Drug Discovery Today
Volume 00, Number 00
January 2018 REVIEWS

Reviews
POST SCREEN

Recent advances in siRNA delivery for
cancer therapy using smart
nanocarriers
Penghui Zhang1,2, Keli An1,2, Xiaoman Duan1,2, Hao Xu1,2, Fei Li1,2 and Feng Xu1,2
1
The Key Laboratory of Biomedical Information Engineering of Ministry of Education, School of Life Science and Technology, Xi’an Jiaotong University, Xi’an,
China
2
Bioinspired Engineering and Biomechanics Center (BEBC), Xi’an Jiaotong University, Xi’an, China

Small interfering RNAs (siRNAs) can selectively target and downregulate disease-causing genes, holding
great promise in treating human diseases, especially malignant cancers. However, how to efficiently
deliver siRNAs into target cell cytosol is a problem that has hindered their clinical application. Here, we
review the recent strategies for siRNA delivery on the basis of smart nanocarriers by using stimuli-
responsive materials. We highlight the rationales of how to design smart nanocarriers responsive to
physiological and external stimuli to improve the delivery efficiency, targeting precision and gene
silencing efficacy. Finally, we provide an outlook on the fundamental limitation for clinical translation
of siRNA-based nanomedicine that should be overcome by the combination of chemistry, biology,
material and medical science.

Introduction The single-stranded miRNA with 19–24 nucleotides always elicits

RNA-based therapeutics have shown great potential for treating functions in an endogenous pathway. It is initially transcribed as
miscellaneous diseases through antisense oligonucleotides (ASOs), primary miRNA in the nucleus, and is processed into precursors
aptamers, microRNA (miRNA) mimics, small-interfering RNA and mature forms through a series of biogenesis machinery in-
(siRNA), synthetic mRNA and CRISPR-Cas9 [1]. Especially in can- cluding the enzymes Drosha and Dicer. The miRNA is recruited to
cers, these drugs bring promise to patients bearing undruggable the RNA-induced silencing complex (RISC) in the cytosol and
mutation genes by knocking down the gene expression, altering regulates the protein-coding genes either by mRNA degradation
mRNA splicing, upregulating target genes and editing the genome or translational repression [5]. By contrast, siRNAs come from
[2]. Different from small-molecule inhibitors or antibodies, once solid-chemistry synthesis and consist of a mRNA sequence (sense
delivered to the specific cell or tissue, the devised RNA-based strand) and its complement (antisense strand), and are 21–23
therapeutics can selectively target the single gene, and off-target nucleotides in length. They can bypass Dicer processing and
effects can be eliminated, bringing great potential in enhancing directly load in the RISC to recognize the target mRNA sequence
therapy efficacy with minimized side effects [3]. through perfect base-pair complementarity, leading to efficient
Since Fire et al. reported that double-stranded RNA exerted 100- cleavage and degradation and consequently gene knockdown. The
fold improved gene silencing as opposed to single-stranded RNAs siRNA therapeutics have experienced booming development since
[4], RNA interference (RNAi) has been widely used to assess the being first administered in 2003 [6], and have attracted tremen-
individual roles of genes in different cellular processes and dis- dous research interest and capital investment. Recently, several
eases, and to develop RNA-based therapeutics to correct disease- siRNA therapeutics have entered clinical trials and demonstrated
causing genes in hepatitis, virus infection, cancer, among others. potent and durable gene silencing in the treatment of solid cancers
So far, the most efficient RNAi effectors are miRNAs and siRNAs. following systemic administration [7].
The challenge in turning siRNAs into drugs is similar to other DNA
or RNA candidates. These large and highly charged macromolecules
Corresponding author: Xu, F. ([email protected])

1359-6446/ã 2018 Elsevier Ltd. All rights reserved.

