THERANOSTIC APPROACH FOR MANAGEMENT OF OSTEOPOROSIS

Abstract

Osteoporosis (OP) is a bone-metabolic disorder, causing micro-architecture degeneration and a decrease in bone density. Nutritional deficiency, i.e., calcium, vitamin D, and hormonal imbalances are the primary cause for the occurrence of OP. Although conventional diagnostic techniques and therapies are available and found to be effective only at a later stage, though still lack prevention strategies. Thus, the patients tend to suffer incidence of fractures and many difficulties to manage their day-to-day activities at an elderly stage. Numerous nanomaterial(s) possessing unique physicochemical, optical, and electrical properties are reported nowadays to be employed for both early-stage detections of disease and its treatment. Amongst these nanomaterials, superparamagnetic iron oxide nanoparticles (SPIONs) possessing strong magnetic susceptibility, less in vivo toxicity, and surface functionalities are extensively employed for MRI contrast imaging agents in the area of disease diagnosis, and drug delivery tools for various therapies. Therefore, this review highlights the pathophysiology of OP, conventional techniques of diagnosis, and the application of SPIONs for diagnostic and treatment purposes of osteoporosis.

Full text

Theranostic Approach for the Management
of Osteoporosis
Anjali Pant,a Joga Singh,a Ravi Pratap Barnwal,b Gurpal Singh,c,* &
Bhupinder Singha,c,*
aUniversity Institute of Pharmaceutical Sciences, Panjab University, Chandigarh, 160014, India;
bDepartment of Biophysics, Panjab University, Chandigarh, 160014, India; cChitkara College of
Pharmacy, Chitkara University, Rajpura, Punjab 140401, India
*Address all correspondence to: Dr Bhupinder Singh Bhoop, Professor, University Institute of Pharmaceutical Sciences,
Panjab University, Chandigarh 160014, India; Tel.: +91-172-2534103; and Chitkara College of Pharmacy, Chitkara
University, Rajpura, Punjab 140401, India; E-mail: bsbhoop@yahoo.com; or Dr Gurpal Singh, UGC Assistant Professor,
University Institute of Pharmaceutical Sciences, Panjab University Chandigarh 160014, India; Tel.: +91-98371393832,
E-mail: gurpalsingh.ips@gmail.com
ABSTRACT: Osteoporosis (OP) is a bone-metabolic disorder, causing micro-architecture de-
generation and a decrease in bone density. Nutritional deciency, i.e., calcium, vitamin D, and
hormonal imbalances are the primary cause for the occurrence of OP. Although conventional diag-
nostic techniques and therapies are available and found to be eective only at a later stage, though
still lack prevention strategies. Thus, the patients tend to suer incidence of fractures and many
diculties to manage their day-to-day activities at an elderly stage. Numerous nanomaterial(s)
possessing unique physicochemical, optical, and electrical properties are reported nowadays to be
employed for both early-stage detections of disease and its treatment. Amongst these nanomateri-
als, superparamagnetic iron oxide nanoparticles (SPIONs) possessing strong magnetic susceptibil-
ity, less in vivo toxicity, and surface functionalities are extensively employed for MRI contrast
imaging agents in the area of disease diagnosis, and drug delivery tools for various therapies.
Therefore, this review highlights the pathophysiology of OP, conventional techniques of diagnosis,
and the application of SPIONs for diagnostic and treatment purposes of osteoporosis.
KEY WORDS: osteoporosis, theranostics, SPIONs, MRI contrast agents, bone-targeting
I. INTRODUCTION
Osteoporosis (OP), the silent bone disease is caused due to the disproportionation of
bone modeling and remodeling process.1,2 The progression of bone formation and re-
sorption is a continuously occurring phenomenon responsible for maintaining skeletal
integrity throughout the lifespan. Moreover, this imbalance tends to increases at the el-
derly stage and is evident in both males and females, therefore, classied into two types,
Type-1 primary OP and Type-2 secondary OP.3 Type-1 OP is related to bone loss due to
the decline in gonadal functions associated with aging in women above 45 years (i.e.,
menopause) and so, termed as postmenopausal OP. However, secondary Type-2 OP may
arise at any age in both males and females.4 Moreover, this type 2 disease starts occur-
ring at an age of 75 years due to chronic disease, nutritional deciency (hyperthyroid-
ism, hypophosphatasia), and prolonged use of medications such as glucocorticoids thus,
also termed as glucocorticoid-induced OP.5,6 The progression of this disease is mostly
allied with typical fractures in the low trauma areas, femoral neck, spinal areas, etc. In
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96 Pant et al.
recent years, the augmented longevity has led to a rapidly increasing number of senior
citizens, worldwide. According to the research in the United States alone, > 54 million
people are diagnosed with reduced bone mass, i.e., development stage of osteoporosis,
and by 2025, approximately increasing the no. of bone fractures to three million per
year. Subsequently, in India, at present, the average lifespan is ~ 67 years and is antici-
pated to upsurge by 2025 to 71 years and by 2050 to 77 years.7 Therefore, as per these
statistics by the end of the century, the old-age population will constitute closely about
34% of the total population in the country.
The World Health Organization (WHO) has conferred that a patient is said to be suf-
fering from OP when the bone mineral density (BMD) measured by dual-energy X-ray
absorptiometry (DEXA) has standard deviations of 2.5, i.e., lower than the peak bone
mass of a young healthy adult.8,9 Various other clinical diagnostic techniques like quan-
titative computed tomography (QCT), X-ray absorptiometry, i.e., single (SXA) and dual
(DXA), and imaging techniques (magnetic resonance and radiography) have also been
used for the detection of OP.10 However, they have only able to quantify the bone den-
sity and changes in the microarchitecture of the bone at later stages, and not at an early
stage. Since, the disease does not show any clinical signs and symptoms, except a per-
tinent change in the biochemical parameters is observed or any fracture exists. Besides,
the elderly population suers from various co-morbidities thus, making it dicult for
clinicians to detect the disease timely. Consequently, an ardent need for highly sensitive,
easily accessible, and cost-eective early-stage detection methods is quite necessary. In
former years, a swift increase has occurred in the eld of pathological detection of the
musculoskeletal system related to aging, altogether the expansion of high-resolution
imaging techniques, in turn, would signicantly lead to improving the management of
OP, too. Apart, from this therapeutic management of OP is quite dicult due to the limi-
tations of the conventional drug delivery systems. Bisphosphonates, selective estrogen
receptor modulators (SERMs) are most commonly prescribed drugs for OP treatment,
in addition, hormone-replacement therapies, nutritional supplements are all unable to
provide complete relief from this disorder. Poor bioavailability, dose-dependent side
eects, toxicity issues, patient compliance all lead to withdrawal of treatment after a
while.11 Thus, to overcome the above limitations of both diagnosis and treatment, exten-
sive research and a systematic approach are needed for the eective management of OP.
