Engineering polyphenol-based carriers for nucleic acid delivery

Abstract

Gene therapy, an effective medical intervention strategy, is increasingly employed in basic research and clinical practice for promising and unique therapeutic effects for diseases treatment, such as cardiovascular disorders, cancer, neurological pathologies, infectious diseases, and wound healing. However, naked DNA/RNA is readily hydrolyzed by nucleic acid degrading enzymes in the extracellular environment and degraded by lysosomes during intracellular physiological conditions, thus gene transfer must cross complex cellular and tissue barriers to deliver genetic materials into targeted cells and drive efficient activation or inhibition of the proteins. At present, the lack of safe, highly efficient, and non-immunogenic drug carriers is the main drawback of gene therapy. Considering the dense hydroxyl groups on the benzene rings in natural polyphenols that exert a strong affinity to various nucleic acids via hydrogen bonding and hydrophobic interactions, polyphenol-based carriers are promising anchors for gene delivery in which polyphenols serve as the primary building blocks. In this review, the recent progress in polyphenol-assisted gene delivery was summarized, which provided an easily accessible reference for the design of future polyphenol-based gene delivery vectors. Nucleic acids discussed in this review include DNA, short interfering RNAs (siRNA), microRNA (miRNA), double-strand RNA (dsRNA), and messenger RNA (mRNA).

Full text

Theranostics 2023, Vol. 13, Issue 10
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Theranostics
2023; 13(10): 3204-3223. doi: 10.7150/thno.81604
Review
Engineering polyphenol-based carriers for nucleic acid
delivery
Mingju Shui1,2*, Zhejie Chen3*, Yi Chen1,2, Qin Yuan1, Hongyi Li1, Chi Teng Vong2, Mohamed A. Farag4,
Shengpeng Wang1,2
1. State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao SAR 999078, China.
2. Macao Centre for Research and Development in Chinese Medicine, University of Macau, Macao SAR 999078, China.
3. Institute of Molecular Medicine (IMM), Shanghai Key Laboratory for Nucleic Acid Chemistry and Nanomedicine, Renji Hospital, School of Medicine,
Shanghai Jiao Tong University, Shanghai 200127, China.
4. Pharmacognosy Department, College of Pharmacy, Cairo University, Kasr el Aini St., Cairo 11562, Egypt.
* Mingju Shui and Zhejie Chen contributed equally to this work.
 Corresponding authors: Mohamed A. Farag, Pharmacognosy Department, College of Pharmacy, Cairo University, Kasr el Aini St., Cairo 11562, Egypt. Email:
mohamed.farag@pharma.cu.edu.eg. Shengpeng Wang, Institute of Chinese Medical Sciences, University of Macau, Avenida da Universidade, Taipa, Macao
999078, China. Email: swang@um.edu.mo.
© The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/).
See http://ivyspring.com/terms for full terms and conditions.
Received: 2022.12.06; Accepted: 2023.05.10; Published: 2023.05.21
Abstract
Gene therapy, an effective medical intervention strategy, is increasingly employed in basic research and
clinical practice for promising and unique therapeutic effects for diseases treatment, such as
cardiovascular disorders, cancer, neurological pathologies, infectious diseases, and wound healing.
However, naked DNA/RNA is readily hydrolyzed by nucleic acid degrading enzymes in the extracellular
environment and degraded by lysosomes during intracellular physiological conditions, thus gene transfer
must cross complex cellular and tissue barriers to deliver genetic materials into targeted cells and drive
efficient activation or inhibition of the proteins. At present, the lack of safe, highly efficient, and
non-immunogenic drug carriers is the main drawback of gene therapy. Considering the dense hydroxyl
groups on the benzene rings in natural polyphenols that exert a strong affinity to various nucleic acids via
hydrogen bonding and hydrophobic interactions, polyphenol-based carriers are promising anchors for
gene delivery in which polyphenols serve as the primary building blocks. In this review, the recent
progress in polyphenol-assisted gene delivery was summarized, which provided an easily accessible
reference for the design of future polyphenol-based gene delivery vectors. Nucleic acids discussed in this
review include DNA, short interfering RNAs (siRNA), microRNA (miRNA), double-strand (dsRNA), and
messenger RNA (mRNA).
Keywords: gene therapy; polyphenols; nucleic acid; RNA; EGCG
1. Introduction
Gene therapy is an intracellular approach of
introducing exogenous therapeutic genes (transgenes)
into specific host cells to elicit therapeutic effects for
the treatment of genetic-based diseases via correcting
the mutated/altered genes or providing the cells with
a new function, which represents a promising and
specific treatment for a series of diseases, such as
cardiovascular, cancer, neurological, infectious disea-
ses, and wound healing [1-5]. Owing to the broad
potential and guaranteed physiological activities,
gene therapy has been increasingly applied for more
than 3 decades, and the first clinical trial of adenosine
deaminase (ADA) gene therapy for the treatment of
severe combined immunodeficiency (SCID) was in
1990 [6]. Generally, gene therapy can be divided into
two major types, somatic and germline therapies,
which brings new possibility and treatment options to
multiple medicinal fields [1,7]. However, as naked
DNA/RNA molecules can be degraded easily by
intracellular lysosomes or extracellular nucleic acid
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degrading enzymes, gene transfer must cross the
multiple cellular and tissue barriers to deliver genetic
materials into the pathological sites and trigger
efficient expression of the therapeutically bioactive
substances or obvious down-regulation of proteins
[8,9]. To achieve effective delivery of gene based-
drugs, innovative preparations that are characterized
by excellent cell targeting specificity, gene expression
regulation, gene transfer efficiency, and vector safety,
are warranted [10].
Currently, gene delivery systems can be
categorized into viral and non-viral systems. Due to
the extreme efficiency at transferring genes, viral
vectors, such as retrovirus, adeno-associated virus,
adenovirus, pox virus, lentivirus, herpes simplex
virus, and human foamy virus (HFV), are regarded as
one of the significant constituent bases of gene
therapy systems [11]. Nevertheless, safety concerns
about severe toxicity and off-target immunogenicity
have severely limited the clinical translation of viral
gene delivery [12]. Although recombinant viruses are
non-pathogenic powerful vectors, it is still possible to
revert the virus to wild-type virus or co-purify it with
replicating virus [13]. In addition, viral vectors can
elicit immunogenic responses and induce the
activation of inflammatory system, toxin production,
and insertional mutations, which can lead to cancer
[14,15]. Other bottlenecks of the low targeting
specificity and high manufacturing costs also further
hinder the application of viral vector-based gene
therapy [13,16]. All these safety issues and existing
challenges motivate the exploration of safer, less
immunogenic and pathogenic, highly efficient, and
stable gene delivery vectors. Non-viral vectors, also
called synthetic vectors, are widely developed to
deliver genetic payloads [13]. Non-viral vectors are
safer and more flexible than viral vectors, and provide
greater structural and chemical versatility for
exploiting physicochemical properties [10,17].
Although the efficiency of non-viral vectors in gene
delivery is less than viral vectors, the lower toxicity,
higher vector stability, larger gene capacity, lower
cost, and less immunogenic response also make them
to be more reliable for gene therapy [9,12]. The
synthetic methods of non-viral vectors include
physical and chemical methods, such as
electroporation, ultrasound, magnetofection, gene
gun, cationic polymers and liposomes [18,19]. In
clinical trials, two non-viral gene delivery methods
are widely utilized, including direct injection of the
naked gene drugs (plasmids containing the transgene)
into the tissues and the preparation of lipofection to
pack nucleic acids [20]. With the advancement of drug
delivery system and nanotechnology, a variety of
non-viral nanocarriers were designed for gene
delivery, such as cationic lipids, polymers, graphene,
dendrimers and other inorganic nanoparticles, which
rely on three main packaging strategies, including
electrostatic interaction, encapsulation and adsorption
[10,21,22]. Additionally, some natural products were
also developed due to the interaction between natural
products and genes, such as gelatin, chitosan and
polyphenols [16,23,24]. Guo et al. described a
hierarchical coating (chitosan/gelatin) through the
assembly of siRNA-loaded nanoparticles on titanium
implants for the synergistic regeneration of skeletal
and vascular tissues. The functionalized nanoparticles
successfully delivered siRNA cathepsin K (siRNA-
CTSK) to bone and showed a high gene silencing
efficiency and therapeutic improvement on osteo-
integration through synergetic effects on bone
regeneration and blood vessel system repair [25].
However, the existing nucleic acid drug delivery
systems are not perfect. For example, the most
commonly used cationic liposomes require expensive
excipients and equipment for their preparation, thus
resulting in high application costs, and they can also
produce high toxicity when a high dose is used
[26,27]. Another obvious disadvantage of liposome
preparation is the lack of targeting when adminis-
tered intravenously, which can lead to adverse
reactions, such as hand-foot syndrome [28,29].
Besides, polymer-mediated drug delivery systems,
such as polyethylenimine (PEI), have a risk of
cytotoxicity, poor molecular weight (MW) control,
and overly slow or fast degradation kinetics [9,30,31].
Inorganic materials are easy to be accumulated in the
body and difficult to exclude [32]. Therefore,
developing new nucleic acid delivery systems with
high efficiency and low toxicity has become an urgent
strategy in the field of nucleic acid delivery.
Among the myriad of natural product classes,
polyphenols are featured by multiple phenolic rings
in their basic structure [33,34]. Over 8000 phenolics
and their derivatives have been identified in various
plants, especially in fruits, vegetables, tea, coffees,
wine, and grains, which play a role in oxidative
stability and sensory properties of food (like flavor,
color, bitterness and astringency) [35-37]. Polyphenols
can be classified into four categories, namely
flavonoids, stilbenes, phenolic acids and lignans,
according to the number and binding structure of
phenolic rings [38]. Among them, flavonoids are the
most abundant found ubiquitous in planta, such as
quercetin, epicatechin gallate (ECG), epigallocatechin
gallate (EGCG) and catechin (CAT). Benefiting from
their fascinating biological activities, such as anti-
oxidant, anti-cancer, anti-bacterial, anti-inflammatory,
anti-viral, and myocardial protection effects, natural
polyphenols have been employed in the field of food,

