Inorganic nanocarriers for siRNA delivery for cancer treatments

Abstract

RNA interference (RNAi) is one of the emerging methodologies utilized in the treatment of a wide variety of diseases including cancer. This method specifically uses therapeutic RNAs (TpRNAs) like small interfering RNAs (siRNAs) to regulate/silence the cancer-linked genes, thereby minimizing distinct activities of cancer cells and aiding in their apoptosis. But, many complications arise during the transport/delivery of these TpRNAs that include poor systemic circulation, instability/degradation inside the body environment, no targeting capacity and also low cellular internalization. These difficulties can be overcome by using nanocarriers to deliver the TpRNAs inside the cancer cells. The following are the various categories of nanocarriers - viral vectors (e.g., lentivirus and adenovirus) and non-viral nanocarriers (self-assembling nanocarriers and inorganic nanocarriers). Viral vectors suffer from few disadvantages like high immunogenicity as compared to the non-viral nanocarriers. Among non-viral nanocarriers, inorganic nanocarriers gained significant attention as their inherent properties (like magnetic properties) can aid in effective cellular delivery of the TpRNAs. Most of the prior reports have discussed about the delivery of TpRNAs through self-assembling nanocarriers; however very few have reviewed about their delivery using the inorganic nanoparticles. Therefore, in this review, we have mainly discussed the delivery of TpRNAs – i.e., siRNA, especially programmed death ligand-1 (PD-L1), survivin, B-cell lymphoma-2 (Bcl-2), vascular endothelial growth factor (VEGF) and other siRNAs using the inorganic nanoparticles – mainly magnetic, metal and silica nanoparticles. Moreover, we have discussed about the combined delivery of these TpRNAs along with chemotherapeutic drugs (mainly doxorubicin) and in vitro and in vivo therapeutic effectiveness.

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Biomedical Materials
TOPICAL REVIEW
Inorganic nanocarriers for siRNA delivery for
cancer treatments
To cite this article: Ganeshlenin Kandasamy and Dipak Maity 2024
Biomed. Mater.
19 022001
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Biomed. Mater. 19 (2024) 022001 https://doi.org/10.1088/1748-605X/ad1baf
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TOPICAL REVIEW
Inorganic nanocarriers for siRNA delivery for cancer treatments
Ganeshlenin Kandasamy1and Dipak Maity2,∗
1Department of Biomedical Engineering, School of Electrical and Communication, Vel Tech Rangarajan Dr. Sagunthala R&D Institute
of Science and Technology, Avadi, Chennai, India
2Department of Environmental and Occupational Health, School of Public Health, Texas A&M University, College Station, TX 77843,
United States of America
∗Author to whom any correspondence should be addressed.
E-mail: dipakmaity@gmail.com
Keywords: magnetic/metal/silica nanoparticles, siRNA, PD-L1, survivin, Bcl-2 and VEGF, chemotherapy, cancer treatment
Abstract
RNA interference is one of the emerging methodologies utilized in the treatment of a wide variety
of diseases including cancer. This method specifically uses therapeutic RNAs (TpRNAs) like small
interfering RNAs (siRNAs) to regulate/silence the cancer-linked genes, thereby minimizing the
distinct activities of the cancer cells while aiding in their apoptosis. But, many complications arise
during the transport/delivery of these TpRNAs that include poor systemic circulation,
instability/degradation inside the body environment, no targeting capacity and also low cellular
internalization. These difficulties can be overcome by using nanocarriers to deliver the TpRNAs
inside the cancer cells. The following are the various categories of nanocarriers—viral vectors (e.g.
lentivirus and adenovirus) and non-viral nanocarriers (self-assembling nanocarriers and inorganic
nanocarriers). Viral vectors suffer from disadvantages like high immunogenicity compared to the
non-viral nanocarriers. Among non-viral nanocarriers, inorganic nanocarriers gained significant
attention as their inherent properties (like magnetic properties) can aid in the effective cellular
delivery of the TpRNAs. Most of the prior reports have discussed about the delivery of TpRNAs
through self-assembling nanocarriers; however very few have reviewed about their delivery using
the inorganic nanoparticles. Therefore, in this review, we have mainly focussed on the delivery of
TpRNAs—i.e. siRNA, especially programmed death ligand-1 (PD-L1), survivin, B-cell
lymphoma-2 (Bcl-2), vascular endothelial growth factor and other siRNAs using the inorganic
nanoparticles—mainly magnetic, metal and silica nanoparticles. Moreover, we have also discussed
about the combined delivery of these TpRNAs along with chemotherapeutic drugs (mainly
doxorubicin) and in vitro and in vivo therapeutic effectiveness.
1. Introduction
Therapeutic nucleic acids have gained enormous
interests in treating many diseases—e.g. cancer,
inflammatory diseases, cardiovascular diseases, neur-
ological disorders, and pathogenic diseases, for more
than two decades [1–8]. Therapeutic nucleic acids
mainly include deoxyribonucleic acid (DNA) and
ribonucleic acid (RNA—for example, short inter-
fering RNA (siRNA)/short hairpin RNA (shRNA))
[9,10]. Among above, the siRNAs with 21–23 nuc-
leotides have been attractive in RNA interference
(RNAi) based cancer therapeutics, as they can effect-
ively regulate/silence specific cancer-linked genes—
i.e., preventing cancer protein translation by binding
with the corresponding messenger RNAs (mRNAs)
[11–13]. This gene regulation/silencing, via thera-
peutic RNAs (TpRNAs—i.e. siRNAs), significantly
influence the one or more of the following activities
in cancer—growth/proliferation, angiogenesis, meta-
bolism, differentiation and apoptosis, resulting in an
efficient cancer cell killing [14,15]. Therefore, the
usage of the TpRNAs is currently dominating major-
ity of the research investigations and cancer clinical
trials [16].
Nevertheless of competent gene-silencing effects,
cancer treatment using TpRNAs is limited due to
their short body circulation time, targeting inability,
poor target accumulation ability, instability, low cel-
lular uptake, and etc [17,18]. To overcome the above
© 2024 IOP Publishing Ltd

Biomed. Mater. 19 (2024) 022001 G Kandasamy and D Maity
hurdles, the following approaches are used in deliv-
ering TpRNAs—viral vectors and non-viral vectors
[19,20]. Viral vectors are classified into (i) gene-
delivery vehicles (e.g. lentivirus, adenovirus, adeno-
associated virus, gamma-retrovirus)—which only act
as a vehicle for delivering TpRNAs [21,22]; and
(ii) oncolytic viruses—which will replicate inside the
cancer cells, resulting in tumor lysis; as the can-
cer cells do not possess active defensive mechan-
isms against viruses [20,23,24]. However, viral
vectors suffer from many disadvantages—e.g. side
effects, inducing inflammations, immunogenicity
and so on. Hence, the non-viral vectors—especially
self-assembling nanoparticles (liposomes/exosomes
and polymers) and inorganic nanoparticles (mag-
netic nanoparticles, metal nanoparticles, silica nan-
oparticles etc) are preferred as better nanocarrier-
cum-delivery agents for TpRNAs [25–29]. In our pre-
vious work, we have comprehensively investigated
about the delivery of specific siRNAs mainly using
self-assembling nanoparticles [30].
As compared to self-assembling nanocarriers,
the inorganic nanoparticles offer a wide variety
of advantages that include stimuli-responsiveness
(thermal/heat, light, magnetic, pH and redox)
because of their inherent properties/characteristics
[31–35]. For instance, magnetic nanoparticles, when
stimulated externally, can magnetically deliver the
TpRNAs—that can be helpful in effective release
of TpRNAs inside the target cancer cells [36–38].
Besides, the inorganic nanoparticles can be surface-
modified with liposomes/exosomes and polymers
to improve their delivery, while providing a multi-
modal approach [39–42]. Previous reports - by
Charbe et al [43], Labatut and Mattheolabakis [44],
and Lee et al [14] - have reviewed about the delivery
of TpRNAs mostly through self-assembling nanocar-
riers; but only few studies have discussed about their
delivery using inorganic nanoparticles. Therefore,
in this review, we have comprehensively discussed
the delivery of TpRNAs—i.e. siRNA using inorganic
nanoparticles. Besides, we have exclusively reviewed
about the applied stimuli that aid in releasing the
TpRNAs. Moreover, we have reviewed also about the
delivery of TpRNAs in combination with chemo-
therapeutic drugs, and further discussed about their
therapeutic effectiveness in in vitro and in vivo cancer
environments.
2. Inorganic nanocarriers
Inorganic nanocarriers/nanoparticles are generally
used as nanomedicines in the delivery of chemo-
therapeutic drugs for cancer treatments; ascribed to
their controllable physicochemical properties, easy
manufacturing, non-immunogenicity and better
biocompatibility. In addition to above, inorganic
nanoparticles provide distinct stimuli-responsive
features, which make them suitable for gene deliv-
ery also. Herein, the major inorganic nanoparticles
are explained in brief.
2.1. Major types of inorganic nanocarriers
2.1.1. Superparamagnetic iron oxide nanoparticles
Magnetic nanoparticles form a significant part in
inorganic nanocarrier/nanoparticle category [45].
Magnetic nanoparticles majorly include: mangan-
ite perovskites (La1−xSrxMnO3) and spinel ferrites
(especially, Fe3O4(iron oxide)) [46,47]. Among
them, spinel ferrites possess attractive superparamag-
netic characteristics, with magnetic and heat stimuli-
responsiveness, for use in imaging and therapeutics
[48,49]. For instance, superparamagnetic iron oxide
nanoparticles (SPIOs) have been utilized as good:
(i) T2 contrast agents for magnetic resonance ima-
ging (MRI) [50–53], and (ii) heating agents in many
magnetic hyperthermia cancer treatments [54–58].
Besides, magnetic/heat-based stimuli responsiveness
in SPIOs is employed in transport and delivery of
anti-cancer drugs and TpRNAs.
2.1.2. Manganese oxide nanoparticles
Manganese oxides (mainly MnO, MnO2, Mn2O3,
Mn3O4and MnOx) in nanoparticle/nanosheet form
have been prepared using different methods by
the groups of Alivisatos, Hyeon and so on [59,
60]. These paramagnetic manganese oxide nano-
particles/nanosheets (MONs) are exploited in differ-
ent applications—bioimaging, drug/TpRNAs deliv-
ery and cancer treatments [61–64]. For instance,
MONs are towards H+/H2O2and/or glutathione
inside the hypoxic/acidic tumor micro-environment,
and degrade to create oxygen and Mn2+ions that are
useful in T1 MRI contrast (alternative to gadolinium
based materials) and cancer treatments [65–67].
2.1.3. Gold and silver nanoparticles
Noble metal nanoparticles, specifically gold and sil-
ver nanoparticles in colloidal form are well-known for
their optical phenomena like localized surface plas-
mon resonance (LSPR) [68–71]. LSPR basically hap-
pens when the surface free electrons of metal nano-
particles oscillate at a specific frequency upon inter-
action with light having an oscillating electromag-
netic field; this result in light scattering or light-to-
heat conversion [72–74]. This is helpful in cellular
imaging, distinct heat-based therapeutics like pho-
todynamic therapy and also light/heat stimuli-based
TpRNAs delivery [75–80]. Besides, due to radiopa-
city, metal nanoparticles are exclusively used as
x-ray/computed tomography (CT) contrast agents
[81–84].
2.1.4. Silica nanoparticles
Silica nanoparticles, especially mesoporous silica
nanoparticles (MSOs) are porous nanocarriers with
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Biomed. Mater. 19 (2024) 022001 G Kandasamy and D Maity
specific pore volume and diameter to accommodate
different nanocargoes—i.e. genes (TpRNAs) and/or
chemotherapeutic drugs, for their delivery [85–89].
Besides, MSOs are easily surface-functionalizable
with ligands and targeting agents due to large sur-
face area and presence of silanol (Si–O) groups
[90–92]. Unlike other inorganic nanoparticles,
MSOs do not possess any inherent responsive (like
heat/magnetic/light) characteristics. So, the com-
mon modes of stimuli-responsiveness such as pH
and redox potential are imparted to release nanocar-
goes inside tumor based on its microenvironment
[93,94].
2.2. Surface modifications
2.2.1. Stability
In gene delivery, colloidal instability, agglomer-
ation or/and reactive oxygen species generation
are the major challenges for the to-be-used inor-
ganic nanocarriers [95]. For instance, SPIOs tend
to agglomerate easily due to magnetic interac-
tions between the neighboring SPIOs. A positive-
charged polymer—i.e. polyethylenimine (PEI) is
usually coated on the surface of the SPIOs to pre-
vent agglomeration and also to facilitate the delivery
of negatively-charged nucleic acids—i.e. TpRNAs
(based on electrostatic interactions). For instance, Jia
et al have modified the surface of SPIOs with PEI
polymer (with prior modifications with oleic acid
and dimercaptosuccinic acid), which allowed the
surface complexation of siRNA molecules; that
later have been effectively delivered to RAW264.7
macrophages [96]. Similar to this, the inorganic nan-
oparticles are surface modified with other polymers
(e.g. polyethylene glycol (PEG)), lipids/liposomes,
and extracellular vesicles (e.g. exosomes) to over-
come the above-mentioned challenges, and also to
impart enhanced permeation and retention (EPR)
characteristics for longer body circulation, and
multi-functionalities including co-encapsulation/-
delivery of inorganic nanocarriers along with
genes and drugs for multi-modal treatments
[97–102].
2.2.2. Targeting
TpRNAs-attached inorganic nanocarriers can reach
tumor locations via leaky blood vessels through pass-
ive targeting and can get accumulated more in those
locations via EPR phenomenon [30]. Apart from this,
stimuli-responsive based targeting including mag-
netic targeting can be applied to deliver the TpRNAs
in hypoxic/acidic tumor environments. Besides, spe-
cific targeting molecules (e.g. small molecules, pep-
tides, proteins, monoclonal antibodies and so on)
can also be surface-attached onto the nanocarriers
to actively reach target tumor cells and deliver the
TpRNAs nanocargoes inside cytoplasm (active target-
ing method).
3. siRNA delivery
3.1. Conventional delivery and challenges
The discovery of siRNA in 1998 has fetched Nobel
Prize for Fire and Mello. In 2001, the siRNA has
been successfully delivered inside the mammalian
cells and silenced a specific gene expression. Later
in 2002, siRNA based RNAi technology has been
named as the ‘Technology of the Year’ by Science.
From there onwards, research works on siRNAs has
ascended, where they have shown high therapeutic
potential in the treatment of many diseases includ-
ing cancer. This is mainly due to their advantages like
high efficacy and safety (as compared to chemothera-
peutic drugs), no DNA interactions (thereby pre-
venting any mutations or teratogenicity), better spe-
cific gene expression knock-down in any single cell
and distinct siRNA for the targets [103]. However,
the naked delivery of siRNA has faced many chal-
lenges because of their inherent properties—i.e. high
molecular weight and negative charge (make them
difficult to enter into cells), instability in plasma,
immunogenicity, intrinsic toxicity and so on [104–
106]. Besides, the major barriers for siRNA-based
RNAi are depending upon two major factors: (i)
the area-of-therapeutic interest—that can be specific
tissues or organs—whose accessibility can pose an
issue for reaching of siRNA; and (ii) the admin-
istration routes (local and systemic)—herein, the
local administration route can pose comparative
lesser threat. The barriers in the systemic adminis-
tration route can be categorized into two, which are
given below along with the corresponding threats
[17,103].
I. Extracellular—endogenous-nuclease/intravascular
based degradation, removal by kidneys, inability
to cross the barriers like biological membranes,
reticuloendothelial system uptake, plasma
protein binding and activation of immune
system.
II. Intracellular—incapable of escaping from lyso-
somes/endosomes and unintended genome tar-
geting
To overcome the above threats, many steps have
been taken and that include chemical modifications
of siRNAs (including their backbones/base/sugars),
PEGylation, receptor-mediated-delivery, antibody-
conjugation, aptamer-conjugation, and so on.
However, the nanoparticles, especially the previously-
mentioned inorganic nanoparticles (i.e. iron oxide/-
manganese oxide/silica/gold) hold high potential in
the siRNA delivery (where siRNA can be conjug-
ated (via different approaches including electrostatic
attraction/covalent binding) onto the inorganic nan-
oparticles (that are surface modified without/with
polymers/lipids), as they can comparatively reduce
3

