Acta Pharmaceutica Sinica B 2023;13(3):955e966

Chinese Pharmaceutical Association

Institute of Materia Medica, Chinese Academy of Medical Sciences

Acta Pharmaceutica Sinica B

w w w. e l s ev i e r. c o m / l o c a t e / a p s b
w w w. s c i e n c e d i r e c t . c o m

ORIGINAL ARTICLE

Nanoparticles (NPs)-mediated systemic mRNA

delivery to reverse trastuzumab resistance for
effective breast cancer therapy
Zhihui Donga,b,y, Zhuoshan Huanga,b,y, Senlin Lia,b, Ying Wanga,b,
Yandan Yaoa,b, Xianzhu Yangc, Xiaoding Xua,b,*

a
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, China
b
RNA Biomedical Institute, Sun Yat-sen Memorial Hospital, Sun Yat-sen University, Guangzhou 510120, China
c
School of Biomedical Sciences and Engineering, South China University of Technology, Guangzhou International
Campus, Guangzhou 511442, China

Received 28 May 2022; received in revised form 31 July 2022; accepted 17 August 2022

KEY WORDS Abstract Monoclonal antibody-based therapy has achieved great success and is now one of the most
crucial therapeutic modalities for cancer therapy. The first monoclonal antibody authorized for treating
Monoclonal antibody
human epidermal growth receptor 2 (HER2)-positive breast cancer is trastuzumab. However, resistance
therapy;
Trastuzumab resistance;
to trastuzumab therapy is frequently encountered and thus significantly restricts the therapeutic outcomes.
Nanoparticle; To address this issue, tumor microenvironment (TME) pH-responsive nanoparticles (NPs) were herein
mRNA delivery; developed for systemic mRNA delivery to reverse the trastuzumab resistance of breast cancer (BCa). This
Cancer therapy nanoplatform is comprised of a methoxyl-poly (ethylene glycol)-b-poly (lactic-co-glycolic acid) copol-
ymer with a TME pH-liable linker (Meo-PEG-Dlinkm-PLGA) and an amphiphilic cationic lipid that
can complex PTEN mRNA via electrostatic interaction. When the long-circulating mRNA-loaded NPs
build up in the tumor after being delivered intravenously, they could be efficiently internalized by tumor
cells due to the TME pH-triggered PEG detachment from the NP surface. With the intracellular mRNA
release to up-regulate PTEN expression, the constantly activated PI3K/Akt signaling pathway could be
blocked in the trastuzumab-resistant BCa cells, thereby resulting in the reversal of trastuzumab resistance
and effectively suppress the development of BCa.

*Corresponding author. Tel./fax: þ86 20 81332833.

E-mail address: [email protected] (Xiaoding Xu).
y
These authors made equal contributions to this work.
Peer review under responsibility of Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences.

https://doi.org/10.1016/j.apsb.2022.09.021
2211-3835 ª 2023 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting
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956 Zhihui Dong et al.

ª 2023 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical
Sciences. Production and hosting by Elsevier B.V. This is an open access article under the CC BY-NC-ND license
(http://creativecommons.org/licenses/by-nc-nd/4.0/).

1. Introduction Therapeutics38. Unlike plasmid DNA that shows the potential risk
of genomic integration and mutagenesis39, mRNA does not insert
Since the first approval of monoclonal antibody, i.e., rituximab into the genome and can directly translate into protein in the
(Rituxan), for cancer therapy in 1997, antibody-based therapy has cytoplasm with higher transfection efficiency40e42. However,
achieved great success and is now one of the most important mRNA is a negatively charged biomacromolecule, and therefore
therapeutic modalities to clinically cure cancer patients1e3. The requires specific delivery vehicles to promote its cytosolic de-
fundamental mechanisms of antibody-based cancer therapy livery. In the past decade, various nanoparticles (NPs) have been
include direct interaction with the receptors on tumor cells to widely developed as mRNA delivery carriers42e45. For example,
inhibit downstream biochemical cascades, activation of immune- Shi group recently rationally designed and developed lipid/poly-
mediated tumor cell killing, and specific inhibitory effect on mer hybrid NPs that could efficiently transport mRNA into tumor
tumor vasculature and stroma4e7. Up to now, approximately 100 tissues and significantly up-regulate the expression of tumor
monoclonal antibodies have been clinically approved and most of suppressors (PTEN and P53) in tumor cells32,33,45. The main
them are used for cancer treatment with high specificity and low principle for designing mRNA delivery NPs is to use poly
adverse effect1,7. Among them, trastuzumab is the first approved (ethylene glycol) (PEG) as outer layer because PEG could impair
monoclonal antibody indicated for breast cancer (BCa) identified the nonspecific interaction of NPs with serum components46.
with human epidermal growth factor receptor 2 (HER2), a subtype Unfortunately, this PEGylation could simultaneously impair
of BCa that accounts for 20%e25% of BCa patients that is cellular uptake and thus weaken therapeutic effect47. In recent
associated with poorer prognosis and higher rate of years, arising from the distinguishing microenvironment (e.g.,
recurrence8e10. However, clinical observations have shown that weakly acidic pH and hypoxia) in solid tumors, more researches
many HER2-positive BCa patients can develop into trastuzumab are carried out to develop tumor microenvironment (TME)-
resistance8,11e13. Therefore, in order to formulate effective treat- responsive NPs, which could respond to a TME signal and
ment approaches, it is vital to unravel the underlying processes significantly improve the cellular uptake of loaded cargo48e50. For
leading to trastuzumab resistant. example, based on the hypoxia-responsive characteristic of azo-
It is known that HER2 belongs to the family of erythroblastic benzene (Azo) or TME pH-responsive characteristic of 2,3-
leukemia viral oncogene homolog (ErbB) and can combine with dimethylmaleamidic acid (DMA), Azo- or DMA-linked poly-
other ErbB family members (e.g., HER1, HER3 and HER4) to mers have been elaborately designed and synthesized to construct
form homologous dimers or heterodimers14. This dimerization hypoxia- or TME pH-responsive NPs as the delivery carriers of
could induce the phosphorylation of intracellular domain of HER2 chemotherapeutic drugs and siRNA51e53. Despite showing the
to promote cell proliferation via activating the biochemical cas- advantages aforementioned, limited efforts have been made to
cades downstream of HER2, such as the RAS/Raf/MAPK and develop effective TME-responsive nanoplatform for systemic
PI3K/Akt cascades9,15e17. Because trastuzumab can inhibit the mRNA delivery and cancer therapy.
dimerization via binding the domain IV of HER2 ectodomain18, We herein developed a new TME pH-responsive mRNA de-
the HER2 downstream signaling pathways are suppressed and livery nanoplatform for reversal of trastuzumab resistance and
tumor cell proliferation is thus inhibited. Based on this biological effective BCa therapy. This nanoplatform comprises of a copol-
function, loss of HER2 expression or extracellular domain of ymer methoxyl-poly (ethylene glycol)-b-poly (lactic-co-glycolic
HER2 (e.g., p95-HER2) has been long considered as the main acid) conjugated with a TME pH-liable linker (Meo-PEG-Dlinkm-
reason for trastuzumab resistance11,19e21. Nevertheless, numerous PLGA) and an amphiphilic cationic lipid (G0-C14) that can
recent researches have revealed that some other factors such as complex mRNA via electrostatic interaction. After mRNA loading
tumor microenvironment (TME) and constant activation of HER2 and then intravenous injection, the NPs accumulated in the tumor
downstream signaling pathways play important roles in trastuzu- site could respond to the tumor acidic microenvironment to detach
mab resistance9,22e24. Among them, the PI3K/Akt signaling surface PEG chains52e54, thereby leading to enhanced cellular
pathway could bypass HER2 blockage in a large number of uptake of encapsulated mRNA. With the intracellular mRNA
HER2-positive BCa patients to maintain constant activation25e28. release to up-regulate PTEN expression, the constantly activated
It is known that phosphatase and tensin homologue (PTEN), a PI3K/Akt signaling pathway could be blocked in the trastuzumab-
tumor suppressor gene that is usually lost or mutated in many resistant BCa cells, ultimately accomplish the goal to reverse
cancer types (e.g., prostate, lung and breast cancer), can negatively trastuzumab resistance and effectively inhibit tumor growth of
regulate the PI3K/Akt signaling pathway29e33. Therefore, block- HER2-positive trastuzumab-resistant BCa.
ing the PI3K/Akt signaling pathway via up-regulating PTEN
expression could be considered as an effective strategy to reverse 2. Materials and methods
trastuzumab resistance and accomplish effective BCa therapy.
In recent years, synthetic messenger RNA (mRNA) has been 2.1. Materials
emerging as a therapeutic for a variety of applications, including
vaccine and cancer therapy34e37. The recent advancement in Trastuzumab (Herceptin) was provided by Genentech and used
mRNA-based therapeutics is the mRNA-1273, a vaccine for directly. Meo-PEG5k-Dlinkm-PLGA11k copolymer and G0-C14
coronavirus disease 2019 (COVID-19) developed by the Moderna were synthesized using our published methods54e56. PTEN
Nanoparticles (NPs)-mediated systemic mRNA delivery for cancer therapy 957

