Journal of Colloid and Interface Science 646 (2023) 370–380

Contents lists available at ScienceDirect

Journal of Colloid And Interface Science

journal homepage: www.elsevier.com/locate/jcis

Enhanced lysosomal escape of cell penetrating peptide-functionalized

metal–organic frameworks for co-delivery of survivin siRNA and oridonin
Mengru Cai 1, Yu Yao 1, Dongge Yin , Rongyue Zhu , Tingting Fu , Jiahui Kong , Kaixin Wang ,
Jing Liu , Aina Yao, Yidan Ruan , Wenjuan Shi , Qian Zhu , Jian Ni *, Xingbin Yin *
School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 102488, China

G R A P H I C A L A B S T R A C T

A R T I C L E I N F O A B S T R A C T

Keywords: In recent years, small interfering RNA (siRNA) has been widely used in the treatment of human diseases,
Metal organic frameworks especially tumors, and has shown great appeal. However, the clinical application of siRNA faces several chal­
Oridonin lenges. Insufficient efficacy, poor bioavailability, poor stability, and lack of responsiveness to a single therapy are
Survivin siRNA
the main problems affecting tumor therapy. Here, we designed a cell-penetrating peptide (CPP)-modified metal
CPP33
organic framework nanoplatform (named PEG-CPP33@ORI@survivin siRNA@ZIF-90, PEG-CPP33@NPs) for
targeted co-delivery of oridonin (ORI), a natural anti-tumor active ingredient) and survivin siRNA in vivo. This
can improve the stability and bioavailability of siRNA and the efficacy of siRNA monotherapy. The high drug-
loading capacity and pH-sensitive properties of zeolite imidazolides endowed the PEG-CPP33@NPs with lyso­
somal escape abilities. The Polyethylene glycol (PEG)-conjugated CPP (PEG-CPP33) coating significantly
improved the uptake in the PEG-CPP33@NPs in vitro and in vivo. The results showed that the co-delivery of ORI
and survivin siRNA greatly enhanced the anti-tumor effect of PEG-CPP33@NPs, demonstrating the synergistic
effect between ORI and survivin siRNA. In summary, the novel targeted nanobiological platform loaded with ORI
and survivin siRNA presented herein showed great advantages in cancer therapy, and provides an attractive
strategy for the synergistic application of chemotherapy and gene therapy.

* Corresponding authors.
E-mail addresses: 602054@bucm.edu.cn (J. Ni), yxbtcm@bucm.edu.cn (X. Yin).
1
These authors contributed equally to this work.