Currently, the advances in the eld of nanotechnology have elicited to systematic
designing and development of vast state-of-the-art organic and inorganic nanostructures,
e.g., polymer-based nanoparticles (NPs), lipid-based NPs, quantum dots, superparamag-
netic nanoparticles (SPIONS), gold nanoparticles (AuNPs), nanodiamonds, nanowires,
nanotubes, whiskers, tubes, pores, and chips, etc.12,13 These nanomaterials are known
to be biocompatible and facilitate site-specic delivery due to distinctive surface func-
tionalities.14 Biomolecules (e.g., gene, antigen, aptamer, peptide, etc.) and drugs either
could be bioconjugated on their surface or encapsulated within these nanomaterials, en-
abling them to be a target-specic delivery system(s).15 Thus, depending upon the term
of applicability, i.e., diagnostic, treatment, and both the surface tuning of nanomaterials
could be done and enrich the potential of disease management safety and ecacy.16
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Theranostic Approach for the Management of Osteoporosis 97
This review presents a biological outline of bone tissue, OP, diagnostic, and treatment
modalities and the application of SPIONs for both.
II. THERANOSTICS
Theranostic nanomedicine is a promising concept for diagnostic and therapeutic which
is emerging nowadays employing nanomaterials due to their well-developed surface
chemistry. Iron oxide nanoparticles (IONPs), quantum dots, carbon nanotubes, gold
nanoparticles, and silica nanoparticles are a few examples that have been previously
reported as suitable theranostic nanoplatforms for early diagnosis and real-time moni-
toring of therapy.17,18 Therefore, a combination of the diagnostic and therapeutic ap-
proaches eventually substantiates the optimization of the individual patient-specic
treatment protocol and early-stage diagnosis of various diseases.19
So, the concept of nanotechnology has conferred to be promising in the area of the
diagnosis and therapeutics together enabling their co-delivery too, for real-time moni-
toring of therapy. However, most of the nanomaterials which do not possess the imaging
functions could be merged by conjugating them on therapeutic moiety.20 Thus, the nano-
theranostic materials would encompass a promising combination of multidisciplinary
areas, i.e., physics, chemistry, biology, drug-delivery, material sciences, etc.,21 to cor-
roborate the diseased site- targeting ability in sucient concentration and progressing
the precision and treatment ecacy. Also, these nanotheranostic agents are the only
distinctive systems helping to overcome the pragmatic variances in the specicity of
conventional imaging agents and their biodistribution. Thus, the amalgamation of di-
agnostic and therapeutic agents in a solo nanocarrier employing nanotechnology is an
utmost footstep for the early-stage detection, treatment, and prevention of life-threat-
ening disease(s).22 One of the common approaches to identify these targeting ligands
or biomarkers is the identication of molecules expressed on the cell surface and, fur-
ther achieving recognition and target to the diseased area via suitable carrier loading.
However, utmost care is needed with the nanosystems to circumvent recognition by
innate immunosystem, and also, they should have a long-circulating half-life to reach
the desired site.23
III. PATHOPHYSIOLOGY OF OSTEOPOROSIS
Micro-architectural degeneration and loss of bone density are two signicant features
possessed by an osteoporosis suering patient. In order to understand the mechanism
of bone mechanism, an insight into bone biology is equally important, as illustrated in
Fig. 1. The progression of bone formation initiates with primary mineralization where
the preosteoblasts undergo proliferation and dierentiation to form matured osteoblasts,
leading to form a layer of unidirectional epithelium-like structure on the surface of an
organic matrix (i.e., osteoid) which is subsequently calcied. Next, the plasma mem-
brane of osteoblasts leads to the transfer of the minerals from bone marrow to the oste-
oid layer. Lastly, either apoptosis of the osteoblasts takes place or they form a at lining
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98 Pant et al.
or osteocytes,24 after which slow secondary mineralization occurs and leads to comple-
tion of bone formation, i.e., bone modeling. In addition, osteoblasts have numerous long
cell processes leading to the formation of thin canaliculi networks. Further leading to
the formation of an interconnection between the osteoblasts (active) and at-lining cells
along with a uid circulating in the extracellular space of the bone marrow. Therefore,
osteocytes play a key role in maintaining homeostasis of the extracellular uid, con-
sequently activating the bone formation and/or resorption in response to mechanical
stress.25 Alkaline phosphatase is an enzyme released at the site of bone formation and is
thus, considered a biochemical marker for bone modeling.26
Secondarily, bone resorption is necessary to maintain skeletal integrity by replacing
the old bone with a newer one throughout the lifespan.27 Herein, osteoblasts cells secrete
two important mediators, i.e., OPG/RANKL (osteoprotegerin and Receptor activated
nuclear factor kappa ligand) which are responsible for regulating the bone resorption
process.