mRNA, EGFP mRNA, and fluorescent dye Cy5-labeled mRNA 2.4. In vitro PEG release
(Cy5-EGFP mRNA and Cy5-PTEN mRNA) were purchased from
APExBIO (Houston, TX, USA). PTEN rabbit mAb (#ab32199), The PBS suspension of EGFP Dm-NPs (1 mL) was placed in a
HER2 rabbit mAb (ab214275), Ki67 rabbit mAb (#ab92742), and dialysis tube (MWCO 100 kDa) and then immersed in 10 mL of
GAPDH rabbit mAb (#ab181602) were purchased from Abcam. PBS solution. Subsequently, 10 mL of the NP suspension was
Anti-rabbit IgG horseradish peroxidase (HRP)-linked secondary withdrawn at different time points and then 100 mL of DMSO was
mAb (#7074), Akt (Pan) (C67E7) rabbit mAb (#4691), p-Akt added. After incubating the above mixture with I2 aqueous solu-
(Ser473) rabbit mAb (#4060), and cleaved caspase-3 rabbit mAb tion (20 mL, 0.05 mol/L) and BaCl2 aqueous solution (20 mL, 5 wt
(#9664) were purchased from Cell Signaling Technology (CST). %) for several minutes, the amount of PEG chains was determined
Alexa Fluor 647-conjugated secondary antibody (#A-11011) and by examining the absorbance of BaI2/Meo-PEG5k-Dlinkm-
Click-iT™ TUNEL Colorimetric IHC Detection Kit (#C10625) PLGA11k complexes at 535 nm57.
were obtained from ThermoFisher. Fetal bovine serum (FBS),
penicillin-streptomycin, Dulbecco’s modified Eagle medium 2.5. In vitro mRNA release
(DMEM), and trypsin were obtained from Invitrogen Corp. All
solvents and reagents are of analytical grade and used directly. The PBS suspension of Cy5-EGFP Dm-NPs (1 mL) was placed in
a dialysis tube and then immersed in 10 mL of PBS solution. At
2.2. Patients and tissue samples different time points, 10 mL of the NP suspension was withdrawn
and the residual Cy5-EGFP mRNA in the NPs was determined by
The surgically resected tumors of HER2-positive BCa patients examining its fluorescence intensity. Before the measurement, the
(n Z 8) who received pre-operative trastuzumab treatment and the NP structure was destroyed by mixing the NP suspension with
needle biopsies of recurrent tumors of the matched patients DMSO at a volume ratio of 1/20.
received standard post-operative trastuzumab treatment (May
2016 and Oct 2019) were collected from the Breast Tumor Center 2.6. Cell culture
of Sun Yat-sen Memorial Hospital. All pathological diagnosis,
including Ki67, and other biomarkers were independently verified Human HER2-positive breast cancer SKBR3 and BT474 cells
by two experienced pathologists. All tumor samples were (denoted SKBR3-DS and BT474-DS) were cultured in DMEM
collected with informed consents from the donors according to the and RPMI 1640 medium with 10% FBS at 37  C in a humidified
International Ethical Guidelines for Biomedical Research atmosphere containing 5% CO2, respectively. Trastuzumab-
Involving Human Subjects (CIOMS). The study was approved by resistant SKBR3 and BT474 cells (denoted SKBR3-DR and
the Institutional Review Board (IRB) of Sun Yat-sen Memorial BT474-DR) were constructed according to the protocol described
Hospital. in our previous study58. SKR3-DR cells were cultured in
trastuzumab-containing DMEM medium (10 mg/mL) in a 37  C
2.3. Preparation and characterizations of mRNA-loaded NPs cell culture chamber with 5% CO2. BT474-DR cells were cultured
in trastuzumab-containing RPMI 1640 medium (5 mg/mL) in a
Nanoprecipitation method was used to prepare the mRNA-loaded similar environment.
NPs. Briefly, 20 mg of Meo-PEG5k-Dlinkm-PLGA11k was dis-
solved in 1 mL of dimethylformamide (DMF). In parallel, EGFP 2.7. Cellular uptake
mRNA (10 mg, 1 mg/mL in deionized water) and G0-C14 (50 mL,
5 mg/mL in DMF) were prepared and mixed with the above Meo- SKBR3-DR and BT474-DR cells were seeded in round-plate at a
PEG5K-Dlinkm-PLGA11K solution. The mixture was added drop- density of 50,000 cells per plate. After 24 h incubation, Cy5-
wise to RNase-free water (5 mL) with continuous stirring EGFP Dm-NPs were added at an mRNA dose of 0.7 mg/mL, and
(1000 rpm). The obtained NP suspension was then transferred to a the cells were allowed to incubate in 2 mL of medium at pH 7.4
dialysis tube (EMP Millipore, MWCO 100 K) for purification via and 6.5, respectively. After 4 h incubation, the cells were viewed
centrifugation (Eppendorf, 2800 rpm  10 min). After three times under a confocal laser-scanning microscope (CLSM, ZEISS 800).
of PBS washing (3  5 mL), the final NPs (denoted EGFP Dm- To further quantitatively examine the cellular uptake, SKBR3-DR
NPs) were suspended in PBS solution at an mRNA concentration and BT474-DR cells were also seeded and then incubated in 2 mL
of 10 mg/mL. The non-pH-responsive NPs loading with EGFP of medium containing Cy5-EGFP Dm-NPs at pH 7.4 and 6.5,
mRNA (PLGA NPs) were also prepared. Dynamic light scattering respectively. After 4 h incubation, the cells were trypsinized and
(DLS, Malvern, USA) was used to examine the NP size and zeta collected for flow cytometry analysis (BD FACSAria™ III).
potential. The NP morphology was visualized on a Tecnai trans-
mission electron microscope (TEM). To examine encapsulation 2.8. In vitro mRNA transfection
efficiency (EE%) of mRNA, Cy5-EGFP mRNA-loaded NPs were
prepared and the fluorescence intensity of Cy5-EGFP mRNA SKBR3-DR and BT474-DR cells in 6-well plate (50,000 cells per
encapsulated into the NPs was measured by BioTek microplate well) were incubated with EGFP Dm-NPs at different mRNA
reader. Before the measurement, the NP structure was destroyed doses. Twenty-four hours later, the culture medium was changed
by mixing the NP suspension with dimethyl sulfoxide (DMSO) at with fresh medium and EGFP expression in the cells was exam-
a volume ratio of 1/20. The EE% of mRNA was determined by ined by CLSM and flow cytometry after 48 h incubation. To
comparing fluorescence intensity of Cy5-EGFP mRNA to its examine the in vitro PTEN mRNA transfection, the PTEN mRNA-
standard curve. Through adjusting the feed amount, the EGFP loaded NPs (denoted PTEN Dm-NPs) were prepared by changing
mRNA-loaded NPs with an mRNA encapsulation efficiency of the EGFP mRNA with PTEN mRNA, and their size and
w80% and average size of w100 nm were used for the following morphology were respectively examined by DLS and TEM. The
experiments. EE% of PTEN mRNA (w80%) was determined by encapsulating
958 Zhihui Dong et al.