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pharmaceutical industries as natural therapeutic
agents in recent years [39-52]. Besides serving as
auxiliary therapeutic drugs, polyphenols can also act
as primary construction modules for multiple func-
tional nanomaterials, due to their special structure
being rich in benzene rings and hydroxyl groups. It
has been demonstrated that natural polyphenols can
interact with various materials (small molecules,
metal ions, polymers, nucleic acids and proteins) via
hydrogen bonding, hydrophobic interactions, π
interactions, metal coordination, electrostatic interact-
ions, and covalent bonding [24,53,54]. Polyphenol-
based functional materials are promising for the
development of adhesive materials, surface coating,
hemostatic applications, and hydrogels [55-61].
Polyphenols have also been functionalized on the
surface of living cells via metal-phenolic complex-
mediated interfacial interactions to create a versatile
cell-based biological platform, in which extrinsic
bioactive molecules (proteins, DNA, mRNA) can be
imparted to the cells [62]. More importantly, they can
bind with genes due to their multi-hydroxyl structure,
thus making them to be promising and excellent gene
delivery vectors. With the employment of poly-
phenols in innovative delivery strategies, the
application of polyphenols in gene delivery systems
has been extensively investigated for the reliable
nucleic acid delivery. At present, polyphenols have
been applied to the construction of delivery systems
for a variety of gene therapies, including DNA,
siRNA, miRNA, dsRNA, and mRNA (Figure 1). Since
polyphenols can complex with nucleic acids to
produce nanostructures, which replace the loose state
of nucleic acid drugs, thereby reducing the risks of
nucleic acid degradation in vitro [59]. Meanwhile,
polyphenols are desired to down-regulate the
molecular weight and dosage of cationic polymers
under the same conditions, thereby improving the
safety of drug delivery systems.
In this review, the most updated status of
polyphenols-assisted gene delivery system is
summarized and the functional roles of natural
polyphenols in the design of gene delivery strategies
are discussed. The description of the preparations of
different polyphenol-based carriers is briefly
reported, while the main part of this review discusses
the efficacy of the polyphenols in promoting nucleic
acids delivery and gene silencing for disease
therapies. In addition, the challenges of polyphenol-
based gene delivery systems and prospects for the
design of future natural polyphenol drug delivery
systems are discussed. Therefore, this review aims to
present the current development of polyphenol-
assisted gene delivery strategies, thus providing an
easily accessible reference and promoting the
application of polyphenol-assisted gene delivery sys-
tems for researchers and pharmaceutical companies.
Figure 1. Schematic illustration of the molecular interactions of dietary polyphenols as structural units with nucleic acids for the construction of gene delivery systems in various
applications.