Osteoclasts, the giant, rued bordered bone-resorbing cells (i.e., 4–20 nuclei) pos-
sess RANK receptors on their surfaces. RANKL secreted by osteoblasts subsequently
binds to these receptors and activates these osteoclasts. They, in turn, secrete hydrogen
ions and expose the organic matrix of bone to the proteolytic enzymes creating the re-
sorption cavities (i.e., Howship’s lacunae) at the cell-bone interface on the calcied bone
surfaces in a sealed-o microenvironment. Further, these enzymes (i.e., collagenases
and cathepsins) cause the dissolution of hydroxyapatite (HA) and the release of calcium
and phosphate minerals for the maintenance of homeostasis. On the other hand, osteo-
blasts also secrete a mediator which too regulates osteoclasts activity which is osteo-
protegerin (OPG), a decoy receptor when binds with RANK receptors and antagonizes
FIG. 1: Pathophysiology of osteoporosis
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Theranostic Approach for the Management of Osteoporosis 99
the osteoclastogenic action thus, stopping the resorption process. OPG and RANKL,
both are thus important signaling molecules promoting cellular communication between
dierent bone cells. Estrogen hormones are known to maintain the interaction between
osteoblasts and osteoclasts. These stimulate OPG production in osteoblasts and its de-
ciency upregulates the RANKL, i.e., an important determinant of resorption. In addition,
estrogen also leads to the regulation of parathyroid hormone for marinating the calcium
and vitamin D levels in the body. Moreover, at an elderly stage the process of calcium
absorption and renal reabsorption is impaired due to various dietary habits and diseases
such as diabetes, renal impairment, etc., thereby decreasing the amount of calcium in
the body eventually leading to decreased bone density, i.e., OP.28 Cytokines and inam-
matory mediators [interleukin (IL-1), TNF-α, prostaglandin E2] also have an important
role in the process of osteoclastogenesis. TNF-α plays an important role in mediating
the increased RANKL expression for osteoclasts stimulation, i.e., bone resorption is
activated.29 Similarly, IL-17 is also known to induce RANKL secretion by bone-forming
cells, leading to resorption of bone. Therefore, when the level of cytokines increases in
the blood it leads to identifying any disturbance in the phenomenon of bone biology
ultimately leads to the development of OP.30,31
A. Conventional Diagnosis and Treatment Approaches of Osteoporosis
Osteoporosis is mostly a disease diagnosed on basis of a linear association between
bone mineral content and slender body mass. Due to lack of any clinical manifestation
and even if the patient suers from mild pain in the legs, hands, back, etc., remains
unnoticed and directs toward OP development. Therefore, making the life of elderly
people more dicult after the incidence of fractures along with other co-morbidities.
Various non-invasive techniques are available for the assessment of bone and skeletal
muscle mass and quality in OP via imaging. Dual-energy X-ray absorptiometry (DXA),
computed tomography (CT), magnetic resonance imaging (MRI), digital X-ray radio-
grammetry (DXR), laser induced break spectroscopy, radiofrequency echographic multi
spectrometry, and ultrasound (US) aids in OP detection and also depict dierent charac-
teristics. Table 1 represents a brief description of diagnostic techniques available.3,10,32–36
However, the densitometry measures provide an incomplete relationship between the
fractures and bone density. Since some patients have normal densitometry results yet
suer from osteoporotic fractures and bone density. In addition, these are also unable to
monitor the bone regeneration process thereby, increasing the uncertainty of the treat-
ment regime too. Therefore, an ideal assessment tool for diagnosis of OP is highly re-
quired which would facilitate testing of mechanical resistance along with histomorphic
examination during and after the treatment too.
B. Therapeutic Management of Osteoporosis
Anticatabolic drugs, including estrogens, SERMS, BPs, and RANKL inhibitors, are be-
ing used for OP therapy37 by reducing bone resorption and promoting bone formation.
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TABLE 1: Various diagnostic techniques for osteoporosis
Technique
Advantage(s)
Disadvantage(s)
Ref. no.
Dual-energy
X-ray
absorptiometry
(DXA)
Low dose of radiation; Evaluation
of sensible anatomical regions of
the fracture. Short acquisition time
Lack of discrimination
between cortical and
trabecular bone
10
Computed
tomography (CT)
Discriminative measurement
of cortical and trabecular bone;
correlation among bone volume
fraction, total volume, and the
trabecular spacing, diagnosis of
early fractures
High dose of radiation
is required
32
Magnetic resonance
imaging (MRI)
Ease of long-term follow-up of
disease progression; Ability to
study the trabecular network of
bone
Expensive technique 33
Quantitative
ultrasonography
(US)
Provides information of
bone mass, mechanical and
microstructural properties,
cheap, portable, and avoidance of
ionizing radiations
— 32
High-resolution
peripheral computed
tomography
Examine the radius, tibia, and
metacarpal bones, Ease of
bone mineral density (BMD)
quantication in both cortical and
trabecular bone
—
2
Digital X-ray
radiogrammetry
(DXR)
Cost-eective vis-à-vis to DXA;
helpful in measurement of BMD
for three middle metacarpal bones
Measurement of
true volume of bone
density is not possible;
texture analysis of
trabecular bone cannot
be performed
33
Laser-induced break
spectroscopy
Calcium and sodium level in hair
samples lead to identication
of BMD for osteoporotic,
osteopenic and healthy women.
Cost-eectiveness, ease of hair
sample collection and avoidance
of exposure of patients to harmful
X-rays
In
situ
diagnosis is not
yet possible
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Radio-frequency
echographic
multi-spectrometry
Analysis of bone quantity and
bone quality via non-ionization
approach; portable device with
high precision and sensitivity
—
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Theranostic Approach for the Management of Osteoporosis 101
BPs or modied BPs, e.g. alendronate (ALN), risedronate, ibandronate, or zoledronic
acid are usually the osteoclasts targeting molecules due to their high anity for bone
mineral causing inactivation and inducing apoptosis in osteoclast.38,39 However, BPs
exhibits poor oral bioavailability (< 1%), limited absorption with a concomitant food or
beverage intake. Therefore, the patients should refrain from oral intake of food (except
plain water) for up to two hours after medication consumption, a directive ignored by
most of the patients.40 In addition, the occurrence of severe side eects, e.g., upper gas-
trointestinal tract irritation and mostly osteonecrosis of the jaw bone is often an associa-
tive dental problem. Moreover, BPs long-term treatment leads to trauma and atypical
fractures of the femoral shaft41 occurring through excess inhibition of bone resorption
and accumulation on compact bones at sites of high tension.
SERMs are the only category used for both, prevention and treatment of OP, These
SERMs interact with intracellular estrogen receptors present in bones as estrogen recep-
tor agonists mostly useful for the treatment of post-menopausal OP.42 Although, poor
oral bioavailability (< 2%), presence of estrogen receptors apart from bone, high hepatic
metabolism, and side eects, in turn, reduces its safety and ecacy.
While enzyme-related treatment measures which include cathepsin K inhibitors,
blocks 24 kDa cysteine protease expressed by osteoclasts43 also lead to skin-related
side eects such as scleroderma-like skin thickening. Odanacatib which was later on
developed oered a better specicity and better toxicity prole to cathepsin K.44 In ad-
dition to these, a monoclonal antibody denosumab also prevented the RANKL binding
to RANK, a cell surface receptor on osteoclasts and preosteoclasts.45 Peptides-based
therapy such as PTH (1-34), NBD (Nemo binding domain peptide), RANKL inhibitory
peptide, OP3–4 (Osteoprotegerin‐like peptide), CGRP (calcitonin related peptide),46
siRNA, bone morphogenetic proteins (BMP), etc., are also reported for treatment of
OP.