https://doi.org/10.1016/j.jcis.2023.04.126
Received 18 February 2023; Received in revised form 20 April 2023; Accepted 24 April 2023
Available online 28 April 2023
0021-9797/© 2023 The Authors. Published by Elsevier Inc. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-
nc-nd/4.0/).
M. Cai et al.
1. Introduction
Cancer has a significant impact on human health, and lung cancer is
the leading cause of cancer death [1]. Traditional tumor treatment
methods mainly include surgical resection, chemotherapy, and radio­
therapy, although still face great challenges, such as poor targeting and
drug resistance [2]. In recent years, immunotherapy [3], gene therapy
[4], hormone therapy [5], stem cell transplantation therapy [6], and
tumor-targeted therapy [7] have become the focus of cancer research. At
the core of RNA interference, small interfering RNA (siRNA) has become
an indispensable tool to verify gene function, and has also been widely
used in research on treating human diseases [8]. Many cancer-related
genes can be used as targets of siRNA, which provides unlimited possi­
bilities for the application of siRNA in tumor therapy. siRNA can knock
down the expression of key genes and have the advantages of high
knockout efficiency, high specificity, and no immunogenicity. Although
siRNA therapy has shown incomparable advantages in anti-tumor ac­
tivity, a single therapeutic agent is still insufficient to achieve satisfac­
tory anti-tumor efficacy. Currently, one of the most promising
combinations for dual delivery is the combination of nucleic acids and
small-molecule drugs. The use of siRNA to inhibit the expression of the
target protein at the gene level combined with chemotherapy drugs such
as doxorubicin (DOX) and cisplatin is an effective strategy to improve
the sensitivity of chemotherapy [9]. The synergistic effects of genes and
chemotherapy can enhance the activity and efficacy of a single drug and
overcome the problem of insufficient efficacy associated with single
drugs.
However, the co-delivery of siRNA and chemotherapeutic agents
brings new problems owing to their easy degradation by nucleases in the
plasma, rapid renal excretion, lack of targeting ability, transmembrane
barriers, lysosomal escape barriers, and differences in biodistribution
[10,11]. The use of nanocarriers to achieve the co-delivery of biological
macromolecules and small-molecule drugs is the most attractive means
of overcoming difficulties in the clinical treatment of tumors [12–14].
Metal-organic frameworks (MOFs) have high porosity, adjustable pore
size, and good biocompatibility, giving them great advantages in anti-
tumor synergistic therapy [15–18] and allowing them to encapsulate
biological macromolecules and small-drug molecules with high drug-
loading capacity. In particular, siRNA encapsulation in the cavity of
MOFs can significantly reduce structural changes in biomolecules, pre­
vent damage and degradation of their structure by the external envi­
ronment, and increase their active sites [19,20]. Zeolitic imidazolate
frameworks (ZIFs) have become a research hotspot in the field of
nanomaterials because of their unique advantages in constructing
siRNA-based drug delivery systems, such as mild synthesis conditions,
good thermal stability, and pH responsiveness [21]. ZIF-8 and ZIF-90 are
the most representative ZIFs [22]. ZIF-90 introduced aldehyde groups
based on ZIF-8; therefore ZIF-90 was more hydrophilic than ZIF-8. The
preparation of siRNA nanoparticles using the two as carriers allows for
the construction of a new RNAi delivery platform, which may be an
effective strategy to improve the inherent defects of siRNA. However,
whether ZIF hydrophilicity induces a protective effect against siRNA has
not yet been reported.
Surface coatings such as chitosan, hyaluronic acid, and polyethylene
glycol play important roles in nanobiomedicine research [23,24]. Sur­
face coatings can provide active sites for the conjugation of targeted
ligands, improve the stability and biosafety of nanoparticles, and pre­
vent recognition and clearance by the immune system [25].
Nanotechnology-based targeted drug delivery systems present another
important challenge. Cell-penetrating peptides (CPPs) are a class of
small-molecule peptides with strong cell membrane penetration abilities
[26]. CPPs are usually non-toxic, and their ability to target tumor cells
and transport various drugs (such as enzymes, RNA, and chemical
molecules) through the cell membrane and into the cell has been
confirmed. Hade et al. used cell-penetrating peptide (CPPs) to deliver
microRNA (miRNAs) into cells, showing good tumor-homing ability
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[27]. Our group also developed a CPP44-modified MOF delivery system
for efficient drug delivery into HepG2 cells [23]. CPP33
(RLWMRWYSPRTRAYG) can specifically penetrate non-small cell lung
cancer A549 cells, overcoming the barrier of the cell membrane to
improve drug absorption, and has great application potential in the
targeted delivery of anti-tumor drugs [28].
Survivin is a tumor-specific gene in the human genome that is highly
expressed in lung cancer cells. As important inhibitors of apoptosis
proteins, translated proteins have anti-apoptotic and cell cycle regula­
tory functions [29]. Oridonin (ORI) has received considerable attention
as an innovative anticancer drug. ORI induces apoptosis in A549 cells,
and its mechanism may be related to the regulation of Bcl-2/Bax, PARP,
caspase 3 and survivin signaling pathways [30]. Co-delivery of survivin
siRNA with ORI may enhance the anti-tumor effect and achieve better
efficacy. Here, we first synthesized two ZIFs (ZIF-8 and ZIF-90) with
different hydrophilicities to investigate their different protective effects
against siRNA, and finally screened ZIF-90 with a better protective effect
for subsequent anti-tumor studies. We constructed a functional, pH-
responsive, nanodrug delivery platform (named PEG-
CPP33@ORI@survivin siRNA@ZIF-90, PEG-CPP33@NPs) to specif­
ically co-deliver ORI with survivin siRNA and enhance the chemo­
sensitivity in vivo (Scheme 1). Specifically, ORI and survivin siRNA were
non-covalently adsorbed in ZIF-90 (named ORI@survivin siRNA@ZIF-
90, NPs) and further coated with polyethylene glycol-CPP (PEG-
CPP33). The obtained PEG-CPP33@NPs had good tumor selectivity and
pH sensitivity and could be effectively internalized into the cytoplasm.
Finally, the nanoformulation exhibited excellent gene-silencing effi­
ciency and anti-tumor effects. Our study provides insights into the
combination of chemotherapy and gene therapy, and we expect to
expand this strategy for a wide range of disease treatment applications.
2. Materials and methods
2.1. Materials
Imidazole-2-formaldehyde (≥98%), 2-methylimidazole (≥98%), and
oridonin (≥98%) were purchased from Shanghai Yuanye Biotechnology
Co., Ltd. Zn(NO3)2⋅6H2O (99.99%) was obtained from Shanghai Aladdin
Bio-Chem Technology Co., Ltd. Zn(CH3COO)2⋅2H2O (98%) was pro­
vided by Shanghai Macklin Biochemical Co., Ltd. 3-(4,5-dimethylth­
iazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT, ≥98%) and
annexin V-FITC/PI were provided by Beijing Beyerdi Biotechnology Co.,
Ltd. DEPC water was obtained from Beijing Rambolide Biotechnology
Co., Ltd. siRNA (antisense sequence: UAAUUCUUCAAACUGCUUCTT,
98%) was purchased from Shanghai Jima Pharmaceutical Technology
Co., Ltd. High rich glucose DMEM was purchased from Sigma-Aldrich
LLC. Fetal bovine serum (FBS) and a penicillin–streptomycin mixture
were obtained from Beijing Sijiqing Biotechnology Co., Ltd. N,N-
dimethylformamide (DMF, ≥99.5%) were purchased from Shanghai
Aladdin Bio-Chem Technology Co., LTD. Dimethyl sulfoxide (DMSO)
was obtained from Tianjin Guangfu Fine Chemical Research Institute.
2.2. Synthesis of ZIF-8
Zn(NO3)2⋅6H2O was weighed and dissolved in a beaker with deion­
ized water. The Zn(NO3)2⋅6H2O solution was poured into a conical flask
containing an aqueous solution of 2-methylimidazole. After continuous
stirring for 15 min, the mixture was transferred into a centrifuge tube,
and the products were collected by centrifugation for 20 min. The
products were washed with deionized water and dried in a vacuum-
drying oven. The prepared product, ZIF-8, was a white solid powder
that was odorless and was stored in a desiccator.
2.3. Synthesis of ZIF-90
Imidazole-2-formaldehyde was dissolved in DMF, Zn
M. Cai et al. Journal of Colloid And Interface Science 646 (2023) 370–380

(CH3COO)2⋅2H2O was added and stirred for 15 min, and the products SDS solution to terminate the reaction. Agarose gel electrophoresis was
were collected by centrifugation for 10 min. The products were washed then performed.
with methanol and dried in a vacuum-drying oven.

