Accepted Manuscript

Title: An Insight into functionalized Calcium based Inorganic

Nanomaterials in Biomedicine: Trends and Transitions
Author: Shweta Sharma Ashwni verma B. Venkatesh Teja
Gitu Pandey Naresh Mittapelly Ritu Trivedi P.R. Mishra
PII:
DOI:
Reference:

S0927-7765(15)00311-2
http://dx.doi.org/doi:10.1016/j.colsurfb.2015.05.014
COLSUB 7087

To appear in:

Colloids and Surfaces B: Biointerfaces

Received date:
Revised date:
Accepted date:

14-11-2014
6-5-2015
8-5-2015

Please cite this article as: S. Sharma, A. verma, B.V. Teja, G. Pandey, N. Mittapelly,
R. Trivedi, P.R. Mishra, An Insight into functionalized Calcium based Inorganic
Nanomaterials in Biomedicine: Trends and Transitions, Colloids and Surfaces B:
Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.05.014
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Highlights of the present review

Focus is more on systemic applications than localized delivery

Discussion on application in gene, protein and small molecules delivery

for diseases like cancer, osteoporosis, diabetes and vaccination.

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Covers all the surface modification or functionalization that has been

done to improve their applicability.

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Discussion on characteristic properties in comparison to other

nanomaterials and method of preparation

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Discussion on Major challenges in clinical application.

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Mineralization of calcium phosphate over other nanosized delivery

systems

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An Insight into functionalized Calcium based Inorganic Nanomaterials in

Biomedicine: Trends and Transitions
Shweta Sharma1, Ashwni verma1, B Venkatesh Teja1, Gitu Pandey1, Naresh Mittapelly1, Ritu
Trivedi2, P.R.Mishra1*
1- Division of Pharmaceutics, CSIR-Central Drug Research Institute

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2-Division of Endocrinology, CSIR-Central Drug Research Institute

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B 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow (U.P.) 226031

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*Corresponding Author
Dr. P.R. Mishra Ph.D
Division of Pharmaceutics,
Preclinical South PCS 002/011,
CSIR-Central Drug Research Institute
B.S. 10/1, Sector-10, Jankipuram Extension,
Sitapur Road, Lucknow-226031,
India
Phone 91-522-2772450 (4537)
Fax: 91-522-2771941 E-mail: prabhat_mishra@cdri.res.in
mishrapr@hotmail.com

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Abstract

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Over the recent years the use of biocompatible and biodegradable nanoparticles in biomedicine
has become a significant priority. Calcium based ceramic nanoparticles like calcium phosphate
(CaP) and calcium carbonate (CaCO3) are therefore considered as attractive carriers as they are
naturally present in human body with nanosize range. Their application in tissue engineering and
localized controlled delivery of bioactives for bones and teeth is well established now, but
recently their use has increased significantly as carrier of bioactives through other routes also.
These delivery systems have become most potential alternatives to other commonly used
delivery system because of their cost effectiveness, biodegradability, chemical stability,
controlled and stimuli responsive behaviour. This review comprehensively covers their
characteristic features, method of preparation and applications but the thrust is to focus their
recent development, functionalization and use in systemic delivery. On the same platform
mineralization of other nanoparticulate delivery system which has widened their application drug
delivery will be discussed. The emphasis has been given on their pH dependent properties which
make them excellent carriers for tumor targeting and intracellular delivery. Finally this review
also attempts to discuss their drawback which limits their clinical utility.

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1. Introduction:
The rapid growth of nanotechnology in the last 35 years of therapeutics can be seen from the
fact that the several products of nano-formulations like Caelyx, Doxil, Transdrug ,
Abraxane etc are already there in the market and apart from that there is an extensive list of
systems which are in clinical trials or being scientifically explored. The reason behind this
unprecedented growth of applications in the area of nanoscience and nanotechnology is the
important and unique capabilities of nanosystems that are not found in the bulk sample of the
same system. It mainly includes their large surface area, intracellular delivery and quantum
properties. Nanomaterials have been used in almost every field of biomedical research, such
as targeted delivery vehicles for therapeutic agents, contrast agents, biosensors and artificial
oxygen carriers. The use of nanomaterial in the delivery of therapeutics provides liberty to
modify their fundamental properties such as diffusivity, solubility, drug release
characteristics, blood circulation half-life, bio-distribution, metabolism and immunogenicity.
Further, tailoring their surface properties allows temporal and site specific delivery of
therapeutics useful in the treatment of variety of diseases and disorders [1]. Nanosized
delivery systems used in biomedical research can widely be classified as polymeric
nanoparticles, polysaccharide nanoparticles, protein nanoparticles, dendrimers, nanoshells,
micelles, engineered viral nanoparticles and metallic nanoparticles. These systems have
shown potential for several branches of medicine such as immunology, neurology, oncology,
cardiology, endocrinology, pulmonary, ophthalmology, orthopedics and dentistry. Polymeric
and lipidic nanoparticles are called as soft nanoparticles and currently the most employed
systems because of their biocompatible and biodegradable nature. On the other side inorganic
particles like metallic and ceramic nanoparticles are known as hard nanoparticles because
of their dense nature. In the current scenario of biomedicine application of inorganic
nanoparticles has advanced rapidly and extensive amount of work is being done in their
synthesis and surface modification. Inorganic nanoparticles covers a wide range of materials
and therefore can further be classified as metallic nanoparticles or ceramic nanoparticles.
Metallic nanoparticles are the ones which are made up of metals like gold , silver or iron
oxide whereas ceramic nanoparticles are made up of alumina (Al2O3), calcium phosphate
(CaP), silica (SiO2), zirconia (ZrO2), titanium oxide (TiO2) or calcium carbonate (CaCO3) [2].
In the list of inorganic nanoparticles, calcium based ceramic nanoparticles are particularly
gaining the interest of researchers as they can be synthesized easily with desired size and
porosity and possess properties that other systems lack. Though, they have been in use for
localized delivery from several decades, however; their systemic and targeted delivery is a
recent topic of interest among researchers. The aim of the present review is to elaborately
discuss their properties, method of preparation, applications and related key issues which
need to be resolved. The review has been covered with most of the reports that have been
published till date.

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2. Constraints of other frequently used nano-particulate delivery systems

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For the past several decades polymers, lipids and proteins are the most commonly used
source materials for the development of nanoparticulate delivery systems. Even being
potential drug carriers they have their own sort of troubles and limitations. Most challenging
limitation with the commonly employed polymeric and lipidic systems is the usage of organic
solvents during their synthesis leading to a big concern for their in-vivo applications.
Moreover, delivery systems like Liposomes, micelles and emulsions suffer the limitation of
stability as well as leakage of drug molecules in systemic circulation before reaching to target
side. Though the limitations have been overcome to a certain extent by the use of polymeric
nanoparticles like PLGA (FDA approved polymer) but it is also known to produce acidic
products on their degradation which may have unwanted side effects on long term
administration. Further, most of these delivery systems are not cost effective and lack
industrial scalability. Cationic delivery systems commonly used in siRNA or pDNA delivery
are toxic in nature and promote unwanted uptake by reticuloendothelial systems (RES). This
review discusses how these limitations can be overcome to a certain extent by the use of CaP
and CaCO3 nanoparticles.

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Ceramic nanoparticles made up of CaP and CaCO3 are completely biocompatible and
biodegradable compared to any other nanoparticles. Unlike carbon nanotubes, quantum dots,
magnetic nanoparticles, silica nanoparticles and metallic nanoparticles they are free of severe
toxicity. Metallic nanoparticles like Cr, Cd, Au, Ag, Se, Te,Co,TiO2, CuO and ZnO have
been shown to increase mutation frequency, produce oxidative lesions, decrease cell viability
and induce damage to DNA. Contrary to this calcium based nanosized carriers belong to the

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3. Nano-structured CaP and CaCO3 particles: Idea behind development

The keen observation of researchers that had attracted great interest of formulation scientists
in developing mineralized biologically inspired delivery systems for therapeutics is that in
biological systems most of the biomaterials such as bones, teeth or shells are composed of
regular hierarchical structure of calcium phosphate and calcium carbonate. These structures
have much superior mechanical and functional properties compared to the artificial materials.
So it was believed that development of delivery vehicles composed of natural bio-mineral
would possess an excellent biocompatibility owing to their chemical similarity to human hard
tissues (bones, teeth) and ions of intracellular signalling pathways. Moreover, they are
already present in blood at relative high concentration. It was also observed that biologically
formed CaP and CaCO3 were often nanocrystals (5-20 nm width by 60 nm length) that are
precipitated under mild conditions and this natural selection as nanostructured materials
provides them the capability of specific interactions with natural proteins. Hence the
development of nanosized bioresorbable inorganic materials such as CaP and CaCO3 in
biomedical research stands to benefit most. In fact, their use in hard tissue engineering and as
a matrix for localized controlled delivery of therapeutic agents to bones and tissues is very
well established [3]. But nowadays they are also being explored for systemic delivery of
therapeutics and this is because of their several attractive features (listed below) over others
which makes them excellent delivery systems.
3.1. Biocompatibility and biodegradability

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safest class of materials known so far where by-products like Ca2+, PO43-or CO32-are already
present in the bloodstream within the concentration range of 1-5mM [4]. If fabricated
appropriately they can possess the same chemistry, size and crystalline structure as the
targeted tissues which enhance the materials activity and bio-acceptability before releasing
the drug. Calcium phosphate per se belongs to a category of GRAS (Generally Regarded as
Safe) as reported by FDA [5]

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3.2. In-vivo Stability

In-vivo stability refers to the stability of nanoparticles in biological matrix. In this respect
most of the inorganic nanoparticles bears the superiority over their counterparts and thus are
relatively much more suitable for in-vivo applications. For example, some of the polymeric
systems swell or their porosity changes on change of the pH and temperature while these
nanoparticles do not. Also, they overcome the major limitation of micelle and liposomes
based carriers, which are subjected to dissipation at below specific critical concentration (a
major obstacle in in-vivo administration). CaP and CaCO3 nanoparticles are much more
robust, particularly those with Ca/P ratio equal to hydroxyapatite (HAP, naturally occurring
calcium phosphate) which is not soluble in blood as blood is itself supersaturated with respect
to HAP. Also polymers like PLGA and PLLA inhibit rapid or prompt release of the loaded
drugs and undergo catalytic degradation with time leading to the generation of acidic
products. These ceramic nanoparticles are not prone to microbial growth, thus its storage
stability is good.

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3.3. Ease of preparation and cost effectiveness

The simplicity in the manufacturing process with minimal raw materials (common salts)
without the use of organic solvents makes them more cost effective and industrially relevant
compared to other nanoparticulate systems. Convenience in fabrication of these nanoparticles
in aqueous media provides a suitable platform for the surface functionalization with
appropriate agents like siRNA, proteins and several polyelectrolytes.