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suffer from the issues of rapid degradation by nucleases in body To further improve the therapy efficacy, modifying the nano-
fluids, fast clearance by kidney and liver scavenger receptors, poten- carriers with specific ligands (i.e., antibodies, aptamers, peptides,
tial threats to trigger innate immune response and inefficient deliv- folic acid, hyaluronic acid, etc.) is a popular and promising strategy
ery to cross the cell membrane [2]. To address this, new RNA to target the malignant tissues [16,17], packaging the charged
chemistries, such as substituting the natural phosphodiester lin- macromolecules, shielding them from damage, delivering the
kages or modifying the 20 -position of the ribose, have been devel- right amount to right location and releasing the cargoes in a
oped to improve the stability of siRNAs, reduce the unintended off- controlled manner. However, a recent review reveals that most
target effects and maximize on-target pharmacologic activity in vivo. nanocarrier systems tend to be trapped in the liver, spleen and
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However, the siRNA macromolecules still lack the capability to kidney, and only 0.7% (median) of the administered dose is
traverse the membrane and escape from the endosomes into the delivered to the solid tumor [18]. This could be beneficial for liver
cytosol, where they initiate gene silencing. To aid cellular uptake, disease, but negatively impact nanomedicine translation for can-
cationic lipid- or polymer-based transfection reagents are widely cer treatment with respect to cost, toxicity and therapeutic efficacy
employed in in vitro experiments, but show poor performance in vivo. (Fig. 1). It should be noted that the poor delivery efficiency in vivo
In fact, 70% of clinical trials in gene therapy carried out so far utilize is attributed to the fact that the nanocarriers with unchangeable
modified viruses to deliver genes, which stagnates for safety issues surfaces or composition cannot adapt to the complex biological
such as carcinogenesis and immunogenicity [8]. Recently, a broad system. To overcome the biological barriers, many smart formula-
diversity of nanomaterials with unique physical and chemical prop- tions have been developed, in which stimuli-responsive moieties
erties has been engineered to construct nanocarriers for delivering translate the chemical or external physical signals into significant
siRNA therapeutics, including polymers, dendrimers, lipids, pro- behavior changes, such as swelling, aggregation, degradation,
tein-, gold-, silica- and iron-oxide-based nanoparticles [9–11]. The surface rearrangement and charge reversal [19]. By virtue of the
most striking characteristic of nanocarriers is that their physico- powerful chemistry and material tools, as shown in Table 1, smart
chemical properties such as composition, size, shape and surface nanocarriers responsive to physiological stimuli (e.g., acidic pH,
chemistry can be precisely modulated [12]. The nanocarriers can not overexpressed enzymes, redox gradient or elevated metabolites)
only efficiently protect the siRNAs from nuclease degradation and and external triggers (e.g., NIR light, magnetic field or ultrasonic
renal clearance but also significantly improve the pharmacokinetics sound) hold great promise in improving delivery efficiency, en-
and biodistribution, thus enabling targeted delivery to specific cells hancing therapy efficacy and reducing side effects [20–33].
and spatiotemporally controlled release on command [13,14].
Recently, the rapid development of stimuli-responsive materials Smart nanocarriers for siRNA delivery based on physiological
has significantly promoted the application of nanotechnology in stimuli
chemotherapy and medical translation to the clinic [15]. Although Significant variations in physiological parameters exist at the
these promising chemical and material tools have been well organ, tissue and cell levels, and are highly correlated with various
studied for drug delivery, there is still lack of comprehensive pathological conditions, which can serve as attractive biomarkers
reviews to summarize the recent application of novel stimuli- for diagnosis and natural endogenous stimuli for controlled drug
responsive materials for siRNA delivery. In this review, we high- release in cancer, infection, diabetes, cardiovascular and degener-
light the recent design and approach to solve the delivery pro- ative diseases.
blems by using smart nanocarriers responsive to the physiological Smart nanocarriers for siRNA delivery based on pH
environment and external stimuli, such as how to improve deliv- The pH gradient in a local tumor is different from that in the
ery efficiency, targeting precision and gene silencing. Then we normal counterpart because increased fermentative metabolism
provide insight to guide the design of future siRNA-based nano- and poor perfusion lead to the accumulation of lactic acid and
medicines for clinical translation. formation of an acid microenvironment, which is hypothesized to
promote tumor invasion and metastasis [34]. The siRNA-based
Recent advances in smart nanocarriers for siRNA nanocarriers will undergo drastic pH variations before exerting
delivery gene knockdown in RISC machinery. They must extravasate from
Efficient delivery and intracellular release are prerequisites for the blood vessels (pH 7.4), diffuse into the tumor center (pH 7.2–
siRNA-mediated gene silencing. Different from small-molecule 6.0), enter the endosomes (pH 5.0–6.5) and lysosomes (pH 4.0–
drugs, siRNA therapeutics exhibit several different characteristics: 5.0), and finally escape into the cytoplasm (pH 7.4). Numerous
(i) they are endowed with high nuclease susceptibility and thus smart pH-responsive formulations have been developed, in which
special packaging is needed to protect them from blood and tissue the drops in pH can protonate the ionizable polymers (e.g., poly-
RNases; (ii) the negatively charged macromolecules cannot tra- acids, cationic lipid and polymers, and poly-amino acids) to
verse the membrane unless it is with the aid of transfection change their solubility or electrostatic interaction, or degrade
reagents; (iii) the condensed siRNAs must be completely released the acid-cleavable bonds (e.g., hydrazine, ketal, ester) and nano-
in the cytosol so that they can load on RNAi machinery. To particles for cargo release [35–37]. In particular, after being inter-
enhance the therapy efficacy in patients, a number of siRNA-based nalized in an acidic endosome, the polyamine-derived polymers
nanomedicines have been approved for clinical trials [7]. Although with pKa range of 5–7 can be rapidly protonated and cause endo-
most cases involve nontargeted lipids or polymers, multiple results somal swelling and rupture via the proton-sponge effect, making
demonstrated these nanocarriers have favored the siRNA accumu- them promising scaffolds to facilitate endosomal escape without
lation in human tumors, presenting great potential in RNAi-based the aid of additional endosomolytic enhancers (e.g., chloroquine),
cancer treatment. which can cause severe side effects in normal tissues.

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Serum adsorption EPR effect

Tumor

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MPS retention Targeting

Vessel Tumor

Renal clearance Endosome escape

Endonuclease Red cell RISC

Macrophage Receptor siRNA

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FIGURE 1
Biological barriers for siRNA delivery in vivo by using nanocarriers. The nanocarriers synthesized in the flask are equipped with PEG corona and targeting ligands.
They are generally administered via intravenous injection, and thus the biological surroundings affect their in vivo circulation and biodistribution. Once in the
bloodstream, distinct serum proteins adsorb onto the nanocarriers to form protein corona and bestow the nanocarriers new biological identity, which could lead
to particle aggregation, MPS retention, liver and renal clearance. After accumulation in tumor sites via the EPR effect, the nanocarriers are internalized into the
target tumor cells via active ligands or passive interaction and are always trapped inside the endosome. Most nanocarriers are designed to escape from the
endosome into the cytosol by means of the proton-sponge effect, and subsequently release the siRNAs. Finally, the siRNAs are loaded in the RISC for gene
silencing.