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2. The binding mechanisms of
polyphenols and nucleic acids
Nucleic acid is a biological macromolecular
compound, which is polymerized from many
nucleotide monomers and plays a role as a carrier of
genetic information in the life system. There are also
some nucleic acids that can be used as enzyme
molecules or other molecular machines with
biological activities, including ribozymes, deoxyribo-
nucleases, and their complexes. Nucleotide is the
basic unit of nucleic acids. A nucleotide molecule is
composed of a nitrogenous base, a five-carbon sugar,
and a phosphoric acid. With the condensation of
phosphoric acids and five-carbon sugars to form the
polymerization skeleton of nucleic acids, the
phosphate backbone containing dense lone pair
electron oxygen is constructed. Notably, the main
characteristics of polyphenols as building blocks for
drug delivery are the dense m-hydroxyl and
o-hydroxyl groups in the trihydroxy phenyl (galloyl)
and dihydroxy-phenyl (catechol) components, such as
tannic acid (TA), CAT, and EGCG. The dense phenolic
hydroxyl groups represent excellent electron donor
groups, which can form intermolecular complexation
with the phosphate skeleton containing dense lone
pair electron oxygen via intermolecular hydrogen
bonds. Besides, nucleic acid molecules contain plenty
of bases whose water solubility is unsatisfactory. The
abundant benzene rings in polyphenols constitute
water-insoluble structure. Through hydrophobic
interaction, the benzene ring structure in polyphenols
can interact appropriately with the bases in nucleic
acids, thus improving the interaction between poly-
phenols and nucleic acids. Zheng et al. investigated
the interaction between EGCG and DNA using
electrochemical techniques, and showed that EGCG
could intercalate into DNA strands and form an
electrochemically inactive complex [63]. Fujiki et al.
found that single-strand 18 mers of DNA or RNA
could bind to 1 to 3 EGCG molecules in surface
plasmon resonance assay (Biacore) and cold spray
ionization-mass spectrometry, suggesting that
multiple binding sites of EGCG were present in DNA
and RNA oligomers [64]. Using enhanced sampling
techniques and molecular dynamics simulations,
Rodrigo et al. observed that both benzopyran moiety
ring and trihydroxyphenyl ring of EGCG could form
hydrogen bonds with the oxygen atoms in the DNA
backbone of the 5’-strand, and a stable complex was
formed between the EGCG ligand and DNA by
intercalating the trihydroxybenzoate aromatic ring
and an ApC step [65]. In summary, the interactions
between polyphenols and nucleic acids are synergis-
tically mediated by both intermolecular hydrogen
bonds and hydrophobic interactions, which are the
basic mechanism of building polyphenol-assisted
drug delivery systems.
3. Polyphenol-assisted nucleic acid
delivery
Nucleic acids, the general name of DNA and
RNA, are a kind of biopolymer and the indispensable
genetic materials of all creatures. The classification of
nucleic acids is mainly determined by the five-carbon
sugars involved in construction. If the pentose is
ribose, the polymer formed is RNA; if the pentose is
deoxyribose, the nucleic acid formed is DNA. As
genetic molecules, nucleic acids are excellent
candidate polymers for biomaterial applications with
precise molecular recognition, superior sequence
programmability, and extensive biological function-
ality [33,66]. Successful delivery of genetic material
plays an important role in gene therapy, and
polyphenols have a strong binding affinity to
macromolecular nucleic acids via forming hydrogen
bonds, thus increasing their in vivo stability, which
can be a safe and high-efficient gene vector.
3.1 Polyphenol-assisted DNA delivery
Various cationic polymers have been employed
to condense plasmid DNA (pDNA) into nanoscale
complexes via electrostatic interactions, especially
PEI, which facilitates endosomal escape and promotes
cell internalization [67,68]. However, cytotoxicity and
agglutination with blood components hinder the
clinical application of pDNA/PEI complex [69].
Therefore, Liang et al. firstly designed a gene delivery
system based on green tea catechin, a nanoscale
self-assembled ternary complex (pDNA/PEI/HA-
EGCG), which exhibited remarkable protection of
pDNA from nuclease hydrolysis and polyanion-
induced dissociation and ascertained high transfec-
tion efficiency for the difficult-to-transfect HCT-116
cells even under serum-supplemented conditions
(Figure 2A-D) [70]. In this DNA delivery system,
hyaluronic acid (HA) did not only improve gene
transfection efficiency through endocytosis mediated
by HA receptor, but rather effectively downregulated
the potential cytotoxicity and pDNA/PEI agglutina-
tion via shielding the surface positive charges.
Besides, the natural polyphenol EGCG could stabilize
pDNA/PEI complexes owing to its strong DNA-
binding affinity and is known to suppress the activity
of various enzymes, such as nucleases, collagenases
and hyaluronidases via blocking their active sites,
thereby protecting DNA against degradation domi-
nated by serum proteases in vivo. Two cell lines were
employed to evaluate gene transfection efficiency of
pDNA/PEI in vitro. In HEK293 cells (cluster of

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differentiation 44 (CD44) low-expressed), the
transfection efficiency was 73.8 ± 5.2% with N/P ratio
of 30, while the transfection efficiency was 19.0 ± 0.8%
in HCT-116 cells (CD44 over-expressed). At the
optimal C/P ratio of 0.5, the transfection efficiency of
pDNA/PEI/HA-EGCG system was improved to 43.7
± 1.5% in HCT-116 cells, which was approximately
2.3-fold higher than that of pDNA/PEI (18.6 ± 0.6%).
In addition, compared to pDNA alone, pDNA/PEI
complexes and pDNA/PEI/HA complex, pDNA/
PEI/HA-EGCG was successfully delivered into
mouse HCT-116 tumor tissues with significantly
increased distribution of pDNA in tumors tissues,
suggesting that this HA-EGCG-stabilized system
could potentially be utilized in CD44-targeted
nucleotide therapeutics delivery.
To co-deliver genes and anti-tumor agents, Yang
et al. developed a general and simple carrier based on
DNA-polyphenol nanocomplex that is characterized
by controllable assembly/disassembly behaviors,
thus exhibiting extremely high gene/drug loading
capacity (Figure 2E-G) [71]. Briefly, two extrinsic
genes antisense DNA and DNAzyme were struc-
turally designed as branched-DNA, thus DNAzyme
was prepared as Y-shape DNA (Y-DNA) while
antisense DNA was programmed into L-DNA. Then,
Y-DNA and L-DNA formed branched DNA via
hybridization, and TA intervened the assembly of
branched-DNA to form nanosized complexes. The
encapsulation efficiencies of antisense DNA,
DNAzyme, and TA in this nanosystem were at 94.4%,
96.2%, and 85.2%, respectively. To improve the
tumor-targeting efficiency, DNA aptamer was
effectively anchored onto this nanocomplex (nano-
complex-apt). The acid microenvironment of
lysosomes triggered the release of TA and branched-
DNA, and branched-DNA was further separated as
antisense DNA and DNAzyme by DNase I and
glutathione (GSH). This nanocomplex exerted specific
cytotoxicity to A549 cells and showed an excellent
biocompatibility with normal cells, suggesting its
safety of use. Antisense DNA conspicuously inhibited
the hyperplasia of A549 cells through reducing C-raf
mRNA expression. Besides, TA facilitated cancer cell
apoptosis via decreasing the expression levels of
apoptosis-related proteins (such as Bcl-2, an
anti-apoptosis protein). The incorporation of DNA
aptamer up regulated the proliferation inhibition of
A549 cells due to the targeting capacity of the
aptamer. DNAzyme also inhibited A549 cell migra-
tion through reducing matrix metallopeptidase 9
protein (MMP-9) expression, thus further improving
the therapeutic efficacy of nanocomplex-apt. In A549
tumor-bearing mice, nanocomplex-apt showed
specific targeting to tumor and achieved the best
medical intervention effects on cancer cells, which
demonstrated the synergistic effects of chemotherapy
and multiple-gene therapy.
Cheng et al. developed a facile, robust, and
efficient strategy in delivering single-strand oligonuc-
leotides (Figure 3A-D) [72]. The natural polyphenol
EGCG was employed to combine with oligonucleo-
tide to prepare a negatively charged core, then
ε-polylysine (PLL) (positive charge) was covered to
the surface of EGCG/oligonucleotide to form the
core-shell nanostructure, which was termed as green
nanoparticles (GNPs). Next, three different oligonuc-
leotides were chosen to estimate the application
potential of GNPs in gene delivery, including
miRNAs, antisense oligonucleotide (ASO), and
DNAzymes. For delivering ASO, GNPs protected
ASO from RNases degradation, which might attribute
to the core-shell structure of GNPs. GNPs,
Lipofectamine 2000 (LPF) and TransExcellent-siRNA
(TE), exhibited prominent gene-silencing efficiency in
delivering siRNA. However, compared to LPF and
TE, GNPs showed higher gene knockdown efficiency
in delivering unmodified ASO (59%) and
phosphorothioate backbone ASO (Ps-ASO) (64%), due
to the good stability of oligonucleotides in GNPs and
efficacious endocytosis and intracellular release. The
successful delivery of DNAzymes could target the
oncogene Bcl-2 in Hela cells and downregulate the
expression of Bcl-2 mRNA, thus effectively inhibiting
Bcl-2 protein expression and increasing apoptosis.
Besides, polyphenol-based hydrogels are also
widely used in innovative drug delivery systems. TA,
a natural polyphenol, is often utilized as a natural
anti-inflammatory, anti-oxidant, and antibacterial
agent. More importantly, it is also an exceptional
crosslinking agent for the preparation of innovative
hydrogel delivery system due to its high content of
natural catechol/pyrogallol moieties. Lee et al.
utilized TA as a molecular glue to produce a DNA
hydrogel, called TNA hydrogel, whose crosslinking
mechanism was the formation of hydrogen bonds
between DNA’s phosphate backbone and TA’s
polyphenol (Figure 3E-H) [73]. At a stoichiometric
ratio of DNA base pairs/TA of 1.3, spontaneous TNA
gelation was observed after TA was added into the
DNA solution, while no gelation was occurred when
the ratio was at 2.6. In contrast, mechanically robust
TNA gels were formed when the ratio was downsized
from 1.3 to 0.9. The TNA gel was degradable due to
the hydrolysable ester bonds between the catechol
and pyrogallol groups in TA, which resulted in the
release of DNA from the gel. The dissociation and
degradation of TNA gel were able to be divided into
two steps. In the first step (0.5-1h), TA in this gel was
degraded to ellagic acid (EA) and gallic acid (GA) at