2.6. ORI loading

2.4. siRNA loading
ZIF-90 (10 mg) was added to the ORI solution (5 mg/mL, 15 mg) and
The ZIFs (1 mg) were added to the siRNA solution (2.5 μmol/L, 9
magnetically stirred for 24 h. The supernatant was collected by centri­
nmol) and stirred on a magnetic stirrer for 24 h. The supernatant was
fugation and the content of free ORI was determined using high-
collected by centrifugation for determination of free siRNA content by
performance liquid chromatography (HPLC). The precipitate was
gel electrophoresis, and the precipitate was freeze-dried and stored at
lyophilized and stored at − 20 ◦ C to obtain ORI@ZIF-90.
− 20 ◦ C. Subsequently, siRNA@ZIF-8 and siRNA@ZIF-90 were
synthesized.
2.7. Synthesis of ORI@siRNA@ZIF-90 (NPs)
2.5. Protective effect of ZIFs against siRNA
ZIF-90 (1 mg) was stirred with the ORI solution (5 mg/mL) for 24 h.
Free siRNA, siRNA@ZIF-8, and siRNA@ZIF-90 were incubated with After centrifugation, siRNA (9 nmol) was added and the mixture was
100 μL of 10% FBS at 37 ◦ C for 1, 2, 4, 6, 8, 10, and 12 h. At the cor­ stirred in DPEC water for 24 h. The precipitate was lyophilized and
responding time point, 10 μL of sample was added to 5 μL of 10 mg/mL stored at − 20 ◦ C to obtain the NPs.

Scheme 1. Schematic Illustration of the siRNA Protection of ZIF-8 and ZIF-90 and Anti-tumor Mechanism of PEG-CPP33@ORI@Survivin siRNA@ZIF-90.

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2.8. Synthesis of PEG-CPP33@NPs
ZIF-90 (1 mg) was stirred with ORI solution (5 mg/mL) for 24 h.
After centrifugation, siRNA (9 nmol) was added and the mixture was
stirred in DPEC water for 24 h. After centrifugation and drying, PEG-
CPP33 was added to the mixture and centrifuged to obtain the PEG-
CPP33@NPs.
2.9. In vitro cytotoxicity test
Cells were seeded in a 96-well plate at a density of 5000 cells/well.
After culturing for 24 h, media containing different concentrations of
NPs were added (siRNA: 10, 30, 50, 100, 150, 200, and 250 nM). After
culturing for 24 h, 20 μL 5% MTT solution was added to each well and
incubated for 4 h. Next, the supernatant was removed, and 150 μL of
DMSO was added to each well. While providing protection from light,
the samples were placed on a shaker and shaken for 5 min. The absor­
bance of each well was measured at an excitation wavelength of 490 nm
using a microplate reader.
2.10. Annexin V-FITC/PI measurement
To investigate the cell apoptosis induced by NPs, cells were treated
with NPs (siRNA: 125 nmol/L) for 24 h at 37 ◦ C. Cells were harvested,
washed with Phosphate buffer solution (PBS) and resuspended in 195 μL
annexin-V FITC binding solution. Next, 5 μL annexin V-FITC and 10 μL
PI were added and incubated with the cells for 20 min in the dark.
Finally, different cells were analyzed by flow cytometry (BD, LSRFor­
tessa SORP).
2.11. Cell uptake study
HepG2, A549, MDA-MB-231, and HT-29 cells were plated at a den­
sity of 1 × 105 cells/well and cultured for 24 h. CPP33-PEG@NPs were
added to the plates at a final coumarin-6 concentration of 0.5 μg/ml.
After 4 h incubation, the cells were washed three times with PBS and
fixed with 4% paraformaldehyde for 10 min at 37 ◦ C. 4′ ,6-Diamidino-2-
Phenylindole (DAPI) was added for 5 min to stain the nuclei. Finally, the
cells were imaged using a confocal laser scanning microscope (CLSM,
Leica Microsystems, LAS X 3.5.5.19976).
2.12. Evaluation of Intracellular Trafficking and Endosomal Escape
To identify the intracellular location of CPP33-PEG@NPs, lysosome
staining was performed on A549 cells. Briefly, A549 cells were incu­
bated with CPP33-PEG@NPs for 0.5, 4, 8, and 12 h and further treated
with LysoTracker Red (50 nM) for 30 min. The cells were imaged using a
CLSM.
2.13. Animal and tumor-bearing mouse model
All animal experiments were performed in accordance with the
guidelines approved by the Institutional Animal Care and Use Com­
mittee of the Beijing University of Chinese Medicine (BUCM-4-
2020123103-4138). BALB/c nude mice (8–10-weeks old, 20 g) were
obtained from Sipeifu Biological Technology Co., Ltd. First, a tumor-
bearing mouse model was established. A549 cells were subcutaneously
injected into the right forelimbs of the mice, and the tumor volume was
monitored daily. The tumors were allowed to grow to an average volume
of 100 mm3 (V = W2 × L/2) prior to the study.
2.14. In vivo fluorescence imaging
NPs were prepared and intravenously injected into tumor-bearing
mice. The mice were imaged using an IVIS imaging system (Caliper,
Hopkington, MA, USA) before injection and at 2, 6, and 12 h after
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Journal of Colloid And Interface Science 646 (2023) 370–380
injection. The hearts, livers, spleens, lungs, kidneys, and tumors were
collected for imaging using an IVIS imaging system. The data were
analyzed using Care Stream software.
2.15. In vivo antitumor effect
Thirty tumor-bearing mice were equally divided into five groups and
treated with normal saline, siRNA@ZIF-90, ORI@ZIF-90, NPs, and PEG-
CPP33@NPs (1.5 mg⋅kg− 1 siRNA and 3 mg⋅kg− 1 ORI) via intravenously
injection, respectively. The body weights and tumor volumes of the mice
were monitored every two days to explore the physiological influences
and treatment efficiency of the NPs. Blood was harvested from the mice
after treatment to detect the blood biochemical parameters.
2.16. Histopathological analysis
The extracted organs (heart, liver, spleen, lungs, and kidneys) and
tumors were preserved in formaldehyde (4%) and stained with hema­
toxylin and eosin (H&E).
2.17. Statistical methods
All experiments were performed with at least three independent
replications, and data were expressed as means ± standard deviations
(SDs). Comparisons were performed using one-way analysis of variance;
*P < 0.05 and **P < 0.001 were considered statistically significant.
3. Results and discussion
3.1. Synthesis of ZIF-8 and ZIF-90
ZIF-8 and ZIF-90 were synthesized by stirring at 25 ◦ C [31]. The
Scanning electron microscope (SEM) images show that the synthesized
ZIF-8 and ZIF-90 are circular in shape, with a particle size of approxi­
mately 100 nm (Fig. S1). The X-Ray Diffraction (XRD) pattern showed
that the synthesized ZIFs were completely crystalline, and the charac­
teristic diffraction peak was consistent with the standard pattern
(Fig. S2). The Fourier Transform Ioncyclotron Resonance (FTIR) results
illustrated the presence of characteristic peaks at the corresponding
band positions of ZIF-8 and ZIF-90 (Fig. S3). ZIF-8 showed the stretching
vibration peak of the imidazole molecule C–– N at 1580 cm− 1, and ZIF-90
showed the characteristic peak of the aldehyde group C– – O at 1680
cm− 1. As shown in Figs. S4–S5, the particle sizes of ZIF-8 and ZIF-90
were 78.82 nm and 68.06 nm, respectively, and the zeta potentials
were 13 mV and 9.99 mV, respectively, consistent with previous studies
[32–34]. The N in the organic ligands of ZIFs is easily protonated, giving
ZIFs a positive charge.
The biosafety of ZIFs must be evaluated before conducting trans­
fection and drug-loading experiments. First, MTT assay showed that ZIF-
8 and ZIF-90 had no obvious cytotoxicity in the concentration range of
0–55 μg/mL, and the survival rate of A549 cells and normal human fi­
broblasts (NHDFS) was more than 85% (Figs. S6–S7). Second, DAPI
staining showed that ZIF-8 and ZIF-90 (0–55 μg/mL) had no significant
effect on nuclear morphology (Fig. S8). In addition, Annexin V-FITC/PI
double staining assay also demonstrated that ZIF-8 and ZIF-90 (0–55 μg/
mL) had no obvious apoptosis-inducing effect (Figs. S9–10). This dem­
onstrates that ZIF-8 and ZIF-90 exhibit good in vitro biological safety at
certain doses.
3.2. The construction of siRNA@ZIFs
siRNA was loaded by solvent adsorption [35]. Briefly, 1 mg of ZIF-8/
ZIF-90 was stirred with 9 nmol siRNA (2.5 mol/L) in DEPC water for 24
h. The drug loading capacity of ZIF-8 and ZIF-90 was 7.91 ± 0.13% and
9.89 ± 0.19%, respectively (n = 3). The morphologies of ZIF-8 and ZIF-
90 were observed using SEM, and no significant changes were observed
M. Cai et al. Journal of Colloid And Interface Science 646 (2023) 370–380