Thus, it could be inferred that all these conventional and newer molecules require
a systematically developed novel delivery system(s) for overcoming all the limitations
of poor bioavailability, side-eects, withdrawal of treatments after a while, and lastly
improved patient compliance.
IV. NANOTECHNOLOGY ROLE AND INTERVENTION FOR OSTEOPOROSIS
Nanotechnology has extensively improved the state of clinical management of diseases
in-eld diagnostics and treatment, both. It has played a signicant part in increasing the
sensitivity of tests for quantifying essential biomarkers of various diseases. Their unique
surface properties can be easily modied with drugs, peptides, etc., increasing the sen-
sitivity and specicity to the target cells. In addition, biodegradable and biocompatible
nature, variable routes of administration, high encapsulation eciency, large surface/
volume ratio, thereby primes to a reduction in therapeutic dose requirement and fewer
toxicity issues. Figure 2 portrays classication of various nanomaterials, based upon
their size, shape, surface characteristics properties and type of materials for biomedical
applications.
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102 Pant et al.
V. SUPERPARAMAGNETIC NANOPARTICLES (SPIONs)
Superparamagnetic nanoparticles (SPIONs) are considered to be idyllic for imaging or
treatment modalities of OP due to their unique magnetic behavior. An overview of the
role of SPIONs is therefore discussed in the following segments.
SPIONs are one of the inorganic nanomaterials possessing excellent magnetic be-
havior, which have been surface-modulated for increasing the drug delivery ecacy.47
The major components of these NPs are the magnetic core, protective coating, and mod-
ied surface functionality. The three iron oxides, i.e., magnetite (Fe3O4), maghemite
(γ-Fe2O3), and hematite (α-Fe2O3) are mostly used for medical applications. The mag-
netic characteristics depend upon various parameters such as shape, size, crystallinity,
and surface functionalization ligands.48,49
The magnetite NPs possessed coercivities of 2.4–20 kA/m in a controlled syn-
thesis manner,50 because at RT it is ferrimagnetic and at high temperatures loses its
stability, and susceptibility with time51,52 and, hence smaller superparamagnetic NPs
of size 10 nm is obtained at RT. Most of the researchers have studied the conse-
quence of synthesis conditions, surface functionalities, size, and precursors on the
magnetic susceptibility and their application modalities leading to an increase in the
physiological solubility and biocompatibility of SPIONs. Zhang et al. reported Fe3O4
NPs with poly (methacrylic acid) attached via coordination bond formation in be-
tween the carboxyl groups and iron. SPIONs with narrow size distribution (20 nm)
were also stabilized by employing a surfactant, i.e., polyvinyl alcohol.53 Similarly,
FIG. 2: Nanotechnology-based materials for biomedical applications
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Theranostic Approach for the Management of Osteoporosis 103
Kohler et al. reported the telechelic polymer having terminal trimethyl silyl group
coated IONPs for the peptide conferring targeting properties.54 Narain et al. reported
the functionalization of ferrite magnetic NPs which were functionalized with Amino
(-NH2), carboxylic acid (-COOH) and poly(ethylene glycol)(-OH-PEG) terminated
silanes bearing dierent functional end groups which resulted in a stable and aque-
ous phase-dispersible system via the inuence of electrostatic and/or steric repulsion.
This work showed that the silane approach by the formation of FeO-Si- bonds thus,
allowed the preparation of stable hybrid NPs over a large range of pH values.55 Kado
et al. studied the synthesis conditions eect on the particle size of NPs, i.e., smaller
NPs than 6 nm were found to be superparamagnetic at RT.56 Similarly, anchored
cysteine-terminated PEG to the surface of IONPs providing an advantage to yield
NPs stable in a biological uid. Also, dierent functional groups such as PEG chains,
-COOH, -NH2, and -SH groups were further used to enhance the colloidal stability in
biological medium (serum and human plasma) for further utilization in biomedical
and sensing applications.57 Surface modication of SPIONs with polyethyleneimine-
DNA complexes, sodium tri-polyphosphate, dendrimers, fatty acids, phosphoric
acid, dopamine, 3-mercaptopropionic acid, 2-Bromo 3-propionic acid, citric acid,
PEG, diethylenetriaminepenta (methylene phosphonic acid) have been reported in
the literature.58–64 This raties the anity of these molecules towards the hydrophobic
surface of magnetic NPs via ligand-exchange processing these with good colloidal
stability in the aqueous medium.
Sayed et al. reported a simple and sustainable protocol for IONP formation. The
atomic arrangements in each crystal facet when exposed lead to shape changes and
eects on its various properties. Iron oxides were synthesized by microwave-assisted
heating at dierent temperatures and hence, six dierent shapes were observed via iron
salts as iron- precursors, a template, i.e., cetyltrimethylammonium bromide (CTAB),
cyclohexane-water-pentanol (reaction solvent), and urea (hydrolyzing agent).65 Sood et
al. reported the surface modication of MNPs with gold using dehydroascorbic acid as
a capping agent imparting stability to these nanocomposites with saturation magnetiza-
tion of IONPs decreased from 42.806 to 3.54 emu/g.66 Similarly, Monterio et al. synthe-
sized hydrophilic magnetite NPs functionalized with cyclodextrin (CD) proved to be an
ecient carrier for irinotecan, along with suitable for magnetic drug targeting via MRI
contrast enhancement due to their superparamagnetic magnetite core.67 Bixner et al.
reported monodisperse SPIONs coated with N-palmityl-6-nitrodopamide for improved
stability of IONPs.68 Peralta et al. studied the magnetic and thermo-responsive be-
havior of poly(N-isopropyl acrylamide-co-3-(methacryloxypropyl) trimethoxysilane)
functionalized NPs for site-specic targeting and controlled delivery of ibuprofen.69
Chee et al. have synthesized bisphosphorylated peptide-coated ultra-SPIONs with high
circulatory uids and tissues dispersibility, colloidal stability and, enhanced magnetic
property.70 Therefore, for enhanced biocompatibility and medical applications, the sur-
face ligands and colloidal stability played an important role and this mechanism is
thus, mostly studied by varying the process of synthesis and further surface chemistry
modications.
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A. Overview of Synthesis Techniques of SPIONs
This review focuses mostly on pure iron oxide NPs with superparamagnetic properties.
However, other chemical routes that have been reported are also mentioned in Table 2.