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low extent owing to the steric hindrance of the
complexed DNA. After > 3h, TA was dissociated from
TA/DNA complexes and then degraded, thereby
increasing the amounts of free EA and GA, which was
accompanied with more DNA release. With excellent
extensibility, biocompatibility and strong mechanical
properties, TNA gel displayed biomedical potential as
controllable adhesives. In a mouse liver bleeding
model, TNA gel exhibited an excellent hemostatic
effect and obviously shortened the hemostatic time
when compared with negative control and single-
component solutions, demonstrating that hemostasis
was enhanced by the adhesive property of TNA gel.
With biodegradability, extensibility, tissue adhesive-
ness and hemostatic ability, this multifunctional TNA
gel could be an innovative DNA-based platform for
future biopharmaceutical applications.
Figure 2. Polyphenol-assisted DNA delivery. (A) The self-assembled formation and cellular uptake process of pDNA/PEI/HA-EGCG ternary complexes. (B) The GFP gene
transfection efficiency in HCT-116 cells treated with different complexes at different C/P ratios. (C) Quantification of GFP expression in HCT-116 cells. (D) Intratumoral

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distribution of Cy5-labeled pDNA delivered by various polyelectrolyte complexes. Adapted with permission from [70], copyright 2016 Elsevier. (E) Schematic illustration of the
assembly and controlled disassembly process of DNA nanocomplex in cells. (F) Ex vivo biodistribution of DNA at major organs and tumors of the mice treated with different
formulations. (G) Tumor size in A549 tumor-bearing mice treated with different formulations. Adapted with permission from [71], copyright 2021 Elsevier.
Figure 3. (A) Schematic illustration of GNPs construction and delivery of single-strand oligonucleotides including anti-miRNA, ASO, and DNAzyme. (B) Efficiency of GNPs in
the delivery of siRNA, Ps-ASO, and ASO into Hela-Luc cells for 24 h. (C) The mRNA expression levels of Bcl-2 in Hela cells after treatment with GNPs loaded with DNAzyme
targeting Bcl-2. (D) Representative Bcl-2 protein expressions in treated cells. Adapted with permission from [72], copyright 2020 Springer Nature. (E) Schematic illustration of
the formation and degradation of TNA gels. (F) The degradation process of TNA gels. (G) Images of the hemostatic effect of TNA gels within 60 s. (H) The required times for
complete hemostasis of the bleeding mouse liver. Adapted with permission from [73], copyright 2015 WILEY.
3.2 Polyphenol-assisted RNA delivery
RNA interference (RNAi) can specifically
down-regulate target genes by delivering small RNA
duplexes, including siRNAs, miRNA mimics, dicer
substrate RNAs (dsiRNAs) and short hairpin RNAs
(shRNAs), which provides alternative treatment
options for various diseases when current drug fails
[74-76].

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3.2.1 Polyphenol-based siRNA delivery
Gene therapy via siRNA has been a promising
technique for the treatment of cancer and other
diseases by preventing the target proteins production
[77]. However, naked siRNA is unstable in
physiological environment and susceptible to enzyme
degradation, and the highly negative charge limits its
penetration through the cell membrane [78]. Thus,
successful RNAi therapies are heavily dependent on
the construction of efficient gene delivery platforms.
Cheng et al. designed a simple and general
supramolecular strategy to fabricate the core-shell
nanostructure for siRNA delivery, in which siRNA
was pre-complexed with EGCG as the negatively
charged core and then coated with low-molecular-
weight cationic polymer as the shell (Figure 4A-F)
[79]. EGCG, that is complexed with siRNA via
hydrogen bonds and hydrophobic interactions,
largely improved the siRNA complexation ability of
low-molecular-weight polymers. The addition of
EGCG facilitated the formation of smaller, more
uniform and stable GNPs, which showed a good
stability in 150 mM sodium chloride (NaCl) solution
and cell culture medium. In addition, six types of
different molecular weight cationic polymers
(including linear, branched, and dendritic polymers)
were tested to balance the transfection efficiency-
toxicity in siRNA delivery. Compared with minimal
toxic low-molecular-weight cationic polymers, the
high-molecular-weight ones exhibited high efficiency
but possessed serious toxicity. In contrast, both low
toxicity and high transfection efficiency were
achieved, when GNPs were fabricated with these 6
screened low-molecular-weight polymers. In HeLa
cells with stable expression of firefly luciferase, all
chosen GNPs exhibited high gene silencing
efficiencies (~80%) compared with the nanoparticles
without EGCG (< 5%). Among these 6 representative
cationic polymers, ε-PLL was preferred for fabricating
GNPs for the in vivo experiments due to the high
siRNA delivery efficiency and good biocompatibility.
In a dextran sulfate sodium (DSS)-induced mouse
intestinal injury model, GNPs exhibited a distinct
reduction of prolyl hydroxylase 2 (PHD2) and TNF-α
gene expressions in the colonic tissues. Compared to
control, GNPs effectively ameliorated intestinal
symptoms and inflammation, including lowered
disease activity index (DAI) score, less body weight
loss, shortened colon length and reduced inflam-
matory cytokines levels. All these results indicated
that this supramolecular strategy of the construction
of GNPs can be a versatile and potential method for
various gene delivery. In addition to cationic
polymers that could condense with siRNA, different
polyphenol moieties can also affect the binding and
deliverable capacity of siRNA. The structure-function
relationship of polyphenolic moieties in facilitating
siRNA condensation and delivery was further
investigated as the natural polyphenol EGCG could
be hydrolyzed into two other polyphenol motifs, EGC
and GA [80]. Their results revealed that EGCG and
EGC exhibited enhanced binding ability to siRNA
through hydrogen bonds and hydrophobic interact-
ions, while GA with a pyrogallol structure could
hardly bind to siRNA and suggestive that it does not
play a role in binding to nucleic acids. In the
experiment of polyphenol assisted PLL delivery of
siRNA targeting luciferase (siLuc), the delivery
capacity was in the following order EGCG > EGC >
GA, with corresponding gene silencing efficiency of
80%, 40%, and almost zero, respectively. As the molar
ratio of EGC/EGCG increased, the gene knockdown
efficiency was significantly increased, which provided
novel insights for the search for other natural
polyphenols with similar chemical structures to
effectively assist gene delivery and further provided
theoretical support for the development of more safe
and efficient RNAi drugs.
Inspired by the structural and functional
characteristics of natural polyphenols, a novel
strategy to simplify the synthesis process into one step
was attempted, in which the functional moieties from
naturally occurring polyphenols were first grafted
onto low-molecular-weight cationic polymers to
fabricate poly-catechol polymers, thus improving
siRNA binding ability (Figure 5A-E) [81]. The phenyl,
phenol, catechol, and pyrogallol-modified PLL
polymers were synthesized and termed P0-P3
separately, then the corresponding polymer/siRNA
complexes were prepared. P2, with catechol groups
showed the highest luciferase gene silencing
efficiency, albeit with certain toxicity in HeLa-Luc
cells. Besides, in HeLa and mouse intestinal epithelial
cells (IECs), P2/siRNA complex successfully
downregulated its targeted glyceraldehyde 3-phos-
phate dehydrogenase (GAPDH) and PHD2 gene
expression. The gene silencing efficiency was related
to the siRNA binding ability of polymers in the order
of P2 > P1 > P3 ≈ P1 > PLL, which was due to the
balance of hydrophobic interactions and hydrogen
bonding between siRNA and polyphenols. As the
number of hydroxyl groups on the aromatic ring
increased, hydrophobic interactions were weakened
while hydrogen bonds became more dominant.
Besides, the interruption of the balance of hydrogen
bonding and hydrophobic interactions resulted in a
decrease of the affinity of siRNA for the polymer. In
DSS-induced intestinal injury or colitis model,
P2/siRNA successfully delivered siRNA to the
injured tissues and significantly relieved the symp-