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3.4. Particles in very small size range

Their synthesis under controlled conditions can result in particles with size range <100 nm.
Therefore, these nanoparticles have the major advantage of enhanced direct endocytosis
uptake which is limited to size range 20-200nm in diameter and can lead to high transfection
efficiency[4].
3.5. pH dependent solubility
Stimuli responsive particles can significantly increase the efficacy of chemotherapeutic drugs
by increasing their accumulation at the tumor site. CaP and CaCO3 as bulk material as well as
nanoparticles show this stimulus responsive property. They are sparingly soluble at normal
blood pH (7.4) and more alkaline conditions but the dissolution accelerates at low pH, which
is generally found in either vicinity of tumor, inflamed regions or endo-lysosomes, making
them useful for the delivery of active molecules in malignant zones[6]. Like some of other
delivery systems they can retain drugs for a longer period of time after administration to
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animals because of longer biodegradation time, a property which is essential for diffusion
controlled kinetics.

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3.6. Suitability as carrier for gene delivery

Escaping the endosome for gene delivery on intracellular uptake is the biggest challenge and
here calcium based nanoparticles play a significant role. They are the most viable option for
gene delivery because of the endosomal escape effect. Their complete dissolution or
disintegration at the low pH of endosome (<5) causes the osmotic imbalance between the
endosome and cytoplasm causing endosome swelling followed by disruption and subsequent
release of the material into the cytosol [7]. Also the presence of positively charged calcium
ions in delivery system provides the effective binding site to negatively charged nucleic acids
making them resistant or stable to attack of RNAse (for siRNA and pDNA) as well as
nuclease. Stability against nuclease for pDNA is essential as it allows the DNA to enter the
nucleus without any degradation. Some of the experimental evidences have also proven that
calcium ions released from the particles also assist DNA uptake from cytosol to nucleus by
deactivation of inositol triphosphate (InP3) which causes the NPC (nuclear pore complexes)
to remain open and thereby allowing the complex of Ca and DNA to pass through the nuclear
membrane [8].
Sometimes it is said that CaP and CaCO3 nanoparticles have poor transfection efficiency and
not appropriate to apply in in-vivo conditions. But this is a myth because most of the time the
observed low transfection efficiency is because of the larger size or aggregation of the
particles obtained after improper or uncontrolled synthesis and can only be avoided by using
a suitable method in the presence of appropriate additive which can produce small size virus
like particle <100nm.
3.7. Ease of surface modification
In the present scenario of drug delivery or diagnostic imaging, targeted delivery is always
desirable as it allows the active molecule to reach the target site only. The application of CaP
and CaCO3 nanoparticles providing a physical entrapment to molecular payload can be
increased by developing composite nanoparticles utilizing lipids and polymers. Their surface
can be easily modified with these molecules which not only enhance physical stabilization
but also provide the binding site for targeting moiety. Ease of surface modification makes
them suitable for targeted delivery of hydrophobic dyes, oligonucleotides and drugs. Also,
they have the advantage that they are capable of loading both hydrophobic as well as
hydrophilic molecules.

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3.8. Relative Reduce macrophage uptake

On intravenous administration most of the delivery systems are rapidly taken up by the
macrophage present in the blood, which reduces the target amount of drug require to a
specific site especially in diseases like Cancer or Alzheimers. CaP being naturally present in
the body are generally not known to evoke macrophages or immune response.
4. Methods of preparation
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Disadvantages

References

Low crystallinity and

rapid aggregation

[9]

Mechanochemical

Spherical and
fibrous
nanostructures

Simple, low cost and high

stirring is not needed

Rapid aggregation ,
need special devices

[10, 11]

Sol-gel
processing

Nanocrystals are
aggregated

Homogeneity, simple
devices are used and high
purity crystals

Time consuming
process and need high
sintering temperatures
Time consuming , low
yield and need special
devices

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Spherical, needle or
rod like

Advantages
Low cost, easy to
synthesize and biomacromolecules can
control the aggregation

Coprecipitation

Morphology

Method

Nanorods, needle
Highly crystallinity, purity
like, spherical and
and homogeneity
fibrous Nanocrystals
Spherical
Size and morphology well
Time consuming , use
Emulsion
nanocrystals, needle
controlled, low aggregation
of organic solvents
technique
like, nanorods and
and homogenous
nanoplates
Needle like or
Low aggregation, narrow
Ultrasonic
Special device needed
spherical
size distribution, low
method
and low yield
Nanocrystals
temperature
Nanoplates,
Time consuming , low
nanorods, flower
yield, employs template
Template
and layer structure
Size can be well controlled
agents and high
method
mesoporous and
sintering temperatures
fibrous
required
nanostructure
High crystalline particles,
Rod, bowknot,
narrow size distribution , Needs device and large
Microwave
needle like and
scale synthesis is
size and morphology can
processing
flower structure
difficult
be well controlled,
synthesis is fast
Synthesis at low
Needs special devices,
Emulsion
Rod, fibrous,
temperatures, narrow size
reaction system is
hydrothermal spherical and strap
distribution , low,
complex, time
combination
like structures
aggregation and well
consuming and low
controlled size and shape
yield
Microwave
Rod, spherical,
High crystalline particles,
Needs special device,
hydrothermal
fibrous and strap
fast synthesis and high
yield is low
combination
like structures
temperatures are not

Hydrothermal

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Method of preparation of nanoparticles always play vital role to get the desired properties.
Therefore, numerous methods have been adapted for synthesis of calcium based
nanoparticles, which are mainly classified as dry or wet synthesis. Dry synthesis includes
solid state reaction, mechano-chemical reaction and plasma spraying whereas wet synthesis
includes wet chemical route, solgel technique, hydrothermal reaction, homogeneous
precipitation, emulsion processing route and recently microemulsions (Table 1). By means of
these methods various kinds of nanoparticles with varying size, structure and crystal
morphology can be synthesized.
Table 1: Different methods of preparation

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[12, 13]

[14-16]

[17, 18]

[19]

[20, 21]

[13, 22, 23]

[24]

[25]

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required

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The main advantage of dry synthesis is that it usually yields well crystallized products with
precise stoichiometry but at the same time needs long time and high temperature for the
processing. On the other hand wet methods generally require low temperature. Nanosized
HAP is a particular type of crystalline calcium phosphate nanoparticles (Ca10(PO4)6(OH)2)
which consist of calcium-to-phosphorus ratio of 1.67 and is the most stable with a least
solubility out of all calcium orthophosphates. It can be prepared in aqueous solutions by
mixing exact stoichiometric quantities of calcium and phosphate ions solutions at pH>9,
followed by boiling at high temperature for several days under a CO2-free atmosphere,
filtration and drying[26].
Several other methods are continuously being developed like Mechanical alloying [27], ball
milling [28], liquid-solid solution synthesis [29], laser induced fragmentation [30],
radiofrequency induction plasma [31], radiofrequency magnetron sputtering [32]however
for all the practical purpose nanoparticles prepared by synthetic and low cost methods are
used. Co-precipitation and microemulsion are the commonly used methods at lab scale level
and are explained in detail (Fig 1.)

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Co-precipitation is one of the most established techniques for the preparation of CaP and
CaCO3 nanoparticles and is also called as wet, chemical or aqueous precipitation. This
technique is widely chosen in contrast to another technique because large amount can be
produced in the absence of organic solvents that too at reasonable cost. The only by-product
of the reaction is water. The reaction involves the precipitation of nanophase CaP/CaCO3
from an aqueous solution containing calcium and phosphate/carbonate ions using various
reactants such as calcium hydroxide, calcium nitrate, calcium citrate, calcium chloride,
sodium carbonate, diammonium phosphate, disodium phosphate, and orthophosphoric etc.
The reaction was first proposed by Yagai and Akoki where they used calcium hydroxide and
orthophosphoric acid as starting materials for the preparation of nanoparticles. Later on, the
same process was modified as the different groups used different starting materials. The
precipitation process is highly dependent upon various critical process parameters like initial
concentration of calcium and phosphate/carbonate ions, addition rate, temperature, stirring
rate and final pH of the medium. The major limitation or disadvantage of this technique is
difficult to control the size by preventing particle aggregation. Though several approaches
have been tried by using different types of polymers, lipids or molecules that can prevent the
size of the particles but still the particle aggregation remains the major concern of this
method. Morphology of the particles is quite sensitive to the presence of additives which also
control the zeta potential of colloidal particles during precipitation[33].Some researchers
have carried out the precipitation process in a simulated body fluid to obtain biomimetic
nanoparticles. Recently advanced techniques like microreactors consisting of microchannels
are also being used to enable the reaction to be performed in several orders smaller than the
conventional reactors. Advantages of using these microreactors are high speed mixing, better
control over the reaction and high yield [34]. Entrapment of molecules can either be carried

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out during the precipitation process (pre-loading) or after the preparation of nanoparticles
(post-loading). Conjugation of proteins, polymers, peptides, cell penetrating moieties or other
functional ligands onto the surface of CaP nanoparticles is usually expected to proceed noncovalently that is via adsorption which is governed by the hydrophobic or vanderwaals
forces.

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Another approach although less explored is microemulsion or reverse micelles technique,

where inverse micelles are used as nanosized vectors. The advantage of this technique is their
capability to produce the particles of uniform shape, controlled size, narrow size distribution,
low degree of agglomeration and high chemical purity. Reverse micelle or microemulsions
are transparent isotropic liquid with nanosized droplets dispersed in continuous phase of oil
and stabilized by a layer of surfactant/polymer at their interface. These water cavities
stabilized by surfactant act like a cage which control the nucleation and growth resulting in
generally very fine and monodisperse particles. Like co-precipitation it also depends upon the
several parameters like ion concentration (Ca2+, PO42-,CO32-),ionic strength, pH, temperature
and concentration of surfactants etc. Microemulsion is also currently the most commonly
used technique for the preparation of nanoparticles at laboratory scale with a wide range of
surfactants. Hexadecyl(cetyl) trimethyl ammonium bromide (CTAB) surfactant has been
frequently used to synthesize these nanoparticles where on altering concentrations of CTAB
nanoparticles with different diameter and morphology has been obtained [35]. Co-surfactants
are also sometimes used in these systems to enhance the stability as well as solubilizing
capacity of microemulsions. Example Co-surfactant like butanol is known to reduce the
rigidity of CTAB/Toulene/water interface systems [36]. However, this method also suffers
from some limitation, unlike co-precipitation which is extremely simple process, this
technique is fairly complicated and involves the removal of organic solvents. Also the yield
of particles is less.