after drug loading (Fig. S11). The dynamic light scattering results also
confirmed that there was no significant change in size after drug loading
(Fig. S12). The FTIR spectra showed that the structure of ZIFs remained
unchanged after drug loading (Fig. S13). The formation of siRNA@ZIFs
was monitored by Ultraviolet–visible spectroscopy (UV–vis) absorption
spectroscopy (Fig. S14). As shown in the figure, siRNA had a maximum
absorption peak at 260 nm, whereas ZIF-8 showed no absorption peak
within 240–480 nm. When siRNA was mixed with ZIF-8, the mixed so­
lution exhibited an absorption peak at 260 nm, indicating that siRNA
was not coated with ZIF-8. Spectral scanning of siRNA@ZIF-8 showed
that the drug-loaded particles had no absorption peak at 260 nm, indi­
cating that siRNA was loaded into ZIF-8. ZIF-90 exhibits a maximum
absorption peak at 290 nm. When siRNA was mixed with ZIF-90, the
maximum absorption peak of the mixed solution was approximately
290 nm, and the shift in the maximum absorption peak was speculated
to be related to the binding of siRNA and ZIF-90. The maximum ab­
sorption peak of the drug-loaded particles was consistent with that of
ZIF-90, indicating that siRNA was successfully loaded into ZIF-90.
siRNA is unstable under physiological conditions and is susceptible
to degradation by nucleases in body fluids [36]. To verify the protection
of siRNA after ZIFs encapsulation, siRNA@ZIFs were dispersed in a
medium containing serum or RNase A for 12 h. The results showed that
after encapsulation with ZIF-8 or ZIF-90, the stability of siRNA in serum
and RNase A was improved compared to that of free siRNA, which may
be due to the physical protection provided by the ZIFs scaffold
(Fig. 1A–D). Interestingly, the protective effect of ZIF-90 against siRNA
was greater than that of ZIF-8, presumably because of the presence of
aldehyde ligands in ZIF-90, which make it more polar than ZIF-8 [37].
ZIF-90 encapsulated hydrophilic siRNA and exhibited a better protective
effect owing to steric hindrance. In addition, the effects of temperature
and organic solvents on the stability of siRNA were investigated
(Figs. S15–16). Neither temperature nor organic solvent affected the
stability of siRNA, which verified the double-stranded nature of the
siRNA structure. Finally, we selected ZIF-90 as the siRNA vector for
subsequent experiments.
3.3. Synthesis of NPs and PEG-CPP33@NPs
ORI regulates multiple signaling pathways in cells, including reactive
oxygen species, Bcl-2/Bax, p53/p21, MAPK, miRNA, and fatty acid
synthase [38]. A large number of studies have emerged on the combi­
nation therapy of ORI with other drugs, such as cisplatin and cetuximab
[39,40]. In this study, the loading capacity of ZIF-90 on ORI was
investigated. The content of ORI was detected using HPLC. The results
showed that the loading capacity of ORI for ZIF-90 was 27.28%.
Furthermore, 8.18% siRNA and 19.56% ORI were incorporated into the
pores of ZIF-90. siRNA and ORI enter the interior of ZIFs via electrostatic
and π-π interactions [41–43]. To verify whether siRNA and ORI entered
the pores of ZIF-90 or were absorbed on the surface of ZIF-90, Bru­
nauer–Emmett–Teller (BET) analyses of ZIF-90 and ORI@siRNA@ZIF-