Cy5-PTEN mRNA into the NPs followed by examining the 2.15. In vivo PTEN mRNA transfection
fluorescence intensity of Cy5. Subsequently, SKBR3-DR and
BT474-DR cells were transfected with the PTEN Dm-NPs at an EGFP Dm-NPs and PTEN Dm-NPs were intravenously injected
mRNA dose of 0.7 mg/mL according to the method of EGFP into the SKBR3-DR tumor-bearing mice (n Z 3, 10 mg of mRNA
mRNA transfection described above, and PTEN expression was per mouse), respectively. After three daily injections, the mice
examined by immunofluorescence (IF) and Western blot. were euthanized at 24 h post the final injection and tumors were
collected for the detection of PTEN expression using Western blot
2.9. Cytotoxicity and immunohistochemistry (IHC).

To determine cell cytotoxicity, SKBR3-DR and BT474-DR cells 2.16. Inhibition of tumor growth
in 96-well plate (5000 cells per well) were transfected with the
PTEN Dm-NPs at an mRNA dose of 0.7 mg/mL. Subsequently, the SKBR3-DR orthotopic tumor-bearing mice were randomly
cells were further incubated with trastuzumab at different con- divided into five groups (n Z 5) and received intravenous injec-
centrations. After 72 h incubation, the cell viability was examined tion of either (i) PBS, (ii) trastuzumab (10 mg/kg), (iii) EGFP Dm-
by AlamarBlue assay. NPs (10 mg of mRNA per mouse), (iv) PTEN Dm-NPs (10 mg of
mRNA per mouse), or (v) trastuzumab (10 mg/kg) and PTEN Dm-
2.10. In vitro cell proliferation NPs (10 mg of mRNA per mouse) once every two days. After all
administration, mice were continuously monitored for weight loss
SKBR3-DR and BT474-DR cells in 6-well plate (30,000 cells/cell and tumor growth. Measurement of tumor was taken by measuring
line) were transfected with the PTEN Dm-NPs at an mRNA dose perpendicular diameters using a caliper and tumor volume was
of 0.7 mg/mL. Subsequently, the cells were further incubated with calculated as Eq. (1):
trastuzumab (1/10 of the half maximal inhibitory concentration 
V ZW2  L 2 ð1Þ
calculated from the cytotoxicity assay) at different times and the
cell viability was determined using AlamarBlue assay. where W and L are the shortest and longest diameters, respectively.
At the end of observation, the tumor tissues were harvested for
2.11. Animals Ki67, cleaved-caspase-3, and TUNEL staining according to the
manufactory’s protocol.
Healthy female mice (4e5 weeks old) were purchased from the
Sun Yat-sen University Experimental Animal Center. All in vivo 2.17. Statistical analysis
studies were performed in designated animal facility in accor-
dance with the rules and regulation of the Institutional Animal Statistical significance was determined by a two-tailed Student’s t
Care and Use Committee at Sun Yat-sen University. test assuming equal variance. A P value < 0.05 is considered
statistically significant.
2.12. Pharmacokinetics study
3. Results and discussion
Free Cy5-PTEN mRNA and Cy5-PTEN Dm-NPs were intrave-
nously injected into healthy female BALB/c mice (n Z 3, 10 mg 3.1. Detection of PTEN expression in HER2-positive BCa
of mRNA per mouse), respectively. At predetermined time points, patients and cells
20 mL of orbital vein blood was withdrawn and mixed with 80 mL
of deionized water. The Cy5-PTEN mRNA in the mixture was To examine the correlation between the level of PTEN expression
determined via examining its fluorescence intensity. and trastuzumab resistance, we established trastuzumab-resistant
HER2-positive BCa cells (SKBR3 and BT474) and then examined
2.13. Construction of orthotopic tumor-bearing mouse model PTEN expression in these cells. The results of IF staining (Fig. 1A)
and Western blot (Supporting Information Fig. S1) show that
Orthotopic tumor-bearing mouse model was constructed by sub- SKBR3-DR and BT474-DR cells show high HER2 expression.
cutaneous injection with the mixture of DMEM medium con- However, both of them are apparently resistant to trastuzumab
taining SKBR3-DR cell and Matrigel in 1/1 volume ratio (200 mL treatment (Fig. 1B). The half maximal inhibitory concentration
with a cell density of 1  107 cells/mL) into the second pair of (IC50) values of trastuzumab against SKBR3-DR and BT474-DR
mammary fat pads of healthy female nude mice. The tumor- cells are around 739.5 and 50.7 mg/L (Supporting Information
bearing mice were used for the following in vivo experiments Table S1), respectively, which are more than 5- and 20-fold higher
when the tumor size reached w100 mm3. than that of SKBR3-DS (w134.5 mg/L) and BT474-DS cells
(w2.52 mg/L). The results of Western blot analysis show that PTEN
2.14. Biodistribution expression is extremely low in both SKBR3-DR and BT474-DR
cells (Fig. 1C) while the PI3K/Akt signaling pathway is activated,
Free Cy5-PTEN mRNA and Cy5-PTEN Dm-NPs were intrave- as demonstrated by the enhanced phosphorylated Akt (p-Akt)
nously injected into the SKBR3-DR tumor-bearing mice (n Z 3, expression. To further validate the correlation between PTEN
10 mg of mRNA per mouse), respectively. Twenty-four hours post expression and trastuzumab resistance, the surgically resected
injection, whole mouse images were observed using an IVIS tumor samples of HER2-positive BCa patients (n Z 8, Supporting
Lumina III (PerkinElmer) imaging system. Major organs and tu- Information Table S2) received pre-operative trastuzumab treat-
mors were also collected and imaged. Quantification of Cy5- ment (denoted trastuzumab-sensitive) and recurrent tumor tissues of
PTEN mRNA in tumors and organs was determined by Image-J. matched patients revived post-operative trastuzumab treatment
Nanoparticles (NPs)-mediated systemic mRNA delivery for cancer therapy 959

