Over cubes, among others (iv) Ease of modulating

Over the last few decades, scientists have actively explored the
synthesis of inorganic nanoparticles (INPs) for applications in various fields.
In this section, we primarily focus on the synthesis of INPs for diagnostic and
therapeutic applications, which require precision engineering of the
nanoparticle properties.

The extensive research in the field has provided ample understanding
to control the attributes at the nano-bio interface, which has led to numerous
successful clinical trials and translations. INPs have also been exploited for
their optical properties, which arise due to the quantum size effect, and have
been shown to be modulated by control over size for application as effective
imaging and contrast agents 69. Similarly, various chemical modulations have
been carried out on their surface, such as polyethylene glycol conjugation
(PEGylation) 70, charge modulation, ligand conjugation, and inclusion of
stimuli-responsive moieties and small-molecule probes for improved drug
efficacy 71. We further discuss the advances in the synthesis and surface
modification of these INPs that have facilitated their successful drug delivery

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3.1.1 Gold nanoparticles (AuNPs)

 First scientific insight into
the property of AuNPs came from Michael Faraday in 1850 72, where through
synthesis he showed the ability to obtain a ruby red colloidal solution of
AuNPs from yellow HAuCl4 gold salt. However, medicinal application of gold salt
was not proposed until 1890, as bacteriostatic against tuberculosis 73.
Subsequently, gold was also found to be effective in the treatment of Rheumatoid
arthritis in 1927 74. This led to prominence of nanogold as therapeutic agent
towards various Rheumatoid diseases. AuNPs have been widely explored for
applications in medicine due to

Biocompatible nature

control over theirsize distribution

Modification in shape that includes spheres,
nanorods, and cubes, among others

 Ease of
modulating surface chemistry through conjugation with various polymers
antibodies, small-molecule therapeutics, and molecular probes 75–76.

 There are diverse methods for the synthesis of
AuNPs, mostly using HAuCl4 as the precursor salt. Most of the methods for the
preparation of AuNPs involve reduction of Au3+ ions to Au0, using various
reducing agents at different concentrations to control the size of AuNPs.
Turkevich et al. introduced citrate salts as the reducing agent for synthesis
of AuNPs in 1951 77. Researchers have constantly been modifying the Turkevich
method to further increase the precision in controlling the size. Furthermore,
thiols have also been widely explored as the reducing agent for the synthesis
of AuNPs. Shiffrin-Brust used thiols in biphasic solvent along with sodium
borohydride and phase transfer reagents to yield 2–4 nm thiol-coated AuNPs 78.
Processes to functionalize AuNPs with different water-soluble surface groups by
ligand exchange have been exploited by using thiolated ligands. In 2000, Murray
et al. developed post-synthesis ligand substitution on AuNP surface 79. This
method has been extensively modified over the years to have a definitive
control on the biophysicochemical interactions of these nanoparticles for low
nonspecific protein adsorption 80, active targeting of the tumor 81, and
enhanced cellular uptake 82. In the last two decades, researchers have
exploited ligand exchange to conjugate nucleic acids and entrapment of toxic
drugs with controlled release. With latest developments in therapeutics, AuNPs
have been widely pursued as effective delivery agents for small interfering RNA
(siRNA)- and DNA-based enzymes (DNAzymes). Kataoka et al. modified the surface
of PEGylated AuNPs with hydroxychloroquine for effi- cient endosomal escape and
increased siRNA distribution 83. Researchers have shown delivery of RNA to
regulatory T cells, glioblastoma, andmesenchymal stem cells along with various
other therapeutic targets 88–90. Broadening their use in drug delivery
applications, Tang et al. have reported the use of AuNPs-stabilized capsules
(AuNPSCs) as an efficient protein delivery system 91. AuNPSCs consisted of an
oil core and were stabilized by amino acids (HKRK) -conjugated cationic AuNPs.
These AuNPSCs fused directly with the cell membrane and efficiently delivered
proteins such as green fluorescent protein (GFP) and caspase-3 into the
cytosol. Further, Ray et al. have demonstrated the versatility of the
AuNPSCmediated delivery system by intracellular targeting of the proteins to
the nucleus 92. In a recent work, Mout et al. used programmed assembly of
AuNPs with modified Cas9 protein for direct cytosolic delivery and efficient
gene editing 93. AuNPs also exhibit interesting absorption and scattering
properties, which can be modulated by control over their size and shape.
Surface Plasmon Resonance (SPR) property has been exploited for numerous in
vivo theranostic applications. Gold nanorods (AuNRs) have been quite
extensively used for these applications as their length and width can be
modulated to obtain SPR bands in the near-infrared (NIR) regions to allow better
contrast in biological tissues 94. Oldenburg et al. synthesized gold
nanoshells (AuNSs) through silica nanoparticle-templated growth of gold, which
have an absorption band in the NIR region and their optical properties can be
tuned by controlling thickness of the gold coating 95. The photothermal
property of the gold nanostructures also allows thermal ablation of solid
tumors if the AuNSs are excited by NIR and they have shown promising results in
clinical studies towards FDA approval under the name of AuroLase 96.
Understanding of size, shape, and surface properties of AuNPs has culminated
into various clinical trials as drug carriers and imaging agents but a
supplementary understanding of their cytotoxicity, bio-distribution, and
clearance from the body is required for improving their clinical translation