Lipid nanoparticles are the most popular platforms for siRNA the same pH distribution is shared inside normal and tumor cells,
delivery [38]. The positive charges on the surface of cationic lipid which means that it is hard to realize specific siRNA release by the
formulations (lipoplexes) can condense the genes and facilitate the acidic endosome.
cellular uptake and endosome escape. Lieberman’s group developed The reticuloendothelial system (RES) in the living body can
a novel imaging approach to visualize the release process of siRNAs rapidly clear the cationic nanoparticles, further deteriorating the
in lipid-based nanocarriers after endocytosis [39]. The results dem- poor delivery. Equipping the nanocarriers with a PEG corona can
onstrated that the free siRNAs could be rapidly released from the minimize the nonspecific interaction and enhance the passive
maturing endosomes during a narrow time window within 5– accumulation at tumor sites via the enhanced permeability and
15 min after uptake. The efficient gene knockdown occurred within retention (EPR) effect [42], but in turn harm the targeted uptake by
a few hours of release with the need for a minimum of 2000 copies in tumor cells. Recently, activatable strategies have attracted great
the cytosol, which established guidelines for designing and optimiz- interest in nanocarrier design, where the functional groups are
ing siRNA delivery strategies. To eliminate the unwanted toxicity, protected by an inert shell in the circulating system and the
more smart motifs should be introduced into the cationic lipid to surfaces are activated in the tumor microenvironment with the
meet the demands for in vivo siRNA delivery. For example, tertiary exposure of targeting ligands or reversal of surface charge. The
amines with tunable pKa values in the physiological range can be minute pH distinction between blood circulation and extracellular
ionized at acidic buffers with sharp phase changes from hydrophobic tumor matrix is one of the most universal and practical tools,
to hydrophilic states [40]. By means of ultrasensitive tertiary amines, which can activate the nanocarriers for specific tumor penetration
Nguyen’s group crosslinked a poly(acrylic acid) shell onto a lipoplex and targeting. As shown in Fig. 2b, Wang’s group incorporated an
core with diamine bonds. The dual pH-responsive behaviors of the extremely pH-sensitive moiety of 2,3-dimethylmaleamidic acid
tertiary ammonium head and acid-cleavable diamine linkers have (DMMA) in a micelleplex delivery system [21]. The PEG corona
facilitated the endosome escape and siRNA release [41]. Ultra pH- collapsed in the acidic tumor microenvironment and exposed the
sensitive nanomicelles on the basis of polycations could enhance the cell-penetrating peptide, enhancing the cellular uptake and endo-
early endosome escape and efficiently improve the transfection some escape, which exhibited superior gene silencing efficiency in
efficiency in vitro (Fig. 2a) [23]. However, it should be noted that vivo. More interestingly, a viral–nonviral chimeric nanoparticle

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TABLE 1
Recent strategies for siRNA delivery using new chemistry and material
Formulations siRNA target Cell type EPR effect Targeting ligands Endosome escape Cargo release Year
Passive targeting
Gold nanoclusters NGF Pancreatic cancer – – Proton sponge – 2017 [31]
(in vivo)
Dendrimers MYC Liver cancer – – Proton sponge Esterase 2016 [28]
(in vivo)
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Polycations SCD1 Liver cancer – – Proton sponge Acidic pH 2016 [23]

(in vivo)
RNA microstructure GFP HeLa – – Proton sponge Dicer 2016 [27]
(in vitro)
Polymer EGFP HeLa – – Proton sponge Acidic pH 2016 [30]
(in vitro)
NIR polymer BRAF Thyroid cancer PEG – Proton sponge Acidic pH 2016 [22]
(in vivo)

Active targeting
pRNA nanoparticles MED1 Breast cancer – HER2 aptamer Proton sponge Dicer 2017 [24]
(in vivo)
pH-responsive polymer PHB1 Prostate cancer PEG ACUPA Proton sponge Acidic pH 2017 [26]
(in vivo)
PolyMetformin BCL-2 Lung cancer PEG Anisamide Proton sponge Acidic pH 2016 [101]
(in vivo)
Polyurethane GFP Osteoblast PEG Periostin-peptide – – 2016 [25]
(in vivo)
CaP Ras Glioblastoma cell – Apolipoprotein E3 Acidic degradation Acidic pH 2017 [29]
(in vivo)
LDH Survivin KB cell – Folic acid Size effect Acidic pH 2016 [33]
(in vivo)

Activatable targeting
Amphiphilic polymer Survivin Prostate cancer PEG iRGD/CRGDR Proton sponge Acidic pH 2016 [99]
(in vivo) (protease)
Coplymer Twist protein Breast cancer PEG PEG/PEI Proton sponge Acidic pH 2016 [20]
(in vivo) (MMP-2/9)
DNA nanotube VEGF Leukemia cell – Aptamer Rigid structure – 2016 [32]
(in vitro) (lock/key)
Copolymer CDK4 A549 cell PEG PEG/R9 Proton sponge Acidic pH 2015 [21]
(in vivo) (acidic pH)

was prepared to simultaneously tackle two key BCR-ABL gene Smart nanocarriers for siRNA delivery based on
regulation pathways by coating the acid-degradable polymeric enzymes
shell on the adeno-associated virus (AAV) core [37]. The sequential The initiation, progression, invasion and metastasis of a tumor are
release of siRNA and virus by acid digestion in endosomes could often associated with elevated expression of certain enzymes. The
avoid the generation of anti-AAV serum, and facilitate the simul- endogenous enzymes are classified into two categories: enzymes
taneous expression of BIM and silencing of MCL-1, synergistically secreted into the ECM of tumors [e.g., matrix metalloproteinases
suppressing the proliferation of BCR-ABL-positive K562 and FL (MMPs), furin, alkaline phosphatase, hyaluronidase] that are wide-
5.12/p190 cells in vitro and in vivo. Besides polymer, the degradable ly investigated as triggers to change the surface states of nano-
inorganic nanoparticles are also promising scaffolds for siRNA carriers for massive retention or targeted recognition; and the
delivery. An acidic-degradable inorganic nanocarrier based on overexpressed intracellular enzymes (e.g., esterase, cathepsin
layered double hydroxide (LDH) demonstrated excellent biocom- and kinase) that can cleave the specific chemical bonds or peptide
patibility, high delivery efficiency and gene silencing, presenting substrates, so as to decompose the nanocarriers for cargo release
as a promising therapeutic system [33]. By virtue of the pH gradi- [43].
ent in tumor tissues, pH-responsive nanocarriers have facilitated MMPs, always overexpressed in tumor tissues with metastatic
the specific cellular uptake, endosome escape and siRNA release. potential, are the most popular cues for designing enzyme-acti-
To promote clinical translation, new chemical and material tools vating nanocarriers [44,45]. Two members of the MMP family:
should be introduced to make the design more simple and robust. MMP-2 and MMP-9, can specifically recognize and degrade the