Figure 1 (A) IF analysis of HER2 expression on the trastuzumab-sensitive (SKBR3-DS and BT474-DS) and trastuzumab-resistant HER2-
positive BCa cells (SKBR3-DR and BT474-DR). (B) Viability of trastuzumab-sensitive and -resistant HER2-positive BCa cells incubated
with trastuzumab at different concentrations (mean  SD; n Z 5). (C) Western blot analysis of PTEN, total Akt, and p-Akt expression in the
trastuzumab-sensitive and -resistant HER2-positive BCa cells. (D) IHC analysis of PTEN expression in the surgically resected tumor tissues of
HER2-positive BCa patients (n Z 8) received pre-operative trastuzumab treatment (Trastuzumab-sensitive) and their recurrent tumor tissues post-
operative trastuzumab treatment (Trastuzumab-resistant). Scale bar, 100 mm.

(denoted trastuzumab-resistant) were collected, and PTEN expres- efficacy using EGFP mRNA as a model mRNA. The TME pH-
sion was examined by IHC. As shown in Fig. 1D, PTEN expression responsive block copolymer Meo-PEG-Dlinkm-PLGA and cationic
level in the trastuzumab-sensitive tumors is much higher than that in lipid G0-C14 we previously developed54,55 were used to prepare
the trastuzumab-resistant tumors, which is similar as the previous the mRNA-loaded NPs. For this system, the amphiphilic cationic
reports that PTEN expression is down-regulated in tumor tissues of G0-C14 could complex negatively charged mRNA in the DMF
trastuzumab-resistant HER2-posotive BCa patients compared to the solution with the hydrophobic alkyl chains positioned on the
trastuzumab-sensitive patients58,59. All these results indicate that surface of G0-C14/mRNA complexes. After adding the DMF
low PTEN expression and activated PI3K/Akt signaling pathway mixture of G0-C14/mRNA complexes and Meo-PEG-Dlinkm-
are closely associated with the trastuzumab resistance and up- PLGA into water, the complexes will be entrapped into the hy-
regulation of PTEN expression could be expected to recover the drophobic cores of NPs via hydrophobic interaction with PLGA
sensitivity of HER2-positive BCa to trastuzumab treatment. segment (Scheme 1)55,60,61. As shown in Fig. 2A and B, through
adjusting the amount of Meo-PEG-Dlinkm-PLGA (Supporting
3.2. Preparation and characterization of mRNA-loaded NPs Information Table S3), well-defined spherical NPs with an
mRNA encapsulation efficiency of w80% and average size of
Having validated the low PTEN expression in the trastuzumab- w100 nm could be obtained, and these NPs show good stability at
resistant tumor samples of BCa patients and cells, we next con- the physiological pH of 7.4 (Supporting Information Fig. S2).
structed mRNA-loaded NPs and evaluated their gene transfection More importantly, because the amide bond of linker between PEG
960 Zhihui Dong et al.

Scheme 1 Schematic illustration of TME pH-responsive mRNA delivery NP platform for the reversal of trastuzumab resistance and effective
BCa therapy. In this NP platform, the amphiphilic cationic lipid-like compound G0-C14 could bind PTEN mRNA to form G0-C14/mRNA
complexes (a), which could be then embedded into the hydrophobic cores of NPs made with the TME pH-responsive block copolymer Meo-PEG-
Dlinkm-PLGA (b). After intravenous administration (c), the PTEN mRNA-loaded NPs could extravasate from leaky tumor vasculature and
accumulate in the tumor tissue. With the TME pH-triggered detachment of PEG chains from the NP surface (d ), the encapsulated PTEN mRNA
could be efficiently internalized by trastuzumab-resistant HER2-positive BCa cells (e, f ), which could thus block the activity of PI3K/Akt
signaling pathway via up-regulating PTEN expression (g), leading to the reversal of trastuzumab resistance (h) and significant inhibition of tumor
growth of trastuzumab-resistant HER2-positive BCa.

and PLGA chains is acid liable (Scheme 1)52e54, the PEG chains SKBR3-DR and BT474-DR cells at an EGFP mRNA dose of
could be gradually removed from the surface of EGFP Dm-NPs at 0.7 mg/mL. This efficient gene transfection at pH 6.5 is further
a TME pH of 6.5 (Fig. 2C), thereby leading to much faster mRNA confirmed by CLSM (Fig. 2H), in which much brighter green
release compared to the physiological pH of 7.4 (Fig. 2D). fluorescence corresponding to EGFP could be observed in
After the successful preparation of mRNA-loaded NPs and SKBR3-DR and BT474-DR cells incubated with the EGFP Dm-
validation of their TME pH response, we next evaluated whether NPs at pH 6.5. To further demonstrate that the efficient gene
this nanoplatform could use its TME pH-responsive characteristic transfection at pH 6.5 is mainly attributed to the TME pH-
to enhance the cellular uptake and gene transfection of encapsu- triggered PEG chain detachment from the Dm-NPs, we encapsu-
lated mRNA. To this end, Cy5-EGFP Dm-NPs were incubated lated EGFP mRNA into the NPs made with the commercial
with SKBR3-DR and BT474-DR cells at pH 6.5 or 7.4. Compared available polymer Meo-PEG-b-PLGA without pH-liable linker
to the cells incubated with the Cy5-EGFP Dm-NPs at pH 7.4 and then examined their gene transfection efficacy. The results of
(Fig. 2E), the stronger red fluorescence corresponding to Cy5- flow cytometry analysis and CLSM observation show that EGFP
EGFP mRNA indicates that more mRNA-loaded NPs could be expression is very weak in SKBR3-DR and BT474-DR cells at
internalized at pH 6.5. The result of flow cytometry analysis shows both pH 7.4 and 6.5 (Supporting Information Fig. S5). This result
the similar tendency (Fig. 2F), in which both SKBR3-DR and strongly demonstrates that the Dm-NPs could use their TME pH-
BT474-DR cells have more than 4-fold higher uptake of Cy5- triggered PEG chain detachment characteristic to promote the
EGFP Dm-NPs at pH 6.5 than that at pH 7.4. All these results cellular uptake and gene transfection efficacy of encapsulated
indicate that the detachment of PEG chains from the Dm-NPs at mRNA.
pH 6.5 could indeed enhance the cellular uptake of encapsulated
mRNA. With this improved cellular uptake and efficient endo- 3.3. In vitro up-regulation of PTEN expression to reverse
somal escape (Supporting Information Fig. S3), the Dm-NPs could trastuzumab resistance
dramatically promote the gene transfection of encapsulated EGFP
mRNA. As displayed in Fig. 2G, much higher percentage of Having validated the efficient gene transfection mediated by Dm-
EGFP-positive cells with stronger EGFP fluorescence (Supporting NPs, we next encapsulated PTEN mRNA into this nanoplatform
Information Fig. S4) could be detected in the flow cytometry and investigated whether it can reverse trastuzumab resistance via
analysis of cells incubated with the EGFP Dm-NPs at pH 6.5. In up-regulating PTEN expression. Notably, the Dm-NPs show the
particular, EGFP could be expressed in more than 80% of both similar ability to encapsulate EGFP or PTEN mRNA and the
Nanoparticles (NPs)-mediated systemic mRNA delivery for cancer therapy 961