Fig. 1. (A) Protective effect of ZIFs against siRNA after different serum treatment times (a: free siRNA, b: siRNA@ZIF-8, c: siRNA@ZIF-90 incubated with 10% FBS at
37 ◦ C); B. relative activity of siRNA after different serum treatment times; (C) protective effect of ZIFs against siRNA after different RNase A treatment times (a: free
siRNA, b: siRNA@ZIF-8, c: siRNA@ZIF-90 incubated with RNase A at 37 ◦ C); (D) relative activity of siRNA after RNase A treatment times; (E) SEM of PEG-
CPP33@NPs; (F) FT-IR spectrum of PEG-CPP33@NPs; (G) zeta potential of PEG-CPP33@NPs; (H) siRNA release from PEG-CPP33@NPs at a pH 5.5 or pH 7.4;
and (I) ORI release from PEG-CPP33@NPs at pH 5.5 or pH 7.4. (n = 3, mean ± SD).

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90 were also performed. The results are presented in Fig. S17. The BET
specific surface area of ZIF-90 was 1419 m2/g, whereas that of
ORI@siRNA@ZIF-90 was 794 m2/g. According to the BET test results,
the specific surface area and pore volume of ZIF-90 were significantly
higher than those of ORI@siRNA@ZIF-90, indicating the successful
loading of siRNA and ORI.
To improve the distribution of ZIF-90 in vivo, we coated ZIF-90 with
a CPP, CPP33, which specifically targets A549 cells [44]. The cysteine at
the end of CPP33 and the maleimide group at the end of mPEG2000-Mal
were used to construct the targeting ligand PEG-CPP33 via the addition
reaction of the sulfhydryl and maleimide groups. As shown in Fig. 1E,
the PEG-CPP33@NPs had a quasi-circular spherical shape, although the
surface was no longer smooth, and the particle size was approximately
100 nm (Fig. 1E). The FTIR spectrum showed that PEG-CPP33 had an-
OH stretching vibration peak at 2880 cm− 1, –NH+ 3 deformation vibra­
tion peaks at 1600–1575 cm− 1 and 1550–1504 cm− 1, and a COO-
symmetric stretching vibration peak at 1412 cm− 1 (Fig. 1F). The
bending and stretching vibration peaks of C–N appeared at 1220–1020
cm− 1 and 1340–1280 cm− 1, and the vibration peak of CH in –CH2–
appeared at 1466 cm− 1, indicating that PEG was successfully connected
to the polypeptide chain CPP33. The infrared spectrum of PEG-
CPP33@NPs was similar to that of PEG-CPP33. PEG-CPP33@NPs
retained the characteristic peaks of ZIF-90 at 1680 cm− 1 and 796 cm− 1,
although the peak intensity became weaker, indicating that PEG-CPP33
was modified on the surface of the drug-carrying particles and the
structure of the nanoparticles was intact. The XRD pattern of PEG-
CPP33@NPs was further examined, and no significant change in the
Fig. 2. CLSM images of the uptake of different drugs by different cells. (A) A549 cells; (B) HepG2 cells; (C) HT-29 cells; and (D) MDA-MB-231 cells. (a) Coumarin 6;
(b) Coumarin 6@ZIF-90; and (c) PEG-CPP33@Coumarin 6@ZIF-90.
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Journal of Colloid And Interface Science 646 (2023) 370–380
characteristic peak of the NPs after PEGylation was found (Fig. S18),
indicating that PEGylation did not affect the crystal form of the NPs.
Zeta potential results showed that PEG-CPP33@NPs transforms into a
negative potential of –23 mv, which is opposite to the positive zeta
potential of blank ZIF-90 (Fig. 1G). We speculated that PEG-CPP33
binding to the ZIF-90 surface caused a change in the zeta potential. A
nanodrop microultraviolet spectrophotometer was used for ultraviolet
full-wavelength scanning (Fig. S19). The siRNA had a maximum ab­
sorption peak at 260 nm, ZIF-90 had a maximum absorption peak at 290
nm, PEG-CPP33 had no UV absorption peak at 240–480 nm, and the UV
absorption peak of the NPs was consistent with that of the drug-carrying
particles, indicating that the structure of the NPs did not change.
The responsive release of drugs at tumor sites is a major research
focus. Therefore, ORI and siRNA release rates in PEG-CPP33@NPs were
evaluated in acidic (pH = 5.5) and neutral (pH = 7.4) PBS at 37 ◦ C. ORI
and survivin siRNA were released faster at a pH 5.0, than at a pH 7.4
(Fig. 1H–I). At a pH 5.5, approximately 70% and 85% of ORI and sur­
vivin siRNA, respectively, were released from the NPs within 24 h. In
contrast, approximately 38% and 40% of ORI and survivin siRNA,
respectively, at a pH 7.4. This is due to the instability of ZIF-90 under
acidic conditions [22,45], allowing the nanocarriers in acidic lysosomes
to release more drugs, which facilitates tumor pH-responsive drug
release [46].
3.4. Cellular uptake and lysosomal escape of NPs
The specific selection of drugs for cells is the key to tumor-targeted
M. Cai et al. Journal of Colloid And Interface Science 646 (2023) 370–380