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Fig 1: Commonly used method for preparation of CaP/CaCO3nanoparticles

5. Biomedical applications:

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Biomedical applications of CaP/CaCO3 can broadly be classified into localized delivery or

systemic delivery. Earlier main applications of these nanoparticles were limited to localized
tissue engineering however their excellent properties make them attractive for other purpose
as well (Fig 2.). The following sections discuss their applications in wide areas.
B

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Fig 2: A) Various Different biomedical applications of CaP and CaCO3 nanoparticles B) Functionalization of CaP/
CaCO3 nanoparticles with different molecules

5.1. Hard tissue engineering

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5.1.1. Bone repair

In hard tissue engineering the material with higher mechanical strength such as metals, alloys
or their composites are always preferred. However, most of these materials are not
bioresorbable and need surgical removal after the implantation. Also, most of them have the
tendency to elicit an unnatural response which leads to the formation of capsule round the
implant. The ideal material for the hard tissue engineering should be the one possessing
sufficient mechanical strength and bioactivity to stimulate active tissue regeneration.
Though there are number of other ceramic materials which also bear these properties but
particularly calcium based ceramic has gained more attention due to the excellent
cytocompatibility, biodegradability and osteoinductive properties in physiological
environment. CaCO3 has also been employed as scaffolds for bone repair and osteoregenerative applications use of CaP is much more extensive than its carbonate counterparts.
This might be due to the major occurrence of phosphate ions in human body than the
carbonate ions. CaCO3 composites with CaP have broader range of applications than the
calcium carbonates alone. In fact in natural systems CO32- is the most common dopant of
hydroxyapatite (HAP) and is known to improve bioactivity compared to pure HAP [37].
Earlier traditional ceramics as implants with grain size > 1micron were only used in
orthopedic and dentistry as filler materials, structural forms and surface coatings but there
biological properties were limited to a great extent because of large grain size which resulted
in variety of implant failures due to insufficient osteo-integration, osteolysis as well as
implant wears. These conventional implants also lack the property of evoking natural cellular
responses to regenerate tissue. The limitations of conventional implants as well as the fact

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that naturally occurring CaP also exist in nanocrystalline form led the necessity for
development of new generation implants comprising of nanophase CaP that can lengthen the
service time of implants and can dramatically reduce the patient pain [38]. They are known to
overcome many limitations of conventional scaffolds and simultaneously exhibits enhanced
bioactivity, resorbability and mechanical strength. This combination of nanotechnology and
bioceramics has resulted in effective biological interactions as bioactivity of selected proteins
is altered on these nanomaterials (important for cell recognition) because of their surface
grain size, pore size, wettability, etc. which is closer to natural bone. Basically nanophase
ceramics show these properties in tissue engineering because of the enhanced adhesion of
osteoblasts compared to the conventional ceramics (Fig 3) [11, 39]. Nanocrystalline HAP is
the most popular choice out of all calcium phosphate categories to be used in tissue
engineering because of its high stability and structural similarity as a major inorganic
component of biological systems [40]. CaP or CaCO3 nanoparticles in its 3D nanocomposites
form with other metals, minerals or polymers are the major developments in hard tissue
engineering. These 3D nanocomposites are composed of CaP or CaCO3 nanoparticles
incorporated into a biodegradable polymer matrix having interconnected porous structure
which serve as support and reinforce tissue regeneration or replacement in a natural way.
These composites improve the mechanical properties as well as incorporate nanotopographic
features that mimic the structure of natural bone. In Injectable hydrogels CaP or CaCO3
nanoparticles mainly serves as tool to control the release kinetics of bioactives by reducing
the fast degradation of hydrogels without affecting their self -healing behaviour of
nanoparticles[41]. In 2014 Thi Phuong Nguyen et al developed the enzyme assisted injectable
chitosan hydrogels consisting of biphasic calcium phosphate nanoparticles and HAP for bone
regeneration and found that incorporation of nanoparticles increased both the mechanical
strength as well as binding to mesenchymal stem cells (MSC) on the hydrogel composite
[42]. Now days synthetic polymers like poly (lactic acid), poly (glycolic acid), poly (lactideco-glycolide) are more widely used in scaffolds than the natural polymers or proteins as they
avoid the risk of virus infection and their mechanical properties are better which can be
controlled during synthesis. CaP also provides the advantage to buffer the acidic degradation
product of some polymers like PLGA and other lactides and glycolides [43].The application
of nanophase CaP/CaCO3 in localized delivery can be divided in two parts. First in tissue
engineering, they can directly act as bone substitutes or regenerate the bone because of
osteoinductive action. Secondly, they can be loaded with specific growth factors (e.g. growth
hormones, bone morphogenetic protein, transforming growth factor-beta and insulin growth
factor),genes or drugs for the local and controlled release specifically useful in the treatment
of bone regeneration, bone tumour, osteoporosis or osteomyelitis [44]. NanoOss(bone void
filler, US FDA approved) and Ostim (injectable paste, CE approved) are approved
nanotechnology based medical devices as bone substitutes. Several groups have worked on
localized delivery of drugs, gene, growth factors etc. by means of CaP nanoparticles
composites. Kreb et al. reported that BMP-3 encoded plasmid DNA loaded in CaP
Nanoparticles and embedded into injectable alginate hydrogel were more efficacious in bone
regeneration applications[45]. They have gained specific attention in bone diseases like
osteoporosis and osteomyelitis because of their capability to augment the osteogenic response
of osteoblasts [46, 47]. Fibres loaded with pDNA-CaP Nanoparticles encoding basic

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fibroblast growth factors (pbFGF) and vascular endothelial growth factor (pVEGF) led to
drastically higher density of mature blood vessels than those containing plain plasmid and
also particles showed higher cell viability as well as comparatively less inflammation
compared to PEI-pDNA complex[48]. Localized delivery of pDNA, siRNA or shRNA by
multishell calcium phosphate nanoparticles embedded into different layers of multilayer films
for gene silencing of osteopontin and osteocalcin is a new recent strategy in tissue
engineering to control the bone formation[49].
5.1.2. Enamel and tooth repair
Further nanocrystalline CaP/CaCO3 has also shown excellent results in enamel and dentin
erosion, gingivitis, dentin hypersensitivity and marginal periodontitis. Like bones,
nanostructured CaP is also analogous to the basic building block of enamel which is needle
like apatite crystals with diameter between 20-40nm. Thus their remineralizing nature,
capability to neutralize lactic acid and their mechanical strength make them better tools
compared to the commercially available dental composites [50, 51]. This was clearly visible
when eroded sample of tooth were treated with both nanophase HAP as well as traditional
HAP crystals where in-vitro repair effects were much effective with nanophase HAP
compared to conventional crystals. MD et al reported enhanced remineralization under nanocomposites of CaP after the demineralization/remineralization treatment as compared to the
commercial fluoride releasing composites[52]. Dental fillers with nanophase CaP have also
shown substantially reduced caries formation and mineral loss from enamel near the margins
of nanocomposite compared to the control composites, which may be due to the release of
calcium and phosphate ions from the composites [53].The efficacy of these composites in
caries or periodontitis can further be improved by encapsulating nanoparticles with molecules
like antibacterial agents chlorhexidine[54] or using them along with Ag nanoparticles having
inherent antibacterial properties[55]. Most of the results show that nanophase CaP ceramics
have enhanced in-vivo bioactivity and thus have prospective application in tissue engineering.

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5.1.3. Coating material for other material implants

Nanostructured CaP as coating material for biomedical metals is another application in hard
tissue engineering. Elements such as cobalt chromium alloy, titanium and its alloy and
stainless steel are coated with nano-CaP to improve their biological attributes. Their coating
on biomedical metals accelerates growth of natural cells and thus enhances their fixation
making them useful for high load sites such as femoral and tibial bones. This approach is
basically used to improve the bonding between the implants and surrounding bones by
enhancing the interaction between the proteins, cells and tissue on the implant surface [56]
[57-59]. Hamdan S Alghamdi et al reported that titanium implants coated with
bisphosphonate (BP)-loaded CaP nanoparticles when implanted for 4 weeks, bone volume
and bone area was significantly increased compared to control implants in healthy and
osteoporotic conditions. They concluded that simultaneous targeting of bone resorption (by
BP) and bone formation (nano-CaP) can be an effective strategy in bone implants for
osteoporotic conditions [60].

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Fig 3: Osteoblast adhesion on conventional implants and nano-calcium based implants. The number of cells
adhered to nanoparticles based implants are more compared to conventional implants

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5.2. Gene and siRNA delivery

Gene therapy is the impending treatment option for some incurable and genetic disorders
(such as cancer, diabetes, hemophilia etc.) which works by substituting the defective gene or
by silencing the undesired mRNA. However, the delivery of these molecules inside the cell
has always been challenging task because of their inherent properties like large size, negative
charge and rapid degradation by enzymes like RNAse and nuclease which inhibit their
penetration in most of the cell membrane. Though viral vectors investigated initially appear
to be the most effective as they have natural tendency to infect cells but these carriers grieve
from some shortcomings like difficulties in pharmaceutical processing, immune response
against the transfection system, low capacity to incorporate exogenous genes, difficulties in
scale up and the possibility of reversion of an engineered virus to the wild type[61]. Despite
low transfection efficiency non viral vectors are favoured alternative to viral vectors owing to
their low cost of preparation procedures and absence of any immunogenicity. However most
of the preferred non viral vectors or delivery systems usually contains cationic polymers like
polyethyleneimine (PEI) or cationic lipids like 1,2-dioleyltrimethylammoniumpropane
(DOTAP) which are not satisfactory to use because of their polycationic nature making them
significantly toxic to normal cells. At present different approaches used in non-viral delivery
systems are aimed at reducing the toxicity and simultaneously increasing the transfection
efficacy. CaP/CaCO3 nanoparticles are attractive option among various non-viral gene
delivery methods because of their good binding capacity with nucleic acids, low toxicity and
endosome disruption effect. The calcium ion (Ca2+) of the carrier provides the binding site for
the phosphate backbone of antisense oligonucleotides. As previously mentioned these
nanoparticles also have endosome disrupting properties which makes them desirable carriers
specifically for DNA /miRNA/siRNA delivery. Calcium phosphate is in use for in vitro
transfection for more than 30 years. Although their ability to encapsulate negatively charged
molecules during precipitation and protection from enzymatic attack make them desirable
systems for the delivery of oligonucleotides but their rapid aggregation precludes its clinical
usage. After the initial mixing of the calcium ion and other inorganic anions the growth of the
precipitates is uncontrollable which results in large size particles triggering unfavourable
effects on cellular internalization which is a main cause for the steep drop in transfection
efficacy. This also causes difficulties in handling and reproduction. However, this limitation