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toms and reduced inflammatory levels. Inspired by
these results, Fan et al. also designed and synthesized
a set of cationic poly-catechols for siRNA delivery by
two different direct polymerization methods. Several
important macromolecular parameters affecting
siRNA delivery efficiency were investigated, such as
catechol content, molecular weight, and backbone
rigidity (Figure 5F-I) [82]. Poly-catechols P1 - P6 were
fabricated via radical polymerization, whereas P8 -
P18 were via typical ring opening metathesis poly-
merization (ROMP) method. Furthermore, polymers
were complexed with siLuc. Polymers P1 and P4 of
high catechol contents (50%) potently downregulated
luciferase gene expression and exhibited better
performance in siRNA condensation, protection, and
cellular uptake, while polymers with lower catechol
contents (40% for P2 and P5; 30% for P3 and P6) failed
to silence luciferase gene at the same molar
concentrations. P8 - P18, with more rigid backbones,
showed poor efficiencies in siRNA delivery, which
revealed that catechol-derived polymers with high
catechol content and flexible backbones (P1 and P4)
were the best for efficient siRNA delivery. In addition,
compared with P4, P1 still maintained high gene
knockdown efficiency even at a low siRNA dose, and
showed high siRNA efficiency and efficiently down-
regulated TNF-α gene expression. In DSS-induced
ulcerative colitis (UC) model, P1/siTNF-α effectively
downregulated TNF-α level and ameliorated UC
symptoms without any adverse effects.
Figure 4. Polyphenol-based RNAi efficient delivery. (A) Schematic formation and intracellular gene silencing mechanism of GNPs. (B) Different polymers mediated RNAi
efficiency in HeLa-Luc cells for 24 h. (C) The mechanism of TNF-α induced cell apoptosis in DSS-induced intestinal injury model. (D) The protein levels of PHD2 and TNF-α in

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the colon tissues. (E) Disease activity index and (F) colon length of mice after different treatments. Adapted with permission from [79], copyright 2018 American Chemical
Society.
Figure 5. Polyphenols with different structures for siRNA delivery. (A) Schematic illustration of the synthetic route of polycatechols with different phenolic moieties for siRNA
delivery. (B) Gene silencing efficiency of PLL and P0-P3 in delivering siLuc to HeLa-Luc cells. (C) Cellular uptake efficiency of polymer/siRNA-FAM complexes in HeLa-Luc cells.
(D) Protein expression levels of PHD2, HIF-1α, and TNF-α in colon tissues analyzed by Western blotting. (E) Colon length of the mice in different groups after treatment.
Adapted with permission from [81], copyright 2021 WILEY. (F) Schematic illustration of the synthesis method of the polymers and the gene silencing mechanism of siRNA
delivery in cells and DSS-induced intestinal injury mice. (G) The structures, molecular weights of polymers P1 - P7 (synthesized by radical polymerization) and polymers P8 - P10
(synthesized by ROMP). (H) The luciferase gene knockdown efficiencies of polymer/siLuc (P1 - P7) in HeLa-Luc cells for 24 h. (I) Colon length of mice after treatments. Adapted
with permission from [82], copyright 2020 Chinese Chemical Society.
Therefore, this study provided strong support
for the synthesis and screening of polyphenols for
siRNA delivery. In addition to gene delivery, the
synthesized polycatechols were also utilized as a
promising drug delivery platform for cytosolic
protein and peptide delivery, which greatly promoted
the delivery efficiency [83,84].
Considering the dual roles as the structural units
and therapeutic agents, polyphenols are used in
combination therapy of gene cancer therapies. Yang et

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al. utilized natural TA as a “sandwich component”
that binds to nucleic acids and tumor-associated-
antigen of cancer cell membranes, thus constructing a
smart system for drug delivery and gene therapy that
delivered therapeutic RNA to target cells (Figure
6A-F) [85]. Firstly, the sticky ends of siRNA were
attached to the complementary sticky ends of Y-DNA
to form branched-DNA/RNA, TA was then added to
mediate both assemblies of branched-DNA/RNA and
A549 cell membrane to form a nanocomplex
(nanocomplex@A549m), which could mediate the
dissociation of the nanocomplex in the lysosomal
acidic microenvironment and exerted pro-apoptotic
effects on cancer cells. In A549 cells, nanocomplex@
A549m showed high cellular uptake efficacy, specific
homotypic targeting capability and reduced
macrophage cell internalization. Furthermore, in A549
tumor-bearing mouse model, nanocomplex/siPKL1@
A549m effectively delivered therapeutic siRNA to
tumor sites and exhibited high RNAi efficiency and
enhanced anti-tumor activity, which were also
confirmed by in vitro assays. Zhu et al. described a
high-effective and low-toxic multi-component vector,
that was capable of facilitating gene and drug
co-delivery for the treatment of drug-resistant breast
cancer overexpressing connective tissue growth factor
(CTGF), which encapsulated both EGCG and siRNA
into a biodegradable nanogel through a self-assembly
process (Figure 6G-I) [86]. In this biodegradable
system, positively charged protamine was employed
as the “adhesive” to encapsulate sufficient amounts of
siRNA and EGCG, and the outer surface was coated
with HA and cell-penetrating peptide PEGA-pVEC.
In this system, HA coating could specifically identify
the overexpressed CD44 receptor on the surface of
cells to induce endocytosis mediated by clathrin and
triggered EGCG release by hyaluronidase (HAase),
while PEGA-pVEC could target the proline amino-
peptidase overexpressing in the blood vessels of
breast tumor. Compared to CTGF-negative MCF-7
cells, siRNA/EGCG/protamine/HA nanogel showed
high selectivity to CTGF-overexpressed MDA-MB-231
cells, due to the specific recognition of CD 44 receptor
and endogenous HAase-induced drug release.
Besides, compared to EGCG, siRNA/EGCG/prota-
mine/HA showed a 15-fold increase in cytotoxicity
against drug-resistant MDA-MB-231 cells, which was
attributed to the synergistic effect of EGCG and
siRNA. In MDA-MB-231 xenograft tumor model,
nanogel@peptide displayed the highest tumor
inhibition effect and little toxicity to normal tissues
and organs. Additionally, the expressions of cellular
apoptosis protection related proteins (phosphor-focal
adhesion kinase (p-FAK), phospho-extracellular
regulated protein kinases (p-ERK), and poly
ADP-ribose polymerase (PARP)) and drug resistance
related proteins (B-cell lymphoma-extra large (Bcl-Xl),
cellular inhibitor of apoptosis protein 1 (cIAP1), and
CTGF) were down-regulated both in vitro and in vivo.
Natural polyphenols have also been utilized as
functional coatings to assist gene delivery. Recently, a
novel type of core-shell structured MSN (MNC@
LPMSA@siRNA@TA) with large pore was proposed
for siRNA delivery, which possessed small particle
sizes, high siRNA loading capacity, magnetic-guided
delivery ability and pH-responsive cellular siRNA
release [87]. The magnetic nanocrystal clusters (MNC)
were firstly synthesized and coated with dendritic
mesoporous silica layer to yield core-shell
MSC@LPMS, which were then functionalized with
(3-aminopropyl)-triethoxysilane (APTES) to obtain a
positively charged silica surface for the loading of
negatively charged siRNA (loading efficiency ca. 2%
w.t.) due to the high pore volume and surface area.
Finally, TA/Al3+ complex, acid-resistant coating
formed via pH-dependent complexation, was coated
to MNC@LPMSA@siRNA to protect siRNA against
degradation, thereby improving the stability of these
nanoparticles. These nanoparticles were then
endocytosed and entered the acidic lysosomes
through the tight binding between Al3+ and the cell
membrane. Then TA/Al3+ coating was disassembled
in the acidic environment of lysosome, which
consumed protons (H+) and facilitated intracellular
endosomal escape through the “proton sponge”
effect. In osteosarcoma KHOS cells, siRNA was
effectively delivered into the cytoplasmic region and
under the magnetic field, it showed the highest
cellular uptake, with negligible cytotoxicity.
Metal-polyphenol networks (MPNs) is an
increasingly attractive topic that is used in gene
delivery in recent years. Chen et al. developed a green
carrier (Mg(II)-Cat NPs) for efficient siRNA delivery
through the chelation of natural polyphenol
((+)-catechin) with metal ions (Mg2+) [88]. Mg(II)-Cat
NPs were prepared by the reaction of Mg2+ with
adjacent hydroxyl groups on catechin at room
temperature. siEIF5A2 was chosen for targeting and
knocking down the oncogene eukaryotic translation
initiation factor 5A2 (EIF5A2), thus inhibiting cancer
cell growth. How would be the enantiomer of
catechin? i.e., epicatechin that performs in siRNA
delivery and its effect of stereochemistry on nucleic
acid binding has yet to be fully explored. In T24 cells,
Mg(II)-Cat/siEIF5A2 successfully down-regulated the
gene expression of EIF5A2 and induced cancer cell
apoptosis. In athymic mice bearing T24 cells xenograft
model, Mg(II)-Cat/siEIF5A2 was effectively accumu-
lated in tumors and exerted significant antitumor
effects via inhibiting phosphoinositide 3-kinase/