3.1.2 Iron oxide nanoparticles

 IONPs have been used since
the 1960s as targeted imaging and therapeutic agents. Meyers et al. used an
external horseshoe magnet to show accumulation of IONPs as contrast agent in
the vascular and lymphatic system of dogs 100. Application of IONPs in drug
delivery was explored by Widder et al. in 1979, by encapsulating IONPs in
albumin microspheres along with a chemotherapeutic agent to establish an in
vivo delivery method 101. Since then encapsulated IONPs have been explored
for site-specific delivery and entered Phase I clinical trial in 1996 for delivery
of epirubicin for treatment of advanced cancer 102. However, the trial was
unsuccessful as most of the IONPs accumulated in the liver. IONPs have emerged
as a successful class of nanoparticles in clinical translation as numerous
IONPs are approved by the FDA for therapeutics and imaging. Magnetite (Fe3O4)
nanoparticles have attained such eminence due to

Extremely low cytotoxicity.

Magnetic responsiveness and its tenability.

Controlled size and surface modification.

Contrast agent for magnetic resonance imaging
(MRI) 103.

IONPs have different magnetic property than bulk magnetite. These
nanoparticles exhibit single-domain magnetic property in the sub-100 nm range,
which has maximum coercivity as compared to the bulk magnetite that is
multi-domain 104. Further reduction in the size of IONPs decreases their
magnetic anisotropy energy. When thermal energy equals the anisotropy energy,
it results in random flipping of the magnetic moment and the nanoparticles
exhibit super paramagnetic nature 105. These superparamagnetic IONPs (SPIONs)
heat up when placed in an alternating electric field (hyperthermia) 106.
Subsequently, these particles have been used in vivo for targeting and thermal
ablation of tumors. Recently, Espinosa et al. synthesized 20-nm iron oxide
nanocube, which were heated with alternating magnetic fields and NIR radiation
to show complete ablation of solid tumor in vivo due to increased heating power
that they termed as dual-mode activation 107. These SPIONs have also been
extensively used in stem cell engineering for their controlled differentiation,
efficient homing, and long-term tracking in cell-based therapies. Researchers
used PEGylated SPIONs that were internalized by the mesenchymal stem cells
(MSCs) to guide them to the site of injury using external magnetic field. This
technique was further exploited by Xu et al. to track the MSCs post-engraftment
108. Surface moieties on the IONPs play an eminent role in their
bio-distribution, cytotoxicity, cellular uptake, and clearance. The rapid
degradation of IONPs results in release of Fe2+/3+ ions that alters levels of
ferritin, and inflammatory cytokines and several other reactive oxygen species
(ROS)-dependent proteins in cells, increasing their cytotoxicity 110. Thus,
efficient surface coating of these IONPs plays a very important role in their
physicochemical property in vivo. For most applications, IONPs are coated with
hydrophilic polymers for controlled degradation, as well as decrease in
immunogenicity and opsonization. Sakhulkhu et al. analyzed the protein corona
on the SPIONs coated with polyvinyl alcohol (PVA) and dextran along with the
effect of charge modulation. They showed that dextran had less adsorption of
protein as compared to PVA. Further, they found that electrostatic charge
played an important role and the negatively charged PVA SPIONs had less protein
adsorption (22%) compared to 36% and 41% on positive and neutral PVA-coated
SPIONs 111. In 2009, FDA approved ‘Ferumtoxyl’, SPIONs for the treatment of
anemia patients with chronic kidney disease. These particles are coated with
polyglucose sorbitol carboxymethylether that controls their degradation and
improves their pharmacokinetics. In further studies, Zanganeh et al. showed
that Ferumtoxyl could polarize macrophages in tumor tissue to pro-inflammatory
M1 phenotype, which could constrain the proliferation of subcutaneous
adenocarcinomas 112. They also showed the tendency of the SPIONs to inhibit
liver metastasis up to 6 times if Ferumtoxyl was pre-injected in vivo 113.
Currently, these materials are being actively explored for imaging the
progression of type 1 diabetes and response of the host to the therapies.
SPIONs-based imaging has also been effective in distinguishing non-diabetic
patients from the ones with recent onset of diabetes due to enhanced accumulation
of SPIONs in pancreases 114, this discovery led the way for drug delivery to
pancreas for diabetes. Due to the small sizes of IONPs, macrophages and
monocytes generally uptake them by phagocytosis or macro-pinocytosis depending
on their surface functionality. These IONPs-loaded macrophages have been
actively pursued for in vivo imaging and as drug delivery agents for diseases
and injuries that involve increased macrophage accumulation. Macrophage-assisted
IONP imaging has been explored for various cancers by taking advantage of
tumor-associated macrophages. They have also been explored for imaging in
myocardial infarction, myocarditis, aortic aneurysm, and atherosclerosis.