therapy. Using Coumarin 6 as a fluorescent indicator, the specific
selectivity of PEG-CPP33 for A549 cells was investigated using a CLSM.
The results showed that green fluorescence appeared in all four cell
types after incubation with coumarin 6 and Coumarin 6@ZIF-90, indi­
cating that the targeting abilities of coumarin 6 and Coumarin 6@ZIF-90
were not obvious (Fig. 2A–D). After incubating the cells with PEG-
CPP33@Coumarin 6@ZIF-90, the intensity of green fluorescence in
the A549 cells was the highest, and no obvious green fluorescence was
observed in the HepG2, HT-29, and MDA-MB-231 cells. This indicates
that PEG-CPP33@Coumarin 6@ZIF-90 is only taken up by A549 cells
and has high cell-targeting ability [47]. CPP33 promotes the internali­
zation of NPs in A549 cells through dynamin-dependent and clathrin/
microcytosis/caveo-independent pathways [26,28].
siRNASur was labeled with Cy3 for fluorescence, and the uptake of
siRNA, siRNA@ZIF-90, and PEG-CPP33@siRNA@ZIF-90 by A549 cells
was measured using flow cytometry. As shown in Fig. 3A, only a small
amount of naked siRNA was taken up by A549 cells, most siRNA@ZIF-90
and PEG-CPP33@siRNA@ZIF-90 were internalized by the cells, and the
drug-loaded nanoparticles showed the strongest fluorescence intensity
in the cells after modification with PEG-CPP33. This suggests that the
encapsulation of ZIF-90 and modification of PEG-CPP33 could promote
cellular uptake of the drug, and CPP33 in particular could further pro­
mote the internalization of the drug.
Effective lysosomal escape is key to the therapeutic effects of siRNA.
After staining with LysoTracker Green, the lysosomes showed green
fluorescence; Cy3-siRNAsur showed red fluorescence. After 0.5 h of in­
cubation with PEG-CPP33@Cy3-siRNAsur@ZIF-90, a small amount of
red fluorescence was observed inside the A549 cells, indicating that a
small amount of siRNA was internalized into the cells (Fig. 3B). After 4 h
of treatment, yellow fluorescence, formed by the superposition of green
and red fluorescence, was observed in the cells, indicating that siRNA
was swallowed by lysosomes. After co-incubation with cells for 8 h, the
yellow fluorescence decreased and the red fluorescence increased,
indicating that the siRNA successfully escaped from the lysosomes and
was released into the cytoplasm, which was more obvious after 12 h. The
colocalization rates of Cy3-siRNA with lysosomes at 0.5, 4, 8, and 12 h
were 0.45%, 94.34%, 58.13%, and 16.52%, respectively, which further
indicated that PEG-CPP33@Cy3-siRNAsur@ZIF-90 could achieve lyso­
somal escape of siRNA. After incubation with siRNA, the co-localization
signal (yellow) was significantly higher than that of PEG-CPP33@Cy3-
siRNAsur@ZIF-90, and the level of co-localization was greater than
90% at different times (Fig. S20), indicating that the escape of siRNA
from the lysosomes was difficult. This enhanced lysosomal escape may
be related to the pH sponging effect of ZIF-90 [23].
3.5. Survivin expression
The mRNA expression level was used as a direct indicator of the gene
silencing effect of siRNA. Survivin mRNA expression was detected by
real-time Polymerase Chain Reaction (PCR) after NPs treatment. The
results showed no significant difference between the cells treated with
siRNA alone and the negative control group (Fig. 3C). The relative
expression of survivin mRNA in cells treated with siRNA@ZIF-90 and
PEG-CPP33@siRNA@ZIF-90 was significantly lower than that in the
negative control group. Among them, siRNA@ZIF-90 reduced the rela­
tive expression of survivin mRNA by 57.41 ± 9.56%; PEG-

Fig. 3. (A) Intracellular fluorescence intensity of A549 cells incubated with siRNA, siRNA@ZIF-90, and PEG-CPP33@siRNA@ZIF-90 for 24 h; (B) CLSM images of the
localization of siRNA in A549 cells treated with PEG-CPP33@Cy3-siRNAsur@ZIF-90 for different times; (C) relative expression of survivin mRNA in A549 cells treated
with different nanoparticles for 24 h; (D) relative expression of survivin protein in A549 cells treated with different nanoparticles for 24 h; (E) images of Western blot:
(a) control; (b) siRNA; (c) siRNA@ZIF-90; and (d) PEG-CPP33@siRNA@ZIF-90; (F) cell viability of A549 cells treated with different drugs for 24 h; and (G) apoptosis
statistics of A549 cells treated with different drugs for 24 h. (n = 3, mean ± SD, *P < 0.05, **P < 0.001).