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protein kinase B (P13K/Akt) signaling pathway. In
addition, Mg(II)-Cat/siEIF5A2 was also found to be
an active anti-tumor agent in a clinically-relevant rat
in-situ bladder cancer model, suggesting that
metal-polyphenol network was a promising platform
for the co-delivery of siRNA and chemotherapeutic
agents.
Figure 6. Polyphenol assisted siRNA delivery. (A) Schematic construction of nanocomplex@A549m. (B) Scheme of the synthesis and controlled disassembly of the
nanocomplex. (C) The expression level of relative GFP mRNA in A549-EGFP cells analyzed by qRT-PCR. (D) The apoptotic percentages of A549 cells. (E) The dissected tumors
after treatment. (F) The expression levels of PLK1 mRNA in tumor-bearing mice. Adapted with permission from [85], copyright 2021 Elsevier. (G) Schematic illustration of the
self-assembly of nanogel/peptide and the enhanced treatment of CTGF-overexpressing TNBC. (H) The antitumor activity of the nanogel in MDA-MB-231 tumor-bearing mice
after different treatments. (I) Corresponding tumor inhibition ratio of different formulations. Adapted with permission from [86], copyright 2018 American Chemical Society.

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In addition, Caruso et al. designed a metal-
phenolic assembly approach to fabricate bioactive
metal-polyphonic nanoparticles (b-MPN NPs) via the
one-pot assembly of biomacromolecules, metal ions,
and polyphenols nanoparticles, which was driven by
metal-polyphenol coordination, hydrogen bonding
and hydrophobic interactions (Figure 7A-D) [89].
Poly(ethylene glycol) (PEG) was acted as the seeding
agent that could locally increase the concentrations of
the metal ion, phenolic ligand, and biomacromolecule
precursors. Various phenolic building blocks (i.e.,
EGCG, CAT, GA and TA), and metal ions with
different valences and coordination states (i.e., CuII,
FeIII, AlIII, ZrIV, and TiIV) could be employed to b-MPN
NP platform, demonstrating its tunability and
versatility. In the delivery of siRNA, EGCG and ZrIV
were selected as the phenolic ligand and metal ion,
respectively, and luciferase siRNA was assembled
into the nanoparticles (Luc-MPN NPs), which was
proved by 10% tris-borate-EDTA (TBE) polyacryl-
amide gel electrophoresis. Luc siRNA was
successfully delivered into PC3 cells expressing the
firefly luciferase gene (PC3-Luc2), and 80% of
luciferase gene expression was downregulated by
Luc-MPN NPs at up to 96 h after transfection, which
was comparable to the commercial cationic lipid-
mediated transfection agent Lipofectamine RNAiMax
(Lipofectamine-Luc). Due to the multi-choice of metal
ions, phenolic ligands and biomacromolecules, this
system was performed in a range of applications, such
as delivering cytochrome C for cell apoptosis, RNase
A for RNA degradation, and glucose oxidase/catalase
co-assembly complex for toxic intermediates
elimination via cascade reactions. Then, they synthe-
sized DNA-functionalized metal-phenolic networks
through the assembly of catechol modified DNA
block copolymer (DBC) and metal ions, in which
assembly process was driven by the electrostatic
interactions between phenolic groups and metal ions
(Figure 7E-H) [90]. Catechol-functionalized DBC
(DNA-b-poly(methyl methacrylate-co-2-methacryl-
oylethyl dihydrocaffeate, DNA-b-poly(MMA-co-
DHCAF)), that was composed of DNA segment,
catechol group and hydrophobic group, was used as a
building block and exhibited molecular recognition
properties of DNA. FeIII was exploited as the metal ion
to construct DBC-based MPN nanoparticles and
capsules (DBC-FeIII capsules), which could be stably
existed in acidic, metal-chelating, and surfactant
solutions due to the multiple assembly interactions
(metal coordination, hydrogen bonding and hydro-
phobic interactions). In HeLa cells, the cellular uptake
of DBC-FeIII MPN particles was increased when the
particle size was decreased, as smaller particles
possessed more dense DNA strands on the surface,
suggesting that high DNA surface density is
beneficial for cell internalization of DBC-FeIII MPN
particles. Therefore, DBC-FeIII MPN particles with a
size of 0.147 μm were used in a subsequent study.
Luciferase siRNA-functionalized DBC-FeIII MPN
particles were designed through conjugating siRNA
to the complementary sequence of DNA1
(DNA1’18-siRNA) for the delivery of siRNA. In
PC3-Luc2 cells, 89% of the luciferase gene was
silenced by siRNA-functionalized DBC-FeIII MPN
nanoparticles, while the free siRNA strands had no
effect on gene silencing, demonstrating that
DNA-functionalized metal-phenolic systems were
potentially biocompatible nucleic acid delivery
vehicles.
3.2.2 Polyphenol-assisted miRNA delivery
The polyphenol-based hydrogel could also be
employed to deliver therapeutic miRNA to disease
tissues. Wang et al. designed and prepared an
injectable gelatin-based inflammatory-responsive
hydrogel for the sustained release of curcumin (Cur)
and cholesterol-modified miRNA-21 inhibitor
(Antagomir-21) to treat intervertebral disc
degeneration (IDD) (Figure 8A-E) [91]. This study
provided a supramolecular host-guest system for
miRNA delivery by grafting β-cyclodextrin (β-CD)
onto Gel-phenylboronic acid (BA) (Gel-BA-CD) to
form hydrogels with TA. Specifically, β-CD-modified
Gel-BA bound with TA possessed a large number of
natural catechol/phthalate groups to form reversible
boronic ester bonds. Cur was encapsulated into the
ROS-responsive micelles that were prepared from the
amphiphilic polymer mPEG-TK-PLGA (MIC@Cur).
Owing to the homogenously porous structures of the
hydrogels, Antagomir-21 and MIC@Cur were loaded
in Gel-BA-CD, which ensured that the hydrogels have
injectable, remolding, and self-healing features. In the
context of low pH and high ROS levels, the hydrogel
was collapsed, Antagomir-21 was sustainably
released due to the reversible host-guest assembly,
and MIC@Cur was further collapsed under the
condition of overexpressed ROS to rapidly release the
anti-inflammatory Cur. In vitro, the hydrogel system
effectively reduced the inflammatory responses in
macrophages and Antagomir-21 down-regulated
extracellular matrix (ECM) degradation, recovering
the synthesis/catabolism balance of ECM in nucleus
pulposus cells (NPCs). Notably, the hydrogel
presented a significant inhibitory activity of ROS
generation owing to the anti-oxidant effects of Cur
and TA, which efficiently promoted the M2
macrophage polarization, thereby relieving the
inflammatory response. In the degenerated disc
model, Hydrogel&MIC@Antagomir-21 exhibited