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of Calcium based nanoparticles has been overcome to some extent by the use of hybrid
nanospheres [62]. Hybrid nanospheres are inorganic nanoparticles coated or stabilized with a
layer of organic molecules like polymer or lipid. The outer coating of polymer or lipid not
only protects the active constituent but also control the size, thermodynamically stabilize as
well as provide the site where a targeting moiety can be attached thus enhancing the delivery
efficiency[63]. The same techniques have also been extended for the delivery of other
immunoactive oligonucleotides like DNAzymes. Some of the groups have reported that small
molecules like PEGylated chelating agents bisphosphonates or inositolpentakisphosphate[64],
adenosine 5-triphosphate disodium salt (ATP)[65] can also be used as potential stabilizers for
CaP nanoparticles[66].
Out of the various approaches used to control particle size and aggregation of CaP and
CaCO3 nanoparticle lipid coated (LCP) are the ones which are most extensively explored.
They consist of a nanosized CaP core coated with a layer of cationic lipid (DOTAP, ()N,N,N- trimethyl-2,3-bis(z-octadec-9-ene-oyloxy)-1-propanaminium chloride ) and pegylated
lipid (DSPE-PEG). The CaP core gets dissolved at low pH in the endosome releasing the
entrapped siRNA whereas the PEG coating at the surface shield the genetic material from
nuclease attack leading to improved gene silencing in-vitro as well as in-vivo. The potential
of LCP in gene delivery can be adjudged from the fact that siRNA loaded LCP were efficient
in reducing 78% B16F10 lung metastasis in C57BL/6 mice that too at a very low dose [67].
Even though the technique is elegant the only concerns is that it involves the use of organic
solvents[68].
Just like lipids, potential of several polymers as stabilizers have also been established.
Conventionally used block co-polymers like Pluronic 127 are the ones that have been used to
some extent [64]. However, these simple classical block co-polymers are not sufficient
enough to produce small size nanoparticles because of which several research groups are
focussing on development of specially designed polymers with improved properties to make
the nanoparticles stable as well as more effective. A Japan based research group developed a
new block-copolymer poly (ethylene glycol)-block-poly (aspartic acid) (PEG-PAA) and used
that for coating of nanoparticles during precipitation technique only to inhibit the crystal
growth. Even though this organic-inorganic hybrid was found to have good colloidal stability
as well as high siRNA loading but the behaviour of these particles in physiological relevant
media needs to be established [69].
PEG modified bisphosphonates are also potential stabilisers due to their strong affinity for
naturally occurring crystalline calcium phosphate (nanophase HAP). These bisphosphonates
form a tight bond with the calcium present in the CaP and thus inhibit aggregation in in-vitro
systems. However, the use of bisphosphonates as stabilizers can raise the question of toxicity.
However, bisphosphonates being itself a therapeutic drug and moreover it can stabilize the
system in micromolar concentration the toxicity issue can be ruled out or can be assessed in
detail. Also the extreme simplicity of the process makes it a safe and attractive option [70].
Some of the groups have specially used positively charged molecules that serve the dual
purpose. They condense with pDNA/siRNA as well as stabilize the particles leading to
improved transfection efficiency. Semi-synthetic polymer DOPA-chitosan conjugate is an
example of it that has been used to prepare stabilised nano-CaP/CaCO3which adsorb at the
surface of growing particles to act as both gene carrier as well as stabilizer [71]. Synthetic

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polymers like protamine, polyallylamine hydrochloride and PEI have been reported to show
better transfection efficiency compared to natural polymers (alginate or chitosan) because of
their self endosomal escape property [72, 73]. Babak Mostaghaci et al reported one step
synthesis of stable amino functionalized calcium phosphate nanoparticles for DNA delivery.
They used novel N-(2-aminoethyl)-3-aminopropyltrimethoxysilane as stabilizing and
modifying agent and reported that different pH values can generate different crystalline forms
like HAP and Brushite. Whereas because of amine functionalization, the zeta potential of the
particles were positively charged leading to enhanced physical stability and pDNA
condensation capability [74, 75].
Another approach that has been used to prevent the aggregation and protection of the loaded
gene is layer-by-layer coating of CaP nanoparticles. In-vitro experiments of multiple shell
CaP nanoparticles have shown enhanced transfection efficiency when loaded with DNA or
siRNA in comparison to traditionally used single shell nanoparticles on different cancer cell
lines [76]. Even though polyelectrolytes like PEI are known to enhance transfection
efficiency of CaP nanoparticles their use in gene delivery should be limited because of
toxicity. Further important consideration must be given to the fact that coating the particles
with addition shells of either polymer or CaP itself may lead to significant increase in size of
the particle which can alter the bio-distribution as well as activity of the particles [77]. To
avoid this toxicity as well as to maintain the high transfection efficiency a group has
developed siRNA loaded hybrid micelles consisting of nanophase CaP and poly (ethylene
glycol)-block-charge conversion polymer. They observed 90% inhibition of luciferase
expression at 200nM compared to control micelles with sequence of siRNA [78]. Sangmok
Jang et al developed a phosphate backbone co-polymer PEG-b-PMOEP (poly (ethylene
glycol)-b-poly(2-methacryloyloxyethyl phosphate) which strongly interacts with calcium ions
of pDNA loaded nanoparticles and prevent aggregation or uncontrolled growth of
nanoparticles. The zeta potential of the particles was close to zero compared to plain CaP
nanoparticles which showed ZP of +33mV at normal pH. The advantage of using either
charge conversion polymer or the polymers which gives almost neutral zeta potential at blood
pH 7.4 is to have reduced unwanted interaction which leads to enhanced potential for in-vivo
applications compared to positively charged system [79]. Also the ease of surface
modification of these nanoparticles makes them acceptable for targeted delivery. Attachment
of molecules like Galactose can result in increased targeting capability of nanoparticle
[80].Treatment of antibody conjugated CaP nanoparticles to dendritic cells in in-vitro mixture
of primary cells resulted in more specific activation of immune system suggesting the
possibility of in-vivo targeting [81]. CaP Nanoparticles are efficient carries for almost all the
types of nucleic based drug which can be inference from the report that DNAzymes delivery
by arginine modified HAP nanoparticles showed much more biological activity in in-vivo
compared to the Fugene HD [82].
CaP nanoparticles are definitely very promising carriers but in recent years researchers have
also shown interest towards calcium carbonate particles for the gene delivery which might be
due to the fact of simplicity in manufacturing process of CaCO3/DNA co-precipitates with an
advantage that no buffer solution is required to adjust the pH compared to approach used for
the preparation CaP/DNA co-precipitates[83]. But the aggregation phenomenon during
preparation is rapid in both a CaP and CaCO3 nanoparticles which results in large increase in

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size. To overcome this issue just like CaP several strategies have been used for calcium
carbonate. Semi-synthetics and water soluble polymers like Carboxylmethyl cellulose
carboxymethyl chitosan, alginate, protamine or lipids[84] have been utilized to form hybrid
particles with CaCO3 with controlled sizes for gene and/or drug delivery. The implication of
calcium carbonate nanoparticles in siRNA delivery can be seen from the fact that these
nanoparticles results in 65 % transfection efficiency without any cytotoxicity in cell culture
experiments or mortality in whole animals unlike lipofectin which although have high
transfection efficiency (approx 70%) but also cause large proportion of cell death [85].CaCO3
nanoparticles loaded with siRNA exhibited significantly reduced vascular endothelial growth
factor-C (VEGF-C) expression in SGC-7901 cells .When activity was further checked in invivo models with subcutaneous xenografts tumor lymphangiogenesis, tumor growth and
regional lymph-node metastasis was dramatically suppressed [85]. Most of the experimental
results that have been published till now prove that nanosized CaP or CaCO3 possess higher
penetration rate in cells and transfection efficiency than conventional particles.

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5.3. Protein and antigen delivery

Protein therapy is the most direct and safe approach for treating disease. Proteins have
enormous biological and medical applications including cancers, vaccination, genetic
diseases and imaging. But their optimal performance depends upon the delivery vehicles
which protect their degradation until it reaches the target site. Proteins cannot be delivered
without the help of carrier system.
5.3.1. Vaccine delivery
Vaccines are also kind of antigenic proteins that when administered into the body stimulates
the production of antibodies[86]. Vaccines given with adjuvants have much more efficacy
than soluble antigens. Alum is one of the most regularly used adjuvant in licensed vaccines
for human use but it suffers from several disadvantages like severity of local tissue irritation,
minimal induction of cell mediated immunity, longer duration of inflammation and elicitation
of undesirable IgE response. Furthermore, the major objective behind the development of
new adjuvants especially against virus vaccine is fact that alum based vaccines are frequently
ineffective for the induction of antiviral immunity. CaP nanoparticles are the new generation
and suitable alternative to alum based adjuvant (especially for viral antigen). They are simple
having good immunogen adsorption efficiency as well as several outstanding immunological
features. Further nanoparticles itself bear the advantage of having relatively higher cellular
uptake. A research group in BioSante Pharmaceuticals, Georgia developed CaP
nanoparticles as adjuvant and compared them with alum for their ability to induce immunity
against herpes simplex virus type 2 (HSV-2) and EBV infection on intraperitoneal
administration. They observed that CaP nanoparticles were more potent as adjuvants, induced
very little inflammation at the site of entry and facilitated high percentage of protection by
inducing high titer of IgG2 antibody and neutralizing antibody. They proposed that CaP
nanoparticles has the potential to reduce the antigen dose by sustaining the release over
extended period of time[87]. The same group in 2002 demonstrated that CaP nanoparticles
can also be effective mucosal adjuvant[88]. Even in immunized mice against influenza virus
infection CaP nanoparticles loaded with immunoactive TLR9 ligand (CpG) combined with a
viral antigen from the influenza A virus hemagglutinin showed efficient uptake by dendritic