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M. Cai et al.
CPP33@siRNA@ZIF-90 reduced the relative expression of survivin
mRNA by 69.34 ± 1.06%. This illustrates that CPP33-modified nano­
particles have a better survivin mRNA downregulation effect, possibly
owing to the enhanced efficacy of efficient delivery.
Survivin is a specific inhibitor of apoptosis in tumor cells. Down­
regulation of its mRNA level can reduce the expression of the survivin
protein and induce apoptosis of tumor cells, which plays an anti-tumor
role. Western blotting revealed that both siRNA@ZIF-90 and PEG-
CPP33@siRNA@ZIF-90 downregulated survivin protein expression. As
shown in Fig. 3D–E, no significant difference was observed in survivin
protein expression in A549 cells treated with siRNA alone compared
with the control group. After 24 h of treatment with siRNA@ZIF-90 and
PEG-CPP33@siRNA@ZIF-90, the survivin protein expression levels in
A549 cells decreased significantly compared with that in the control
group, and the targeted drug-loaded nanoparticles reduced the protein
expression more significantly. These results demonstrate that CPP33
modification could further downregulate the expression of anti-
apoptotic proteins in A549 cells, thereby showing a more significant
pro-apoptotic effect.
3.6. In vitro cytotoxicity of nanoparticles
The cytotoxicity of ORI, survivin siRNA and the different nano­
particles in A549 cells was determined using the MTT assay. As shown in
Fig. 3F, survivin siRNA showed little cytotoxicity, whereas survivin
siRNA@ZIF-90 showed dose-dependent cytotoxicity, which was attrib­
uted to the enhanced uptake of survivin siRNA by ZIF-90 encapsulation.
Both ORI and ORI@ZIF-90 showed cytotoxicity, although survivin
siRNA combined with ORI significantly improved the antitumor effects
of individual chemotherapeutics. No significant difference was observed
in the cytotoxicity of NPs before and after coupling with CPP33 peptide
chains, which may be due to similar intracellular drug content after 24 h
of incubation. Since both ORI and survivin siRNA inhibited survivin
protein expression, annexin V-FITC staining and FACS analysis were
used to detect the apoptosis rates induced by the different drugs. After
ORI and survivin siRNA treatment, the apoptosis rates were 35.2% and
10.9%, respectively (Fig. 3G). The apoptosis rates of survivin
siRNA@ZIF-90, ORI@ZIF-90, NPs, and PEG-CPP33@NPs were 30.13%,
36.45%, 51.37%, and 60.73%, respectively. The combination of ORI and
survivin siRNA resulted in a higher apoptotic rate, which was consistent
with the results of the MTT assay.
Fig. 4. In vivo biodistribution of nanoformulations. (A) In vivo fluorescence imaging at 2 h, 6 h and 12 h after injection of different nanoparticles; (B) fluorescence
imaging of major organs and tumors in mice 12 h after injection of different nanoformulations; (C) fluorescence intensity statistics of main organs and tumors of mice
12 h after injection of different nanoformulations. (a: Cy3-siRNAsur@ZIF-90; b: PEG-CPP33@Cy3-siRNAsur@ZIF-90; n = 3, mean ± SD, *P < 0.05, **P < 0.001).
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Journal of Colloid And Interface Science 646 (2023) 370–380
3.7. In vivo biodistribution of nanoformulations
Having determined the in vitro biological processes of the nano­
formulations, we evaluated their tissue selectivity of the nano­
formulations. The biodistribution of nanoparticles in mice was observed
at 2, 6, and 12 h after tail vein injection of Cy3-siRNAsur@ZIF-90 and
PEG-CPP33@Cy3-siRNAsur@ZIF-90 (siRNA, 2 nmol). Two hours after
administration, the mice injected with PEG-CPP33@Cy3-siRNAsur@ZIF-
90 showed strong fluorescence at the tumor site, whereas the fluores­
cence of Cy3-siRNAsur@ZIF-90 was mainly distributed in the abdomen
of the mice (Fig. 4A). Twelve hours after injection, PEG-CPP33@Cy3-
siRNAsur@ZIF-90 still accumulated the most at the tumor site, which
proved that PEG-CPP33 modification could retain the nanoparticles at
the tumor site for a long time. In addition, as shown in Fig. 4B, the
unmodified PEG-CPP33 nanoparticles mainly accumulated in the liver,
which may be due to their uptake by the mononuclear phagocytic sys­
tem [48].
After administration, the fluorescence intensity of each tissue and
tumor was analyzed. The tumor sites of the mice in the Cy3-siRNA
sur
@ZIF-90 group showed a certain degree of fluorescence intensity,
although the liver and kidney showed stronger fluorescence (Fig. 4C).
Fluorescence at the tumor site in the PEG-CPP33@Cy3-siRNAsur@ZIF-
90 group was the strongest. There was also fluorescence accumulation in
the liver and kidney, which was mainly related to the liver and kidney
being important metabolism and excretion organs for drugs [49,50].
Compared with Cy3-siRNAsur@ZIF-90, the PEG-CPP33@Cy3-
siRNAsur@ZIF-90 group showed stronger tumor accumulation,
whereas other organs showed lower fluorescence values. This is due to
the ability of CPP33 to penetrate the cell membrane of human non-small
cell lung cancer A549 cells while minimizing transduction to normal
tissues [47].
3.8. Anti-tumor efficacy in vivo
The in vivo anti-tumor effects of PEG-CPP33 were investigated in
A549 tumor-bearing nude mice. A549 tumor-bearing nude mice were
randomly divided into five groups and injected with saline, siRNA@ZIF-
90, ORI@ZIF-90, NPs and PEG-CPP33@NPs (1.5 mg⋅kg− 1 siRNA and 3
mg⋅kg− 1 ORI per injection) via the tail vein every 2 days for four
consecutive days. The body weight and tumor volume of nude mice were
measured for 8 consecutive days. As shown in Fig. 5A, no significant
change was observed in the body weights of the mice in each group,
M. Cai et al.
Fig. 5. (A) Changes of body weight over time after injection of different nanoparticles (n = 6, mean ± SD); (B) changes in the tumor volumes of mice after injection
of different nanoparticles (n = 6, mean ± SD); (C) photo of the tumor after administration (a: saline, b: siRNA@ZIF-90, c: ORI@ZIF-90, d: NPs, and e: PEG-
CPP33@NPs); (D) tumor weight at the end of administration (n = 6, mean ± SD); and (E–I) expression of Bcl-2, Bax, PARP, caspase 3 and survivin in tumors (n
= 3, mean ± SD, *P < 0.05, **P < 0.001).
indicating that the nanoparticles had good biosafety and no significant
toxicity. Changes in tumor volume in each group are shown in Fig. 5B–D.
Compared with the control group, siRNA@ZIF-90, ORI@ZIF-90, NPs,