Biomed. Mater. 19 (2024) 022001 G Kandasamy and D Maity
Scheme 1. Illustrates the timeline of significant events in siRNA based RNAi technology including the involvement of siRNA
delivery using inorganic nanocarriers.
the nuclease/intravascular degradation and renal
clearance, enhance the blood circulation time, spe-
cifically reach the intended target cells and deliver
the siRNAs intracellularly. This is mainly due to the
high surface area-to-volume ratio (that aids in better
siRNA loading) and easy-modifiable physicochem-
ical properties (size/shape/surface-charge/formation
of core-shell/core-satellite structures) of the inor-
ganic nanoparticles [43]. Besides, the inorganic nan-
oparticles can also offer surface-functional groups
that aid in conjugating them with distinct fluor-
escent molecules (for tracking) and this can help
in improving the overall therapeutic efficacy of the
siRNA based RNAi therapy. Moreover, the surface
modification of inorganic nanoparticles with poly-
mers like PEI and PEG can prevent the endosomal
and immune (opsonization) traps by exhibiting ‘pro-
ton sponge’ and ‘stealth’ effects respectively, while
maintaining their unique properties (magnetic/op-
tical). Though one of the major concerns is tox-
icity and this has been investigated limitedly [107,
108], the inorganic nanoparticles are successfully
employed in siRNA delivery from 2006 onwards and
their major events are given in a timeline format in
scheme 1.
3.2. siRNA-inorganic nanocomplexes
Generally, siRNAs and inorganic nanoparticles com-
bine to form nanocomplexes. In this section, we have
mainly focused on the delivery of four major siRNAs
based nanocomplexes—programmed death ligand-1
(PD-L1) siRNA, survivin siRNA, B-cell lymphoma 2
(Bcl-2) siRNA and vascular endothelial growth factor
(VEGF) siRNA.
3.2.1. PD-L1 siRNA
Programmed death ligand-1 (PD-L1), a protein
(an immune check-point), is located on the cell
membrane. PD-L1 is comparatively more expressed
on the cancer cells than some normal healthy
cells. PD-L1 interacts and binds with PD-1 trans-
membrane protein in T-immune cells, thereby pre-
venting them from killing the cancer cells, resulting
in further proliferation of cancer cells. So, the PD-L1
protein expression on cancer cells can be influenced
by delivering the corresponding siRNAs.
Luo et al have focused on the treatment of gast-
ric cancers having high expression of PD-L1 [109]. In
this work, initially complex molecules made of folic
acid (FA)—PEG-disulfide-PEI are formed. Then,
they are surface-attached to the SPIOs and finally
formed a nanocomplex by conjugating with PD-L1
siRNA. The to-be-used amount of siRNA molecules
is selected based on the amount of nitrogen (N)
on the delivery vehicle and also on the amount of
phosphate (P) on the siRNA. Herein, an N:P ratio
of 10 is found suitable for complete the complex-
ation and better transfection as compared to lower
N:P ratios, and it is also found to be less cyto-
toxic contrast to nanocomplexes with higher N:P
ratios. Besides, among 4 designed PD-L1 siRNAs
(that are individually complexed with SPIOs), the
siRNA having the following sequence: i.e., (sense) 5′-
CCAGCACACUGAGAAUCAATT-3′and (antisense)
5′-UUGAUUCUCAGUGUGCUGGTT-3′, has effect-
ively knocked down the PD-L1-expression on SGC-
7901-based gastric cancer cells by ∼90.9%.
Liu et al have chosen MnO2nanoparticles as
delivery agent for PD-L1 siRNA as these nanoparticles
4