Figure 2 (A, B) Size distribution (A) and TEM image (B) of EGFP Dm-NPs in aqueous solution at pH 7.4. (C, D) Cumulative PEG chain
release profile of EGFP Dm-NPs (C) and mRNA release profile (D) of Cy5-EGFP Dm-NPs incubated in aqueous solution at pH 7.4 and 6.5,
respectively. mean  SD; n Z 5. (E) CLSM images of SKBR3-DR and BT474-DR cells incubated with the Cy5-EGFP Dm-NPs at pH 7.4 and 6.5
for 4 h, respectively. Scale bar, 30 mm. (F) Flow cytometry profile and mean fluorescence intensity (MFI) of SKBR3-DR and BT474-DR cells
incubated with the Cy5-EGFP Dm-NPs at pH 7.4 and 6.5 for 4 h, respectively. (G) Flow cytometry analysis of EGFP expression in SKBR3-DR
and BT474-DR cells treated with the EGFP Dm-NPs at different EGFP mRNA doses. (H) CLSM images of SKBR3-DR and BT474-DR cells
treated with the EGFP Dm-NPs at an EGFP mRNA dose of 0.7 mg/mL. Scale bar, 30 mm.

resulting PTEN Dm-NPs have an average size of w100 nm and EE Information Fig. S7). Similarly, from the result of IF analysis
% of PTEN mRNA of w80% (Supporting Information Fig. S6). (Fig. 3B), much brighter red fluorescence corresponding to PTEN
More importantly, these PTEN Dm-NPs could significantly up- could be observed when treating the cells with the PTEN Dm-NPs
regulate PTEN expression is in both SKBR3-DR and BT474-DR at pH 6.5. Due to the up-regulation of PTEN expression demon-
cells at an mRNA dose of 0.7 mg/mL (Fig. 3A). In addition, due strated above, the p-Akt expression is significantly suppressed
to the TME pH-triggered PEG detachment from the surface of (Fig. 3A), demonstrating the important biological function of
Dm-NPs to improve cellular uptake (Fig. 2E and F), the cells show PTEN to block the activity of PI3K/Akt signaling pathway31e33.
around 3-fold stronger PTEN expression after treatment with the After verifying the ability of PTEN mRNA-loaded NPs to up-
PTEN Dm-NPs at pH 6.5 compared to pH 7.4 (Supporting regulate PTEN expression and block the activity of PI3K/Akt
962 Zhihui Dong et al.

Figure 3 (A) Western blot analysis of PTEN, total Akt, and p-Akt expression in SKBR3-DR and BT474-DR cells treated with the PTEN Dm-
NPs or EGFP Dm-NPs at an mRNA dose of 0.7 mg/mL. (B) CLSM images of SKBR3-DR and BT474-DR cells treated with the PTEN Dm-NPs at a
PTEN mRNA dose of 0.7 mg/mL. Scale bar, 30 mm. (C) Viability of SKBR3-DR and BT474-DR cells treated with the PTEN Dm-NPs or EGFP
Dm-NPs at an mRNA dose of 0.7 mg/mL followed by trastuzumab at different concentrations. mean  SD; n Z 5. (DeF) Proliferation profile (D,
mean  SD; n Z 5), colony formation assay (E), and clone number (F) of SKBR3-DR and BT474-DR cells treated with trastuzumab, PTEN Dm-
NPs at an mRNA dose of 0.7 mg/mL, EGFP Dm-NPs at an mRNA dose of 0.7 mg/mL followed by trastuzumab, or PTEN Dm-NPs at an mRNA
dose of 0.7 mg/mL followed by trastuzumab. ns, no significance; *P < 0.05.

signaling pathway, we next investigated whether this NPs- followed by trastuzumab. The above results indicate that the NPs-
mediated PTEN up-regulation could reverse trastuzumab resis- mediated PTEN up-regulation could efficiently reverse trastuzu-
tance. To this end, SKBR3-DR and BT474-DR cells were treated mab resistance via blocking the activity of PI3K/Akt signaling
with the PTEN Dm-NPs and then further incubated with trastu- pathway.
zumab. As shown in Fig. 3C, the up-regulation of PTEN expres-
sion could indeed reverse the trastuzumab resistance of SKBR3- 3.4. In vivo mRNA transfection and inhibition of trastuzumab-
DR and BT474-DR cells. In particular, compared to the cells resistant tumor growth
incubated with the PTEN Dm-NPs at pH 7.4, the treatment with
the mRNA-loaded NPs at pH 6.5 is much more effective to Having confirmed the ability of Dm-NPs to up-regulate PTEN
enhance the cytotoxicity of trastuzumab, leading to more than 5- expression and reverse trastuzumab resistance in vitro, we next
or 16-fold decrease in the IC50 value of trastuzumab (Supporting examined whether this nanoplatform could efficiently deliver
Information Table S4) against SKBR3-DR (from 739.5 to PTEN mRNA into the tumor tissues to up-regulate PTEN
139.4 mg/L) or BT474-DR cells (from 50.7 to 3.1 mg/L). With expression in vivo. The pharmacokinetics was first examined. As
this improved sensitivity to trastuzumab, the proliferation of displayed in Fig. 4A, naked mRNA is rapidly cleared and only
SKBR3-DR and BT474-DR cells is significantly suppressed around 1% of residual mRNA could be detected in the blood at
(Fig. 3D). Within an evaluation period of 6 days, around 2-fold 30 min post injection. In contrast, with the presence of PEG outer
increase in the cell number could be found after treatment of shell46, the Cy5-PTEN Dm-NPs show much longer blood circu-
the cells with the PTEN Dm-NPs at pH 6.5 followed by trastu- lation time and around 10% of injected NPs remains in the blood
zumab. In contrast, the cells proliferate by around 4- or 6-fold at 8 h post injection. With this long-circulating characteristic, the
after treatment with the PTEN Dm-NPs or EGFP Dm-NPs at pH Cy5-PTEN Dm-NPs could highly accumulate in the tumor tissues
7.4 followed by trastuzumab. Similarly in the clone formation via the enhanced permeation and retention (EPR) effect after
assay (Fig. 3E and F), the cells show the weakest ability to form intravenous injection into the SKBR3-DR orthotopic tumor-
colonies after treatment with the PTEN Dm-NPs at pH 6.5 bearing mice (10 mg of mRNA per mouse, n Z 3) (Fig. 4B)62.
Nanoparticles (NPs)-mediated systemic mRNA delivery for cancer therapy 963

