhibit tumor growth.
form into large amorphous aggregates and small size particles,
The amphiphilic polymer, methoxyl-poly(ethylene glycol)-b- which may possibly correspond to the ionized polymer and
poly(2-(pentamethyleneimino)ethyl methacrylate) (Meo-PEG- exposed saporin/G0–C14 complexes.25 This morphological
b-PPMEMA), was synthesized via atom-transfer radical change is mainly due to the NP dissociation induced by rapid
polymerization (ATRP) (Fig. S1 and S2, ESI†).17 The pKa value protonation of the Meo-PEG-b-PPMEMA polymer at a pH below
of this polymer was determined to be B6.9 (Fig. S3, ESI†), its pKa (e.g., pH 6.8). The TME pH-triggered rapid NP dissocia-
which is close to the pH of tumor extracellular fluid (6.5–6.8),23 tion is further proven by dynamic light scattering (DLS) analysis
suggesting that a TME pH-responsive protein release could be (Fig. S5, ESI†), in which particles ranging from several
achieved when using a carrier made with this polymer. To verify nanometers to thousand nanometers could be detected after
our hypothesis, the classic nanoprecipitation method was incubating the NP50 platform in aqueous solution at pH 6.8 for
used to prepare the NPs.24 As shown in Fig. 2A, when mixing several minutes. With this rapid NP dissociation, the NP50
2564 | Chem. Commun., 2021, 57, 2563À2566
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platform shows a fast protein release at a TME pH. As shown in 7.4, the percentage of apoptotic cells reaches around 34.5%,
Fig. 2D, more than 60% of the loaded saporin has been released which is more than 3-fold higher compared to the cells treated
within 8 h at pH 6.8. In comparison, less than 20% of the with free saporin. Moreover, because the TME pH-triggered
loaded saporin is released at pH 7.4.
NP dissociation could significantly improve saporin uptake
After validating the TME pH response, we next investigated (Fig. 3A and B), the saporin-mediated apoptosis (B46.6%) is
whether this TME pH-triggered NP dissociation could enhance further enhanced at pH 6.8 (Fig. 3D). With this improved
saporin uptake and improve its anticancer effect. Human apoptosis, the cell death rate reaches B50% (IC50 = 12.8 nM)
breast cancer cells (MDA-MB-231) were incubated with the after 24 h treatment with the saporin-loaded NPs at a saporin
NP50 platform and the saporin uptake was viewed using a concentration of 10 nM (Fig. 3E). However, more than 70% of
confocal laser-scanning microscope (CLSM). As shown in the cells are still alive after 24 h treatment with the NP50
Fig. 3A, the bright red fluorescence demonstrates that the platform at pH 7.4 (IC50 = 36.3 nM). In comparison, there is no
NP50 platform could indeed improve saporin uptake compared obvious difference in the cell viability after 24 h treatment at
to free saporin (Fig. S6, ESI†). More importantly, with the TME pH 7.4 or 6.8 with the saporin-loaded NPs made with the
pH-triggered NP dissociation to expose saporin/G0–C14 com- methoxyl-poly(ethylene glycol)-b-poly(lactic acid-co-glycolic
plexes, MDA-MB-231 cells show a higher saporin uptake at pH acid) (MeO-PEG-b-PLGA) (denoted as PLGA NPs), demonstrat-
6.8 than at pH 7.4. The similar tendency could be also found in ing the importance of the TME pH response to improve the
the flow cytometry analysis (Fig. S7, ESI†), in which the saporin cytotoxicity of saporin. Herein, via consideration of the particle
uptake at pH 6.8 is more than 3-fold higher compared to that of size, zeta potential, saporin encapsulation efficiency, and IC50
the cells treated with the NP50 platform at pH 7.4 (Fig. 3B). It is value, the NP50 platform was selected for the next pharmaco-
noteworthy that these internalized saporin molecules are kinetics (PK) and biodistribution (BioD) experiments due to its
mainly distributed in the cytoplasm (Fig. S8, ESI†), indicating relatively small size, moderate zeta potential, high encapsula-
that encapsulation of saporin into the TME pH-responsive NPs tion efficiency, and low IC50 value (Fig. S9, ESI†).