Biomed. Mater. 19 (2024) 022001 G Kandasamy and D Maity
can modify the hydrogen peroxide in tumor microen-
vironment into oxygen and water molecules [110]. In
this work, MnO2nanoparticles are primarily pre-
pared and then coated with calcium carbonate (to
improve the presence of oxygen molecules inside
tumors), while encapsulating an FDA-approved
photodynamic therapy (PDT) agent—indocyanine
green. Then, anionic PD-L1 siRNA molecules, that
are surface-attached with these positive-charged nan-
oparticles to form nanocomplexes with a binding
ratio of 0.64, have resulted in a 85%–27% drop of PD-
L1 expression in Lewis lung cancer cells. Moreover,
the in vitro PDT (under 808 nm laser irradiation)
and siRNA based gene silencing have revealed better
therapeutic effects by killing ∼91% of cancer cells,
while imaging through T1-MRI (via MnO2) and
computed tomography (via calcium carbonate). The
in vivo results have revealed inhibited tumor growth,
while preserving biosafety.
Liu et al have designed gold nanoprisms to act
as a delivery agent for human PD-L1 siRNA for
human lung cancer (HCC827) treatments—as shown
in figure 1[111]. Herein, primarily the gold nano-
prisms are surface-modified sequentially with the fol-
lowing polymers: (i) negative-charged poly (sodium
4-styrenesulfonate) and (ii) positive-charged poly (-
diallyldimethylammonium chloride). Later, the PD-
L1 siRNA molecules are electrostatically attached on
the surface of the gold nanoprisms to form the nano-
complex. This nanocomplex has shown good cellu-
lar uptake, and comparatively higher PD-L1 knock-
down in HCC827 cancer cells. Besides, the authors
have shown better cell killing effects in the lung can-
cer cells under in vitro and in vivo environment using
photoacoustic imaging-guided photothermal therapy
(via gold nanoprisms) in combination with gene
silencing effects. In in vivo scenario, the nanocomplex
has been proved to be safe as it has not accumulated
in major organs.
3.2.2. Survivin siRNA
Survivin, a baculoviral inhibitor of apoptosis repeat-
containing 5 (BIRC5)-gene encoded protein, is
responsible for inhibiting the apoptosis in cells.
Survivin is selectively and highly expressed in dif-
ferent cancer cells to prevent them from dying to
result in further proliferation, evasion and meta-
stasis. The siRNA with the following sequence:
(sense, 5′-CACCGCAUCUCUACAUUCATT-3′; anti-
sense, 5′-UGAAUGUAGAGAUGCGGUGTT-3′) spe-
cific to survivin can be used to down-regulate
it in cancer cells to improve the apoptosis in
them.
For instance, Wu et al, have initially covered
the SPIOs with cationic amylose (a biopolymer)
molecules, and then conjugated them with a tar-
geting agent, folic acid [112]. Later, survivin siRNA
molecules are electrostatically attached on the sur-
face of SPIOs to form the nanocomplexes, which
exhibited better transfection (confirmed via MRI
investigations) and down-regulation of survivin
expression in hepatocellular carcinoma (HepG2)
cells. In a recent analogous investigation, Zhang et al
have utilized the SPIOs along with tetraphenylethyl-
ene molecules and quaternary ammonium cationized
amylose to form nanocarriers; which are conjug-
ated further with peptide—SP94 with the sequence
of SFSIIHTPILPL through amide bond formation,
and finally with survivin siRNA [113]. The SPIOs
plus tetraphenylethylene, and SP94 peptides are
included respectively to impart dual imaging (MRI
plus aggregation induced emission based fluores-
cence) functionality, and to bind/target to the short-
peptide-based-receptor-protein (i.e. GRP78) on the
membranes of hepatocellular carcinoma (Huh-7)
cells. In in vitro carcinoma cells, the nanocarriers
with siRNA has shown high transfection efficacy (at
N:P ratio of 20), good MRI (53.0 mM−1s−1) plus
fluorescence imaging capacities, better internaliza-
tion (due to endocytosis via binding of SP94 with
cancer cell receptors) and enhanced toxicity against
carcinoma (more than 80% killing of Huh-7 cells).
The in vivo studies with MRI and fluorescence guid-
ance have revealed that the suppression of Huh-7
tumor size in mice models is comparatively bet-
ter for the nanocarriers with survivin siRNA (i.e.
240 mm3) rather than control group (1823 mm3)
and naked siRNA (1599 mm3) on the 14th day of
observation. Similarly, PEI-conjugated SPIOs are
prepared and a nanocomplex with survivin siRNA
is formed (having 0.6 µg of SPIOs and 0.2 µg of
siRNA—used for further purposes) by Jin et al [114].
This nanocomplex has relatively inhibited the corres-
ponding mRNA levels higher in oral cancer (Ca9-22)
cells, after their incubation under magnetic field for
30 min, and also enhanced the anti-tumor activity.
Arami et al have covered the magnetic core of SPIOs
layer-by-layer with PEG-lactide, chitosan and PEI,
which provided good scope for conjugating adequate
amount of siRNA against the survivin protein to form
the nanocomplexes [115]. The as-formed polymeric
layers have increased colloidal stability and biocom-
patibility, provided safe cellular internalization with
good nuclear distribution, and decreased the agglom-
eration and toxicity issues. Besides, the siRNA has
particularly lowered the protein levels in leukemia
cancer (K562) cells. Same authors (i.e. Arami et al)
have utilized the same nanocomplex with a 2:1 ratio
of nanoparticles to survivin siRNA molecules and
achieved better transfection and enhanced apoptosis
in the breast carcinoma (MCF-7) cells also [116]. In
a recent work, Wang et al have formed nanocom-
plexes by modifying the surface of the SPIOs with
the PEI polymer to facilitate the electrostatic inter-
action based attachment of survivin siRNA [117].
The nanocomplexes with good cytocompatibility are
introduced to the glioblastoma multiforme (U251)
based brain cancer cells, which revealed the following
5

Biomed. Mater. 19 (2024) 022001 G Kandasamy and D Maity
Figure 1. (i) A schematic illustration shows the sequential coating of gold nanoprisms (GNPs) with poly (sodium
4-styrenesulfonate) (PSS) and poly (-diallyldimethylammonium chloride) (PDADMAC) and conjugation of human program
death-ligand 1 (PD-L1) siRNA for gene silencing in HCC827 cells along with photoacoustic (PA) imaging-guided photothermal
therapy (PTT) using laser, and (ii) (A) in vivo PA images—before and after the injection of PD-L1 siRNA conjugated GNPs and
(B) PTT images of HCC827-tumor bearing mice after treatment with PD-L1 siRNA conjugated GNPs in comparison with other
controls. Reprinted from [111], Copyright (2019), with permission from © 2019 Acta Materialia Inc. Published by Elsevier Ltd.
All rights reserved.
results: cytotoxicity up-to 40% and apoptosis rate
of ∼60%—comparatively better than free siRNA.
Similarly, Bruniaux et al (from Stephanie David
group) have utilized SPIOs with specific surface
modifications for treating breast cancer, especially
human epidermal growth factor receptor-2 (HER2)
overexpressing breast cancer (e.g. BT-474) [118].
In this work, silane and PEG molecules attached
SPIOs are prepared and then conjugated with anti-
HER2 single chain antibody fragments (for targeting
HER2-overexpressing BT-474 cancer cells). Survivin
siRNA pre-attached polymer (poly-L-arginine) and
chitosan are later added to the surface of SPIOs to
form the complete nanocomplexes. Herein, 30 and 10
6

Biomed. Mater. 19 (2024) 022001 G Kandasamy and D Maity
charge-ratios are maintained among chitosan/siRNA
and polymer/siRNA (i.e. positive-charged/negative-
charged) molecules, where a mass-ratio of 10 is main-
tained for iron/siRNA. The targeted and non-targeted
nanocomplexes are incubated separately with HER2-
overexpressing BT-474 and HER2-negative MDA-
MB-231 cells. In comparison, the targeted nanocom-
plexes have down-regulated the protein expression
better by 90% in BT-474 (due to high-intake of nano-
complexes) and 70% in MDA-MB-231 cells. Hou et al
have followed a slightly different targeted strategy for
delivering the survivin shRNA plasmid [119]. Herein,
the authors have formed nanospheres, via desolva-
tion cross-linking process, by using a combination
of bovine serum albumin, Fe3O4(iron oxide) nan-
oparticles and survivin shRNA plasmid, and then
attaching with a targeting agent cetuximab (an anti-
epidermal growth factor receptor monoclonal anti-
body). These targeted nanospheres have revealed high
transfection efficacy and cytotoxicity (∼66%), under
the presence of external magnetic field, in epidermal
growth factor receptors overexpressed lung cancer
(GLC-82) cells.
Wang et al have formed hydrazine hydrate-
mediated gold nano-flowers with bovine serum albu-
min (also worked as a bio-template during synthesis
process) surface coatings [120]. The albumin layer has
facilitated a layer-by-layer electrostatic-interaction
to conjugate the fluorescein amidites-labelled sur-
vivin siRNA onto the surface of the nano-flowers.
The labelled-fluorescein molecules have helped in
efficient fluorescent imaging-cum-tracing of the
siRNA inside the human pancreatic adenocarcinoma
(BXPC-3) cells during their internalization process.
Besides, the siRNA has effectively silenced the corres-
ponding protein the BXPC-3 cells even at its minimal
concentration of 200 nM; while gold nano-flowers
have displayed better contrast effects under computed
tomography (CT =∼1.5 times higher hounsfield
unit compared to commercial CT contrast agent—
Ultravist).
3.2.3. Bcl-2 siRNA
The B-cell lymphoma 2 (Bcl-2) genes mainly encode
for the proteins linked to the regulation of cell
death—i.e. apoptosis. The overexpression of Bcl-2
genes and the corresponding proteins can result in
suppression of apoptosis, thereby lowering the death
of cells and thus resulting in proliferation of dif-
ferent cancers including lymphoma, melanoma and
so on. Bcl-2 siRNA can be delivered through metal
and magnetic nanoparticles for Bcl-2 gene down-
regulation. For instance, Shen have made a mag-
netic non-viral vector to deliver the Bcl-2 siRNA
inside human neuroblastoma, which is usually a solid
tumor that form in extracranial region [121]. In this
work, PEG-grafted-PEI polymer has been attached
with the SPIOs, whose surface is then conjugated with
a neuroblastoma-specific ligand GD2 single-chain
antibody through PEG-COOH molecules. Later, the
Bcl-2 siRNA molecules are complexed with PEI on
the surface of the SPIOs through electrostatic interac-
tions at various N:P ratios; where N:P ratio of 10 has
resulted in better transfection efficacy up-to ∼56%.
The confocal laser scanning microscopic results have
revealed an effective uptake of these siRNA-loaded
SPIOs by neuroblastoma (SK-N-SH) cells, which is
further confirmed via MRI images. These SPIOs have
reduced the expression of Bcl-2 to ∼46% in vitro, and
also the in vivo tumor size by promoting the apoptosis
in the neuroblastoma tumors.
Bcl-xL proteins, responsible for anti-apoptosis,
belong to the family of Bcl-2 proteins and they
are over-expressed due to the up-regulation of
interleukin-4 receptors based on the interactions
between the interleukin-4 and the receptors in the
tumor cells. In a recent investigation, Guruprasath
et al have loaded the siRNA against the Bcl-xL
genes on the surface of the branched PEI polymer
(BPEI) coated SPIOs to form nanocomplexes (at N:P
ratio of 25 with 60 pmol of siRNA concentration),
while labeling them with a targeting interleukin-4
receptor binding peptide [122]. Through interleukin-
4 receptor targeting, the nanocomplexes have been
able to induce high cytotoxicity in drug-resistant
breast cancer (MDA-MB-23) cells by suppressing the
expression of Bcl-xL genes. This has created sensitiv-
ity in the cancer cells for further getting treated with
chemotherapy. The in vivo biodistribution and tumor
accumulation of the siRNA loaded SPIOs are invest-
igated and confirmed via NIR fluorescence imaging
(by priorly attaching the Cy5.5 dye to the siRNA)
and MRI imaging (based on T2 weighted images)—as
shown in figure 2.
Choi et al have formed gold nanorods (with
3.25 aspect ratio) that are surface functionalized
with CTAB molecules, which have been replaced by
thiol-modified bovine serum albumin molecules (as
the surface coatings) via exchange method [123].
Later, the Bcl-2 siRNA molecules have been encap-
sulated inside the bovine serum albumin to form
nanocomplexes via desolvation technique using eth-
anol and glutaraldehyde. Finally, through NH2-
PEG-biotin, anti-erythroblastic oncogene B (ErbB)-2
antibodies–streptavidin molecules are conjugated to
the nanocomplexes, which have exhibited a temperat-
ure increase up-to 47 ◦C under photothermal applic-
ation on exposure to near-infra red (NIR) waves hav-
ing a wavelength of 810 nm. Anti-ErbB-2 antibod-
ies conjugated nanocomplexes are internalized 5-fold
greater by ErbB-2 overexpressing breast cancer cells,
i.e., SK-BR-3 than the MCF-7 cells. As a result, this
helped in minimizing the corresponding mRNA and
protein levels in SK-BR-3 cancer cells.
Li et al (from Hongchen Gu group) have pre-
pared MSOs with a magnetic core in the middle
7