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cells in-vivo and elicit sufficient immune response with high production of effector T-cells
[89-91].Vladimir M Temchura et al very recently reported that how antigen coated CaP
nanoparticles can be efficient carriers to induce humoral immunity. They observed that in invitro conditions Hen Egg Lysozyme (HEL) antigen functionalized nanoparticles were
preferentially internalized by HEL-specific B-cells and were specifically able to increase the
surface expression of B cell activation markers. Nanoparticles were 100 folds more efficient
in activation of B cells compared to soluble antigen [92]. Flagellin, an immune activator,
generate more pronounced immunostimulation when loaded in CaP nanoparticles producing
higher levels of IL8 after intraperitoneal administration in mice compared to free flagellin
[93]. University of Washington researchers worked on on demand vaccines. They used an
engineered protein which mimics the antigen and can bind to CaP nanoparticles. After 8
months of immunization in mice there was 3 fold increase in the levels of protective killer T
cells compared to plain protein. This approach has potential application specifically in
developing countries against diseases which are hard to vaccinate [94]. HAP nanoparticles
loaded with malarial merozoite surface protein-1(19) (MSP-1(19)) was able to induce
vigorous immunoglobulin G (IgG) response, with higher IgG2a than IgG1 titre. Considerable
amount of IFN-g and IL-2 was observed in spleen cells of mice immunized with
nanoparticles compared to plain antigen or with alum[95]. To completely explore the
potential of CaP nanoparticles in antigen delivery several other animal models have been
tested. In a fish model Labeo rohita H the protein adsorbed on CaP particles elicited both
innate and adaptive immune responses. This parentral immunization persisted for 63 days and
also gave cross protection [96]. Results of vaccine delivery by CaP nanoparticles through
oculo-nasal route was found better than commercial available vaccines in chickens against
Newcastle Disease Virus (NDV)[97].
5.3.2. Insulin and other therapeutic protein delivery
Apart from the antigenic properties the other protein which has been extensively explored in
nanotechnology is Insulin. Insulin delivery other than the subcutaneous injection has always
been an elusive goal for many investigators. Even though the two FDA approved dosage
forms (buccal delivery through Oral-lyn and pulmonary delivery by Exubera) have shown
much improvement in diabetic patients but still the oral delivery of either proteins or drugs is
always the preferred choice. Exubera though being potential, later on withdrawn from the
market because of poor sales. So, the cost of the product is always important for market
applicability. Oral delivery of insulin also provides the advantage that oral absorption occurs
through the portal circulation mimicking the natural physiological secretion. Number of
polymeric carriers have been developed however none of them were successful enough to be
used commercially but the many beneficial properties of CaP nanoparticles makes them
potential carriers that can make it reach to the market. Rukmani Ramachandran et al
synthesized CaP nanoparticles core with PEG coating for the oral delivery of insulin. The
particles were able to sustain the release at physiological pH of intestine without any
degradation and conformational change in secondary structure of protein. The nontoxic
nature of the particle was also established by MTT assay and found to be non-toxic [98].
Later on lauric acid conjugated and alginate coated CaP nanoparticles were prepared with the
hypothesis that conjugating lauric acid will improve the intestinal uptake across the intestinal
epithelium [99]. Transdermal delivery of insulin is another non-invasive technique to attain

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sustained physiological levels of basal insulin. CaCO3 nanoparticles system has shown
successful delivery of insulin transdermally by significantly sustaining the decrease in blood
glucose level of normal mice as well as diabetic mice supporting the feasibility of developing
transdermal systems for human application. Efficacy of these nanoparticles in delivery of
antibodies has also been tested by in-vitro as well as in-vivo assays[100]. Lipid coated CaCO3
nanoparticles loaded with a therapeutic peptide EEEEpYFELV (EV) and conjugated with a
targeting ligand for lung cancer cells provoked higher apoptosis in H460 lung cancer cell line
as well as significant retardation in tumour growth on in-vivo administration with minimal
uptake by other organs[101]. Xia Cao et al. used the CaP nanoparticles as the delivery vector
of Yamanaka factors into HUMSCs which was capable of establishing virus free human
iPSCs [102].

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Fig 4: (A) Fate of CaP/CaCO3 nanoparticles after parenteral administration. The image represents how
CaP/CaCO3 nanoparticles behave on coming in contact with different pH conditions of blood, tumor extracellular
fluid and tumour intracellular compartment. (B) The Stability of the pH sensitive calcium carbonate core studied at
5, 10, 15, 20, 30 and 45 min exposure to different pH conditions (pH 5.5, 6.5 or 7.4). The disruption of the
formation of calcium cores was measured using dynamic light scattering (average mean value of detected
particle intensity per second (%)) Reproduced with permission from Elsevier [101](C) Kinetics of calcium
dissolution from mineralized micelles under pH control Reproduced with permission from Elsevier [103].

5.4. Small molecules delivery

Nanotechnology is spreading widely in drug delivery. Many drugs are being investigated for
therapeutic efficacy using nanoparticles for several diseases especially cancer. Use of
nanoparticles in cancer have specific advantage is that they can access to tumor site
selectively because of small size and modifiability [104]. Therefore just like gene and protein
delivery CaP and CaCO3 nanoparticles can be potential carriers for small molecules as well.
In contrast to inorganic nanoparticles like mesoporous silica, which are known to have porous
structure and thus higher loading, little work has been done on these alternative nanoparticles.
These nanoparticles also possess the advantage that they can be made sufficiently porous by
enhancing the drug loading which is one of the most important parameters for drug delivery
systems. In fact CaCO3 nanoparticles are very good carrier for drugs because of their porous
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nature and high surface area. These nanoparticles compared to mesoporous silica
nanoparticles have superior biocompatibility, biodegradability and also offers pH dependant
solubility. In our lab itself multilayered CaCO3 microparticle had been used for the oral
delivery of kaempferol in osteoporosis [105] [106]. These particles resulted in improved
outcomes like enhanced bone mineralization, bone mineral density and anabolic effects.

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5.4.1. Anticancer drugs delivery

In comparison to other diseases their use in cancer as nanoparticulate forms provides the
advantage of having stimuli based responsive targeted delivery where the encapsulated drug
is released only in the acidic environment of tumours (Fig 4). The small size, easy adsorption
of anticancer drugs on the surface through electrostatic interactions and pH tunable properties
makes them versatile nanoparticulate carriers for the targeted delivery of anticancer drugs.
CaP nanoparticles have been explored for the delivery of various anticancer drugs like
Cisplatin [107, 108], Methotrexate [109], docetaxel[110] and Gemcitabine triphosphate [111]
and in almost all the cases better efficacy observed with nanoparticles. In case of cisplatin
loaded CaP nanoparticles almost equivalent anti-tumor effectiveness was observed with
respect to plain drug solution but the main advantage was reduction in side effects where
weight loss and kidney damage was minimized. [107, 108]. The cytotoxicity studies
performed on A2780cis human ovarian cancer cell line indicated that conjugation procedure
did not adversely affect cisplatin structure and release [107]. CaP nanoparticles also provide
the advantage of intracellular delivery which can be implicated from the reports where
Methotrexate loaded nanoparticles prepared using reverse micelles approach showed more
than 90% release at endosomal pH and enhanced uptake of dextran FITC loaded particle
The particles were also found stable for the period of 90 days at 2-8 C but significant
agglomeration occurred at room temperature[109]. For cancer targeted delivery systems
reduce macrophage uptake is primary requirement and though CaP itself bear the advantage
of reduce macrophage uptake several groups are working on development of PEGylated
systems which can further prolong the circulation time in body. A research group has
developed PEGylated phospholipids (DSPE-PEG2000-COOH) stabilized and oleic acid
coated HAP nanoparticles as a vehicle for the delivery of docetaxel in hormone refractory
prostate cancer (HRPC) with the aim of enhancing the encapsulation efficiency. The in-vitro
activity studies conducted on PC3 and DU cell lines showed enhanced cytotoxicity as
compared to the free docetaxel (DTX)[112]. Later on another group developed multilayered
DTX loaded CaP nanoparticles prepared by precipitation using oppositely charged
polyelectrolytes ((poly(diallyldimethylammonium chloride) (PDADMAC) and poly(acrylate
sodium) (PAS)) as dual templates) and found much higher anticancer efficacy of
nanoparticles [113] .pH dependant profile of DTX was also reported by Chao Yang et al.
where they observed that only 20% of the adsorbed drug was released in 120h at pH 6.8 in
comparison to pH 5 (endosome pH) at which release was increased up to 90% in same
duration. Increase in cytotoxicity to A549 cells was also observed at 24 h by nanoparticles
which was almost 29.37% compared to free DTX [114]. Earlier reported LCP for siRNA
delivery have also been recently used for the delivery of anticancer drugs like gemcitabine
triphosphate (GTP, a bioactive form of gemcitabine & a nucleoside analog) and acyclovir
monophosphate (ACVP) for the treatment of non-small cell lung cancer and pancreatic

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cancer. The phosphorylated analogs of the corresponding drugs were develops to enhance the
drug loading in the CaP core with encapsulation efficiency over 60 %. Moreover use of GTP
or ACVP avoids the activation required in cell. [111]. With both the drugs encapsulation in
CaP nanoparticles resulted in enhanced efficacy against inhibiting tumor cell proliferation
and cell cycle progression[115].CaP nanoparticles itself are also potential apoptogenic agents
against cancer cells and this is because either they elevate the levels of Ca2+ in cytosol or may
be due to their nuclear localization on intracellular uptake which results in growth inhibition
or induction of apoptosis [116, 117]. HAP nanoparticles have been found toxic to several
cancer cell lines MGC80-3 > HepG2 > HeLa but had no toxicity on normal hepatic cell line
(L-02)[118]. Just like CaP nanoparticles CaCO3 nanoparticles are also potential candidates
for drug delivery particularly due to their porous nature. Several modifications have been
done to improve their usage and applicability for in vitro and in vivo purpose. Carboxy
Methyl Chitosan (CMC) modified CaCO3 micro and nanoparticles showed 60 %
encapsulation efficiency of doxorubicin (DOX) because of its porous structure and
electrostatic interaction between negatively charged CMC and positively charged DOX [62].
Low molecular weight drug betamethasone or bioactive protein loaded CaCO3 nanoparticles
showed enhanced chemical stability and sustained release on subcutaneous injection [122].
The nanoparticles also show potential application as calcium supplement where enhanced
serum calcium concentrations has been achieved than conventional formulations [123].
5.4.2. Ocular hypotensive agents delivery
Apart from the delivery of anticancer drugs the other major area where CaP nanoparticles
have been explored are in delivery of ocular hypotensive agents like timolol [119], 7hydroxy-2-dipropyl-aminotetralin [120] and methazolamide [121] with the major aim is to
sustain the drug levels in anterior tissue for a period of few hours and reduce the dosing
frequency as compared to the solution. Unlike CaP nanoparticles CaCO3 nanoparticles have
not been investigated much for systemic delivery.