Figure 4 (A) Blood circulation profile of naked Cy5-PTEN mRNA and Cy5-PTEN Dm-NPs. mean  SD; n Z 3. (B) Overlaid fluorescence
image of SKBR3-DR orthotopic tumor-bearing mice at 24 h post injection of naked Cy5-PTEN mRNA or Cy5-PTEN Dm-NPs. Tumors are
indicated by ellipses. (C) Biodistribution of naked Cy5-PTEN mRNA and Cy5-PTEN Dm-NPs in the tumors and major organs of SKBR3-DR
orthotopic tumor-bearing mice sacrificed at 24 h post injection. mean  SD; n Z 3. (DeF) Western blot analysis (D, E) of PTEN, total Akt,
and p-Akt expression and IHC analysis (F) of PTEN expression in the tumor tissues of SKBR3-DR orthotopic tumor-bearing mice treated with the
EGFP Dm-NPs or PTEN Dm-NPs. Scale bar, 100 mm; **P < 0.01; ***P < 0.001.

Through examining the fluorescence intensity of collected tumor We finally assessed whether the NPs-mediated PTEN up-
tissues (Supporting Information Fig. S8), the Cy5-PTEN Dm-NPs regulation could enhance the anticancer effect of trastuzumab by
show around 10-fold stronger tumor accumulation than that of intravenous injection of PTEN Dm-NPs into the mice bearing
naked mRNA (Fig. 4C). Notably, the release of loaded mRNA SKBR3-DR orthotopic tumors (Fig. 5A). As shown in Fig. 5BeD,
from the NPs (w30% of accumulative release within 24 h, due to the ability to block the activity of PI3K/Akt signaling
Fig. 2D) might be the reason for the observed higher accumulation pathway32,33, the single administration of PTEN Dm-NPs could
of Cy5-PTEN Dm-NPs in the kidney than liver in Fig. 4C. To inhibit the tumor growth to some extent. More importantly,
examine whether these NPs accumulated in the tumor tissues because this NPs-mediated PTEN up-regulation could reverse
could respond to the TME acidic pH to enhance mRNA uptake by trastuzumab resistance, the tumor growth is significantly sup-
tumor cells, we further established orthotopic tumor model using pressed when combining the PTEN Dm-NPs with trastuzumab
the EGFP-expressing SKBR3-DR cells and then intravenously treatment (10 mg/kg). Within 22 days, the tumor size increases by
injected the Cy5-PTEN Dm-NPs into the tumor-bearing mice less than 3-fold (from 90 to 230 mm3). In contrast, for mice
(10 mg of mRNA per mouse, n Z 3). As expected, compared to treated with the PTEN Dm-NPs or trastuzumab alone, a 6- or 13-
the non-pH-responsive PLGA NPs, the EGFP-expressing SKBR3- fold increase was seen in their tumor growth (Supporting
DR cells show much stronger uptake of Cy5-PTEN Dm-NPs Information Fig. S10). This enhanced anticancer effect is further
(Supporting Information Fig. S9). This result clearly demonstrates confirmed by histological analysis (Fig. 5E), in which the com-
that the Dm-NPs could respond to the TME pH to improve the bination of PTEN Dm-NPs and trastuzumab is much effective than
uptake of loaded mRNA by tumor cells in vivo. We next assessed other therapeutic formulas in suppressing proliferation (decreased
the ability of Dm-NPs to in vivo up-regulate PTEN expression. As Ki67 staining) and inducing apoptosis (cleaved-caspase-3 and
shown in Fig. 4D and E, after three daily injections, PTEN TUNEL-staining) in the tumor tissues (Supporting Information
expression is up-regulated by around 5-fold in the tumor tissues Fig. S11). Notably, the administration of PTEN Dm-NPs does
compared to the mice treated with the EGFP Dm-NPs. Similarly, not influence the mouse weight (Supporting Information Fig. S12)
much stronger PTEN expression in the IHC staining could be and no apparent histological change could be observed in the
observed in the tumor tissues of mice received the treatment with tissues of major organs (Supporting Information Fig. S13),
the PTEN Dm-NPs (Fig. 4F). More importantly, with this up- implying the low in vivo toxicity of PTEN Dm-NPs. This result is
regulated PTEN expression, the activity of PI3K/Akt signaling further verified by blood routine analysis (Supporting Information
pathway is blocked, as demonstrated by the decreased p-Akt Fig. S14), in which the main parameters of liver and kidney
expression in the tumor tissues (Fig. 4D and E). function are in the normal range.
964 Zhihui Dong et al.

Figure 5 (A) Schematic illustration of SKBR3-DR orthotopic tumor-bearing mice treated with PBS, trastuzumab, EGFP Dm-NPs, PTEN Dm-
NPs, or the combination of trastuzumab and PTEN Dm-NPs. (BeD) Tumor size (B), tumor weight (C), and photograph of collected tumors (D) of
SKBR3-DR orthotopic tumor-bearing mice after systemic treatment in each group. mean  SD; n Z 5. (E) Ki67, cleaved caspase-3 (C-caspase-
3), and TUNEL staining of SKBR3-DR orthotopic tumor tissues after systemic treatment in each group. Scale bar, 100 mm; ns, no significance;
***P < 0.001.

4. Conclusions Acknowledgments

In summary, we have developed a new TME pH-responsive mRNA This work was supported by the National Natural Science Foun-
delivery NP platform for the reversal of trastuzumab resistance and dation of China (82173392 and 81874226), the Thousand Talents
BCa therapy. This NP platform could respond to tumor acidic Program for Distinguished Young Scholars, the International
microenvironment to dramatically improve PTEN mRNA uptake by Scientific and Technological Cooperation Program from Guang-
tumor cells. With the up-regulation of PTEN expression, the dong Science and Technology Department (2018A050506033,
constantly activated PI3K/Akt signaling pathway in the China), the Natural Science Foundation of Guangdong Province
trastuzumab-resistant HER2-positive BCa cells could be effectively (2019B1515120006, China), and Guangzhou Science and Tech-
blocked, inducing the reversal of trastuzumab resistance and dra- nology Bureau (201902020015 and 20210303004, China), and the
matic suppression of tumor growth of trastuzumab-resistant HER2- “Three million for Three Years” Project of the High-level Talent
positive BCa. This new mRNA delivery nanoplatform could be Special Funding Scheme of Sun Yat-sen Memorial Hospital.
employed to up-regulate other tumor suppressors such as P53 and
RB1 for cancer treatment. However, unlike lipid nanoparticles Author contributions
(LNPs) that are the preferential tools for clinical mRNA delivery
due to their mature synthesis technology and good reproducibility, Xiaoding Xu, Zhihui Dong, and Zhuoshan Huang conceived and
much more efforts need to be paid to promote the clinical translation designed the experiments. Zhihui Dong, Zhuoshan Huang, and
of TME-responsive NPs-mediated mRNA delivery. Senlin Li performed the experiments. Xiaoding Xu, Zhihui Dong,
Nanoparticles (NPs)-mediated systemic mRNA delivery for cancer therapy 965