Biomed. Mater. 19 (2024) 022001 G Kandasamy and D Maity
Figure 2. (i) Near infrared fluorescence (NIRF) and (ii) magnetic resonance imaging (MRI) images of the mice-models injected
with interleukin-4 receptor binding peptide-1 (IL4RPep-1) attached branched polyethyleneimine-superparamagnetic iron oxide
nanoparticles (BPEI-SPION) conjugated with cy5.5 fluorescence molecule-modified Bcl-xL siRNA molecules and their
corresponding controls. Reprinted from [122], Copyright (2017), with permission from Elsevier.
[89]. In this work, initially Fe3O4magnetic nano-
particles are prepared via chemical co-precipitation
and modified with cetyltrimethyl ammonium brom-
ide (CTAB) surface coatings (by replacing the ori-
ginal oleic acid coatings). Further, the mesoporous
silica is formed on the surface of magnetic nano-
particles by using the self-assembly based reaction
between CTAB (as a template) and tetraethyl ortho-
silicate (TEOS) under basic conditions; where the
template has been removed later by utilizing acet-
one to form the mesopores. These mesopores are
efficient in accommodating the Bcl-2 siRNA under
the dehydration environment created by ethanol and
guanidine-based salt. Finally, the PEI-surface covered
Bcl-2 encapsulating magnetic MSOs have effectively
knocked down ∼80% of the corresponding protein in
the adenocarcinomic human alveolar basal epithelial
(A549) cells.
8

Biomed. Mater. 19 (2024) 022001 G Kandasamy and D Maity
3.2.4. VEGF siRNA
VEGF promotes angiogenesis by enhancing the pro-
liferation of vascular endothelial cells in normal cells.
But, VEGF is highly up-regulated in cancer tissues
and form new tumor vasculatures. Apart from their
major expression in leukemia and lymphoma, VEGF
is also highly expressed in solid tumors such as breast
cancer, lung cancer and etc.
Dalmina et al have made magnetic-responsive
gene nanocarriers using the SPIOs, which have been
covered with calcium phosphate via layer by layer
technique [124]. This specific covering has later
accommodated the attachment of PEG-poly(aspartic
acid) copolymer (for stabilizing the nanocarriers) and
then with the VEGF siRNA. Based on the real-time
quantitative polymerase chain reaction (PCR) stud-
ies and western blot analysis confirmation, it has been
identified that the expression of VEGF in breast can-
cer (MDA- MB 231) cells has been suppressed by 60%
after 48 h incubation with the siRNA carrying nano-
carrier at 100 nM concentration. The same author
group has delivered VEGF siRNA using hybrid nan-
oparticles made of calcium phosphate and methoxy-
PEG -block-poly(L-glutamic acid) and achieved 67%
reduction in corresponding protein levels [125].
Bae et al have chosen and made hollow manganese
oxide nanoparticles for siRNA delivery due to their
small size for good cell internalization and T1 MRI
contrast effects [126]. In this work, 3,4-dihydroxy-L-
phenylalanine is initially bound with PEI molecules
and then immobilized on the surface of manganese
oxide nanoparticles while further conjugating the act-
ive targeting agent, Herceptin or biocompatible and
blood circulation enhancing material, PEG. Later,
VEGF siRNA molecules and the manganese oxide
nanoparticles with HER or PEG are mixed at differ-
ent N:P ratios in buffer solution. It has been found
through agarose gel electrophoresis process that the
complexation of VEGF siRNA has been completed for
manganese oxide nanoparticles with Herceptin at N:P
ratio of 24 due to their high positive surface-charge,
but it has been completed only at N:P ratio of 32
for nanoparticles PEG, as PEG molecules might have
covered more surface. Besides, the nanoparticles with
Herceptin and siRNA are internalized more in HER2-
overexpressing SK-BR-3 cells (at-least 4 fold increase)
and correspondingly displayed a brighter T1 MRI sig-
nal as compared with HER2-less-expressing MCF-7
cells.
Chen et al (from Hongchen Gu group) have fol-
lowed their prior work and prepared magnetic MSOs
possessing VEGF siRNA inside their mesopores (via
dehydration process) and covered them with PEI
and maleimide-PEG-N-hydroxysuccinimide (NHS),
which helped in further conjugation of a fusogenic
peptide (KALA)—as shown in figure 3[127]. Herein,
30 mg of VEGF siRNA per gram of the MSOs is used
in in vitro studies, where high transfection and VEGF
knockdown efficacies are obtained in lung cancer
(A549) cells, while maintaining less cytotoxicity
towards human normal hepatic (L02) cells. Moreover,
the combined MRI and fluorescence imaging mod-
alities have confirmed the in vivo tumor accumula-
tion of the magnetic MSOs with siRNA. Besides, the
in vivo studies have revealed that these VEGF siRNA-
attached MSOs have down-regulated up-to 70% of
VEGF proteins and decreased the tumor volume in
VEGF-overexpressed A549 cells-based tumor mice
models (as shown in figure 3), where the metastasis
is completely inhibited. In a similar investigation, the
same authors utilized the same above nanoformula-
tion with VEGF siRNA molecules and have repressed
the human ovarian adenocarcinoma (SKOV3) cells-
based orthotropic mice models [128]. MRI stud-
ies have displayed the accumulation of these MSOs
around the tumor peripheral regions after 24 h of
intravenous administration. Besides, the growth of
the ovarian tumors in mice has been significantly
delayed by obstructing the angiogenesis process, in
comparison to the control mice groups. Analogously,
Li et al (from Yi Yao Liu group) have formed a layer
of silica (SiO2) on the 10 nm sized iron oxide nano-
particles and then coated their surface with polyether-
imide polymer, which has been helpful in the attach-
ment of VEGF shRNA (at 30:1 ratio) to finally form
a nanocomplex [129]. This nanocomplex has been
highly up-taken by the breast cancer (MCF-7) cells
(confirmed via in vitro MRI), and resulted in better
gene knockdown efficacy.
3.2.5. Other siRNAs
Recently, Sun et al have initially prepared PEI
(1.8 kDa)-attached SPIOs and then covered them
with the cationic liposomes made of dioleoyl-
3-trimethylammoniumpropane, 1,2-dioleoyl-sn-
glycero3-phosphoethanolamine and cholesterol (at
a ratio of 2:1:1) [130]. The siRNA-Luc molecules and
enhanced green fluorescent protein (EGFP) siRNA
molecules are separately attached, which silenced the
respective reporter genes in lung cancer (Luc-SPC-
A1 and EGFP-SPC-A1) cells having luciferase and
green fluorescence protein expressions. Moreover,
the SPIOs exhibited good MRI contrast effects. In
another study, the surface of the SPIOs is modi-
fied with heptafluorobutyryl-PEG-PEI molecules
and complexed with siRNA specific to CXCR4 (C-
X-C chemokine receptor 4) [131]. Usually, CXCR4 is
overexpressed in many cancers (especially breast can-
cers) as it involves in promoting metastasis, forming
new tumor blood vessels and developing resistance to
chemotherapeutic drugs. The fluorinated nanocom-
plexes (with 0.75 µg of nanoparticles and 40 pmol of
CXCR4 siRNA) have achieved high transfection effic-
acy, under the influence of magnetic field, in breast
cancer (4T1) cells, and better knockdown of CXCR4
in these cancer cells.
9

Biomed. Mater. 19 (2024) 022001 G Kandasamy and D Maity
Figure 3. (i) A schematic representation of sequential conjugation of vascular endothelial growth factor (VEGF) siRNA,
polyethylenimine (PEI) and fusogenic peptide KALA (via polyethylene glycol (PEG)) onto the as-synthesized magnetic
mesoporous silica nanoparticles (M-MSN) based nanocarriers (NC), and (ii) comparison of controls with the in vivo intravenous
(i.v.) injection (A) of siRNA/KALA conjugated M-MSN based NCs and their corresponding effects on tumor volume (B) and (C)
and the intratumor VEGF protein content (D). Reprinted from [127], Copyright (2014), with permission from Elsevier.
In a similar study, Panday et al have modified
the surface of SPIOs with PEI-PEG polymer and then
complexed with the siRNA molecules that are specific
to the trans-membrane proteins—a disintegrin and
metalloproteinase 10 [132]. Generally, these pro-
teins can translocate from cellular membrane to the
inside nucleus, and can enhance tumor formation,
progression and migration. This nanocomplex has
10

Biomed. Mater. 19 (2024) 022001 G Kandasamy and D Maity
Figure 4. (a) A scheme representation of (a) preparation of Fe3O4(iron oxide) nanoparticles loaded polyethylenimine
(PEI)-polyethylene glycol (PEG) based nanogels (NGs) in conjugation with transforming growth factor-β1 (TGF-β1) siRNA
molecules, and (b) the usage of as-prepared NGs in in vivo gene therapy and MRI. Reprinted with permission from [135].
Copyright (2021) American Chemical Society.
induced cytotoxicity (up-to 74%) in the prostate
cancer (PC3) cells. Analogously, Wang et al have
delivered the repressor element 1-silencing transcrip-
tion factor (REST) siRNA molecules by employing
the PEI-coated SPIOs based nanocarriers [133]. REST
is responsible for the replication and migration of
the glioblastomas. The glioblastoma (U-87 and U-
251) cells have effectively up-taken the nanocarri-
ers along with the siRNA molecules, which caused
good REST silencing (confirmed via western blot-
ting method), cytotoxicity (CCK-8 assay) and anti-
migration (transwell assay) effects. In a recent invest-
igation, SPIOs are coated with galactose to target asia-
loglycoprotein receptor, and PEI for attaching siRNA
against c-Met that is responsible for the growth of
hepatocytes [134]. Generally, c-Met is over-expressed
in hepatocellular carcinoma and help them prolifer-
ate, form tumor blood vessels and etc. In this study,
the c-Met expression is efficiently down-regulated
by the siRNA molecules after their delivery inside
the in vitro (Hepa1–6 cells) and in vivo (ortho-
tropic luc-Hepa1–6 based hepatic tumor) envir-
onments through nanocarriers, while reducing the
tumor growth in mice models. For gene therapy
using transforming growth factor-β1 siRNA, Peng
et al have used a reverse microemulsion method and
prepared PEI (800 Da)—PEG (400 Da)-diacrylate
based nanogels while simultaneously encapsulating
the ultra-small SPIOs (as shown in figure 4) [135].
These nanogels are then loaded with the transforming
growth factor-β1 siRNA molecules (refer figure 4) at
N:P ratio of 60:1. Epithelial-to-mesenchymal trans-
ition in the tumor cells and their invasion/migra-
tion are mainly regulated by the transforming growth
factor-β1, which has been down-regulated in in vitro
up-to ∼51% by the siRNA loaded nanogels, with 2.5
times reduction in cell migration. Besides, the ultra-
small SPIOs in nanogels have provided T1 bright MRI
signals (refer figure 4) about the in vivo subcutaneous
sarcoma; whereas the nanogels have attained ∼41%
apoptosis rate in sarcoma tumors while maintaining
the bio-safety.
In a recent study, the authors have altered the
surface of the SPIOs with PEI polymer possessing
different alkylation to check their binding capacity
with the siRNA molecules [136]. The alkyl chains
that have been used are hexacyl (6 chains), dodecyl
(12 chains) and octadecyl (18 chains), which have
possessed different thermal properties and critical
micelle concentration values. Among above chains,
dodecyl (12 chains) and octadecyl (18 chains) based
PEI polymer have formed stable self-assembling
micelles, while encapsulating the SPIOs; that dis-
played good relaxation signals in MRI. Besides, the
magnetic nanocarriers with higher number of alkyl
chains (i.e. 18 chains) have effectively bound with
11