1. Co-Precipitation Method
The co-precipitation of ferrous chloride and ferric chloride (1:2) aqueous solution under
alkaline and inert condition was performed rst by Massart et al.77 This process leads to
the formation of black precipitates, i.e., spherical magnetite NPs having uniform sizes.
The following equation no.1 shows the reaction;
Fe2+ + 2Fe3+ + 3 OH–1 → Fe3O4 + 4H2O (1)
In the past few years, Honary et al. considered the eect of ferrous/ferric ion ra-
tio, pH, and ionic strength of the media as critical parameters inuencing the size of
TABLE 2: Synthesis of SPIONs using various methods
Method of
preparation
Temperature
Particle size
Remarks
Ref. no.
Co-precipitation
~ 25°C
7.2 nm
Co-precipitation of aq.
ferrous and ferric chloride
71
Electrochemical
decomposition
25°C
5–40 nm
Polarization of iron at anode
in dierent composition of
water and ethanolic mixture
of 0.1 M lithium chloride
72
Solvothermal
200°C
58–250 nm
and a width
of 8–64 nm
Single crystalline Fe
3
O
4
nanorods using (Fe(CO)5),
oleic acid, and hexadecyl
amine
73
Green synthesis
~ 35°C
50 nm
Extracellular synthesis by
induction of proteins for
biochemical reactions when
reacting with ferric chloride
74
Flow injection
synthesis
80°C
2–7 nm
Reaction mixture was
mixed in laminar ow
pattern continuously in a
capillary reactor
75
Thermal
decomposition
300°C
3–50 nm
Decomposition of fatty acid
iron salt in non-aqueous
solvent
76
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IONPs by Box-Behnken experiments and multivariant analysis.78 Shahid et al. reported
the IONPs possessed a narrower size distribution, stable morphology at pH (8.0–8.3),
and the saturation magnetization, and the coercive eld was 68 emu/g with reverse co-
precipitation method.79 Khalil reported a quantitative, synthetic, and ecofriendly method
for preparation of magnetite nanocrystals (7.84 ± 0.05 nm) and nanorods (6.3 ± 0.2
nm) by reduction of ferric and ferrous salt (2:1) in an alkaline solution (pH = 9–11).80
Similarly, Dolores et al. represented simple preparative methods producing dierent
magnetic IONPs via co-precipitation of aqueous ferrous salt solutions and the aque-
ous alkaline ethylene glycol (EG) solution. This process also involves sonosynthesis of
cisplatin-loaded IONPs under uniform acoustic power and varying ultrasound frequen-
cies (i.e., 581, 861, and 1141 kHz). These NPs possessed saturation magnetization of
60–93 Am2 kg–1 without any alteration of size (diameter of 21–31 nm) and hence, it was
conferred that there was a proportionate increase in the drug-loading with ultrasound
frequency.81 Similarly, Aliramaji et al. reported the synthesis employing co-precipitation
under sonication. This resulted in the narrow size distribution of IONPs and good mag-
netic behavior.82 Sood et al. further synthesized by coprecipitation using dehydroascor-
bic acid (DHA) as a capping agent. The gold coating resulted in enhancing the optical
properties, e.g., enhanced CT and MRI contrast, site-specic drug delivery, and also
assisted in improving the colloidal stability of NPs.14
2. Electrochemical Decomposition
Electrochemical decomposition is a redox reaction of metal ion species in the presence
of a stabilizer. Park et al. synthesized crystalline maghemite NPs by cathodic electrode-
position at room temperature (RT) using FeCl3. Magnetic saturation of maghemite NPs
was determined to be 66 emu g–1 at 300 K.83 Cabrera et al. reported polypyrrol–magne-
tite NPs composite with good magnetic and electric properties of size 20 nm followed by
encapsulation of the NPs in a polymer matrix during its formation by chemical oxidation
of the monomer.84 Karimzadeh et al. optimized the synthesis of uncoated and in situ
PEG coating of SPIONs via electrochemical decomposition having saturation magneti-
zation of 62.9 emu g–1, and 37.5 emu g−1.85
3. Solvothermal Method
Nanocrystalline metal oxide particles of uniform size, the large surface area is synthe-
sized under auto-generated pressure upon heating at 100–374°C, i.e., boiling point and
critical point of water to achieve a crystalline phase at low temperature in a closed en-
vironment. Employing this route of synthesis, the phase, particle size, and crystallinity
could be easily controlled and this method is also termed hydrothermal due to the use
of water as a solvent for preparation.86 Li et al. synthesized sodium citrate functional-
ized SPIONs resulting in enhanced colloidal stability and preventing their aggregation.
These functionalized SPIONs possessed a higher magnitude of saturation magnetiza-
tion, i.e., 69.641 emu/g and low retentivity, i.e., 0.8 emu/g.87 Kozakova et al. reported a
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106 Pant et al.
fast reaction process employing both the solvothermal and microwave irradiation, i.e.,
prevention of Ostwald ripening, yet leading to formation of uniformly shaped NPs in
size range of 20–130 nm and saturation magnetization of range 8–76 emu g−1.88
4. Green Synthesis Route
Green synthesis is a route of synthesizing magnetic NPs by using bacteria, fungi, pro-
teins, etc., which are termed magnetosomes. These magnetosomes are formed by the
biomineralization of magnetotactic bacteria having a single domain and uniform par-
ticle size (20–45 nm). Geobacter sulfurreducens bacteria was employed for reduction
the ferric salt by Coker (2007) for the synthesis of IONPs.89 However, it is a large-scale
process yet, leads to formation of magnetic NPs via green technology and zero-usage
of harmful chemicals. Bharde et al. carried out the extracellular synthesis of SPIONs
size = 50 nm at 35°C by induction of proteins for biochemical reactions when reacting
with ferric chloride.74 El-Kassas et al. studied the formation of IONPs employing brown
seaweed water extracts, i.e., Halophila stipulacea, Padina pavonica (Linnaeus) Thivy
and Sargassum acinarium (Linnaeus) for reduction of ferric chloride, i.e., (Fe3+ to Fe2+)
resulting in dierent sizes of NPs, i.e., 10–19.5 nm (P. pavonica) and 21.6–27.4 nm (S.