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5.5. In-vitro/ in-vivo imaging and photodynamic therapy

Diagnosis of particular disease is very essential and for that several imaging methods have
been in practice. The broad spectrum profile, photochemical instability and low bleaching
threshold restricts the direct use of organic dyes and lanthanide chelates for bio-sensing and
imaging purposes[124]. Quantum dots which are stable and produce bright fluorescence are
frequently accepted as an alternative but their heavy metal toxicity limits their in-vivo use.
Nanocarriers which can encapsulate fluorescent molecules in their rigid matrix of colloidal
carrier can be a better substitute to shields interaction of dyes with solvent molecules as well
as to improve photostability and in vivo stability [125]. The use of surface decorated
particulate carriers for diagnostic imaging gives the additional advantage of cellular targeting
and therapeutic activity. The encapsulation of fluorophores in CaP/CaCO3 nanoparticulate
systems retains the stability associated with silica and polymeric nanoparticles but
simultaneously eliminates the problems associated with other systems and therefore results in
bright and stable fluorescent particles like quantum dots. Several reports are available CaP
nanoparticles have been used as carriers for fluorescent probes either by doping with
lanthanides or by surface functionalizing the organic dyes. CaP nanoparticles doped with
lanthanide were prepared by precipitation method at low temperature and stabilised with
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DNA to give a stable colloid. These particles were tested on in vitro HUVEC cell where
much enhanced uptake of particles were observed by confocal laser scanning microscopy. In
this study though the results were good but some morphological changes also observed which
raises the concern regarding future use of lanthanide doped particles on in-vivo use due to
potential toxicity[126, 127]. In another study conducted by Erhan I. Altnog lu et al
Indocyanine green (ICG, NIR emitting fluorophore) doped PEGylated CaP nanoparticles
prepared for in vivo studies of breast adenocarcinoma tumors. The dye loaded particles
showed 2-fold higher quantum efficiency per molecule and photostability which was 4.7-fold
longer relative to free molecule making these ICG doped particles an attractive fluoroprobe
for sensitive diagnostic imaging applications[128]. Number of other dyes such as rhodamine
WT, fluorscein, Cy3 loaded particles have also been developed along with drugs for
simultaneous imaging and drug delivery. Just like encapsulation surface functionalization is
also an approach for loading of actives in CaP nanoparticles than involves either
incorporation in coating layers or stabilisation by the actives only. In 2008 Kathirvel Ganesan
et al prepared CaP nanoparticles surface functionalized with water soluble p-TPPP
(5,10,15,20-tetrakis(4-phosphonooxyphenyl) porphine dye for photodynamic therapy in
cancer. Photodynamic therapy is a technique useful in the treatment of tumours and bacterial
biofilms in which light-sensitive dye is brought into the malignant tissue and irradiated with a
laser source. The excited dye destroys the malignant cells or bacteria by forming singlet
oxygen species. The goal of the study was to demonstrate that surface functionalization of
organic dyes on CaP can also be a biocompatible and harmless technique for the delivery of
fluorescing molecules[129]. They showed enhanced uptake of particles into NIH 3T3
fibroblast cells after a few hours of administration. Organic dyes can also be loaded on to
polymeric shell decorated nanoparticles. Epple and co-workers did the experimental analysis
by loading m-THPP (porphyrin dye) dye and methylene blue dye into the polymeric shell
functionalized CaP nanoparticles. The final charge of the particle was adjusted by selecting
an appropriate polymer. The efficiency of these positively charged nanoparticles were
compared to free dye on HT- 22(human colon adenocarcinoma cells), HIG-82 (synoviocytes
from rabbits) and J774A.1 cells (murine macrophages). The different activity profile was
obtained depending upon the cell line, like for J774 A the particles were toxic even in dark
and moderate activity was observed for HT29 epithelial cells but contrastingly particles
showed a good performance with HIG-82 synoviocytes[130]. Thus though they were able to
prepare water-dispersible system for dye by avoiding the alcoholic solution, the final
efficiency/activity was dependent upon the number of other parameters, e.g. particle charge,
kind of polymer, types of cell and cell culture medium (e.g. the presence of proteins) which
needs further studies for complete justification[131]. Incorporation of a pH sensitive dye
SNARF-1 and MRI probes in CaP nanoparticles has further widened the application of CaP
nanoparticles in imaging[132]. Peng Mi et al observed higher amount of contrast agent at
tumor position after intravenous injection of diethylenetriaminepentaacetic acid
gadolinium(III) Gd-DTPA (an MRI probe) encapsulated CaP nanoparticles than free GdDTPA which was due to EPR effect leading to higher tumor accumulation as well as higher
molecular relaxivity of Gd-DTPA than the free Gd-DTPA [133]. Recently, Yu-Cheng Tseng
et al reported how lymphotropism can be achieved by LCP with particle size ~25nm.
Intravenous administration of Indium (111In) loaded LCP by SPECT/CT imaging showed

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~70% ID/g accumulation in lymph nodes compared to ~25% ID/g accumulation in liver and
spleen. However they also found that particles with size >67nm were less lymphotropic.
These particles were sufficiently able to visualize 4T1 breast cancer lymph node metastasis
model [134]. Potential of CaP nanoparticles have also been shown through multi-modal
imaging where combined delivery of nuclear (99m-Technetium-methylene diphosphonate
99mTc-MDP), magnetic (Gadolinium Gd3+) and near infrared imaging (indocyanine green
ICG) in in-vivo models without showing any toxicity on healthy human mononuclear cells,
red-blood cells and platelets [135].

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5.6. Multifunctional nanoparticles

A recent advancement in nanomedicine-mediated therapy is the development of
multifunctional carriers which involves combined delivery of two or more active molecules
to serve the dual purpose. In fact these multifunctional nanoparticles have become a hot
topic in recent times for biomedical applications. So far several promising systems for codelivery have been developed based on liposomes, polymeric/lipidic and silica-based
nanoparticles[83]. Advantage of co-delivery of a gene and drug is that it can overcome the
multidrug resistance (MDR) to main line drug and thus can increase its therapeutic efficacy.
Just like other nanosized CaP and CaCO3 has also been investigated for simultaneous
delivery of multiple molecules and as expected in this arena also they had proven themselves
as potential candidates. On in vivo administration Alginate and KALA peptide modified
CaCO3 nanoparticles showed enhanced tumor cell apoptosis on simultaneous incorporation of
Doxorubicin (DOX) and gene plasmid (pGL3Luc) compared to unmodified nanoparticles.
The observed enhanced efficacy with Alginate modified CaCO3 nanoparticles was because of
decreased size and improved physical stability [136] whereas better activity observed with
KALA modified particle was because of endosomolytic and fusogenic property of the peptide
[137, 138]. Another example of such includes the combined incorporation of cytotoxic drug
(PTX, DOX) and fluorophores like Rhodamine-WT (Rh-WT)/fluorescein for theranostic
purpose. A research group in The Pennsylvania State University used microemulsion
approach to encapsulate the Cer6 and fluorescein in CaP nanoparticles. The nanoparticles
induced 80% growth inhibition in human vascular smooth muscle cells at 25 fold lower
concentration than ceramide solution in DMSO. The same research group also prepared
nanoparticles loaded with Cer10 and WT rhodamine and performed in vitro studies in UACC
903 melanoma cells. Results revealed 5% lower survival of melanoma cells as compared to
free drug dissolved in DMSO at a concentration of 5M. Also no morphological changes
were observed in control wells (particle without dye and ceramide) in both cases implying
that blank particle themselves were not toxic. The Cer10 doped particles were also found to
be significantly effective in sensitive as well as resistant MCF-7 breast cancer cell lines [139,
140]. Feng Chen et al developed dual doped Eu3+ (NIR probe) and Gd3+ (MRI probe) CaP
vesicle like nanospheres in the presence of PLA-mPEG amphiphillic block copolymer as
multifunctional delivery system for in-vivo bio-imaging and therapeutic activity. The
particles were found non-toxic in in-vitro. Further the particle when loaded with drug
(ibuprofen as a model drug) they observed that release of drug was sustained for a very long
time with more than 80 days [141].Gemcitabine monophosphate and c-myc siRNA codelivery by anisamide conjugated LCP nanoparticles triggered apoptosis in ~28% of H460

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subcutaneous and A549 orthotopic xenografts whereas only drug and only siRNA loaded
LCP triggered apoptosis in ~ 23 % and ~ 3 % tumor cells respectively. Tumour volume was
also found to be significantly less compared to the control on treatment with co-loaded
nanoparticles [142].

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5.7. As inert templates for polyelectrolyte capsule

The calcium based inorganic microparticles or nanoparticles can also be used as inert
templates for the preparation of drug loaded hollow nanocapsules in place of particles like
polystyrene latex and gold which are not biocompatible and biodegradable [106] [105].
Basically in this technique CaP or CaCO3 nanoparticles can serve as core template which
carries the bioactive molecule and over which alternate layers of oppositely charged polymers
are deposited as per the necessity followed by the removal of core in the presence of acidic
condition (Fig 5)[143]. The formed nanocapsules serve as containers for drug, enzymes or
other bioactive molecules. Since they are biodegradable they have an edge over other
frequently used non-biodegradable templates (like polystyrene nanoparticles) as it has been
reported that for some microcapsule systems core cannot be removed completely. These
systems have also been used as template for the synthesis of porous silica nanoparticles[144]
.

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Fig 5: Layer by layer deposition of oppositely charged polyelectrolyted on CaP/CaCO3nanoparticles followed by

the core removal

5.8. Mineralization

Mineralization is deposition of minerals like calcium phosphate or calcium carbonate over

other organic particles to develop robust and pH sensitive carrier systems. This kind of
deposition of inorganic material over organic material has widened the choice of new
functional nanoparticles. In the field of drug delivery stability of liposomes and micelles has
always been a great concern. They suffer the limitation of low structural ability and therefore
disassemble upon injection into blood stream and thus cannot be used for the preferential
targeting the drug to a site or for sustained effect. Chemical cross linkers used to enhance
their stability are mostly organic for which their toxicities are not well defined. Moreover the
process of crosslinking requires complex chemistry and also acts as permanent barriers which
reduce the drug release even at the target site. Earlier in inorganic deposition, silicication
was the only choice to enhance the stability of micelles against disrupting chemical agents.
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But recently CaP or CaCO3 has found to be better alternative showing superiority in respect
of biocompatibility [145].Apart from enhancing the stability, coating of the CaP and CaCO3
over other nanoparticles also makes them to show pH dependent dissolution behavior and
thus increasing their application in targeted or intracellular delivery without compromising
the net payload (Fig 6). This kind of mineralization at the surface of organic nanoparticles
does not change their structure since the process of deposition occurs without any chemical
reaction. There are several publications reporting the deposition of CaP over micelles
containing especially anticancer drugs loaded. It was observed that the release of doxorubicin
from polymeric micelles of poly(ethylene glycol)-b-poly(l-aspartic acid)-b-poly(lphenylalanine) (PEG-PAsp-PPhe) was almost negligible in extracellular condition and
significantly enhanced in the intracellular acidic conditions. Similarly, in another study
release of doxorubicin was sustained at normal blood pH-7.4 but rate was much higher at
mildly acidic conditions from mineralized PEGylated hyaluronic acid nanoparticles. The size
of the mineralized particle (153.7 4.5 nm) was also less than bare nanoparticles
(265.1 9.5 nm), which means that mineralization allows the formation of compact
nanoparticles[146].