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(a) (b)

5n
M
W siR
/W NA

PC ited
=3
0:1

B
by hib
In
PCB-P(DPA-co-DMA)-PG(GDDC polycations)
Self-assembly i)

Low internalization iii)

ii)

em sive

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bly
n
iv)

dis respo
ass
-
pH
se
elea
Disa al r
sse osom
mbly End
pH<
pH d
pH> pH dis is 78% targeted
mRNA cleavage
Early endosome

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FIGURE 2
Recent advances in siRNA package and release with pH-responsive polymers. (a) pH-sensitive nanomicelles for high-efficiency siRNA delivery in vitro and in vivo
[23]. (b) Tumor acidity-sensitive polymeric vector for activated siRNA delivery. (i) Self-assembly of PEG-Dlinkm-R9-PCL into nanoparticles to form Dm-NPsiRNA for
siRNA package. (ii) Systemic injection of Dm-NPsiRNA. (iii) Prolonged circulation of Dm-NPsiRNA with PEG layer. (iv) Enhanced recognition of Dm-NPsiRNA by tumor
cells following degradation of the Dlinkm labile linkage [21].

peptide sequence of Pro-Leu-Gly-Leu-Ala-Gly (PLG). In a typical hydrogen peroxide (H2O2), singlet oxygen (1O2), hydroxyl radical
design (Fig. 3a), the PLG peptide was inserted in the middle of the (
OH) and superoxide radical (O2), playing important parts in
PEG-p-PDHA copolymer and the MMP/pH dual-sensitive nano- different cell signaling pathways. The normal cells must maintain
carriers were prepared for co-delivery of paclitaxel and siRNA in the ROS homeostasis because overproduction of ROS causes oxi-
breast tumor cells. In a tumor microenvironment rich of MMP-2, dative stress, and subsequent cellular damage and organ function
the PEG layer was pulled off and the positively charged polyethy- decline [49]. Redox balance between oxidizing and reducing spe-
lenimine (PEI) enhanced the penetration and cellular uptake, cies is achieved by various enzymes such as superoxide dismutase
where simultaneous release of drugs and siRNAs synergistically and glutathione peroxidase. Accumulating evidence suggests that
inhibited the tumor growth and pulmonary metastasis [20]. the dysregulation of aggressive metabolism and ROS scavengers
Intracellular enzymes are also attractive triggers for tuning the leads to detrimental accumulation of ROS in tumor cells, which
release profiles of the nanocarriers. For instance, Meade et al. will adapt to oxidative stress by upregulating glutathione (GSH) [50].
synthesized short interfering ribonucleic neutrals with phosphate Ironically, the tumor cells utilize ROS to drive proliferation and
backbones that were neutralized by phosphotriesters, allowing invasion, but they are more susceptible to the reagents that damage
good serum stability and efficient delivery into cells [46]. Once the redox balance, whereas the normal cells are less sensitive owing
inside the cell, the neutralized groups as well as the cell-penetrat- to the low basal ROS and high antioxidant capacity [51].
ing peptides were efficiently cleaved by intracellular thioesterases, The ROS level in tumor cells (100 mM) reaches 10- to 100-fold
creating native siRNAs capable of gene silencing, with apparent levels higher than that in normal cells, which can be engaged for
EC50 values in the low nanomolar range. Shen’s group reported an controlled drug release by cleaving thiolketal and aryl boronic acid
esterase-responsive gene carrier for potent cancer therapy with bonds, or oxidizing thiolester and ferrocene units [52–54].
quaternary amines carrying N-propionic-4-acetoxybenzyl ester Murthy’s group incorporated a ROS-sensitive thioketal bond into
substituents [47]. The ester group underwent a fast esterase-cata- the polymer backbone to package the siRNA for oral delivery to
lyzed hydrolysis, subsequently triggering charge reversal from intestinal tissue [55]. The thioketal linkages were stable to acid-,
cationic to zwitterionic and cargo release. To better understand base- and protease-catalyzed degradation, but were broken down
the influence of the molecular structures on delivery efficacy, by elevated ROS at inflamed intestinal tissues, triggering the siRNA
Anderson and co-workers prepared 1400 esterase-degradable lipi- release. Similarly, Peng et al. reported a ROS-responsive fluorinated
doids and evaluated their transfection efficiency and structure– bola-amphiphilic dendrimer for delivering siRNA in cancer cells
function activity [48]. Four structural and pKa criteria were identi- on command [56]. The ROS-sensitive thioacetal linkage in the
fied that could robustly predict the ability of nanocarriers to dendrimer promoted the specific and efficient dissociation of the
mediate >95% gene silencing in vivo without any prior biological siRNA/vector by endogenous ROS in tumor cells for effective
testing. Different from other stimuli, the enzymes can specifically siRNA delivery and gene silencing.
cleave their substrate to control targeting and triggering release. In To maintain the redox balance, tumor cells generate massive
the future, more tumor-associated enzymes should be screened for intrinsic antioxidants, such as GSH, to counteract ROS-induced
personalized clinical use. oxidative stress. The intracellular concentration of GSH in tumor
Smart nanocarriers for siRNA delivery based on redox cells is 2–10 mM, which is fourfold higher compared with that in
Reactive oxygen species (ROS) are a group of highly reactive normal cells, but the extracellular GSH level drastically decreases
chemical species generated by all aerobic organisms in forms of to 2–20 mM in the tumor microenvironment. The distinct GSH

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(a) (d)
PEG-p-PDHA

ii iii

PEI-PDHA siTwi
PEG corona RGD shell Y-motif

PTX PPM PPTN

i
MMP-2/9 iii
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ii

iii
Cleaved mRNA Dox

siRNA Integrin

miRNA ATP

RISC: RNA-induced silencing complex

(b) (c)
siRNA 1. Complexation
TEGGu4
+ Salt-bridge 2. Interlayer
mPEG-PAsp(MEA)-PEI crosslinking

siRNA release
pH 5.0

Intracellular
SH HS
siRNA thiol
Oxidative Reductive
polymerization depolymerization

pH 7.4
S S pH 6.8 Long circulation &
siRNA-nanocaplet
(PTEGGu4⊃siRNA) disulfide Endocytosis “on” endocytosis “off”