3.1.3 Silica nanoparticles (SiNPs)

SiNPs provide a new modality to the inorganic nanoparticles due to
their rapid degradation in vivo, regulated pore sizes (2–10 nm) for drug
encapsulation 115, incorporation of metals for theranostic applications 51,
and ease of camouflage by chemical conjugations 116. Since their invention in
the 1960s, as catalysts due to their large surface-to-volume ratios, they have
been actively modified to enhance their absorption properties. In 1968, Stöber
et al. proposed synthesis of solid SiNPs by a process based on hydrolysis in ammonium
oxide of silyl ethers 117. This method has been adapted by many laboratories
to synthesize SiNPs with sizes from 50 to 3000 nm. Further, researchers
recognized the niche realm of SiNPs in drug delivery since the synthesis of
mesoporous silica nanoparticles (MSNPs) in 1990’s 118. They have filled the
gap that wouldn’t otherwise be covered by metallic or other inorganic
nanomaterials with regards to their degradability, biocompatibility, and drug
release rates. MSNPs provide an interesting and alternative route to drug
delivery as they have nanopores that can encapsulate hydrophobic drugs for
efficient delivery, unlike other inorganic nanoparticles. The pore size of
MSNPs can be modulated by using various templates, surfactant concentrations,
pH, and solvents during the synthesis; this control allows sustained
degradation of the particles to deliver various payloads 50. MSNPs have also
been explored as stimuli-responsive drug release systems, where various chemical
entities on the surface of MSNPs can be used to control the release of
encapsulated drug by a trigger reaction 119. Essentially, release rates of
vancomycin and adenosine triphosphate (ATP) from mesoporous silica
nanosphere-based drug delivery systems was controlled by using disulfide
bond-reducing molecules, such as dithiothretol (DTT) and mercaptoethanol (ME),
as release triggers. This concept is termed as gatekeeping drug delivery
system. In the past years, various systems such as AuNPs, IONPs, CdS
nanoparticles, and polymers have been used as gatekeepers for controlled
release 120. MSNPs have hydrophilic surfaces, which is attributed to the
presence of hydroxyl groups that can be further modified using (3-aminopropyl)
triethoxysilane (APTES) to replace with more versatile amine groups. Since
early 2000s, many research groups have functionalized MSNPs with antibodies,
nucleic acids, and cell membranes to control the biodistribution and reduce the
systemic toxicity of MSNPs. In 2012, Parodi et al. used advanced techniques to
show enhanced delivery of doxorubicin using cell membranecloaked MSNPs . They
demonstrated that MSNPs coated with leucocyte membranes (termed as ‘cloaking’)
have reduced cytotoxicity and they efficiently extravasated into the blood
vasculature and accumulated near the tumor site in vivo. Recently,
physicochemical properties of PEGylated SiNPs have been further exploited by
Kim et al. 121. They used Cornell dots, which completed Phase I trial and is
currently under FDA-investigational new drug (IND) for actively targeting
melanoma by conjugating v?3-integrintargeting peptides to induce ferroptosis in
cancer-bearing mice. As researchers proceed to understand the fate of SiNPs and
actively pursue their interactions at the nano-bio interface, we can foresee
several clinical translations in the near future.


3.1.4 Quantum dots (QDs)

In the 1980s, researchers discovered that confined SiNPs emitted red
light upon illumination with laser 122. This led to further research in the
field of inorganic nanomaterials to produce a range of light-emitting
fluorescent INPs known as Quantum Dots (QDs) are very small semiconductor
particles, only several nanometres in size, with different optical and
electronic properties as compared to larger particles. They are crystalline in
nature and comprise of two different periodic group elements with the size varying
in the range of 2–10 nm 123. Understanding of their material composition and
size modulation has led to the development of a broad spectrum of fluorescent
QDs, with high quantum yield and low photobleaching. Thus, QDs have found
various applications in photodynamic therapy, in vivo imaging, and tracking
drug biodistribution 124, 125. Over the years, QDs have been widely explored
in theranostics alone and as an adjunct with wide variety of nanomaterials 126.
Cai et al. used pH-responsive ZnO-QDs coordinated with doxorubicin for targeted
drug delivery using hyaluronic acid conjugation 127. They have shown
efficient anti-cancer effect of the ZnO-QDs due to dual effect of Zn2+ and
doxorubicin. QDs have been explored for various applications in vitro and in
vivo but they are yet to be approved in clinical trials due to their potential
cytotoxicity 128. This is due to two main issues with QDs

Use of heavy metals in their composition

Their clearance from the body

Oh et al. analyzed the published literature to elucidate the
dominant role of surface properties and size of the QDs on the cytotoxicity
129. They also assessed the dataset to understand the role of different
core-shell QDs and their surface coating and their half maximal inhibitory
concentration (IC50) value. This sequential data-mining gives researchers a
broad database to comprehend the plethora of work done on QDs for designing more
particles that can be used for clinical translation in drug delivery
applications and as theranostic agents. To minimize the cytotoxic effect,
researchers are exploring Cd-free QDs 130, which have come up as less
cytotoxic nanoparticles along with advances in more efficient polymeric coating
of the QDs 131,132.