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Fig 6: Schematic representation of mineralization of nanoparticles showing sustained release at pH 7.4 but burst
release at endosomal pH.

In another study mineralized chitosan-grafted-p(ethylene glycol)-dodecylamine (CMC-gPEG-DDA) micelles were developed which exhibited much enhanced serum stability as well
pH dependant release at endosomal pH (<5) [147]. In osteoporosis model CaP coating on
deoxycholate micelles loaded with simvastatin resulted in improved whole body therapeutic
effect [148].Another major recent application of mineralization by calcium phosphate is as
pore blocking agent for mesoporous silica nanoparticles. End-capped mesoporous silica
nanoparticles (MSPs) are attractive carriers for controlled drug delivery especially in cancer
due to their high surface area, low toxicity and large accessible pore volumes. These porous
particles uncap the pore blockers and release the drug at the target site by using specific
external stimuli like pH, temperature, photo-irradiation, and change in redox state or
enzymatic activity. Although these particles are potential delivery vehicles, many of the pore24
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blocking species have crucial drawbacks for clinical applications like poor applicability in
aqueous solutions and their body toxicity which has not been well defined. The
biocompatibility and pH dissolution behaviour of calcium phosphate makes it an exciting
pore blocking agent for mesoporous silica nanoparticles [149, 150]. It increases the
structural rigidity to otherwise fragile systems in addition to providing tailorable diffusive
barrier. The application of these mineralized nanoparticles has also been extended to protein
delivery. Cyclodextrin nanoparticles having size 310 nm releases the protein in 3 days on the
other hand mineralized cyclodextrin nanoparticles were much smaller in size (121nm) and
release the drug upto 21 days in sustained fashion due to the strong barrier of calcium
phosphate which inhibit the drug release. So another advantage that can be derived from this
data is sustaining the release at blood pH [151, 152]. Mineralization has also been used to
enhance the stability of nanogels in blood stream. Nanogels are drug delivery systems
composed of hydrophobically modified polysaccharides which form self assembled structures
in aqueous environment. These nanogels can trap drugs, proteins or semiconductor
nanocrystals or adsorbed liposomes but suffers from the stability issues in blood stream.
Hybridization of nanogels like liposomes adsorbed nanogels with inorganic compounds may
result in the fabrication of new hybrid and stable pH responsive drug carrier [153]. Definitely
controlled mineralization of polymeric carriers is a rational choice for successful
development of biocompatible, biodegradable, robust intracellular delivery systems. These
hybrid systems enhance the cytocompatability and biomedical application of other inorganic
nanoparticles. The approach has been used for Au and iron oxide nanoparticles where the
theranostic hybrid nanoparticles showed selective release of drug after cellular uptake [154].
Zhaomin Tang et al developed multifunctional drug and DNA encapsulated CaP coated
SPIONS (also called as magnetite nanoparticles SPIONs@PEI-CPs) for the magnetic guided
delivery to the tumor area. The particles displayed pH dependent property as well as high
transfection efficiency in A549 and HepG2 cells under external magnetic field [155].When
cellular viability of CaP coated amphiphilic self assembled gelatin iron oxide nanoparticles
(AGIO@CaP-DOX) was compared to bare nanoparticles it was found that HeLa cells were
viable over wide range of nanoparticles concentration for those with calcium phosphate
shell[156]. In tissue engineering this coating provides excellent cell attachment and possibly
induces growth of new bone. These kinds of delivery systems would provide more efficient
local delivery of higher drug payloads to the site of infection and in parallel minimize the risk
of systemic toxicity. Limitations like low encapsulation efficiency and poor controlled
degradation makes the use of mineralization favourable over directly used CaP/CaCO3
nanoparticles. Coating would provide all their advantages with the expense of their
limitations [157]. Many biodegradable polymers such as collagen, chitosan, proteins,
polyhydroxyalkanoates are considered as best choices for the mineralization because of their
biological speciality[158, 159]. This process of mineralization can be modified or greatly
enhanced depending upon the functional groups of the polymers which may act as nucleating
site for the deposition[160].

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5.9. Nanocomposite with other metals

Substitution of some of the ions in a matrix of CaP nanoparticles is an approach generally
used by material scientist to overcome some of its limitations. In case of CaP nanoparticles
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usually lower levels of transfection are observed because of low amount of DNA
condensation and poor endosomal escape and this is basically due to its physicochemical
properties. Apart from using as an efficient stabilizer, this property can also be altered with
the substitution of ions which are naturally present in the body. These ions for substitution
can be either cation or anion where cations include Mn2+, Mg2+, Zn2+, Na+,Sr2+ whereas
anions include F-,CO32-,HPO42-. Significantly enhanced transfection efficiency was reported
by A. Hanifi et al when Ca2+ ions were substituted with Sr2+ ions. As reported by them this
enhancement could either due to decrease in particle size and crystallinity or increase in
cationic strength which was leading to higher binding of DNA as well as improved
endosomal escape [161]. Other than these ions silver is also common dopant for CaP
nanoparticles. Silver in ionic form or particulate form is a proven bactericidal agent and
usually used in medicine by coating it on catheter, bandages or as silver containing implant
coatings. Alexander Peetscha, et al developed silver doped CaP nanoparticles and reported
that this kind of doping can be biocompatible approach for the delivery of silver ions and can
be easily be incorporated in bone cements, paste like formulations or to coat the implant
surface[162]. Silica doped CaP nanoparticles loaded with cisplatin in polymeric scaffolds
showed approximately 50% reduction in tumor volume (Hepatocellular carcinoma) after five
days of implantation [163].
Well-tailored gold nanostructures show Surface Plasmon Resonance (SPR) effects (means
that they can strongly absorb the NIR and destroy the cancer cells by localized heating
without affecting the normal tissues). Jinyoung Kang, et al developed the biocompatible CaP
gold nanocomposites conjugated with antibody Erbitux as a bimodal treatment which can
kill the cancer cells by inhibiting the EGFR signalling as well by localized photothermal
effect by the NIR laser. However in-vivo studies of these particles have not been done which
are essential for the complete evaluation of their clinical potential[164].
5.10. Other applications in medicine
Apart from their primary application these delivery systems are also known to be useful in
many other ways. For example, hollow structured CaP nanospheres activated by ultrasound
can be used for delivery of actives as they are known to collapse and transform into pinshaped crystallites under ultrasonic treatment to release the encapsulated drug [165]. Further
Calcium based nanoparticles have also proven their benefit in wound healing process after
intravenous administration by providing ionized calcium ions. Although intravenous
administered calcium chloride also enhance healing process but this cause significant
elevation in the total serum calcium level causing calcium related side effects[166]. Coating
of CaP or CaCO3 over other systems like emulsion or micelles can be useful artificial oxygen
carriers as they would combine the strength of polymersomes with permeability characteristic
of liposomes. Hydroxyapatite nanoparticles are used as particulate emulsifiers specifically for
oils containing ester groups. These emulsions are called as pickering emulsions stabilised by
solid particles at the interface [167].

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6. Drawbacks and challenges

Certainly CaP and CaCO3 nanoparticles possess many advantages and are attractive delivery
systems in biomedicine but they also suffer from some major drawbacks or limitation which
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constricts their wide utilization in clinical systems. The present section deals with major
issues associated with theses nanoparticles as well as discuss that how they can be overcome.

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6.1. Low drug loading

Loading or amount of encapsulation of therapeutic molecules is one of the most essential
parameters in a delivery system and therefore should be the primary focus while developing
the same for particular drug. Here comes one of the major limitations of these biologically
accepted ceramic nanoparticles. In comparison to other polymeric or lipidic delivery systems
they possess low drug loading capacity. In this case drug is basically adsorbed on particles
and this is further dependent upon many factors like the method of preparation used,
conditions at the time of preparation, size and morphology of the particles. Although
preloading of drug in particles is generally expected better than post loading but still it is very
less. CaCO3 nanoparticles are little better in this aspect to CaP nanoparticles as they possess
more porous structure thereby providing more space for drug binding. While several
techniques has been tried by researchers which includes making the amorphous or porous
particles to improve the drug loading still they have not reached to the point where the drug
loading in these particle become comparable to other soft nanoparticles.

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6.2. Rapid Aggregation

Rapid aggregation is another serious draw back in the fabrication process of these
nanoparticles. Co-precipitation is the most commonly employed technique but it bears
uncontrollable reaction rate which results in the formation of large sized particles, producing
unfavourable effects on cellular internalization and transfection efficiency. This crystal
growth also causes difficulties in handling and reproduction during the pharmaceutical scale
up. Various modification techniques like the use of synthesized polymers or lipids to control
the reaction rate have been tried but still researchers lag in approach which can control the
reaction as per the requirement. Though other commonly used techniques i.e. micro-emulsion
is much better with respect in controlling size and polydispersity, but the use of organic
solvents like cyclohexane which are difficult to remove completely limits the widespread use
of this technique.

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6.3. Toxicity
Biomedical advantages of nanoparticles are widely known but knowledge of their potential
threats or risk are just emerging. For examples, particles with size <100nm are expected to
cross the blood brain barrier which can lead to several brain diseases [168]. Even though
these ceramic nanoparticle are completely acceptable in biological system however, their
toxicity profile is yet to be addressed before reaching to clinical settings. Since it is known
that atherosclerotic plaques in the human body are largely composed of cholesterol along
with its derivatives and hydroxyapatite, a probable toxicity would be the atherosclerotic
complications associated with their undesirable deposition along the arterial walls. Another
possible toxicity may be attributed to the rapid increase in intracellular Ca2+ above
physiological concentrations. Even though their apoptogenic action on cancer cells is also
because of elevation of Ca2+ in cytosol but this toxicity may also occur in normal cells also
leading to their death. Generally many studies conducted on different cell lines revealed that
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the calcium based nanoparticles did not produce any cytotoxicity. In fact HAP nanoparticles
are known to inhibit proliferation of osteosarcoma cells but stimulate the proliferation of
bone marrow mesenchymal stem cells. Cao et al observed that even very high dose
(0.56mg/ml) of HAP nanoparticles was non-toxic to normal hepatic cells [169]. In contrast,
CaP nanoparticles showed toxicity at concentration more than 250g/ml in human
monocytes-derived macrophages (HMMs) implying that intervention with intracellular
[Ca2+] homeostasis would be the prime reason of nanoparticles toxicity. Slow degradation of
CaP nanoparticle at normal pH is also expected to raise concerns on in vivo application. For
example if used in implants it is possible that broken particles may migrate to other tissues
and may alter the gene expression of the cell. During the toxicity studies we must consider
this fact that any kind of toxicity associated with nanoparticles is very much dependent upon
the size as well as dose and therefore needs to be established for complete understanding of
biological effects of nano-sized particles [11].