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sustained release of Antagomir-21 and on-demand
release of Cur, thus exerting anti-inflammatory
activities, reversing the ECM metabolic imbalance and
effectively inhibiting the deterioration of IDD, this
suggested that this hydrogel system was a potential
and effective platform for gene delivery and could
also be utilized in other inflammatory tissue repairs.
Figure 7. Metal-phenolic networks for siRNA delivery. (A) Schematic illustration of the assembly of b-MPN NPs driven by metal–phenolic interactions. (B) TBE polyacrylamide
gel electrophoresis of free Luc siRNA (Luc control), Luc-MPN NPs, and Luc-free NPs (Luc-free control). (C) Schematic illustration of luciferase siRNA working mechanisms. (D)
The luciferase gene silencing efficiency in PC3-Luc2 cells at 48 and 96 h after transfection. Adapted with permission from [89], copyright 2022 WILEY. (E) Schematic illustration
of the preparation of DBC-FeIII MPN particles and capsules through the assembly of catechol-modified DBC and FeIII ions. (F) Quantitative and qualitative analysis of the cellular
uptake of different sized DBC-FeIII particles after 24 h. (G) Schematic illustration of the preparation of siRNA-functionalized DBC-FeIII MPN particles and working mechanism. (H)
Luciferase gene knockdown efficiency in PC3-Luc2 cells after 48 and 96 h transfections. Adapted with permission from [90], copyright 2022 American Chemical Society.

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Figure 8. Polyphenol-assisted miRNA delivery. (A) Schematic illustration of the formation of inflammation-responsive hydrogels and the mechanisms of facilitating IDD repair.
CD206 expression levels in different groups analyzed by (B) immunofluorescence staining and (C) flow cytometry. (D) X-ray and (E) MRI images of rat coccygeal vertebral discs
in different groups at 4 and 8 weeks. Adapted with permission from [91], copyright 2022 Elsevier.
3.2.3 Polyphenol-assisted dsRNA delivery
Besides, RNAi-based methods are also being
explored for controlling insects that carry diseases
(such as malaria) and damaging crops. Palli et al.
designed a simple, effective, and easy-to-synthesize
dsRNA nanosystem (NPPLL/EGCG/dsRNA) aiming at

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controlling pests [92]. NPPLL/EGCG/dsRNA was composed
of a negatively charged core complexed by dsRNA,
EGCG and a PLL-coated shell. Compared with
NPPLL/dsRNA, a compact spherical nanoparticle with a
small size was formed via enhancing dsRNA
compressibility, and NPPLL/EGCG/dsRNA showed
superior efficiencies of dsRNA delivery owing to the
addition of EGCG. It also showed better stability in
Spodoptera frugiperda (Sf9) cell conditioned medium
containing nuclease and improved target gene
knockdown efficiency compared to naked dsRNA or
PLL/dsRNA. In Sf9 cells, NPPLL/EGC G/dsRNA could
effectively protect dsRNA from double-stranded
ribonuclease (dsRNase) degradation, increase the
tolerance of dsRNA to dsRNase, enhance cellular
uptake and endosome escape, thereby resulting in a
stronger gene silencing effect.
3.2.4 Polyphenol-assisted mRNA delivery
A Previous study has established an easy,
effective, and feasible polyphenol-assisted gene
delivery system for UC therapy, in which EA was
utilized as a carrier to directly deliver mRNA (Figure
9A-E) [93]. This nanosystem (mRNA/EPHB) was
constructed layer-by-layer. The negatively charged
core was prepared by the binding between IL-10
mRNA and EA (mRNA/E), then the core was
complexed with linear polyetherimide (mRNA/EP),
and the outermost layer was covered by
bilirubin-modified hyaluronic acid (HA-BR). Due to
the layer-by-layer structure and the targeting of CD44
of HA, this novel IL-10 mRNA delivery system
(mRNA/EPHB) was endowed with active targeting
and self-protection, showing a remarkable therapeutic
effect in DSS-induced acute UC models. Firstly,
mRNA was mixed with 6 frequently used
polyphenols (EA, gallic acid (GC), punicalagin (PC),
CAT, TA and EGCG) to obtain mRNA/polyphenol
complexes. Among phenolics, EA showed the
strongest protective effect on mRNA through the
assessment of Tyndall effects, ethidium bromide (EB)
competitive binding experiment and RNase
degradation assay. The hydrodynamic size of
mRNA/EPHB was at 229.0 ± 4.8 nm. In NCM460 and
Raw264.7 cells, the expression level of IL-10 was
significantly enhanced by mRNA/EPHB, which was
8-fold higher than the control group in Raw264.7 cells
and 4-fold higher in NCM460 cells. In DSS-induced
acute UC model, mRNA/EPHB treatment showed
lower DAI score, less weight loss, and longer colon
length when compared to the model, EPHB, and
5-aminosalicylic acid (5-ASA) groups. In addition, the
successful delivery of IL-10 mRNA through this
nanosystem evidently promoted the up-regulation of
IL-10 expression and inhibited inflammation in acute
UC, showing a restorative and anti-apoptotic effect on
the colonic epithelium. Similar results were also
confirmed in the chronic UC model. This layer-by-
layer core-shell delivery system possesses a great
feasibility in clinical transformation which provides a
new multifunctional design scheme for gene therapy
of IBD.
4. Conclusions and future perspectives
As one of the most widely distributed plant
bioactive compounds, polyphenols possess excellent
biomedical activities, such as anti-tumor, anti-oxidant,
anti-inflammatory, and anti-viral effects, and serve as
nutraceuticals and adjuvant therapeutic agents in
food and pharmaceutical industries. Besides, their
unique physicochemical features of dense benzene
rings and hydroxyl groups prompt them to be a
promising drug carrier, which can interact with
different materials, including nucleic acids. In this
review, we have summarized the recent progress in
natural polyphenol-based gene delivery systems
which can be divided into two categories, DNA and
RNA delivery systems. Besides, natural polyphenols
can also serve as the building blocks to form
nanoparticles or hydrogels to assist effective gene
transfer, thus possessing high gene silencing
efficiency or significantly modulating the expressions
of bioactive proteins for disease therapy. Studies have
shown that the dense phenolic hydroxyl groups and
benzene of polyphenols can combine with nucleic
acid drugs through intermolecular hydrogen bonds
and hydrophobic interactions, respectively [80,81,90],
thereby achieving effective preparation of nucleic acid
drug nanoparticles. In the process of cellular uptake,
nanoparticles formed by the complexation of nucleic
acids and polyphenols have the advantage of its size,
which is more conducive to the passive targeted
uptake of nanodrugs by the cells. In addition,
nanostructures produced by the complexation of
polyphenols with nucleic acid drugs can also be
actively targeted through the coating of functional
materials, such as HA with CD44 active targeting [86].
Generally, loose nucleic acid nanostructures can be
easily hydrolyzed by extracellular rich nucleic acid
degrading enzymes. For the stability of nucleic acid
drugs, the usual strategy is to use polymers with high
charge density and molecular weight, such as PEI, to
encapsulate nucleic acid drugs. Although the binding
affinity and transfection efficiency of nucleic acid
drugs have been improved in these efforts, the
efficiency-toxicity correlation of these polymers is
unsatisfactory. Currently, there is no direct evidence
showing that the involvement of polyphenols in
nucleic acid drug delivery systems contributes to
intracellular escape. The main role of polyphenols in