acinarium), respectively.90 Martínez-Cabanas et al. reported novel magnetic hybrid NPs
using a green reducing agent, i.e., eucalyptus extract, and further encapsulation within
the chitosan matrix.91 Rajiv et al. also prepared nanorods shaped SPIONs (size 10–20
nm) with good stability and biological activity using Lantana camera for reduction of
ferrous sulfate.92 Kanagasubbulakshmi et al. also reported the use of Lagenari asicer-
aria leaves extract for green synthesis of IONPs having good stability, cubical shape
with the size range of 30–100 nm.93
5. Flow Injection Synthesis
Flow injection system (FIS) was reported by Alvarez et al. synthesized magnetic NPs in
which Fe2+and Fe3+ were co-precipitated in an alkaline medium by continuous mixing
under laminar ow in a capillary reactor leading to formation of small NPs of size 2–7
nm.75
6. Thermal Decomposition
Metal fatty acid salts are thermally decomposed to produce uniform size and shaped
NPs with high crystallinity and magnetic behavior. Many researchers have used the
various fatty acid salt combinations as shown in the table. In past years the researchers
have prepared SPIONs with sizes ranging between 5–50 nm by this thermal decomposi-
tion process which was hydrophobic and non-biocompatible due to the use of organic
solvents. However, the hydrophilic nanocrystals formed by the co-precipitation method
are hydrophilic and less stable with low magnetization. Therefore, to increase the stabil-
ity of these nanocrystals in biological systems and surface functionalization to convert
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them into hydrophilic systems, some measures have been reported in the earlier section
which would eventually improve the colloidal stability in an aqueous medium. Jana et
al. reported a non-aqueous solvent-based thermal decomposition method to synthesize
SPIONs of 3–50 nm size range at 300°C.76 Hufschmid et al. reported SPIONs prepared
using iron-oleate as a precursor having a tunable size range 2–30 nm.94 Unni et al. re-
ported SPIONs with a magnetic diameter of 10.7 ± 5.6 nm in presence of molecular
oxygen, thereby diminishing the magnetic dead layer and single-crystalline SPIONs
with fewer defects.95 Orsini et al. synthesized IONPs (7–12 nm) with decomposition of
ferric acetate (Fe(acac)3) at high temperature with observing the eect of type of solvent
(1-eicosene and 1-octadecene) at a xed concentration of Fe ions.96 Thus, it was con-
cluded that the heating rates were the critical process parameter for desired particle size
determination.
7. Polyol Process
IONPs possessing a uniform morphology and desired magnetic properties were suc-
cessfully synthesized by employing an up-scalable method called the polyol process.97
Various researchers have reported the eect of doping of other metals such as gold,
the nature of precursors, and the inuence of numerous experimental parameters. This
process was considered to be versatile, ecofriendly and less-time consuming process. Yu
et al. carried out a polyol-microwave-assisted synthesis of monodisperse gold nanotri-
angles (5 nm) coated SPIONs of size = 280 nm possessing good colloidal stability and
magnetic susceptibility.98 Hachani et al. reported the eect of high pressure and tempera-
ture on the formation of SPIONs. This was a simple and economical process and did
not require an inert environment, resulting in the formation of biocompatible SPIONs
of size 8 nm and high magnitude of saturation magnetization, i.e., 84.5 emu g−1.99 Later,
Hemery et al. investigated that the amount of water and temperature as critical param-
eters when injected either with a polyol solvent or an admixture of poly(hydroxy)amine,
both in a reux system. NPs formed by this method were spherical shaped of size range
4–37 nm. Thus, these MNPs were termed as nanoowers due to denite narrow size
ranged multi-core assemblies, which would be applicable as contrast agents in MRI
study, magnetic hyperthermia and/or both.100
8. Microwave-Assisted Synthesis
Scale-up synthesis of SPIONs is necessary for its vast application in the eld of bio-
medicine. Thus, microwave (MW)-assisted synthesis has various advantages over other
synthesis protocols. Earlier, Parsons et al. and Pascu et al. synthesized iron oxide/oxy-
hydroxide nanophases employing ferric salt titrated with aq. sodium hydroxide (alkaline
medium) at 100 to 250°C with pulsed MW resulting in the formation of spherical NPs
with size 17–28 nm and low anisotropy.101,102 Pascu et al. reported that MW-assisted
synthesis is a fast, easy operation, energy consumption ecient, and environmentally
friendly approach for SPION synthesis.102 In addition, Gonzalez et al. studied eect of
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108 Pant et al.
critical process parameters for reaction, i.e., temperature, power, time. The usage of
MW was reported to be a multi-mode apparatus resulting in formation of uniform sized
SPIONs with good colloidal stability and magnetic behavior. In addition, on comparison
with lab-scale there was no variation in the size, colloidal stability and magnetization
on increasing the reaction volume up to 10 times, i.e., 4.5–50 mL (scale-up), in turn
resulting in higher percent yield.103 Other microwave-assisted synthesis includes the use
of PEG and β-cyclodextrin (β-CD)/aqueous solutions iron chlorides in the presence of
ammonia solution resulting in obtaining NPs of size 10.3–19.2 nm and zero contami-
nation.104 Also, magnetic hybrid NPs derived from wood were synthesized employing
thermal decomposition and microwave-assisted synthesis integrating their magnetic
functionality and isotropic behavior.105
So far, the above description of the synthesis and functionalization of SPIONs shows
that these nanocrystals can be prepared and eectively used for various biological ap-
plications. Therefore, being regarded as a theranostic tool, i.e., oers the possibility of
both diagnosis and treatment. There is a wide application of these NPs for numerous
diseases majorly those which are fatal for humans and the latest technology is still inad-
equate to detect them at early stages. Collectively, mentioned below are the applications
of SPIONs as imaging, i.e., diagnosis as well as a treatment tool.