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6.4. Difficult to predict release kinetics

Release kinetic involves the prediction of release of encapsulated active molecule from a
delivery system. pH dependent accelerated release of drug from these nanoparticles is well
known and this also makes it suitable for delivery of anticancer drugs and gene but yet
release at normal pH from them has not been assessed in detail. It has also been observed that
even though the particles are suitable for encapsulation of both water soluble and insoluble
drugs, sometimes it becomes difficult to control the rapid release at normal pH especially of
water soluble drugs.

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Applications

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7. Summary of applications in biomedicine:

Modifications

Remarks

References

Particles show strong aggregation behaviour in aqueous dispersion

[170]

p-DNA

No modification

GFP-DNA

poly(ethylene glycol)-functionalized
bisphosphonate stabilized

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Gene delivery

Nanoparticles were stable over time and could transfect efficiently

cells in vitro using physiologically relevant media.

Particle size and the size distribution were virtually unchanged even
delivery by lipid coated nanoparticles (LNCP)after 21 days of storage. AFM revealed that LNCP have a 5-fold higher
rigidity than common cationic liposomes.
block copolymer, poly(ethylene glycol)oligodeoxynucleotide (ODN) or
Good colloidal stability and high ODN- or siRNA-loading capacity
block-poly(aspartic acid) (PEG-PAA),
siRNA
reaching almost 100 %
stabilized nanoparticles
GFP encoding DNA (pEGFPDopa-chitosanconjugate stabilized
Increased serum stability and showed high cellular uptake efficiency
C1) / GFP-silencing siRNA
and trans-gene expression

[172, 173]
[69]
[71]

Hybrid micelles siRNA loaded

poly(ethylene glycol)-block-chargeconversional polymer (PEG-CCP)/

Low toxicity, inhibition of luciferase expression at low dose

,preferential tumor accumulation

[174]

pDNA loaded nanoparticles

phosphate back co-polymerPEG-b-PMOEP,

pH dependant polymers, particle size <100nm

[79]

DNAzymes loaded nanoparticles

Arginine modified HAP nanoparticles

Higher biological activity in in-vivo compared to the Fugene HD

pcDNA3-EGFP
(CaP/DNA/CaP/DNA)

ce
pt

ed

Plasmid DNA and Antiluciferase siRNA

[66, 171]

PEI stabilized

High Transfection of corneal endothelial cells

Drug delivery

Methotrexate
Docetaxel
Paclitaxel

Sustained release of drug from the nanoconjugates over time and The
nanoconjugates stabilised by DARVAN 811 cytotoxicity of the nanoparticles compared to that of the free drug in an
in vitro cell proliferation assay
Prepared by reverse emulsion
Sustained drug release with > 90% in 3-4 hrs at endosomal pH

Ac

Cisplatin

Gemcitabine triphosphate
(GTP),Acyclovir
monophosphate (ACVP)

Coated with oleic acid and DSPE-PEG

Sustained release profile in vitro and enhanced therapeutic efficacy on

prostate cancer cell lines

folic acid (FA) modified polyethylene glycol

(PEG) functionalized
LCP

[107]
[109]
[112]
[175-177]

Dramatic tumour inhibition with little in-vivo toxicity

[111, 178]

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[119-121]

Vaccine adjuvant

High titre of IgG2 antibody and neutralizing antibody, dose can be

reduced

[33]

PEG or fatty acid conjugationfor the oral

delivery of insulin

Prevention from degradation and conformational change

[98, 179]

Multishell nanoparticles

Nanoparticles were capable of inducing both innate and adaptive

immunity

[90]

Immunoactive oligonucleotide
with hemagglutinin
Hen Egg Lysozyme (HEL)
antigen functionalized
nanoparticles
HAP nanoparticles with MSP1(19) protein

PEI functionalized

Imaging and photodynamic therapy

Carboxy SNARF-1
Lanthanides including europium
and yttrium

[92]

High immunoglobulin response with nanoparticles compared to plain

antigen

[95]

Immunogen-controlled adjuvant mineralization for just-in-time

manufacturing of effective T cell vaccines

[180]

2-fold higher quantum efficiency

[128]

Only dye functionalised nanoparticles

Polymeric functionalised particles

Activities were dependent upon the cell lines

[130]

Demonstrated pH measurements in complex biological fluids and

[132, 181]

Enhanced fluorescence properties

[126, 127]

LCP

Lymph node metastasis (lymphotropic)

[134]

Polyethylene glycol capping

Improved diagnosis by synergistic effect

[182]

In-vitro and in-vivo imaging of breast and pancreatic cancer

[139, 140]

block copolymer polylactideeblockemonomethoxy(polyethyleneglycol)

(PLA-mPEG).

Dual imaging with combined drug release

[141]

LCP

Potent antitumor activity in both subcutaneous and orthotopic models of

NSCLC

[183]

Acicular hydroxyapatite with silica shell

Ac

Indium (111In) Loaded

Nuclear , magnetic and NIR coloaded multimodal
Multifunctional nanoparticles
ceramide (Cer6 and Fluorscein)
Ceramide 10-WT Rhodamine
Eu3+ (NIR probe) Gd3+(MRI
probe) and ibuprofen drug
loaded CaP vesicle like
nanospheres
c-myc siRNA and gemcitabine
monophosphate coloaded
particles
Inert templates

PEGylated nanoparticles

ce
pt

p-TPPP (5,10,15,20-tetrakis (4phosphonooxyphenyl) porphine

m-THPP (porphyrin dye)

Efficient in activation of B cells and humoral immunity

ed

Just in time vaccines ( model

antigen ovalbumin)
ICG ,NIR emitting dye

cr

Sustain the drug levels and reduce the dosing frequency as compared to
the solution

us

Insulin

Ocular drug delivery of hypotensive agents

M
an

timolol ,7-hydroxy-2-dipropylaminotetralin and

methazolamide
Protein delivery
Antigen against herpes simplex
virus type 2 (HSV-2) and EBV

30

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cr

[143]
[77]

[146, 149, 150, 153, 154, 158, 160,

184, 185].

Ac

ce
pt

ed

M
an

us

As template for layer by layer 150-250 nm inert core coated with alternate
LBL technology useful for the delivery of multiple kind of therepeutics
technique
layers of oppositely charged polymers
Double and triple shell calcium
Additional layers of calcium phosphate
Transfection efficiency better than single shell nanoparticles
phosphate nanoparticles
Mineralization
Hyaluronic acid micelles,
Cyclodextrin nanoparticles,
Enhanced physical stability, stability in blood, prevent the leaching as
Mesoporours silica nanocarriers
Calcium phosphate coating outside
well as to provide site specific delivery
gold nanoparticles, iron oxide
nanoparticles Nanogels

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8. Conclusion and future outlook

Use of bulk calcium based ceramics is well known especially in tissue engineering since they
are highly biocompatible, bioactive, non-immunogenic and possess homology to natural
inorganic material such as bones and teeth. They are being used successfully in therapeutics
for various applications in the form of macroscale (cements and scaffolds),microscale
(coatings, scaffolds) and nanoscale (colloidal particulate system, targeted therapy). Their use
in nanosize range is specifically gaining interest because in this range they exhibits
significant biological activity along with enhanced biological affinity and also respond well
to the natural physiological environment which is highly required in tissue
engineering.Several in-vitro and in-vivo reports are available demonstrating their potential in
wide areas including gene delivery, drug delivery and protein delivery through different
route. They have also shown potential application in diagnostic imaging of different diseases
especially cancers. This enhanced inclination of researchers towards calcium based ceramic
nanoparticles for therapeutic delivery of bioactives is because of their several exceptional
features over the existing organic and inorganic counterparts. First of them is their cost
effectiveness, biodegradability and ability to encapsulate various kinds of molecules like
proteins, dyes, drugs or oligonucleotides. Second is their pH sensitive behavior which helps
in targeted delivery of therapeutics to tumour as well as their endosome disrupting effect
which is mandatory in gene delivery. Third and more recent advancement which is receiving
considerable attention is their use as coating material over other metallic implants or
nanosized delivery systems like micelles, liposome or self assembled nanoparticles that not
only enhance their usefulness by enhancing the physical stability in in-vivo system but also
make them to show pH dependent behavior.
Although Calcium phosphates and carbonate nanoparticles seems to be attractive carriers,
their major limitation which hampers the clinical usage is to control the size during synthesis,
insufficient colloidal stability, low drug loading and unpredictable release kinetics in
physiological environment. Currently several groups working on these delivery systems have
addressed these problems as well as try to resolve them especially size control and rapid
aggregation by adapting different numbers of methods for their synthesis. Controlled Coprecipitation and microemulsion in the presence of different polymers, lipids or small
molecules as stabilizers have been investigated extensively out of which some of them have
shown good potential in preventing the rapid aggregation of nanoparticles. But apart from
their aggregation issues we must notice that even though they have predictable fate and
limited adverse effects in comparison to quantum dots, carbon nanotubes and some
polymeric/lipidic systems, detailed toxicity profile of these carriers is yet to be assessed and
should not be underestimated as biological properties of nanoparticle are altogether different
from their bulk properties. A complete understanding of interaction between living cells and
nanoparticles needs to be required to completely asses their biological activity and their
influence on human health. Further, issues like behaviour of surface-functionalized
nanoparticles in biological milieu, efficiency of on-site or targeted delivery and change in
physicochemical properties and biological activity of therapeutic molecules after
encapsulation require attention.
Much work has been done, but more efforts still needs to be taken on the above discussed
issues to meet challenges. In summary, after everything said we believe that despite several
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challenges calcium based nanoparticles definitely have much more potential to reach the
market with probabilities of improved outcomes as they are likely to face fewer regulatory
hurdles in comparison to newly designed organic or inorganic materials. But to be successful
in these endeavors we need to resolve the problems associated with them which can only be
done by bridging the gap between material scientist and life scientist. We need to understand
the importance of correlation between engineering aspects of particles to that of biomedical
performance of particles. In conclusion, it is implied that if we succeed in doing this than in
near future calcium based nanoparticles will be having much more advance application in
medicine comparison to any other delivery system.

an

us

Acknowledgement
The authors would like to thank CSIR-CDRI Government of India for their generous support
to this work.

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Graphical abstract

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