could enhance its endosomal escape via the ‘‘proton sponge’’
The PK study was conducted via intravenous injection of the
effect.21 With the improved saporin uptake and efficient endo- saporin-loaded NPs into healthy mice (0.5 mg kgÀ1 saporin
somal escape, the NP50 platform shows a stronger ability to dose, n = 3). Compared to free saporin that is rapidly cleared
induce apoptosis compared to free saporin. As shown in Fig. 3C from the blood (Fig. 4A) due to the protection of the PEG outer
and D, for the cells treated with the saporin-loaded NPs at pH layer,19 the NP50 platform shows long blood circulation with a
half-life (t1/2) of around 1.94 h. The BioD study was examined by
intravenous injection of the saporin-loaded NPs into MDA-MB-
231 xenograft tumor-bearing mice (0.5 mg kgÀ1 saporin dose,
n = 3). As shown in Fig. 4B, the NP50 platform shows much
higher accumulation in the tumor tissues compared to free
saporin. This is mainly attributed to the long-circulating char-
acteristic of the NP50 platform to improve its tumor accumula-
tion via the EPR effect.20 The major organs and tumor tissues
were harvested at 24 h post-injection (Fig. S10, ESI†), and the
NP distribution is shown in Fig. 4C. Free saporin mainly
accumulates in the liver and kidney but not tumor tissues. In
comparison, the saporin-loaded NPs show more than 5-fold
higher tumor accumulation compared to free saporin.
We finally evaluated the in vivo anticancer effect of the NP50
platform via intravenous injection of the saporin-loaded NPs
into MDA-MB-231 tumor-bearing nude mice (once every two
days at a 0.5 mg kgÀ1 saporin dose, n = 5). After three
consecutive injections, due to its high accumulation in the
tumor tissues (Fig. 4B), the NP50 platform shows a much
stronger tumor inhibition effect compared to other formula-
tions (Fig. 4D–F). After 16 days post-treatment, there is around
2-fold increase in tumor size (Fig. 4D), which is lower than
Fig. 3 (A and B) CLSM images (A) and mean fluorescence intensity (MFI, B)
the tumor size increase of the mice treated by free saporin
(4.1-fold), saporin-loaded PLGA NPs (3.1-fold), or the TME
pH-responsive NPs without loading of saporin (control NPs,
7.3-fold). The similar tendency could be also found in the
histological analysis of tumor tissues (Fig. 4G), in which the
NP50 platform is the most effective in reducing cell prolifera-
tion. Notably, the administration of the NP50 platform shows
determined by flow cytometry analysis of MDA-MB-231 cells treated with
the NP50 platform at pH 7.4 or 6.8 for 4 h. (C and D) Flow cytometry
analysis (C) and quantification of apoptosis (D) of MDA-MB-231 cells
treated with free saporin or NP50 platform at a saporin concentration of
10 nM for 24 h. **P o0.01; ***P o0.001. (E) Viability of MDA-MB-231 cells
treated with free saporin, NP50 platform or the saporin-loaded PLGA NPs
for 24 h. The TME pH-responsive NPs without loading of saporin were
used as a control.
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for the Care and Use of Laboratory Animals issued by the Ministry
of Science and Technology of China and approved by the Institu-
tional Animal Care and Use Committee at Sun Yat-sen University.
Conflicts of interest
There are no conflicts to declare.
Notes and references
1 F. Chiti and C. M. Dobson, Annu. Rev. Biochem., 2017, 86, 27–68.
2 D. Hanahan and R. A. Weinberg, Cell, 2011, 144, 646–674.
3 C. A. Ross and M. A. Poirier, Nat. Med., 2004, 10, 10–17.
4 B. Leader, Q. J. Baca and D. E. Golan, Nat. Rev. Drug Discovery, 2008,
7, 21–39.
5 D. S. D’Astolfo, R. J. Pagliero, A. Pras, W. R. Karthaus, H. Clevers,
V. Prasad, R. J. Lebbink, H. Rehmann and N. Geijsen, Cell, 2015,
161, 674–690.
6 S. Mitragotri, P. A. Burke and R. Langer, Nat. Rev. Drug Discovery,
2014, 13, 655–672.
7 J. Shi, P. W. Kantoff, R. Wooster and O. C. Farokhzad, Nat. Rev.
Cancer, 2017, 17, 20–37.
8 M. Yu, J. Wu, J. Shi and O. C. Farokhzad, J. Controlled Release, 2016,
240, 24–37.
9 M. Amidi, E. Mastrobattista, W. Jiskoot and W. E. Hennink,
Adv. Drug Delivery Rev., 2010, 62, 59–82.
10 R. Mo, T. Jiang, J. Di, W. Tai and Z. Gu, Chem. Soc. Rev., 2014, 43,
3595–3629.
11 M. Wang, K. Alberti, S. Sun, C. L. Arellano and Q. Xu, Angew. Chem.,
Int. Ed., 2014, 53, 2893–2898.
12 S. Reinhard, H. Han, J. Tuma, J. J. Røise, I. C. Li, J. Li, H. Y. Lee and
N. Murthy, Chem. Commun., 2020, 56, 14207–14210.
13 Y. Lu, A. A. Aimetti, R. Langer and Z. Gu, Nat. Rev. Mater., 2016,
2, 16075.