Zhuoshan Huang, Ying Wang, Yandan Yao, and Xianzhu Yang length receptor is associated with nodal metastasis in human breast
analyzed the data and co-wrote the paper. Zhihui Dong and cancer. Clin Cancer Res 2002;8:347e53.
Zhuoshan Huang contributed equally to this work. All the authors 20. Lennon S, Barton C, Banken L, Gianni L, Marty M, Baselga J, et al.
have read and approved the final manuscript. Utility of serum HER2 extracellular domain assessment in clinical
decision making: pooled analysis of four trials of trastuzumab in
metastatic breast cancer. J Clin Oncol 2009;27:1685e93.
Conflicts of interest 21. Mittendorf EA, Wu Y, Scaltriti M, Meric-Bernstam F, Hunt KK,
Dawood S, et al. Loss of HER2 amplification following trastuzumab-
The authors have no conflicts of interest to declare. based neoadjuvant systemic therapy and survival outcomes. Clin
Cancer Res 2009;15:7381e8.
Appendix A. Supporting information 22. Pernas S, Tolaney SM. HER2-positive breast cancer: new therapeutic
frontiers and overcoming resistance. Ther Adv Med Oncol 2019;11:
1e16.
Supporting data to this article can be found online at https://doi.
23. Oh DY, Bang YJ. HER2-targeted therapies-a role beyond breast can-
org/10.1016/j.apsb.2022.09.021. cer. Nat Rev Clin Oncol 2020;17:33e48.
24. Salkeni MA, Rizvi W. Neu perspectives, therapies, and challenges for
References metastatic HER2-positive breast cancer. Breast Cancer 2021;13:
539e57.
1. Mehren MV, Adams GP, Weiner LM. Monoclonal antibody therapy 25. Morrow PK, Wulf GM, Ensor J, Booser DJ, Moore JA, Flores PR,
for cancer. Annu Rev Med 2003;54:343e69. et al. Phase I/II study of trastuzumab in combination with Everolimus
2. Adams GP, Weiner LM. Monoclonal antibody therapy of cancer. Nat (RAD001) in patients with HER2-overexpressing metastatic breast
Biotechnol 2005;23:1147e57. cancer who progressed on trastuzumab-based therapy. J Clin Oncol
3. Nelson AL, Dhimolea E, Reichert JM. Development trends for human 2011;29:3126e32.
monoclonal antibody therapeutics. Nat Rev Drug Discov 2010;9: 26. Chandarlapaty S, Sakr RA, Giri D, Patil S, Heguy A, Morrow M, et al.
767e74. Frequent mutational activation of the PI3K-AKT pathway in
4. Melero I, Hervas-Stubbs S, Glennie M, Pardoll DM, Chen L. Immu- trastuzumab-resistant breast cancer. Clin Cancer Res 2012;18:
nostimulatory monoclonal antibodies for cancer therapy. Nat Rev 6784e91.
Cancer 2007;7:95e106. 27. Liu R, Chen Y, Liu G, Li C, Song Y, Cao Z, et al. PI3K/AKT pathway
5. Ellis LM, Hicklin DJ. VEGF-targeted therapy: mechanisms of anti- as a key link modulates the multidrug resistance of cancers. Cell
tumour activity. Nat Rev Cancer 2008;8:579e91. Death Dis 2020;11:797.
6. Weiner LM, Surana R, Wang S. Monoclonal antibodies: versatile 28. Berns K, Horlings HM, Hennessy BT, Madiredjo M, Hijmans EM,
platforms for cancer immunotherapy. Nat Rev Immunol 2010;10: Beelen K, et al. A functional genetic approach identifies the PI3K
317e27. pathway as a major determinant of trastuzumab resistance in breast
7. Scott AM, Wolchok JD, Old LJ. Antibody therapy of cancer. Nat Rev cancer. Cancer Cell 2007;12:395e402.
Cancer 2012;12:278e87. 29. Nagata Y, Lan K-H, Zhou X, Tan M, Esteva FJ, Sahin AA, et al. PTEN
8. Vogel CL, Cobleigh MA, Tripathy D, Gutheil JC, Harris LN, activation contributes to tumor inhibition by trastuzumab, and loss of
Fehrenbacher L, et al. Efficacy and safety of trastuzumab as a single PTEN predicts trastuzumab resistance in patients. Cancer Cell 2004;6:
agent in first-line treatment of HER2-overexpressing metastatic breast 117e27.
cancer. J Clin Oncol 2002;20:719e26. 30. Lu CH, Wyszomierski SL, Tseng LM, Sun MH, Lan KH, Neal CL,
9. Wang J, Xu B. Targeted therapeutic options and future perspectives for et al. Preclinical testing of clinically applicable strategies for over-
HER2-positive breast cancer. Signal Transduct Targeted Ther 2019;4: coming trastuzumab resistance caused by PTEN deficiency. Clin
34. Cancer Res 2007;13:5883e8.
10. Schlam I, Swain SM. HER2-positive breast cancer and tyrosine kinase 31. Pandolfi PP. Breast cancer-loss of PTEN predicts resistance to treat-
inhibitors: the time is now. NPJ Breast Cancer 2021;7:56. ment. N Engl J Med 2004;351:2337e8.
11. Pohlmann PR, Mayer IA, Mernaugh R. Resistance to trastuzumab in 32. Lin Y-X, Wang Y, Ding J, Jiang A, Wang J, Yu M, et al. Reactivation
breast cancer. Clin Cancer Res 2009;15:7479e91. of the tumor suppressor PTEN by mRNA nanoparticles enhances
12. Modi S, Saura C, Yamashita T, Park YH, Kim S-B, Tamura K, et al. antitumor immunity in preclinical models. Sci Transl Med 2021;13:
Trastuzumab deruxtecan in previously treated HER2-positive breast eaba9772.
cancer. N Engl J Med 2019;382:610e21. 33. Islam MA, Xu Y, Tao W, Ubellacker JM, Lim M, Aum D, et al.
13. Murthy RK, Loi S, Okines A, Paplomata E, Hamilton E, Hurvitz SA, Restoration of tumour-growth suppression in vivo via systemic
et al. Tucatinib, trastuzumab, and capecitabine for HER2-positive nanoparticle-mediated delivery of PTEN mRNA. Nat Biomed Eng
metastatic breast cancer. N Engl J Med 2019;382:597e609. 2018;2:850e64.
14. Moasser MM. The oncogene HER2: its signaling and transforming 34. Weng Y, Li C, Yang T, Hu B, Zhang M, Guo S, et al. The challenge
functions and its role in human cancer pathogenesis. Oncogene 2007; and prospect of mRNA therapeutics landscape. Biotechnol Adv 2020;
26:6469e87. 40:107534.
15. Goutsouliak K, Veeraraghavan J, Sethunath V, De Angelis C, 35. Pardi N, Hogan MJ, Porter FW, Weissman D. mRNA vaccines-a new
Osborne CK, Rimawi MF, et al. Towards personalized treatment for era in vaccinology. Nat Rev Drug Discov 2018;17:261e79.
early stage HER2-positive breast cancer. Nat Rev Clin Oncol 2020;17: 36. Sahin U, Karikó K, Türeci Ö. mRNA-based therapeutics-developing a
233e50. new class of drugs. Nat Rev Drug Discov 2014;13:759e80.
16. Rimawi MF, Schiff R, Osborne CK. Targeting HER2 for the treatment 37. Cheng Q, Wei T, Farbiak L, Johnson LT, Dilliard SA, Siegwart DJ.
of breast cancer. Annu Rev Med 2015;66:111e28. Selective organ targeting (SORT) nanoparticles for tissue-specific
17. Martı́nez-Sáez O, Prat A. Current and future management of HER2- mRNA delivery and CRISPR-Cas gene editing. Nat Nanotechnol
positive metastatic breast cancer. JCO Oncol Pract 2021;17:594e604. 2020;15:313e20.
18. Cho H-S, Mason K, Ramyar KX, Stanley AM, Gabelli SB, 38. Baden LR, El Sahly HM, Essink B, Kotloff K, Frey S, Novak R, et al.
Denney DW, et al. Structure of the extracellular region of HER2 alone Efficacy and safety of the mRNA-1273 SARS-CoV-2 vaccine. N Engl
and in complex with the Herceptin Fab. Nature 2003;421:756e60. J Med 2020;384:403e16.
19. Molina MA, Sáez R, Ramsey EE, Garcia-Barchino M-J, Rojo F, 39. Anguela XM, High KA. Entering the modern era of gene therapy.
Evans AJ, et al. NH(2)-terminal truncated HER-2 protein but not full- Annu Rev Med 2019;70:273e88.
966 Zhihui Dong et al.