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FIGURE 3
Novel smart nanocarriers for siRNA delivery with endogenous molecules as triggers. (a) The tumor-microenvironment-sensitive nanoparticle co-loading siTwi
and PTX [20]. (b) Oxidative polymerization of TEGGu4 with siRNA as the template to form siRNA-containing nanocaplet [59]. (c) Formation of charge-reversible
polyplex for long circulation, tumor-specific cell uptake and easy intracellular siRNA release [58]. (d) Schematic design of the smart nanocarriers triggered by
miRNA and fueled by ATP. Amplified dissociation of Y-motifs based on TMSD cascade reactions with miRNA as the trigger and ATP as the fuel: (i) Toe1-TMSD
triggered by miRNA; (ii) ATP recognition by aptamer; (iii) Toe2-TMSD for miRNA regeneration [71].

gradient makes it convenient to design GSH-responsive nanocar- vesicles for rapid drug release and gene transfection [62]. Further-
riers for on-command drug release (Fig. 3b,c) [57–59]. The disulfide more, the degradation of the redox-responsive nanocarriers con-
bond is the most promising motif that is stable in the extracellular sumes massive redox species and breaks the intracellular redox
space, but can be cleaved into thiol by reductive species or through imbalance, which is neglected in most studies but would be
thiol–disulfide exchange with thiol-bearing molecules [60]. To promising to be combined with other strategies in tumor therapy
ensure deep tissue penetration through endothelial fenestrations [63].
(60–80 nm) and intercellular gaps (10 nm), a series of nanoca- Smart nanocarriers for siRNA delivery based on other
plets with uniform size (7 nm) were synthesized by oxidative metabolites
polymerization of a water-soluble dithiol monomer carrying mul- The aggressive proliferation and malignant metastasis of tumors
tiple guanidinium ion (Gu+) pendants (Fig. 3b) [59]. The packaged also led to the dysregulation of other metabolites, such as oxygen,
siRNA could be liberated by GSH-mediated cleavage. Besides di- glucose, ATP and nucleic acids, which can be used to trigger on-
sulfide bonds, diselenide bonds also can be cleaved by GSH but command controlled drug release. Hypoxia is a general character-
undergo slower reduction kinetics because the sulfur is much more istic during tumor progression. Altered vasculature and conse-
active as an electron acceptor than selenium. Gu’s group designed quent chaotic blood flow cause the gradients of oxygen tension
a hierarchically ternary system to compact genes with diselenide and acute hypoxia (<1.4% oxygen) in fast-growing tumors, which
and disulfide polymers [61]. These smart nanocarriers showed poses a survival pressure to favor the upregulation of genes associ-
stepwise responses to the dual reduction gradients in the tumor ated with tumor initiation, metastasis and resistance to chemo-
sites and intracellular conditions, and exerted efficient genome therapy and radiotherapy. Nitroaromatic, quinone and
release. In addition, GSH was also utilized to reduce the ferroce- azobenzene derivatives undergo reduction-mediated cleavage in
nium cation to ferrocene with a polarity shift from hydrophilic to the hypoxia microenvironment, which is inhibited by molecular
hydrophobic, which could drive the disassembly of siRNA-loaded oxygen owing to radical depletion [64]. Torchilin’s group

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introduced a hypoxia-sensitive moiety of azobenzene in a PEG- and release processes in vitro and in vivo owing to noninvasiveness
ended cationic copolymer for hypoxia-induced siRNA uptake and and high controllability. The absorption of light with different
gene silencing, representing a promising tumor-environment-re- wavelengths can induce physical or chemical property changes of
sponsive modality for tumor targeting and therapy [65]. the nanocarriers through photoisomerization, photocleavage,
As the molecular unit of currency for energy transfer in the photosensitization or photothermal effect.
living body, ATP is the essential biogenic molecule for cellular Ultraviolet (UV) and visible (Vis) light has sufficient energy to
metabolism and signaling, which is highly concentrated within initiate photochemical reactions. For instance, the azobenzene
the intracellular cytosol (1–10 mM) but much lower in the extra- derivatives conjugated on the mesoporous-silica-based nanocar-
cellular niche (<5 mM). This great concentration gradient is a