B. Role of SPIONs as Bone Imaging Agents and in Targeted Bone Drug
Delivery
MRI is a prevailing, imaging-based non-invasive technique for the diagnosis of various
diseases. SPIONs possess an excellent magnetic property and are thus considered a good
MRI contrast agent, thereby enhancing the diagnostic and treatment ecacy. Contrast
agents employed during MRI are categorized as T1-positive (very bright) or T2-negative
(very dark).106,107 T1 contrasts helps to acquire the images only for morphological ex-
amination via induction of water molecules and spinning of electrons, thus determining
the rate of protons to acquire equilibrium. On the other hand, T2-negative represents the
transverse relaxation time constants at which the excited protons tend to lose the phase
contrast.108 SPIONs have been commonly used as contrast agents for MRI due to their
magnetic properties and spin disarrays leads to suppression of T2 contrast and enhances
the T1 contrast for good image resolution, e.g., Ultra-small SPIONs (USIONPs) are re-
ported to be biocompatible, highly sensitive, good relaxivity properties, and, thus used
as T1 and T2 contrast agents for image-based detection.109,110
A novel multimodal imaging system was thus, developed to study the bone regen-
eration process using pamidronate (PM) of BPs which have a high binding anity for
HA. Pullulan a biodegradable polymer of varying molecular weights was conjugated
with PM and administered inside a hydrogel and monitored with two probes for uores-
cence and MRI.111 Similarly, the anity of ALN conjugated PLGA-PEG functionalized
USPIONs was 8- to 10-fold higher for synthetic and biogenic HA in comparison with
ALN-NPs. Thus, enabled the T2-MRI imaging of the bone mineral phase.112 Panahifar
et al. also studied the outcome of BPs-conjugated to non-ionizing SPIONs to be used as
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Theranostic Approach for the Management of Osteoporosis 109
a bone-targeting contrast agent which was reported to be a relatively simple, rapid, and
economical technique.113
In addition to BPs, tetracycline, an antibiotic, was employed for bone-targeting due
to its calcium-binding property and on bioconjugation with poly(lactic-co-glycolic acid)
(PLGA) copolymer promoted the delivery of simvastatin (SIM). These SIM-loaded
NPs were able to release SIM within 72 hours up to 80% and had excellent cellular
(MC3T3-E1 cells) uptake capacity and biocompatibility, thereby reducing the cytotoxic
eects of SIM.114 Hu et al. reported the eect on tissue repair via SPIONs loaded gelatin
sponge (GS) scaold. In vivo MRI study in rats showed a consistent new bone forma-
tion process and scaold degeneration. In addition, a correlation was observed with the
rapid decrease in SPIONs and degradation of GS, as the endocytic SPIONs number in
the cells increased with time, eventually leading to the detection of bone by MRI after
4 weeks indicating their role in active osteogenesis without an application of external
magnetic eld.115
C. Treatment Application of SPIONs for Osteoporosis
Apart from the perspective of diagnostic purposes, the role of SPIONs is also been stud-
ied for the treatment of OP. Earlier, Tran et al. reported the role of HA, a bone mineral
for application in eld of bone-health (i.e., reversing OP). In this study, hydrothermal
process-controlled HA-coated IONPs were prepared and used for targeting and treating
bone defects. The ability of these HA-SPIONs was eective for treating bone defects
corroborated by the long-term osteoblast experiments for up to 21 days which resulted
in enhanced activity of alkaline phosphatase, total protein, collagen production, and cal-
cium deposition with up to a high conc. of 200 μg/mL.116 The same authors, Tran et al.,
reported that HA-coated IONPs enhanced the osteoblast proliferation and dierentiation
in calcium depositing cells. HA-coated IONPs conc. was increased from 12.5–200 μg/
mL which led to a four-fold increase in adsorption of protein was observed along with
greater osteoblast gene regulation.117
Another BP, medronate was conjugated with SPIONs which had a size of 17 nm.
It was thus, conrmed by in vitro binding studies that these NPs have a higher anity
towards HA which seemed to be eective for bone imaging and treatment. Moreover,
to understand the eect of HA, Tran et al. studied the cellular uptake in osteoblasts and
concluded that internalization of HA was via receptor-mediated endocytosis which re-
sulted in elevated levels of intracellular calcium and also mediated the osteoblast func-
tionalities for the treatment of OP as well as other bone-disorders.118 Shi et al. prepared
chitosan, a biocompatible and biodegradable polymer-coated SPIONs for the treatment
of OP by enhancing osteoblasts proliferation, decreasing cell membrane damage, and
promotion of cell dierentiation which eventually led to a rise in alkaline phosphatase
enzyme levels and extracellular calcium deposits.119
Magnetic hyperthermia, another treatment approach for disease management utilizes
magnetic NPs, as a heating mediator which was rst introduced in the 1950s to be used
as a treatment modality for tumor suppression.120,121 It is most eective at a temperature
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range of 40–43°C inducing cancer cell death.122 However, this therapy is limited due to
its low ecacy for malignant cells. Thus, hyperthermia could probably show enhanced
eects with improving cell-targeted therapy. Thus, this signicant property of SPIONs
was studied by Lee et al.123 to explore the potential of ALN, conjugated on dextran-
coated water-dispersible SPIONs (20 nm size) in OP by reducing the increased no. of
osteoclasts by thermolysis. Since ALN can target osteoclasts eectively and with the us-
age of hyperthermia via a radiofrequency system (electromagnetic frequency = 42 kHz
and current = 450 A) the temperature of NPs was raised for time = 20 minutes showing
the heat eect, i.e., thermolysis, by destroying the osteoclasts which have been consid-
ered as a synergistic eect for treatment of osteoporosis. In addition, in vivo studies also
proved that these IONPs were non-toxic and were indeed good MRI contrast agents as
they accumulated into the body of rats after injection.
Further, a treatment therapy using pulsed electromagnetic elds (PEMFs) was
reported by Wu et al. for improving bone fracture healing and treating OP. The
SPION-labelled, low PEMF-exposed cells showed synergistic eects by increased os-
teogenesis-related gene levels and protein expression (e.g., ALP, OCN, and RUNX2) in
a polymerase chain reaction and western blot studies.124 Quan et al. studied the impetu-
ous assembly on HA nanocrystals via ALN and IONPs for regeneration of osteoporotic
bone. The in vitro and in vivo activity of these functionalized HA nanocrystals proved
that the presence of combining two dierent functional groups inhibited the osteoclasts
and also promoted osteoblastic cell proliferation and cell dierentiation. In addition,
correspondingly enhanced the osseointegration of implants eventually leading to accel-
erated bone remodeling under osteoporotic conditions and, hence would be considered
as a desirable biomaterial for osteoporotic fracture treatment, too.125
Marycz et al. investigated the eect of magnetic eld and γ-Fe2O3 and α-Fe2O3
NPs (190–220 nm) on TRAP and Cathepsin K which are vital regulators of osteoclasts
metabolic process and increase the osteoclasts apoptosis, i.e., a key factor for osteopo-
rotic bone regeneration. In addition, a signicant reduction in the expression of TNF-α
can be only be observed via a combination of the magnetic eld with IONPs which
would trigger the RANKL-induced osteoclast formation.126 Figure 3A and 3B, depicts
the diagrammatic representation of diagnosis and treatment of OP and various routes of
administration.