Biomed. Mater. 19 (2024) 022001 G Kandasamy and D Maity
the P-gp siRNA and also transfected better in the
drug-resistant breast cancer (MCF-7/ADR) cells at
a lower concentration of 50 nM. This enhancement
in transfection could have been due to improved
interactions between the long alkyl chains with the
membrane of cancer cells, which resulted in better
in vitro cancer MRI imaging also. Moreover, the local
delivery of the siRNA attached nanocarrier to the
in vivo cancer site has resulted in 62%–71% reduc-
tion in the P-gp protein levels. Similarly, You et al
have developed hybrid nanocarriers made of SPIOs
that are particularly coated with the polymer combin-
ations such as PEI25000 modified with 14 alkyl chain
group (i.e. 1,2-epoxytetradecane (C-14)) and PEG-
poly (caprolactone) [137]. For this work, the authors
have initially examined the expression of different
circular RNAs in hepatocellular carcinoma (SMMC-
7721 cells) through transcriptome analysis, where
they found a total of 173 circular RNAs, with 118 cir-
cular RNAs as down-regulated and 55 circular RNAs
as up-regulated. Herein, to treat, they have chosen
one of the significant up-regulated circular RNAs—
i.e. circ_0058051 that has negative correlation with
the prognosis of the carcinoma. For treatment,
they devised 3 different siRNAs and among them
circ_0058051-3 siRNA with the following sequence—
sense: CUCCAGAAAUGCUGUUACUAATT and
antisense: UUAGUAACAGCAUUUCUGGAGTT has
shown high inhibition of carcinoma cells. Hence,
they complexed this circ_0058051 siRNA with nano-
carriers at various mass ratios; wherein 1:80 ratio
of siRNA to nanocarrier has been found to be suit-
able as these siRNAs have completely complexed on
the surface of the nanocarriers. This nanocomplex
has inhibited the malignancy and also the growth of
SMMC-7721 based hepatocellular carcinoma cells
in in vitro (under magnetic targeting) and in vivo
conditions. In another study, through magnetic tar-
geting, the authors have effectively delivered the
siRNA specific to lifeguard-based anti-apoptotic
protein [138]. Herein, the magnetic (FexOy) nano-
particles are enclosed by niosomes made of span60,
1,2-distearoyl-sn-glycero-3-phosphoethanolamine-
N-[maleimide(PEG)-2000] maleimide, and choles-
terol, while simultaneously complexing with the life-
guard siRNAs that are priorly attached to the prot-
amine molecules. This niosomes-based nanocom-
plex has highly down-regulated the lifeguard pro-
tein in HER2 +ve breast cancer (BT-474) cells and
induced cytotoxicity also. Similarly, Cristofolini et al
initially surface modified the SPIOs with caffeic acid
for preventing the agglomeration [139]. Then, these
SPIOs are covered with calcium phosphate and poly-
mer PEG5K-poly(aspartate)50 , while simultaneously
complexing with the HER2 siRNA (sense:
5′-TCCGTTTCCTGCAGCAGTCTCCGCA-3′and
anti-sense: 5′-AGAGAGCCAGCCCTCTGACGTCCAT-
3′). Herein, calcium phosphate can aid in improving
endocytosis and also endosomal escape, whereas the
polymer prevents the agglomeration during calcium
phosphate surface-layer formation. Under magnetic
field, the calcium-based nanocomplexes have effi-
ciently transfected the HER2 +ve breast cancer
(HCC1954) cells and induced gene silencing effects
up-to 38% in these cells.
Ben Djemaa et al have treated triple negative
(including HER2-ve) breast cancer (MDA-MB-231)
cells by using the SPIOs-based nanocarriers that
are decorated with a cell-penetrating-peptide (i.e.
gH625) and covered with cationic polymers such
as chitosan and poly-L-arginine (for proton spon-
ging effects and escape from the endosomes) [140].
At mass ratio of 10, anti-green fluorescence protein
siRNA is complexed with the SPIOs through poly-
L-arginine, while the chitosan did not have any dis-
turbing effect on the complexation. The siRNA has
inhibited the corresponding protein in the MDA-
MB-231/GFP cells, analyzed through transfection
studies. In a similar investigation, Na et al have
modified the surface-chemistry on the SPIOs with
3 different dopamine-conjugated molecules, which
are tertiary amine, methoxy PEG and sulfonic acid
[141]. The positive charged dopamine-tertiary amine
surface-attached SPIOs have helped in the deliv-
ery of negative charged nucleic acid—i.e. NOTCH1
siRNA; this resulted in 75% down-regulation of
corresponding mRNA expression in T-cell leuk-
emia (Jurkat) cells. Similarly, the lipids and PEG-
coated SPIOs are utilized in the delivery of fluores-
cein amidites (FAM)-siRNA by forming a nanocom-
plex, where the ratios (0, 15 and 50 mol%) of the
lipid (1,2-dioleoyl-3-trimethylammonium propane)
to the PEG-lipid (i.e. 1,2-dimyristoyl-sn-glycero-
3-phosphoethanolamine-N-[methoxy(polyethylene
glycol)-2000] (ammonium salt)) has been investig-
ated as the influencing factor in MRI r2 relaxivity val-
ues, gene knockdown and cytotoxicity [142]. Herein,
the r2 relaxivity has incremented to 61.4 mM Fe−1s−1
till 50 mol% of surface lipid: PEG-lipid ratio, which
has been better than the commercial r2 based MRI
contrast agents. However, the binding capacity of the
FAM-siRNA has been noted lower at high surface
lipid: PEG lipid ratio (i.e. 50 mol%) compared to the
0 and 15 mol%; which could be due to a major decline
in non-specific adsorption of siRNA under the pres-
ence of more amount of PEG molecules. Besides,
∼60% cytotoxicity and 80% suppression of genes in
HeLa cancer cells are observed for the 0 mol% of lipid:
PEG-lipid ratio based nanocomplexes, and these per-
centages have modified to lower for the increasing
mol% of surface lipid: PEG-lipid ratios (i.e. 15 and
50).
In an investigation, Xing et al have formed man-
ganese oxide nanoparticles and then converted them
into nanoclusters by using an alkyl-modified poly-
mer, PEI having a low molecular weight of 2KDa [60].
12

Biomed. Mater. 19 (2024) 022001 G Kandasamy and D Maity
By utilizing, the surface present positive charged PEI,
firefly luciferase siRNA is complexed with the nano-
particles at different N:P ratios, confirmed through
agarose gel electrophoresis method. The firm bind-
ing of the siRNA with the nanoparticles has been
confirmed by using heparin molecules via a decom-
plexation assay. At N:P ratio of 10, the luciferase
siRNA-attached nanocomplex has greatly decremen-
ted the corresponding expression in 4T1-luciferase
cells. Recently, Zuo et al have efficiently delivered
the cell death siRNA by using manganese-aluminum
based layered-double-hydroxide nanoparticles inside
N2a cells (resulting in high cell death), while attain-
ing pH-responsive in vitro MRI [143]. In a similar
study, the manganese-doped magnetic nanoparticles
are prepared by Zhang et al [144]. These nan-
oparticles along with NIR-responsive indocyanine
green are enclosed inside a polymeric vehicle made
of poly vinyl alcohol and PEI- poly(lactic-co-glycolic
acid) and then the yes-associated protein 1 (YAP1)
siRNA molecules are conjugated via PEI. YAP1 pro-
tein in exogenous scenario has been mainly associ-
ated with the increment of malignancy in immor-
tal hepatocytes, drug-resistance, and progression of
hepatocellular cancers. So, the authors have focused
and developed this specific YAP1-siRNA nanocom-
plex, which demonstrated a considerable inhibi-
tion of YAP1 protein in hepatocellular carcinoma
(HepG2) cells, confirmed via real-time quantitat-
ive PCR. Herein, it has also been found, through
the western blot analysis, that the nanocomplex has
down-regulated the expression of β-catenin that is
also involved in the HepG2 progression. Besides this,
808 nm laser-based heat induced photothermal ther-
apy has resulted in ∼75% of in vitro cancer cell killing
at 30 µg ml−1concentration. Under the in vivo con-
ditions, the nanocomplex has accumulated higher
inside the HepG2 tumor regions which is confirmed
via dual imaging i.e. near infrared and MRI, and
also significantly suppressed the growth of HepG2
tumors in the mice models via siRNA therapy and
low-temperature photothermal therapy.
In melanoma, signal transducer and activator of
transcription 3 (STAT3) protein is overexpressed,
thus causing an increase in the proliferation/survival,
angiogenesis, metastasis, and also anti-apoptosis in
melanoma cells/melanocytes; and this protein can be
suppressed to improve melanoma treatment. Labala
et al have focused their study to deliver STAT3 siRNA
using gold–based nanocarriers [145]. For this pur-
pose, they have formed chitosan coated gold nan-
oparticles that are further stabilized by covering
them with a layer of poly(vinylpyrrolidone) poly-
mer. Lastly, the STAT3 siRNA molecules are com-
plexed with the nanoparticles at different weight
ratios—i.e. 5:1 and 10:1, which correspondingly led
to the encapsulation with efficacies of ∼21.6% and
∼58%. The up-take of nanoparticles by melanoma
cells has been confirmed via fluorescence micro-
scopy, after labelling the surface chitosan with the
fluorescein isothoicyanate molecules. The 0.25 and
0.5 nM STAT3 siRNA concentrations on the nano-
particles have respectively decremented the expres-
sion of STAT3 by ∼45% and ∼47% in melanoma
(B16-F10) cells, while improving apoptotic events.
The nanoparticles have effectively penetrated the por-
cine ear skin up-to a depth of 70 µm via ionto-
phoresis, which is comparatively better than 30 µm
penetration through passive application.
Thus, the major siRNAs are transported through
different metal and magnetic nanocarriers with dif-
ferent surface coatings for better cancer treatments.
The above-discussed recent developments in the
siRNA nanocomplexes are consolidated in table 1.
3.3. Co-delivery of siRNA with chemotherapeutic
drugs
3.3.1. Co-delivery with doxorubicin
Since 1960, doxorubicin, an anthracycline-based
chemotherapeutic molecule and mainly derived from
Streptomyces peucetius bacteria, has been used in
cancer treatments individually and in combination
including siRNA molecules.
Glioblastoma stem cells are a subpart of glio-
blastoma cells, and responsible for recurrence and
resistance to treatments. Wang et al have focused
to treat glioblastoma stem cells using doxorubi-
cin in combination with siRNA molecules [146].
Herein, the SPIOs (γ-Fe2O3) are sequentially coated
with carboxymethyl chitosan and PEI—which have
provided higher affinity towards the attachment
of anti-proliferative survivin siRNA and heparin-
conjugated targeting agent (epidermal growth factor)
that further have formed the nanocomplexes (refer
figure 5). Epidermal growth factor is mainly attached
for easy targeting of the epidermal growth factor
receptors that are overexpressed in glioblastoma stem
cells and glioblastoma cells. It has been noted that
negative-charged heparin have mainly influenced
the attachment of siRNA, where even a ratio of
1/50 heparin did not affect the binding of siRNA.
Finally, the as-formed targeted nanocomplexes are
loaded with doxorubicin molecules (refer figure 5).
The in vitro and in vivo tumor proliferation stud-
ies based on glioblastoma U251 cells have revealed
that the synergy between the RNAi therapy via siRNA
(i.e. the attachment of siRNA with RNA-induced
silencing complex and further cleavage of survivin
mRNA—as shown in figure 5) and chemotherapy
via doxorubicin has resulted in enhanced apop-
tosis rate (∼37%) and also decreased tumor size.
Like their previous work, recently Eljack et al (from
Stephanie David group) have complexed survivin
siRNA with SPIOs (that are surface-modified with
13