14 B. Chen, W. Dai, B. He, H. Zhang, X. Wang, Y. Wang and Q. Zhang,
Theranostics, 2017, 7, 538–558.
15 H. Xu, Y. Wang, Z. Pei, W. Ji and Y. Pei, Chem. Commun., 2019, 55,
14930–14933.
16 C. Lin, Y. Tao, P. E. Saw, M. Cao, H. Huang and X. Xu,
Chem. Commun., 2019, 55, 13987–13990.
17 P. E. Saw, H. Yao, C. Lin, W. Tao, O. C. Farokhzad and X. Xu,
Nano Lett., 2019, 19, 5967–5974.
Fig. 4 (A) Blood circulation profile of free saporin and the NP50 platform.
(B) Overlaid fluorescence image of MDA-MB-231 xenograft tumor-bearing
mice at 24 h post-injection of free saporin or the NP50 platform. Tumors
are indicated by ellipses. (C) Biodistribution of free saporin and the NP50
platform in the tumors and major organs of MDA-MB-231 xenograft
tumor-bearing mice sacrificed at 24 h post-injection. (D and E) Tumor
size (D) and weight (E) of MDA-MB-231 xenograft tumor-bearing mice
treated with PBS, free saporin, NP50 platform, saporin-loaded PLGA NPs,
and the TME pH-responsive NPs without loading of saporin (Control NPs).
The intravenous injections are indicated by the arrows. **P o0.01;
***P o0.001. (F) Representative photograph of MDA-MB-231 xenograft
tumor-bearing mice in each group at day 16. Tumors are indicated by
ellipses. (G) H&E staining of MDA-MB-231 tumor tissues after systemic
treatment in each group.
negligible toxicity, as demonstrated by no influence on mouse
body weight (Fig. S11, ESI†) and no noticeable histological
changes in the tissues of heart, liver, spleen, lungs, and kidneys
(Fig. S12, ESI†). This good biocompatibility is further con-
firmed by blood serum analysis, in which TNF-a, IFN-g, IL-6,
and IL-12 levels are within the normal range at 24 h post-
injection (Fig. S13, ESI†).
18 Z. Bi, Q. Li, X. Dinglin, Y. Xu, K. You, H. Hong, Q. Hu, W. Zhang,
C. Li, Y. Tan, N. Xie, W. Ren, C. Li, Y. Liu, H. Hu, X. Xu and H. Yao,
Adv. Sci., 2020, 7, 2000915.
In conclusion, we have developed a new TME pH-responsive
NP platform for systemic saporin delivery and effective cancer
therapy. This NP platform could highly accumulate in the 19 K. Knop, R. Hoogenboom, D. Fischer and U. S. Schubert,
Angew. Chem., Int. Ed., 2010, 49, 6288–6308.
20 H. Maeda, Adv. Drug Delivery Rev., 2015, 91, 3–6.
21 W. He, X. Xing, X. Wang, D. Wu, W. Wu, J. Guo and S. Mitragotri,
tumor tissues and then rapidly respond to TME pH to expose
the saporin/G0–C14 complexes, which subsequently transport
saporin into the cytoplasm to achieve efficient inhibition of
tumor growth. The TME pH-responsive NP platform developed
herein could be used as an effective vehicle for the systemic
Adv. Funct. Mater., 2020, 30, 1910566.
22 I. Barthelemy, D. Martineau, M. Ong, R. Matsunami, N. Ling,
L. Benatti, U. Cavallaro, M. Soria and D. A. Lappi, J. Biol. Chem.,
1993, 268, 6541–6548.
delivery of various cytotoxic proteins (e.g., cytochrome C and 23 Y. Wang, K. Zhou, G. Huang, C. Hensley, X. Huang, X. Ma, T. Zhao,
B. D. Sumer, R. J. DeBerardinis and J. Gao, Nat. Mater., 2014, 13,
204–212.
24 L. Zhang, J. M. Chan, F. X. Gu, J.-W. Rhee, A. Z. Wang, A. F. Radovic-
apoptin) and cancer therapy.
This work was supported by the International Scientific and
Technological Cooperation Program from Guangdong Science and
Technology Department (2018A050506033) and the grant from
Guangzhou Science and Technology Bureau (201902020015). The
animal experiments were conducted in accordance with the Guide
Moreno, F. Alexis, R. Langer and O. C. Farokhzad, ACS Nano, 2008,
2, 1696–1702.
25 X. Xu, P. E. Saw, W. Tao, Y. Li, X. Ji, M. Yu, M. Mahmoudi,
J. Rasmussen, D. Ayyash, Y. Zhou, O. C. Farokhzad and J. Shi,
Nano Lett., 2017, 17, 4427–4435.
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