and PEG-CPP33@NPs showed tumor inhibition. The tumor inhibition
rates of siRNA@ZIF-90, ORI@ZIF-90, NPs, and PEG-CPP33@NPs groups
were 7.28%, 27.54%, 49.87%, and 61.04%, respectively. In contrast, the
tumors in the NPs and PEG-CPP33@NPs groups were significantly
smaller than those in the other groups, which may have been due to the
combined therapeutic effect of siRNA and ORI, making the anti-tumor
effect more significant. In addition, PEG-CPP33@NPs showed the
strongest anti-tumor effect, which may have been due to the specific
targeting of PEG-CPP33@NPs to A549 cells to promote the local accu­
mulation of siRNA and ORI in tumor tissues. In conclusion, PEG-
CPP33@NPs exhibited good siRNA and DOX synergy and excellent
targeting capability against A549 tumors.
ORI can reportedly induce tumor cell apoptosis through multiple
pathways, such as caspase-3, survivin, and Bcl-2/Bax [51]. The syner­
gistic application of ORI and other drugs can achieve an additive effect
on cancer treatment [52]. Fan et al. reported that ORI reversed cisplatin
resistance in non-small cell lung cancer [53]. To investigate apoptosis in
tumor tissues, survivin, Bcl-2/Bax, PARP, caspase 3 levels were
measured by western blotting. In accordance with the above results,
survivin levels were significantly decreased in the NPs and PEG-
CPP33@NPs groups, while the Bax/Bcl-2 ratio and PARP and caspase
3 levels were significantly higher than those in the other groups
(Fig. 5E–I). In addition, PEG-CPP33 had a more significant effect on
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Journal of Colloid And Interface Science 646 (2023) 370–380
promoting tumor apoptosis. Therefore, we determined that ORI exerts
anti-tumor effects through multiple targets and enhances the tumor
suppressive effect of siRNA through multiple pathways, such as caspase-
3, survivin, and Bcl-2/Bax.
3.9. In vivo biosafety evaluation
Owing to the inevitable accumulation of nano-formulations in the
liver and kidney of mice, the physiological and biochemical parameters
of the mice were measured to evaluate possible liver and kidney dam­
age. No significant differences were observed in ALT, AST, ALB, and
BUN concentrations among the groups, indicating no significant damage
to the liver and kidney function in each preparation (Fig. S21). In
addition, hematological parameters were measured in the mice after
treatment (Fig. S22). No obvious abnormalities were observed on
routine blood examinations in any group. The tumor, heart, liver,
spleen, lungs, and kidneys of the mice in each group were collected and
stained with H&E for observation, as shown in Fig. S23. No significant
histological differences were observed between the major organs in the
treatment and control groups. No significant histopathological changes
were observed in any of the major organs (heart, liver, spleen, lungs, or
kidneys), indicating that the nano-preparation was safe in vivo. In
contrast, tumor morphology significantly changed after treatment with
NPs and PEG-CPP33@NPs, further demonstrating the therapeutic effect
of the nano-preparations.
M. Cai et al.
4. Conclusion
We constructed a nanoplatform with tumor-homing and enhanced
lysosomal escape capabilities for synergy with chemotherapy and gene
therapy. The high porosity of MOFs enables the co-delivery of survivin
siRNA and ORI. The targeting ability of A549 cells was determined by
coating a PEGylated CPP (CPP33) onto the surface. The results showed
that ZIF-8 and ZIF-90 exerted protective effects against siRNA. In
addition, ZIF-90, with an aldehyde structure, showed better siRNA
protection in serum-containing media and RNase. This protective effect
resulted in a significant increase in the stability of the siRNA, thereby
alleviating the vulnerability of the siRNA to circulation in vivo. In
contrast to previous studies, survivin siRNA enhanced lysosomal escape
and was specifically taken up by A549 cells [54,55]. In addition, we
found that co-delivery of ORI with siRNA significantly increased the
apoptotic index of A549 cells and decreased the expression of survivin
compared with the application of siRNA alone, indicating a good syn­
ergistic effect between survivin siRNA and ORI [56]. The coating of PEG-
CPP33 improved the tumor-homing ability of the nanoplatform, and the
anticancer effect was significantly improved without obvious damage to
normal tissues. Most importantly, this study provides a simple method
for the simultaneous delivery of siRNA and small-molecule drugs to
achieve effective tumor suppression. Notably, PEGylated CPP-coated
MOFs offer a promising low-side-effect strategy for synergistic chemo­
therapy/gene therapy and may accelerate the clinical translation of
MOFs for cancer therapy.
Availability of data and materials:
The datasets used and/or analyzed during the current study are
available from the corresponding author on reasonable request.
Declarations:
Ethics approval and consent to participate.
This study is approved by the Institutional Animal Care and Use
Committee of Beijing University of Chinese Medicine.
Consent for publication:
All authors agree to submit the manuscript for publication.
CRediT authorship contribution statement
Mengru Cai: Conceptualization, Investigation, Methodology,
Writing – original draft, Writing – review & editing. Yu Yao: Concep­
tualization, Investigation, Methodology, Data curation. Dongge Yin:
Visualization, Investigation. Rongyue Zhu: Validation. Tingting Fu:
Validation. Jiahui Kong: Investigation, Visualization. Kaixin Wang:
Investigation, Visualization. Jing Liu: Investigation, Validation. Aina
Yao: . Yidan Ruan: Investigation, Validation. Wenjuan Shi: Formal
analysis, Investigation. Qian Zhu: Formal analysis, Investigation. Jian
Ni: Supervision, Project administration. Xingbin Yin: Project adminis­
tration, Funding acquisition.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Data availability
Data will be made available on request.
Acknowledgements
This research was funded by the Beijing Natural Science Foundation
(No. 7202121), Young Elite Scientists Sponsorship Program by CACM
(2021-QNRC2-A03), and the National Natural Science Foundation of
China (No. 81703715).
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Journal of Colloid And Interface Science 646 (2023) 370–380
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.jcis.2023.04.126.
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