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nucleic acid delivery systems is to improve the
affinity between nucleic acid drugs and delivery
carriers, reduce the risk of hydrolysis of nucleic acid
drugs in vitro and the safety risks of high potential
density and high molecular weight carriers. In this
review, the preparation of polyphenol-based gene
delivery system was described briefly. Besides, the in
vitro gene delivery efficiency and in vivo therapeutic
effects for various diseases were highlighted, this
provided an easily accessible guide for the research
and application of natural polyphenol-based gene
delivery.
Despite considerable potential outcomes that
have been achieved in polyphenol-assisted gene
delivery systems, there are some challenges that need
to be solved: 1) Nucleic acids are susceptible to
enzyme degradation in the physiological environment
due to their circulatory instability. Besides, it is a
double-edged sword that polyphenols could interact
with biomacromolecules (nucleic acids, proteins, and
polysaccharides). Polyphenols can bind with genes to
form polyphenols-based gene delivery vectors, but
they can also interact with the extracellular matrixes
of organs and tissues, thus leading to decreased
circulatory stability. 2) The difficulty in storage.
Natural polyphenols are unstable and can be easily
oxidized into oligomers and polymers in the air [60].
Therefore, polyphenol-gene complexes should be
freshly used or not be stored for a long time after
fabrication. 3) The difficulty in achieving clinical
Figure 9. Polyphenol-assisted mRNA delivery. (A) Schematic illustration of the polyphenol-assisted IL-10 mRNA delivery system with self-protective and active-targeted
mechanisms. (B) Screening of polyphenols in protecting mRNA degradation. (C) The expression level of IL-
10 in NCM460 cells of different groups. (D) The expression level of
IL-10 in Raw264.7 cells of different groups. (E) Photos of colon length of different groups after 9-day administration in a chronic UC model. Adapted with permission from [93],
copyright 2022 Elsevier.

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application. Although natural polyphenols are
demonstrated as a safe, easy-obtained, and cheap
vector for gene therapy, their low water solubility,
instability, and poor targeting are the most obvious
disadvantages of polyphenols as a building block for
gene delivery systems. At present, only a few
polyphenols are found to be used as gene carriers, and
the carrier type is relatively undiversified (nano-
particles and hydrogels), which heavily hinder their
clinical applications [59]. In addition, polyphenol-
based gene delivery systems are complex and usually
consisted of several components. Therefore, the
mechanisms of cellular uptake, adsorption, metabo-
lism, and excretion of polyphenol-based gene delivery
systems in vivo are not fully understood, which may
affect the safety of clinical applications. In conclusion,
polyphenols-based gene delivery is a promising
strategy for effective delivery of nucleic acid drugs,
but there is still a long way to be applied in the clinic.
Abbreviations
ADA: adenosine deaminase; Antagomir-21:
cholesterol-modified miRNA-21 inhibitor; APTES:
(3-aminopropyl)-triethoxysilane; 5-ASA: 5-aminosali-
cylic acid; ASO: antisense oligonucleotide; BA:
boronic acid; β-CD: β-cyclodextrin; Bcl-xL: B-cell
lymphoma-extra large; b-MPN NPs: bioactive
metal-polyphonic nanoparticles; CAT: catechin;
CD44: cluster of differentiation 44; cIAP1: cellular
inhibitor of apoptosis protein 1; CTGF: connective
tissue growth factor; Cur: curcumin; DAI: disease
activity index; DBC: DNA blocking copolymer;
dsiRNAs: dicer substrate RNAs; dsRNA: double
strand RNA; dsRNase: double-stranded ribonuclease;
DSS: dextran sulfate sodium; EA: ellagic acid; EB:
ethidium bromide; ECG: epicatechin gallate; ECM:
extracellular matrix; EGC: epigallocatechin; EGCG:
epigallocatechin gallate; EIF5A2: eukaryotic
translation initiation factor 5A2; GA: gallic acid;
GAPDH: glyceraldehyde 3-phosphate dehydroge-
nase; GNPs: green nanoparticles; GSH: glutathione;
HA: hyaluronic acid; HAase: hyaluronidase; HFV:
human foamy virus; IDD: intervertebral disc
degeneration; IECs: intestinal epithelial cells; LPF:
Lipofectamine 2000; miRNA: microRNA; MMP-9:
matrix metallopeptidase 9 protein; MNC: magnetic
nanocrystal clusters; MPNs: metal-polyphenol
networks; mRNA: messenger RNA; NaCl: sodium
chloride; NPCs: nucleus pulposus cells; PARP: poly
ADP-ribose polymerase; PC: punicalagin; pDNA:
plasmid DNA; PEI: polyethylenimine; p-ERK:
phospho-extracellular regulated protein kinases;
p-FAK: phosphor-focal adhesion kinase; PHD2: prolyl
hydroxylase 2; PLL: ε-polylysine; Ps-ASO:
phosphorothioate backbone ASO; P13K/Akt:
phosphoinositide 3-kinase/ protein kinase B; RNAi:
RNA interference; ROMP: ring opening metathesis
polymerization; SCID: severe combined immuno-
deficiency; Sf9: Spodoptera frugiperda; shRNAs: short
hairpin RNAs; siLuc: siRNA targeting luciferase;
siRNA: short interfering RNAs; siRNA-CTSK: siRNA
cathepsin K; TA: tannic acid; TE: TransExcellent-
siRNA; UC: ulcerative colitis; Y-DNA: Y-shape DNA.
Acknowledgements
This work was funded by the Science and
Technology Development Fund, Macao SAR
(00151/2020/A3 & 001/2023/ALC), Major Basic and
Applied Basic Research Projects of Guangdong
Province of China (2019B030302005), and the
University of Macau (MYRG2022-00009-ICMS).
Competing Interests
The authors have declared that no competing
interest exists.
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