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riers underwent reversible photoisomerization between trans and
promising biological rationale for designing an ATP-triggered drug cis on alternating irradiation by UV and Vis light, presenting
release system [66,67]. Phenylboronic acid (PBA) can form revers- photoregulated targeting activation and drug release [74]. UV
ible covalent esters with 1,2-cis-diols incorporated on a ribose ring, and Vis light can also irreversibly cleave the photolabile groups
which has been used for capturing RNA in affinity chromatogra- such as o-nitrobenzyl and coumarin-4-yl-methyl groups to change
phy. Kataoka’s group has conjugated the PBA pedants on a cationic the hydrophilic–hydrophobic balance and the stability of the
polymer, which could efficiently condensate the siRNAs via elec- polymers, release the drug molecules anchored on nanoparticles
trostatic interaction and firmly lock them via PBA–diol recogni- or remove the caging groups [75]. For instance, Garcia et al.
tion [68]. Once exposed in cytosol, the excess diols of ATP would designed light-controlled cell-adhesive interfaces by protecting
competitively bind with the PBA motifs and release the siRNAs for the cyclic RGD peptide with a 3-(4,5-dimethoxy-2-nitrophenyl)-
gene silencing. 2-butyl ester (DMNPB) photolabile caging group [76]. On exposure
Another kind of attractive stimuli in tumors is the gene targets to UV light (350–365 nm), the caging groups were cleaved to
including DNA fragments, mRNA and especially miRNAs. miRNAs liberate the active cyclic RGD peptide, demonstrating a noninva-
play important parts in the gene regulation network in all the sive, transdermal time-regulated technique to activate the cell-
cellular processes. Particularly during cancer initiation, progres- adhesive interfaces on implanted biomaterials so as to regulate in
sion and metastasis, tumors exhibit different miRNA expression vivo cell adhesion, inflammation, fibrous encapsulation and vas-
patterns compared with normal tissues, providing promising bio- cularization. In early studies, different photoresponsive groups of
markers for cancer diagnosis and potential therapeutic targets. In DMNPE [1-(4,5-dimethoxy-2-nitrophenyl)ethyl], NPE [1-(2-nitro-
our previous work, we have focused on the design of miRNA- phenyl)ethyl], NPP [2-(2-nitrophenyl)propyl] and CD-DMNPE (cy-
responsive smart nanocarriers for controlled drug release and clo-dodecyl DMNPE) were introduced on the terminal phosphate
cancer detection [69–71]. To recruit low copies of endogenous to sterically block the binding of siRNAs with RISC, whereas the
miRNA, a Y-shaped DNA nanomachine was developed in Fig. 3d, UV irradiation could shed the caging groups and activate the gene
by which the drugs and siRNAs could be efficiently released with silencing [77]. However, the UV and Vis light not only induced
oncogenic miR-21 as triggers and ATP as fuels, resulting in high severe tissue injury but also showed poor penetration depth
tumor inhibition and minimum damage to normal organs. (<10 mm) in soft tissues owing to the strong adsorption by tissue
Endogenous stimuli can selectively activate the targeting and chromophores (e.g., hemoglobin, myoglobin and melanin),
trigger the drug release in the tumor sites. However, genetic and resulting in poor light-triggered drug delivery in vivo.
phenotypic heterogeneities are common features of tumors, lead- Near-infrared (NIR) light with wavelengths of 650–900 nm can
ing to the dynamic changes of the tumor microenvironment [72]. penetrate the tissue at the centimeter scale with lower scattering
The extracellular matrix, metabolites, chemokines, growth factors, and minimal harm to tissues, making the NIR-responsive system
as well as surrounding cells (e.g., fibroblasts, immunes cells, epi- promising for clinical application [78]. But the NIR photoenergy is
thelial cells, etc.) can vary from patient to patient, and even too low to initiate the photochemical reactions. The upconversion
dynamically change during tumor progression and therapy in nanoparticles (UCNPs), consisting of an inorganic crystalline host
the same person [73]. These uncertain factors make it difficult matrix and trivalent lanthanide ions embedded in the host lattice,
to predict the drug dosage and release kinetics, and more-efficient are powerful tools to convert the absorbed NIR light to UV or Vis
and smart formulations are needed to meet the demands of light through the unique ladder-like energy level structures of
translation to the clinic. lanthanide ions [79,80]. Bian’s group encapsulated DMNPE-caged
siRNA in the mesoporous-silica-coated UCNPs for the remote
Smart nanocarriers for siRNA delivery based on external stimuli controlling and real-time monitoring cell differentiation
The external stimuli, such as light, temperature, magnetic fields, (Fig. 4a) [81]. The NIR light illumination cleaved the DMNPE
ultrasound and electric fields, can be applied with a high spatial groups and activated the gene silencing, which could be visualized
and temporal resolution to mediate active targeting and con- by the aggregation-induced emission (AIE) of organic dyes.
trolled drug release, presenting great potential in enhancing ther- Kohane’s group have designed polymeric micelles with a photo-
apeutic efficacy and minimizing off-target effects. Herein, we will sensitizer (PS) in the hydrophobic core to manipulate cell targeting
introduce some external-stimuli-responsive strategies in drug de- in vitro and in vivo by using gentle laser irradiation (green light at
livery, which could also be beneficial to the translation to the 200 mWcm2 for 5 min) [82,83]. The energy of green light would
clinic of siRNA therapeutics. be efficiently transferred to cleave a covalent bond by combination
Smart nanocarriers for siRNA delivery based on light of triplet–triplet annihilation-based upconversion and Förster res-
Light with a broad spectral window from 300 nm to 900 nm is the onance energy transfer (FRET), with the releasing of RGD peptide
most versatile tool as an external stimuli to modulate the targeting for specific cell binding.

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(a) Cell membrane (b) NRP-1

RP Endosome

RNAi

RGD mediated
cell entry
Modular NIR hv
design
MMP13
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Cytosol
“On”
Nucleus
Stem cell

(d) (c)

PFP vaporization Shell fragmentation

Liquid PFP droplets

Ultrasound

Chitosan shell
Deoxycholic acid
siRNA Iron oxide nanocrystal

Drug Discovery Today

FIGURE 4
Novel smart nanocarriers for cancer therapy controlled by external stimuli. (a) Mechanism of controlling and detecting differentiation of hMSCs by UCNP-
peptide-AIE-siRNA [81]. (b) Hollow gold nanoshell (HGN)-siRNA-targeting peptide (RP) architecture with original or modular design and schematic of
nanoparticle uptake, laser-activated siRNA delivery pathway in PPC-1 cells [87]. (c) Magnetic tandem apoptosis trigger (m-TAT) and its tandem capability for
magnetic clustering and Dox release [94]. (d) Schematic representation of chitosan-deoxycholic acid coated perfluoropentane nanodroplets (CNDs) [97].