Biomed. Mater. 19 (2024) 022001 G Kandasamy and D Maity
Table 1. Non-targeted and targeted inorganic nanocarriers used in delivery of major siRNAs for cancer treatments.
Type of nanocarriers
Additional nanocarrier coating +any
therapeutic molecule Targeting agent Treated cancer
Knockdown efficiency without/with cytotox-
icity +Image/physical guidance Reference
Programmed death ligand-1 (PD-L1) siRNA
SPIOs PEG-disulfide-PEI Folic Acid SGC-7901 Gastric cancer cells 90.9% knockdown [109]
MnO2 Calcium carbonate +indocyanine green
for PDT
— Lewis lung cancer cells 73% knockdown with 91% cytotoxicity+T1 MRI [110]
Gold nanoprisms Poly (sodium 4-styrenesulfonate) and
poly (-diallyldimethylammonium
chloride)
— HCC827 Lung cancer Higher PD-L1 knockdown [111]
Survivin siRNA
SPIOs Amylose Folic Acid Hepatocellular carcinoma (HepG2) Better transfection with good down regulation [112]
Tetraphenylethylene and quaternary
ammonium cationized amylose
SP94 Peptide Hepatocellular carcinoma (Huh-7) High suppression with 80% cytotoxicity +T2 MRI
(53.0 mM-1s-1) plus aggregation induced emission
based fluorescence
[113]
PEI — Oral cancer (Ca9-22) cells Better miRNA level reduction under magnetic field
for 30 min
[114]
PEG-lactide, chitosan and PEI — Leukemia cancer (K562) cells Lowered protein levels [115]
Breast carcinoma (MCF-7) Better transfection with enhanced apoptosis [116]
PEI — Glioblastoma multiforme (U251) Cytotoxicity up-to 40% [117]
PEG and silane anti-HER2 single chain
antibody fragments
Human epidermal growth factor
receptor-2 (HER2) overexpressing
breast cancer (e.g., BT-474) and
HER2-negative breast cancer
(MDA-MB-231) cells
90% and 70% protein down-regulation in BT-474
and MDA-MB-231 cells respectively
[118]
Bovine serum albumin Cetuximab Epidermal growth factor receptors
overexpressed lung cancer (GLC-82)
cells
High transfection efficacy and ∼66% cytotoxicity on
exposure to external magnetic field
[119]
Gold nano-flowers Bovine serum albumin — Human pancreatic adenocarcinoma
(BXPC-3) cells
Better silencing effects +Fluorescein
amidites-guidance and computed tomography
[120]
(Continued.)
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Biomed. Mater. 19 (2024) 022001 G Kandasamy and D Maity
Table 1. (Continued.)
B-cell lymphoma 2 (Bcl-2) siRNA
SPIOs PEG-grafted-PEI polymer GD2 single-chain
antibody
Neuroblastoma (SK-N-SH) cells ∼46% suppression +T2 MRI [121]
SPIOs Branched PEI polymer Interleukin-4 receptor
binding peptide
Breast cancer (MDA-MB-231) cells Better gene silencing +NIR fluorescence imaging
(via Cy5.5 dye attached to siRNA) and T2 MRI
[122]
Gold nanorods Thiol-modified bovine serum albumin
and NH2-PEG-biotin
anti-erythroblastic
oncogene B (ErbB)-2
antibodies
ErbB-2 overexpressing breast cancer
(SK-BR-3) cells
Minimized oncogenic corresponding mRNA and
protein levels and increase cytotoxicity via
photothermal therapy at 47 ◦C using NIR at 808 nm
wavelength
[123]
MSOs with Fe3O4
magnetic core
PEI Adenocarcinomic human alveolar
basal epithelial (A549) cells
80% knockdown [89]
Vascular endothelial growth factor (VEGF) siRNA
SPIOs calcium phosphate and
PEG-poly(aspartic acid) copolymer
— Breast cancer (MDA- MB 231) cells 60% suppression [124]
SPIOs Calcium phosphate and methoxy-PEG
-block-poly(L-glutamic acid)
— Breast cancer (MDA- MB 231) cells 67% suppression [125]
Hollow MnO2
nanoparticles
3,4-dihydroxy-L-phenylalanine modified
PEI
Herceptin HER2-overexpressing breast cancer
(SK-BR-3) cells
Good suppression +T1 MRI [126]
MSOs with Fe3O4
magnetic core
PEI and maleimide-PEG-
N-hydroxysuccinimide
Fusogenic peptide
(KALA)
Lung cancer (A549) cells High transfection and 70% knockdown +MRI and
fluorescence
[127]
Human ovarian adenocarcinoma
(SKOV3)
Better obstruction of angiogenesis [128]
Silica coated iron oxide
nanoparticles
Polyetherimide polymer — Breast cancer (MCF-7) cells Higher gene knockdown +MRI [129]
15

Biomed. Mater. 19 (2024) 022001 G Kandasamy and D Maity
Figure 5. (A) A scheme representation of the preparation of a nanocomplex possessing Fe2O3(iron oxide) nanoparticles (MNNS)
that are coated with carboxymethyl chitosan (CMCS), polyethylenimine (PEI), then conjugated with heparin-mediated epidermal
growth factor (EGF) and finally loaded with survivin (Sur) siRNA and doxorubicin (DOX) molecules, and (B) the targeted
delivery of the nanocomplex in a mice model having brain glioblastoma stem cells (GSCs) and further treatment via gene therapy
using survivin siRNA. Reproduced from [146] with permission from the Royal Society of Chemistry.
silane, PEG5000 anti-HER2 single chain antibody
fragments, polymer (poly-L-arginine) and chitosan)
and encapsulated the drug, doxorubicin [147]. The
siRNA and drug attached nanocomplexes have down-
regulated 50% of survivin protein (relatively) and
killed HER2-positive breast cancer (SK-BR-3) cells
effectively.
Rajendrakumar et al have attempted to impart
stimuli-responsive multi-modal therapies includ-
ing siRNA based nucleic therapy and chemothera-
peutic therapy by using multifunctional inorganic
nanocarriers [148]. In this work, Bcl-2 shRNA is
made in the form of plasmid and this is inter-
calated with a chemotherapeutic drug, doxoru-
bicin. Simultaneously and separately grape-seed
proanthocyanidin coated—iron oxide (Fe3O4)
nanoparticles (for T2 MRI) and also manganese
(Mn3O4) oxide nanoparticles (for T1 MRI) are
prepared via green synthesis process, and then
combinedly attached to the disulfide-cross-linked
PEI polymer. Then, the plasmid containing shRNA
and doxorubicin is conjugated to the PEI poly-
mer. Herein, the disulfide bonds are mainly added
for its reduction-responsiveness on interaction
with glutathione in the cancer cells, and to fur-
ther effectively release the nanoparticles and nuc-
leic acids inside the cells. Later, the entire surface
of the nanocarrier is covered with anionic hyalur-
onic acid molecules for selective uptake by CD44
receptors via N-terminus binding. This multifunc-
tional nanocarrier has been effectively internalized by
CD44 receptors-overexpressing multidrug-resistant
breast cancer (MCF7/ADR) cells, and consequently
released plasmid and nanoparticles on the degrad-
ation of hyaluronic acid by hyaluronidase. Besides
this, the transfection studies show that the nanocar-
rier is highly accumulated inside the 4T1 cells with
high CD44 receptors in comparison to CT26 cells
16

Biomed. Mater. 19 (2024) 022001 G Kandasamy and D Maity
with low CD44 receptors. Moreover, glutathione-
mediated redox-responsive release of shRNA and
doxorubicin has resulted in high Bcl-2 gene silen-
cing and cytotoxicity effects, while the nanoparticles
are proven to be efficient T1 and T2 MRI con-
trast agents in MCF7/ADR cells with respective
r1 =1.566 mM−1s−1and r2 =52.2 mM−1s−1.
In a similar work, redox and pH responsive targeted
nanocarriers are specifically designed and made to
deliver the Bcl-2 siRNA molecules [149]. In this
work, the following are done in a step-wise manner:
(a) initially, MSOs are formed with cleavable thiol
surface groups, which have been further attached
with carboxylic acid functional groups by using reac-
tions with 2,2′-dipyridyl disulfide and then with
3-mercaptopropionic acid; (b) then the preformed
copolymer having PEI and poly lysine molecules and
folate-conjugated PEG molecules are sequentially
linked with the silica nanoparticles using 1-ethyl-3-
(3-dimethylaminopropyl)carbodiimide (EDC)/NHS
chemistry; (c) later, doxorubicin drug molecules are
encapsulated inside the nanoparticles with 8% load-
ing content and ∼49% loading efficiency; and (iv)
finally the Bcl-2 siRNA molecules are complexed with
the nanoparticles through electrostatic interaction
with PEI polymer. On incubation with the aggressive
breast cancer (MDA-MB-231) cells, the encapsulated
drug and siRNA molecules are co-released from the
nanoparticles and entered into the nucleus through
pH and glutathione triggering inside the cancer cells.
The western blot analysis has displayed a ∼59% Bcl-2
gene silencing effects in MDA-MB-231 cells at 10:1
weight ratio of nanoparticles to siRNA. The cytotox-
icity and apoptosis assay results have revealed very low
cancer cell viability with high apoptotic rate for the
nanoparticles that co-delivered siRNA and doxorubi-
cin (with a concentration of 4 µg ml−1). Analogously,
Chen et al have initially formed the MSOs loaded with
doxorubicin molecules and then covered them with
2nd generation polyamidoamine dendrimer for fur-
ther complexation of Bcl-2 siRNA molecules [150].
This nanocomplex has achieved ∼80% suppression
of Bcl-2 expression and high cellular toxicity (∼132
fold increase compared to free doxorubicin) in multi-
drug resistant human ovarian (A2780/AD) cancer
cells.
In a similar fashion, Li et al (from the Yi Yao Liu
group) have formed MSOs (with 3.4 nm diameter
sized pore having volume of ∼0.5 cm3g−1) encap-
sulating a magnetic core via sol-gel process (alike
their previous work) [151]. Then, the drug, doxoru-
bicin has been loaded inside the pores of silica nan-
oparticles (after incubating them at specific weight
ratios), where a ∼12% drug loading efficiency has
been observed for 0.4:1 weight ratio. After that, the
surface of the silica nanoparticles has been covered
with folic acid conjugated PEI polymer, which helped
in effective condensation of VEGF shRNA molecules
at 30:1 nanoparticle-to-shRNA ratio. In drug release
study, the amine molecules in the surface PEI poly-
mer are protonated under acidic pH, thereby creat-
ing repulsion among the surface groups and result-
ing in PEI layer dissociation to further release the
doxorubicin from the pores of silica nanoparticles.
The presence of FA has resulted in enhanced uptake
of the nanoparticles by the HeLa cells and this
uptake has been further boosted by applying an
external magnetic field. These nanoparticles have
revealed a ∼90% down-regulation of correspond-
ing VEGF genes in HeLa cancer cells, while inhib-
iting the cancer cell migration rate (by ∼64%) and
the microtubule formation responsible for angiogen-
esis in human umbilical vein endothelial cells. Yang
et al have developed 10 nm-sized citrate-capped gold
based nano-cage vehicles [152]. Through thiol group
bonding, the surface of these nanovehicles has been
modified with anti-VEGF siRNA and single-strand
DNA-2 which then hybridized with complement-
ary strand DNA-1 containing NH2groups that are
used in further alterations with poly (acrylic acid).
Later, the poly (acrylic acid) layer has been con-
jugated with aptamer- AS1411 for targeting nucle-
olin in cancer cells, and oligonucleotides (single-
strand DNA-3 and single-strand DNA-4) connec-
ted to matrix metalloproteinase-2 cleavable pep-
tides. Finally, the nano-cage vehicles are loaded with
the chemotherapeutic doxorubicin molecules, sta-
bilized via guanine-cytosine pair based intercala-
tion among the double-stranded DNA. The in vitro
studies have revealed an 80% gene silencing effic-
acy after the internal uptake by the lung cancer
(NCI-H889) cells and a ∼69% decrease in the apop-
tosis rate. Moreover, in the in vivo studies, a 12-
fold increase in the lung tumor tissue accumula-
tion has been observed for AS1411-targeted nano-
cage vehicles in orthotopic mouse model express-
ing NCI-H889 lung cancer, after inhalation based
local-delivery. Besides, the expression of VEGF has
been reduced to 70% in tumor tissues present in
lungs, and the tumor cells are effectively killed by
doxorubicin, thereby decreasing the tumor volume
and increasing the survival rate of the tumor-bearing
mice. In another similar study, the direct and indirect
suppressions of VEGF along with hypoxia inducible
factor-1a (induced by the hypoxic condition inside
the tumor microenvironment) have been studied
using manganese oxide based nanocarriers carrying
the corresponding siRNA [153]. In this work, initially
manganese oxide nanoparticles are prepared using a
reaction inside the bovine serum albumin to form
nanocarriers. The nanocarriers are then attached
with specific amount of siRNA (i.e. 1:36 siRNA-to-
nanocarrier ratio after optimization using gel retard-
ation assay) with encapsulation efficacy of 89%, and
17