A compilation of various literature reports on diverse nanomaterials for bone target-
ing and bone regeneration is presented as Table 3.
One of the major challenges towards the real-time monitoring and precision of dis-
ease site-targeting and treatment is the inability of conventional diagnostics and drug de-
livery systems. Since they are partly able to provide the progression of disease treatment
thus, a newer approach is the demand of the current scenario in the eld of biomedicine.
Moreover, the recent advancement in the dynamics of material-based nanocarriers, i.e.,
inorganic and organic, both have led to evolving a newer domain of surface engineering
in turn providing the self-assembling biomaterial(s) to circumnavigate the theragnos-
tic pathway. Therefore, the nanomaterials with enhanced surface characteristics would
surely play a substantial role in the therapeutic management of osteoporosis, nally
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Theranostic Approach for the Management of Osteoporosis 111
leading to improved quality of life at elderly stages. Also, a clear progression of disease
at early stages would help the clinicians to manage the therapy based on individual pa-
tient needs. Thus, the overall application of SPIONs as a theranostic agent because of
FIG. 3: (A) Applications of SPIONs for osteoporosis diagnosis and treatment. (B) Various routes
of administration of SPIONs in the treatment of osteoporosis. Also shown in the right side are the
structure and micro-architecture of the osteoporotic site of the bone, before and after the treat-
ment using drug-conjugated SPIONs, delineating regeneration of cells.
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TABLE 3: Various literature instances on SPIONs for bone-targeting and bone-regeneration
Drug
Nanocarrier
Size
Mechanism proposed
In vitro/In vivo study outcomes
Ref. no.
—
Calcium phosphate coated
γ-Fe
2
O
3
magnetic NPs
125 ± 25 nm
Coating of calcium phosphate
improved anity for bone
Increased osteoblasts density
127
—
Silica-coated magnetic-
cobalt ferrite NPs
50 nm
Enhancement of osteoblast
mineralization
Bone mineral density (BMD), bone
volume, and biochemical markers
of bone formation were increased
128
Zoledronic
acid (ZOL)
Hydroxyapatite (HA) NPs
100–130 nm
Synergistic eect of HA and
ZOL
In vivo evaluation in ovariectomized
(OVX) rats leads to improved levels
of biochemical parameters
129
—
PEG-modied magnetic
NPs
50 nm
Facilitation potent inhibition
of osteoclasts and stimulation
of osteoblasts cells
Bone mineralization was
signicantly enhanced
130
Alendronate
(ALN)
Dextran/Fe3O4 magnetic
NPs
20 nm Thermolysis targeting reduce
the OCs proliferation and
use of nanoplatforms could
limit/reverse the osteoporotic
process
1. Enhanced biocompatibility of
NPs after dextran coating and ALN
grafting
123
2. Increase in conc. of NPs showed
an increase in negative contrast
signal corroborating the good
contrast imaging during therapy
ALN PLGA-PEG-coated
ultrasmall SPIONs
139 nm Successful imaging of bone
to measure the bone mineral
density
Good serum stability and
cytocompatibility with 8–10-fold
higher anity for HA
112
ALN Nanodiamonds 64.74 ±
20.76 nm
Specicity to bone cells
due to the presence of
hydroxyapatite
Accumulated of ALN
functionalized- nanodiamonds in
bone tissues after i.v. injection
resulted in high HA anity, cellular
uptake, and synergistic eect
131
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TABLE 3: (continued)
Anti-
miR214
Polyurethane (PU)
nanomicelles- Aspartic
acid (Asp)8
80 nm
Asp
8
-PU help in osteoclasts
targeting and eective
delivery of anti-miR214
Bone microarchitecture improved
and bone mass increased
132
ALN
Fe
3
O
4
- hydroxyapatite
nanocrystals-scaolds
150 ~ 400
μm pore
size
Synergistic combination of
HA-ALN promoted eective
bone-targeting
1. Multi-functionalization of HA
nanocrystals inhibited osteoclastic
activity and promoted osteoblast
proliferation and dierentiation
125
2. Enhancement of implant
osseointegration and acceleration
of bone remodeling during
osteoporosis conditions
—Magnetic-HA scaold 52–168 nm miRNA-enriched exosomes,
produced by osteoclasts cells,
successfully delivered miR-
214 to osteoblasts
1. Osteoblasts were down-regulated
by membrane fusion, leading
to decreased exosome uptake
eciency
2. Inter-communication of
osteoblasts and osteoclasts was
weakened
133
— Ferumoxytol,
ferucarbotran
19 nm and
61 nm
Intrinsic inhibition of
RANKL causes induction of
osteoclastogenesis of bone
marrow-derived monocytes
1. Inhibition of TRAP-positive
multinucleated osteoclasts, ringed
actin structures during in vitro
structures
134
2. Increased bone-regeneration
observed in OVX mice
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114 Pant et al.
their signicant magnetic attributes, i.e., saturation magnetization, magnetocrystalline
anisotropy, and, coercivity leads to an interplay of optimization for eective imaging
and therapy purposes. Therefore, highlighting their role as for nanoparticles-based MRI
contrast agents with high sensitivity, specicity, and image-resolution properties.
VI. CONCLUSION
Osteoporosis, a debilitating disease has raised concern worldwide due to the growing
aging population and their enhanced susceptibility to suering from the same. From
the economic viewpoint, this is the area where the pharmaceutical industry will focus
to deliver newer therapies. SPIONS is the latest oshoot from nanotechnology that has
proven ecacy in the eective treatment as well as diagnosis of osteoporosis and other
skeletal disorders. SPIONS can be eectively labeled to reach the bone-forming site to
deliver the drug moiety and inhibit the osteoclast activity. Simultaneously, these NPs
will aid in identifying the sites where already the bone decay process has initiated and
the timely drug delivery at the site will ensure less damage to the skeletal. In the coming
years, the SPIONS would be able to integrate osteo-chips which would trigger the drug
release in turn minimizing the side eects and concomitantly keeping a record of the
bone density. Thus, the future of osteoporotic treatment lies in the nano bone regenera-
tion techniques where targeted drug therapy, diagnosis of stage of disease, multiple drug
loading, and concurrent imaging will be combined in a single platform.
ACKNOWLEDGMENT
A.P. acknowledges the Department of Science & Technology, New Delhi, India, for
the requisite funding to perform the present work as a DST INSPIRE Research Fellow
(IF170942).
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