The PS can adsorb the NIR light to produce ROS to kill cancer two TSPs (PEG-tailed P39PEG with LCST at 39  C and RGD-conju-
cells, which is called photodynamic therapy (PDT) [84]. The recent gated P43RGD with LCST at 43  C) onto gold nanorods and revers-
studies demonstrated that if the PS-containing nanoparticles are ibly switched the surface states between PEG and RGD for specific
irradiated with NIR light for a short time, the PS energized sur- cell uptake of nanocarriers carrying siRNA and doxorubicin (Dox),
rounding molecular oxygen (3O2) to generate ROS that could which showed preferred uptake and enhanced cell death in irradi-
induce lipid peroxidation, further leading to an increase of cell ated tumor tissues with minimal damage to normal organs [71].
membrane permeability for photochemical internalization and Similarly, Reich and colleagues conjugated the siRNA on the
endosome escape. Zhou’s group constructed an all-in-one proto- hollow gold nanoshells by Au–thiol bonds to construct plasmonic
porphyrin-based polymer nanoplatform with a step-by-step mul- nanocarriers, which could target the tumor cells using RPARPAR
tiple stimuli-responsive function for enhancing the combined peptide at the end of the siRNAs (Fig. 4b) [87]. By femtosecond
chemotherapy and PDT [85]. Short-time irradiation facilitated pulses of NIR light at 800 nm, the gold nanoshells converted the
the photochemical-aided uptake and endosome escape, whereas light to heat, which thermalized the thiol bonds and released the
the long-time irradiation generated massive ROS species, which siRNAs. At the same time, vapor bubbles were rapidly formed and
cleaved the ROS-responsive bonds and released the drugs, simul- ruptured the endosomes for siRNA release with little damage to cell
taneously inducing ROS- and drug-mediated cell death. structure and function.
NIR light can also be adsorbed by photoabsorbing agents, such Smart nanocarriers for siRNA delivery based on
as different gold nanostructures, carbon nanomaterials and organ- magnetic field or ultrasound
ic nanoparticles, to generate heat, leading to thermal ablation of Compared with light, magnetic fields have excellent tissue pene-
cancer cells and subsequent cell death in terms of photothermal tration and have been widely applied in whole-body MRI, which is
therapy (PTT) [84]. Here, we emphasize the applications of photo- one of the most popular medical imaging techniques. In 2007,
thermal conversion to control targeting and drug release. Thermo- Moore and co-workers first described the application of dual-
sensitive polymers (TSPs), usually poly(N-isopropyl acrylamide) purpose probes for in vivo siRNA delivery and simultaneously
(PNIPAM), undergo reversible phase transition from the hydro- monitored its accumulation in tumors by high-resolution MRI
philic to hydrophobic state at lower critical solution temperature and near-infrared in vivo optical imaging (NIRF) [88]. Zhang and
(LCST). By incorporating different ratios of other monomers, the co-workers assembled the siRNAs on an iron oxide core, which
LCST of the PNIPAM-copolymer can be finely controlled between served as a MRI agent for treatment monitoring [89]. At the same
32  C and 50  C, which provides a promising way to adjust the time, the magnetic manipulation of the magnetic nanoparticles,
surface composition of the nanocarriers, thereby allowing selec- usually the iron oxide nanoparticles (IONs), is useful for drug
tive cell entry in a controlled manner [86]. We have conjugated delivery, which can be externally guided to the desired cells or

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tissues by means of a remote magnet [90]. Plank and co-workers these techniques have been utilized in siRNA delivery, these
developed a protocol to deliver nucleic acids into the cells by pioneering designs accumulate a wealth of chemical, material
magnetic-force-assisted transfection using magnetic nanoparti- and biological experiences, which will be beneficial to promoting
cles. The nanoparticles were deposited onto the surface of the the translation into the clinic of siRNA therapeutics in the future.
cells with the aid of a magnetic gradient field, showing 1000-fold
transfection efficiency compared with nonmagnetic gene vectors Concluding remarks and future outlook
[91]. Recently, many novel strategies have been developed in With the development of chemistry, material science and biotech-
cancer treatment by magnetic manipulation, which also showed nology, thousands of novel stimuli-responsive nanocarriers have