Biomed. Mater. 19 (2024) 022001 G Kandasamy and D Maity
doxorubicin drug (having encapsulation efficacy and
loading rate of 94% and 19.4%, respectively) via
desolvation cross-linking process. Finally, folic acid
is conjugated to the nanocarriers using EDC/NHS
chemistry. Due to the presence of manganese oxide
nanoparticles, the nanocarriers have displayed good
T1 MRI characteristics with r1 =11.871 mM−1s−1
under 5 pH in 100 µM hydrogen peroxide (H2O2).
Besides in the release studies in mild acidic condi-
tions having H2O2, the release of siRNA and drug
has been enhanced to ∼83% and ∼81% respect-
ively based on the degradation of manganese oxide
to manganese ions. This aided in effective up-take
of nanocarriers by the drug-resistant breast cancer
(MCF-7/ADR) cells and in production of oxygen
inside the cancer cells, thus resulting in the decre-
ment of hypoxia inducible factor-1a while silen-
cing the VEGF expression. In in vivo experiments,
the 4T1-cancer induced mice models have shown a
reduction in tumor volume, hypoxia and metastasis
after being treated with doxorubicin/siRNA loaded
nanocarriers.
Peng et al have focused on treating the gastric
cancer by using drug/siRNA loaded thermo-sensitive
nanocarriers [154]. In this work, liposomes are ini-
tially made with 80:5:5:10 ratio of 1,2-dipalmitoyl-
sn-glycero-3-phosphocholine, 3b-[N-(N’,N’-
dimethylaminoethane)-carbamoyl]cholesterol,
dimethyldioctadecylammonium bromide and
cholesterol, and then encapsulated the iron oxide
nanoparticles. Later, the magnetic liposomes are
loaded with doxorubicin (with loading efficiency of
∼73%) and the shRNA against special AT-rich bind-
ing protein (SATB1), which is a chromatin organizer
responsible for maintaining the gene expressions,
and also plays an important role in cancer malig-
nancy. Under the exposure to the magnetic field,
the magnetic liposomes have attained significantly
higher transfection efficacy (i.e. 34%), better cellular
up-take and low cell viability (∼22.3%) as compared
to other controls in the gastric cancer (MNK-28)
cells, through the delivery of doxorubicin and the
SATB1 shRNA. In in vivo studies, the tumor volume
also has been reduced in MNK-28 cancer induced
mice models. In another recent investigation, unique
magnetic nanocarriers are formed by sequentially
encapsulating the iron oxide nanoparticles with a
combination of PLGA polymer, chitosan and the
cell membranes derived from the MCF-7 cancer
cells to deliver the myeloid cell leukemia-1 (Mcl-1)
siRNA (against the breast cancer), and doxorubicin
together [155]. Priorly, iron oxide nanoparticles are
loaded/attached with a photothermal agent, indocy-
anine green (ICG), with loading and encapsulation
efficacies of 14.4% and ∼72%. Here, FDA-approved
PLGA polymer, chitosan and the cell membranes
are utilized due to their characteristics including
biocompatibility, biodegradability, better immune
system evasion, enhanced protection against the drug
leakage in the blood-stream, improved blood cir-
culation and homotypic targeting. The presence of
chitosan along with PLGA has improved the encap-
sulation efficiency of iron oxide nanoparticles, Mcl-1
siRNA and doxorubicin; while the entire surface cov-
ering has enhanced the stability against the serum
proteins for the siRNA that has further effectively
incremented the knock down the corresponding pro-
teins. The application of magnetic field has caused
better internalization of the magnetic nanocarriers
inside the drug-resistant breast cancer (MCF-7/ADR)
cells; which further resulted in increased cytotoxicity
(up-to 75%) and apoptotic rate, while irradiating
with 808 nm laser also. In vivo magnetic targeted
therapeutic study results have revealed ∼79% inhibi-
tion of tumor volume with 90% survival rate compar-
ing to the controls, on treatment with the siRNA, and
synergistic chemotherapy and photothermal therapy.
3.3.2. Co-delivery with other chemotherapeutic drugs
Babaei et al have focused on the concurrent delivery
of survivin shRNA and camptothecin drug molecules
[156]. For this purpose, amino-modified mesopor-
ous silica based nanorods have been synthesized
with 3.56 nm sized pores that have accommodated
camptothecin molecules at an encapsulation effi-
ciency of 32%. Then, the surface of the nanorods is
modified with HOOC-PEG3500 -maleimide molecules
(via EDC/NHS chemistry) which facilitated conjug-
ation of targeting agent, AS1411 aptamer and the
condensation of survivin shRNA molecules (with
a mass ratio of 6). The aptamer targeted nanorods
have displayed a relatively improved (i) transfection
efficacy and (ii) apoptotic rate (∼35% higher) with
low viability for mouse colon carcinoma (C26) cells.
Under in vivo scenario in C26 tumor-bearing mice,
the aptamer-based active targeting has enhanced
the tumor penetrating capability of the nanocom-
plex, while camptothecin and shRNA molecules
have significantly reduced the progression of the
tumor growth. In another recent investigation, the
MSOs are loaded with: (i) small, chemo-preventive
and anti-tumor molecules named myricetin hav-
ing low aqueous solubility (at a loading efficiency of
∼37%)—already pre-combined with the multidrug
resistance protein (MRP-1) siRNA, in their nano-
pores and (ii) targeting folic acid molecules (refer
figure 6(i)) [157]. The proliferation of the non-small
cell lung cancer in in vitro and in vivo conditions has
been reduced after exposing them to the myricet-
in/ MRP-1 siRNA loaded nanoparticles; wherein
the tumor volume has decremented by 4-folds as
compared to the control in A549 tumor-bearing
mice models (refer figure 6(ii)). Similarly in another
18

Biomed. Mater. 19 (2024) 022001 G Kandasamy and D Maity
Figure 6. (i) A scheme representation of the preparation of folic-acid conjugated myricetin (Myr) and multidrug resistance
protein (MRP-1) loaded mesoporous silica nanoparticles based nanocomplex, and (ii) in vivo anti-cancer efficacy after treating
with the nanocomplex (in comparison with controls)—(A) tumor tissue images, (B) mice tumor volume and (C) tumor weight
of mice. Adapted from [157]. © 2019 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND
license (http://creativecommons.org/licenses/BY-NC-ND/4.0/). Reproduced with permission from [157]. © 2019 Published by
Elsevier Masson SAS.
study on lung cancer, 3-mercaptopropyl/pyridyl-
thiol surface-modified MSOs are utilized for multi-
functional purposes: to encapsulate and deliver the
chemotherapeutic drugs—i.e. cisplatin or doxor-
ubicin through their pores, in combination with
thiolated-siRNAs that work against MRP-1 and Bcl-2
genes [158]. Besides above, the nanoparticles are dec-
orated with a targeting peptide, thiolated luteinizing
hormone-releasing hormone receptor based peptide
conjugated with PEG molecules through pyridyl-
thiol surface groups. It has been found that the pore
encapsulation efficacy of the nanoparticles is com-
paratively higher for of cisplatin (i.e. 30%) than dox-
orubicin (i.e. 8%). Besides, these nanoparticles have
effectively delivered the siRNAs in combination, and
have achieved 58% and 56% efficacies in knockdown
of MRP-1 and Bcl-2 genes respectively in non-small
cell lung cancer (A549) cells along with high cyto-
toxicity via the anti-cancer drug. Lee et al have pri-
orly formed an amphiphilic cationic block polymer,
poly(2-(dimethylamino)ethyl methacrylate)-block-
poly(ε-caprolactone) to form micelles; which have
combinedly encapsulated the ultra-small SPIOs and
a high cytotoxic drug, 7-ethyl-10- hydroxycamp-
tothecin (SN-38) [159]. Then, the anionic-natured
VEGF siRNA molecules (attached with carboxyl PEG
via an amine group located on the sense strand
(5′end)) have been complexed with the cationic
polymer. Among different polymer to VEGF siRNA
ratios (i.e. till 30), the siRNA has been completely
complexed with the polymer at a weight ratio of 8
and beyond, which is established via neutral charge
19