40. Leonhardt C, Schwake G, Stögbauer TR, Rappl S, Kuhr JT, Ligon TS, 53. Du JZ, Li HJ, Wang J. Tumor-acidity-cleavable maleic acid amide
et al. Single-cell mRNA transfection studies: delivery, kinetics and (TACMAA): a powerful tool for designing smart nanoparticles to
statistics by numbers. Nanomedicine 2014;10:679e88. overcome delivery barriers in cancer nanomedicine. Acc Chem Res
41. Xiong Q, Lee GY, Ding J, Li W, Shi J. Biomedical applications of 2018;51:2848e56.
mRNA nanomedicine. Nano Res 2018;11:5281e309. 54. Xu CF, Zhang HB, Sun CY, Liu Y, Shen S, Yang XZ, et al. Tumor
42. Hou X, Zaks T, Langer R, Dong Y. Lipid nanoparticles for mRNA acidity-sensitive linkage-bridged block copolymer for therapeutic
delivery. Nat Rev Mater 2021;6:1078e94. siRNA delivery. Biomaterials 2016;88:48e59.
43. Kowalski PS, Rudra A, Miao L, Anderson DG. Delivering the 55. Xu X, Wu J, Liu S, Saw PE, Tao W, Li Y, et al. Redox-responsive
messenger: advances in technologies for therapeutic mRNA delivery. nanoparticle-mediated systemic RNAi for effective cancer therapy.
Mol Ther 2019;27:710e28. Small 2018;14:1802565.
44. Guan S, Rosenecker J. Nanotechnologies in delivery of mRNA ther- 56. Xu X, Xie K, Zhang XQ, Pridgen EM, Park GY, Cui DS, et al.
apeutics using nonviral vector-based delivery systems. Gene Ther Enhancing tumor cell response to chemotherapy through nanoparticle-
2017;24:133e43. mediated codelivery of siRNA and cisplatin prodrug. Proc Natl Acad
45. Kong N, Tao W, Ling X, Wang J, Xiao Y, Shi S, et al. Synthetic mRNA Sci U S A 2013;110:18638e43.
nanoparticle-mediated restoration of p53 tumor suppressor sensitizes p53- 57. Cheng TL, Chuang KH, Chen BM, Roffler SR. Analytical measure-
deficient cancers to mTOR inhibition. Sci Transl Med 2019;11:eaaw1565. ment of PEGylated molecules. Bioconjugate Chem 2012;23:881e99.
46. Shi J, Kantoff PW, Wooster R, Farokhzad OC. Cancer nanomedicine: 58. Gong C, Yao Y, Wang Y, Liu B, Wu W, Chen J, et al. Up-regulation of
progress, challenges and opportunities. Nat Rev Cancer 2017;17:20e37. miR-21 mediates resistance to trastuzumab therapy for breast cancer. J
47. Mishra S, Webster P, Davis ME. PEGylation significantly affects Biol Chem 2011;286:19127e37.
cellular uptake and intracellular trafficking of non-viral gene delivery 59. Gallardo A, Lerma E, Escuin D, Tibau A, Muñoz J, Ojeda B, et al.
particles. Eur J Cell Biol 2004;83:97e111. Increased signalling of EGFR and IGF1R, and deregulation of
48. Saw PE, Yao H, Lin C, Tao W, Farokhzad OC, Xu X. Stimuli-responsive PTEN/PI3K/Akt pathway are related with trastuzumab resistance in
polymereprodrug hybrid nanoplatform for multistage siRNA delivery HER2 breast carcinomas. Br J Cancer 2012;106:1367e73.
and combination cancer therapy. Nano Lett 2019;19:5967e74. 60. Bi Z, Li Q, Dinglin X, Xu Y, You K, Hong H, et al. Nanoparticles
49. Lu Y, Aimetti AA, Langer R, Gu Z. Bioresponsive materials. Nat Rev (NPs)-meditated lncRNA AFAP1-AS1 Silencing to block Wnt/b-cat-
Mater 2016;2:16075. enin signaling pathway for synergistic reversal of radioresistance and
50. Chen B, Dai W, He B, Zhang H, Wang X, Wang Y, et al. Current effective cancer radiotherapy. Adv Sci 2020;7:2000915.
multistage drug delivery systems based on the tumor microenviron- 61. Zhu X, Xu Y, Solis LM, Tao W, Wang L, Behrens C, et al. Long-
ment. Theranostics 2017;7:538e58. circulating siRNA nanoparticles for validating Prohibitin1-targeted
51. Perche F, Biswas S, Wang T, Zhu L, Torchilin VP. Hypoxia-targeted non-small cell lung cancer treatment. Proc Natl Acad Sci U S A
siRNA delivery. Angew Chem Int Ed 2014;53:3362e6. 2015;112:7779e84.
52. Sun CY, Shen S, Xu CF, Li HJ, Liu Y, Cao ZT, et al. Tumor acidity- 62. Fang J, Nakamura H, Maeda H. The EPR effect: unique features of
sensitive polymeric vector for active targeted siRNA delivery. J Am tumor blood vessels for drug delivery, factors involved, and limitations
Chem Soc 2015;137:15217e24. and augmentation of the effect. Adv Drug Deliv Rev 2011;63:136e51.