Reviews
POST SCREEN

potential application in siRNA-based therapy. Alternating mag- been designed during the past decade for in vivo drug delivery,
netic fields (AMFs) cause the magnetic moment to rotate and along with a large capital investment and research interest. Espe-
return to equilibrium by dissipating thermal energy through Neel cially in recent years, the nanocarrier-based delivery strategies
(dipole rotation) and Brownian (physical rotation) relaxations, have significantly promoted the application of siRNA in cancer
which induce hyperthermia for tumor ablation or thermally con- therapy: (i) the siRNA therapeutics are efficiently protected after
trolled drug release [92]. Bhatia and co-workers encapsulated systematic administration and specifically delivered into the tu-
different protease substrates into thermosensitive liposomes to mor cells for function; (ii) the biodistribution and pharmacoki-
design a protease-activity nanosensor [93]. This sensor was remote- netics of siRNAs are highly controlled by means of stimuli-
ly activated at disease sites via an AMF at 515 kHz and 15 kA/m, responsive motifs in the nanocarriers; (iii) the combination of
which could determine the tumor protease activity in vivo and siRNA-based gene silencing and other treatment methods is widely
identify the different profiles in substrate cleavage between two studied to treat complex and malignant cancer. However, despite
mouse models. Interestingly, Cheon and colleagues developed a the booming scientific publications, few of these technologies
magnetic tandem system to overcome multidrug-resistant cancer have been successfully translated into clinical disease diagnosis
at a single-cell level (Fig. 4c) [94]. The magnetic tandem apoptosis and treatment. Several issues should be addressed in the future.
trigger (m-TAT) consisted of magnetic nanoparticles conjugating First, existing smart nanocarriers have been designed to solve one
death receptor 4 (DR4) antibody and Dox. Individual magnetic or two problems during siRNA delivery (Table 1), and this is hard to
nanoparticles attached onto the tumor membrane by recognizing adapt to the complex microenvironment in the living body.
extracellular receptors such as DR4, which were induced to aggre- Passive targeting and active targeting strategies cannot meet the
gate under an external magnetic field, activated the extrinsic death demands in complicated biological systems alone owing to mas-
signal and induced receptor-mediated uptake. The internalized sive nonspecific retention or poor cellular uptake issues. To further
nanocarriers released the Dox owing to GSH cleavage and initiated enhance treatment precision and efficacy, all-in-one activatable
the intrinsic death, presenting a new platform concept for cancer strategies responsive to multiple triggers (endogenous and exter-
therapy. nal) are recommended during nanocarrier design: the synergistic
Ultrasound is another noninvasive technique that can spatio- stimuli-responsive systems in which either trigger A or trigger B
temporally control drug release at disease sites with deep penetra- initiates the tumor recognition and drug release to realize specific
tion but slight harm to healthy tissues. It is convenient to regulate targeting and harness the tumor heterogeneity, and the sequential
the penetration depth by tuning frequency, exposing time and stimuli-responsive systems in which trigger A changes the behav-
cycles. Ultrasound can trigger the drug release through different ior of the outer layer to activate the targeting and then the trigger B
mechanisms. The tissues and nanocarriers can absorb the energy of acts on the inner core for drug release [98]. For instance, a typical
a small fraction of ultrasound and convert to heating, leading to smart nanocarrier has been developed for in vivo siRNA delivery:
thermally triggered release. Otherwise, the ultrasound can cause the iRGD-encoded PEG shell prolongs the blood circulation and
mechanical variations in terms of acoustic cavitation, where the facilitates passive accumulation and active tumor targeting; the
nucleation, growth and collapse of gas bubbles destabilize the iRGD peptide is then cleaved by cell-surface proteases to expose
nanocarriers for drug release. Liu and colleagues prepared nano- the CRGDR sequence, which binds to neuropilin-1 for tumor
droplets of perfluorocarbon as an oxygen shuttle to modulate the penetration; after cellular uptake, the rapid protonation of the
tumor hypoxic microenvironment by ultrasound-triggered oxy- ternary amine segment induces the endosomal swelling for rapid
gen delivery [95]. To minimize the side effects and prevent tumor escaping and efficient gene silencing [99]. By contrast, to precisely
recurrence in high-intensity focused ultrasound (HIFU), Shi’s localize the lesion, assess the pharmacokinetics and monitor the
group constructed an organic–inorganic nanohybrid for co-deliv- treatment response, an imaging agent is a highly important com-
ery of perfluorooctyl bromide (PFOB) and anticancer drug camp- ponent in nanocarrier design for clinical use. By incorporating
tothecin (CPT) [96]. The PFOB could enhance the HIFU ablation, quantum dots, gold nanoparticles, carbon nanotubes, UCNPs or
as well as the HIFU-mediated drug release, which synergistically magnetic nanoparticles, the multifunctional theranostic nanocar-
inhibited tumor growth. Lee et al. replaced the chemotherapeutic riers can not only possess different imaging modalities (e.g., opti-
drugs with siRNA for magnetic-guided delivery and ultrasound- cal imaging, positron emission tomography, MRI, photoacoustic
controlled release (Fig. 4d) [97]. The siRNAs were electrostatically imaging, etc.) but can also be endowed additional powerful tools
bound onto the chitosan–deoxycholic-acid nanoparticles contain- for combination treatment (e.g., photodynamic or photothermal
ing perfluoropentane and iron oxide, which promoted the cell therapy) [100]. However, these exquisite designs are always con-
uptake and apoptosis. So far, other external stimuli have also been sidered as ‘overdesigned’ in the perspective of commercialization,
studied for controlled drug release, including microwave, Cher- which prefers simple structures with ease of scaling up and robust
enkov radiation, electrical field, among others. Although few of clinic safety and efficacy, thus requiring a trade-off between

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January 2018

function and design. Actually, only a few nontargeted nanome- release will be greatly improved to adapt the complex clinical
dicines (e.g., Abraxane1 and Doxil1) have been approved for application.
clinical treatment, and limited improvements in therapy and Last but not least, the poor delivery efficiency of 0.7% (median)
diagnosis are obtained. To face the dilemma between commercial makes us rethink the current underlying principles of nanocarrier
drugs and fascinating proof-of-concepts, new ideas should be targeting. Most in vivo studies are built on the theory that the
introduced. Recently, Huang’s group utilized the antidiabetic drug nanocarriers are passively accumulated at tumors via the EPR
metformin to synthesize a guanidination polymer for a siRNA effect. But what if it is wrong? Current results indicate that the
package [101]. This interesting work inspires us to explore the new majority of the nanocarriers is sequestered by the MPS system and
Reviews
POST SCREEN

applications of FDA-approved materials (e.g., degradable PLGA the EPR effect is not an efficient pathway for in vivo delivery in
polymer and fluorescent ICG dye) to fabricate multifunctional mice, let alone in humans. Meanwhile, it seems that the targeting
nanocarriers, which is much easier to solve the safety and transla- ligands do not favor the desired active delivery because it is either
tion issues. buried by the protein corona or damaged by assorted enzymes in
Second, the majority of bioresponsive nanocarriers has been body fluids. If the nanocarriers cannot accumulate in solid tumors
designed and tested in vitro or in animal models, and the large gap and reach their target tumor cells, all our work will be meaningless.
between laboratory and hospital hinders the clinical translation. Thus, in the future, it is urgent to explore new modification
Before translating the delicate nanocarriers in human trials, it strategies to improve the EPR effect or find new mechanisms to
should be noted that: (i) the levels of biological cues might be favor accumulation in tumor tissues.
different between humans and the animal models, and also vary
from patient to patient; (ii) the local biological stimuli are highly Acknowledgments
heterogeneous and dynamically changing at different stages of This work was financially supported by the National Natural
disease progression. These heterogeneities provide potential Science Foundation of China (21605118, 11372243, 11522219,
chances for personalized treatment, as well as great challenges 11532009), the China Postdoctoral Science Foundation
for clinical translation. In the future, more-specific endogenous (2015M580829, 2017T100735), the Open Funds of SKLACLS
stimuli, such as enzymes and gene targets, should be screened to (1508), Postdoctoral Science Foundation of Shannxi Province
achieve specific targeting and drug release. Meanwhile, by com- (2016BSHTBSHTDZZ01) and the Fundamental Research Funds for
bining with the external stimuli, which can be manually con- the Central Universities (xjj2015081).
trolled, the precision of tumor targeting and controlled drug

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