Biomed. Mater. 19 (2024) 022001 G Kandasamy and D Maity
based on zeta potential studies. The stability of the
nanocomplex has been examined through heparin
based decomplexation assay (as heparin can destabil-
ize the nanocomplexes), and no decomplexation
of siRNA has been found. VEGF-overexpressing
human colon adenocarcinoma (LS174T) cells have
been utilized for in vitro studies and their VEGF
expression has been significantly suppressed by the
nanocomplexes. After radio-labeling with 111In, it
has been found that the nanocomplexes are amassed
inside in vivo tumors within 4 h after administra-
tion and sustained for 24 h, which has been con-
firmed via MRI also. The synergy between the VEGF
siRNA and SN-38 drug molecules has reduced the
growth and volume of the LS174T tumors in mice
models.
Thus, the chemotherapeutic drugs are combined
with the siRNAs for improving the cancer treatments.
Moreover, table 2summarizes the delivery of siRNAs
in combination with chemotherapeutic drugs using
inorganic nanocarriers.
4. Clinical trials, current challenges and
future prospects
The first clinical trial based on siRNA
(NCT00154934) has started in 2004 focusing on the
treatment of pre-eclampsia, a hypertension based
critical disorder in pregnant women. Another clin-
ical study (NCT00363714) has also been started in
the same year for treating the age-related macular
degeneration via VEGF targeted siRNA. The num-
ber of clinical trial studies (as per ‘clinicaltrials.gov’)
has increased from 18 (during 2004–2009) to 76
(during 2010–2019) for treating different diseases
that include amyloidosis, acute hepatic porphyria,
primary hyperoxaluria, atherosclerotic cardiovas-
cular disease, hypercholesterolemia, acute coronary
syndrome, haemophilia and so on [105,160,161]. In
most of them, the delivery of siRNAs is aided by N-
acetyl-d-galactosamine based nanocarrier. Besides,
the number of clinical studies that have been started
during 2020–2023 is above 25, and the rapid initiation
within 3 years of this decade is mainly due to the food
and drug administration (FDA’s) recent approval
to the following siRNA complexes—(i) Patisiran
(ONPATTRO ™), Givosiran (GIVLAARI ™)
and vutrisiran (AMVUTTRA ™), (ii) Lumasiran
(OXLUMO ™) and (iii) Inclisiran (LEQVIO ™) for
treating genetic-neurological, renal and cardiovascu-
lar disorders, respectively [11,160].
Unlike others, cancer is one of the most com-
plex diseases. According to our search in https://
clinicaltrials.gov/ (a global database for all clinical
trials) using the words—cancer as condition/disease
and siRNA as intervention/treatment, 18 clinical trials
(from 2005—till 2023) are shown, where the status of
these studies are as follows: completed: 7, recruiting:
4, terminated: 3, active not recruiting: 2, withdrawn:
1 and unknown: 1. The first observational clinical
study has been started with treating chronic myel-
oid leukemia using siRNA loaded in SV-40 vector,
and then interventional studies on treating distinct
metastatic/recurrent cancers—melanoma, pancreatic
carcinoma, colorectal carcinoma, kidney cancer and
advanced solid tumors are performed. The results of
most of these studies are not submitted/published or
unknown. Besides, as per our further search in clinic-
altrials.gov, the following is observed: cancer clinical
trials that utilize inorganic (iron oxide/gold/silica)
nanoparticles exist; but the trials using inorganic
nanocarriers based siRNA delivery-cum-treatment
are not available.
Nevertheless, for the past two decades, a lot
of researches are done on the in vitro and/or
in vivo cancer therapeutics via RNAi technology
using siRNA as a bio-drug and inorganic nano-
particles as nanocarriers. But, their clinical trans-
lation from research lab to the patients is facing
many hindrances that include a lack of detailed
and accurate information about majority of the fol-
lowing: controlled/sustained release of the siRNAs
(without/with stimuli-responsiveness), response of
the immune system, safe and specific administration
routes, pharmaco-kinetics/-dynamics, biodistribu-
tion, biodegradability, short-/long-term side effects
and so on. However, excellent research works are on-
going and encouraging results are published. This
article highlights some of their specific recent devel-
opments. We believe that the rigorous works that
are done currently/going-to-be-done in near-future
by the scientific community on the inorganic nano-
particles based siRNA delivery-cum-treatments will
be able to obtain the required information (needed by
the regulation authorities) for their successful clinical
translation in future.
20

Biomed. Mater. 19 (2024) 022001 G Kandasamy and D Maity
Table 2. Delivery of siRNAs in combination with chemotherapeutic drugs using inorganic nanocarriers.
Drug siRNA type Type of nanocarrier +coating Targeting agent Treated cancer
Knockdown efficiency without/with
cytotoxicity +image/physical guidance Reference
Doxorubicin Survivin siRNA SPIOs +carboxymethyl chitosan
and PEI
Heparin-conjugated
epidermal growth factor
Glioblastoma stem cells and
glioblastoma cells
Enhanced apoptosis rate (∼37%) and
decreased tumor size
[146]
SPIOs +silane, PEG5000,
polymer (poly-L-arginine) and
chitosan
Anti-HER2 single chain
antibody fragments
HER2-positive breast cancer
(SK-BR-3) cells
50% down-regulation of survivin
protein (relatively) and effective cancer
cell killing
[147]
Bcl-2 shRNA Grape-seed proanthocyanidin
coated—iron oxide (Fe3O4)
nanoparticles and manganese
(Mn3O4) oxide nanoparticles
Hyaluronic acid to
target CD44 receptors
CD44
receptors-overexpressing
multidrug-resistant breast
cancer (MCF7/ADR), 4T1
breast cancer and CT26
murine colorectal cancer cell
High Bcl-2 gene silencing and
cytotoxicity effects via
glutathione-mediated redox-responsive
release +T1 and T2 MRI contrast
effects with respective
r1 =1.566 mM−1s−1and
r2 =52.2 mM−1s−1
[148]
Bcl-2 Thiol-modified MSOs attached
with 2,2′-dipyridyl disulfide,
3-mercaptopropionic acid and
covered with copolymer having
PEI-poly lysine and PEG
Folate Breast cancer
(MDA-MB-231) cells
∼59% gene silencing effects and high
apoptotic rate using pH and glutathione
triggering
[149]
MSOs +2nd generation
polyamidoamine dendrimer
— Multi-drug resistant human
ovarian (A2780/AD) cancer
cells
∼80% suppression and high cellular
toxicity (∼132 fold increase)
[150]
VEGF shRNA MSOs with magnetic core +PEI Folic acid HeLa cancer cells and human
umbilical vein endothelial
cells
∼90% gene down-regulation and
cancer cell migration rate inhibition by
∼64%
[151]
VEGF siRNA Citrate-capped gold
nano-cage +poly (acrylic acid)
Aptamer- AS1411 Lung cancer (NCI-H889)
cells
80% gene silencing efficacy and ∼69%
decrease in apoptosis rate in in vitro and
∼70% VEGF reduction in tumor tissues
[152]
MnO2 nanoparticles +bovine
serum albumin
Folic acid Drug-resistant breast cancer
(MCF-7/ADR) cells
Decrement of hypoxia inducible
factor-1a and better silencing of VEGF
expression +T1 MRI characteristics
with r1 =11.871 mM−1s−1
[153]
(Continued.)
21

Biomed. Mater. 19 (2024) 022001 G Kandasamy and D Maity
Table 2. (Continued.)
Drug siRNA type Type of nanocarrier +coating Targeting agent Treated cancer
Knockdown efficiency without/with
cytotoxicity +image/physical guidance Reference
Special AT-rich
binding protein
(SATB1) siRNA
Iron oxide
nanoparticles +liposomes made
of 1,2-dipalmitoyl-sn-glycero-3-
phosphocholine,
3b-[N-(N’,N’-
dimethylaminoethane)-
carbamoyl]cholesterol,
dimethyldioctadecylammonium
bromide and cholesterol
— Gastric cancer (MNK-28)
cells
High transfection efficacy (34%) under
magnetic field exposure, and better cell
toxicity (∼78%)
[154]
Myeloid cell
leukemia-1 (Mcl-1)
siRNA
Iron oxide nanoparticles and
indocyanine green +PLGA,
chitosan and cancer cell
membrane
Magnetic targeting Drug-resistant breast cancer
(MCF-7/ADR) cells
Increased cytotoxicity (up-to 75%) and
apoptotic rate, and ∼79% tumor
volume inhibition
[155]
Camptothecin Survivin shRNA Mesoporous silica based
nanorods +HOOC-PEG3500-
maleimide
Aptamer- AS1411 Mouse colon carcinoma
(C26) cells
Improved transfection efficacy and
apoptotic rate (∼35% higher)
[156]
Myricetin Multidrug resistance
protein (MRP-1)
siRNA
MSOs Folic acid Human non-small cell lung
cancer (A549) cells
4-fold decrement in tumor volume in
in vivo
[157]
Doxorubicin and
cisplatin
MRP-1 and Bcl-2
siRNAs
MSOs with
3-mercaptopropyl/pyridyl-thiol
surface-modification +PEG
Luteinizing
hormone-releasing
hormone receptor based
peptide
— 58% and 56% knockdown of MRP-1
and Bcl-2 genes
[158]
7-ethyl-10-
hydroxycamp-
tothecin
VEGF siRNA SPIOs +poly(2-
(dimethylamino)ethyl
methacrylate)-block-poly(ε-
caprolactone)
— human colon
adenocarcinoma (LS174T)
cells
Significant VEGF suppression and
reduced growth and volume of
tumors +radio-labeling with 111In
[159]
22

Biomed. Mater. 19 (2024) 022001 G Kandasamy and D Maity
5. Conclusion
To summarize, in this work, the recent delivery of
TpRNAs—i.e. siRNA, specifically PD-L1, survivin,
Bcl-2, VEGF and other siRNAs using magnetic, metal
and silica (MSOs) nanoparticles has been majorly
discussed. Moreover, the combined delivery of these
TpRNAs along with chemotherapeutic drugs (e.g.
doxorubicin) has also been reviewed along with the
in vitro and in vivo therapeutic effectiveness. In
majority of the investigations, it can be identified that
the polymers—PEG and PEI respectively enhance
the biocompatibility or/and systemic-circulation, and
the affinity of the nanocarriers towards the siRNA
molecules—wherein the ratio of nitrogen in PEI and
phosphate in siRNA play a significant role in form-
ing nanocomplexes and also the siRNA transfec-
tion efficacy. Moreover, the inherent characteristics
of the inorganic nanoparticles like magnetic/metal-
oxide and metal nanoparticles have imparted molecu-
lar imaging (e.g. MRI and computed tomography)
capabilities that have effectively aided in the intra-
cellular delivery of the siRNA molecules (along
without/with magnetic-guidance). Furthermore, the
porous structure of the silica nanoparticles has pre-
dominantly improved the delivery of the siRNAs
along with the chemotherapeutic drug molecules.
After delivery, the siRNA molecules have attained
maximum therapeutic efficacy, resulting in better
cancer killing effects. Thus, the delivery of siRNA
molecules via inorganic nanoparticles holds a high
promising potential for future clinical investigations.
Data availability statement
The data cannot be made publicly available upon
publication because no suitable repository exists for
hosting data in this field of study. The data that sup-
port the findings of this study are available upon reas-
onable request from the authors.
Acknowledgments
We are highly thankful to the researchers who have
published their works, and they have been cited in
this review article. This work is supported by the seed
funding project (VTU SEED (FY 22-23)-02) from
Vel Tech Rangarajan Dr Sagunthala R&D Institute
of Science and Technology, Avadi, Chennai. D Maity
would like to thank University of Petroleum and
Energy Studies for all the support.
Conflict of interest
The authors declare that they have no known compet-
ing financial interests or personal relationships that
could have appeared to influence the work reported
in this paper
ORCID iDs
Ganeshlenin Kandasamy https://orcid.org/0000-
0002-6849-6538
Dipak Maity https://orcid.org/0000-0001-9792-
0281
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