an insight

Accepted Manuscript

Title: An Insight into functionalized Calcium based InorganicNanomaterials in Biomedicine: Trends and Transitions

Author: Shweta Sharma Ashwni verma B. Venkatesh TejaGitu Pandey Naresh Mittapelly Ritu Trivedi P.R. Mishra

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

To appear in: Colloids and Surfaces B: BiointerfacesReceived date: 14-11-2014Revised date: 6-5-2015Accepted date: 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 InorganicNanomaterials in Biomedicine: Trends and Transitions, Colloids and Surfaces B:Biointerfaces (2015), http://dx.doi.org/10.1016/j.colsurfb.2015.05.014This is a PDF file of an unedited manuscript that has been accepted for publication.As a service to our customers we are providing this early version of the manuscript.The manuscript will undergo copyediting, typesetting, and review of the resulting proofbefore it is published in its final form. Please note that during the production processerrors may be discovered which could affect the content, and all legal disclaimers thatapply to the journal pertain.

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Highlights of the present review 1

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Discussion on characteristic properties in comparison to other 3nanomaterials and method of preparation 4

Focus is more on systemic applications than localized delivery5

Discussion on application in gene, protein and small molecules delivery 6for diseases like cancer, osteoporosis, diabetes and vaccination.7

Covers all the surface modification or functionalization that has been 8done to improve their applicability.9

Mineralization of calcium phosphate over other nanosized delivery 10systems 11

Discussion on Major challenges in clinical application.12

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An Insight into functionalized Calcium based Inorganic Nanomaterials in 18Biomedicine: Trends and Transitions 19

20Shweta Sharma1, Ashwni verma1, B Venkatesh Teja1, Gitu Pandey1, Naresh Mittapelly1, Ritu 21Trivedi2, P.R.Mishra1*22

231- Division of Pharmaceutics, CSIR-Central Drug Research Institute24

2-Division of Endocrinology, CSIR-Central Drug Research Institute25

B 10/1, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow (U.P.) 22603126

27282930

*Corresponding Author31Dr. P.R. Mishra Ph.D32Division of Pharmaceutics,33Preclinical South PCS 002/011,34CSIR-Central Drug Research Institute35B.S. 10/1, Sector-10, Jankipuram Extension,36Sitapur Road, Lucknow-226031,37India38Phone 91-522-2772450 (4537)39Fax: 91-522-2771941 E-mail: [email protected] [email protected]

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Abstract

Over the recent years the use of biocompatible and biodegradable nanoparticles in biomedicinehas become a significant priority. Calcium based ceramic nanoparticles like calcium phosphate(CaP) and calcium carbonate (CaCO3) are therefore considered as attractive carriers as they arenaturally 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:60The rapid growth of nanotechnology in the last 35 years of therapeutics can be seen from the 61fact that the several products of nano-formulations like Caelyx, Doxil, Transdrug , 62Abraxane etc are already there in the market and apart from that there is an extensive list of 63systems which are in clinical trials or being scientifically explored. The reason behind this 64unprecedented growth of applications in the area of nanoscience and nanotechnology is the 65important and unique capabilities of nanosystems that are not found in the bulk sample of the 66same system. It mainly includes their large surface area, intracellular delivery and quantum 67properties. Nanomaterials have been used in almost every field of biomedical research, such 68as targeted delivery vehicles for therapeutic agents, contrast agents, biosensors and artificial 69oxygen carriers. The use of nanomaterial in the delivery of therapeutics provides liberty to 70modify their fundamental properties such as diffusivity, solubility, drug release 71characteristics, blood circulation half-life, bio-distribution, metabolism and immunogenicity. 72Further, tailoring their surface properties allows temporal and site specific delivery of 73therapeutics useful in the treatment of variety of diseases and disorders [1]. Nanosized 74delivery systems used in biomedical research can widely be classified as polymeric 75nanoparticles, polysaccharide nanoparticles, protein nanoparticles, dendrimers, nanoshells, 76micelles, engineered viral nanoparticles and metallic nanoparticles. These systems have 77shown potential for several branches of medicine such as immunology, neurology, oncology, 78cardiology, endocrinology, pulmonary, ophthalmology, orthopedics and dentistry. Polymeric 79and lipidic nanoparticles are called as soft nanoparticles and currently the most employed80systems because of their biocompatible and biodegradable nature. On the other side inorganic 81particles like metallic and ceramic nanoparticles are known as hard nanoparticles because 82of their dense nature. In the current scenario of biomedicine application of inorganic 83nanoparticles has advanced rapidly and extensive amount of work is being done in their84synthesis and surface modification. Inorganic nanoparticles covers a wide range of materials 85and therefore can further be classified as metallic nanoparticles or ceramic nanoparticles. 86Metallic nanoparticles are the ones which are made up of metals like gold , silver or iron 87oxide whereas ceramic nanoparticles are made up of alumina (Al2O3), calcium phosphate 88(CaP), silica (SiO2), zirconia (ZrO2), titanium oxide (TiO2) or calcium carbonate (CaCO3) [2].89In the list of inorganic nanoparticles, calcium based ceramic nanoparticles are particularly 90gaining the interest of researchers as they can be synthesized easily with desired size and 91porosity and possess properties that other systems lack. Though, they have been in use for 92localized delivery from several decades, however; their systemic and targeted delivery is a 93recent topic of interest among researchers. The aim of the present review is to elaborately 94discuss their properties, method of preparation, applications and related key issues which 95need to be resolved. The review has been covered with most of the reports that have been 96published till date.97

9899

2. Constraints of other frequently used nano-particulate delivery systems100

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For the past several decades polymers, lipids and proteins are the most commonly used 101source materials for the development of nanoparticulate delivery systems. Even being 102potential drug carriers they have their own sort of troubles and limitations. Most challenging 103limitation with the commonly employed polymeric and lipidic systems is the usage of organic 104solvents during their synthesis leading to a big concern for their in-vivo applications.105Moreover, delivery systems like Liposomes, micelles and emulsions suffer the limitation of 106stability as well as leakage of drug molecules in systemic circulation before reaching to target 107side. Though the limitations have been overcome to a certain extent by the use of polymeric 108nanoparticles like PLGA (FDA approved polymer) but it is also known to produce acidic 109products on their degradation which may have unwanted side effects on long term 110administration. Further, most of these delivery systems are not cost effective and lack 111industrial scalability. Cationic delivery systems commonly used in siRNA or pDNA delivery 112are toxic in nature and promote unwanted uptake by reticuloendothelial systems (RES). This 113review discusses how these limitations can be overcome to a certain extent by the use of CaP 114and CaCO3 nanoparticles.115

1163. Nano-structured CaP and CaCO3 particles: Idea behind development117The keen observation of researchers that had attracted great interest of formulation scientists 118in developing mineralized biologically inspired delivery systems for therapeutics is that in 119biological systems most of the biomaterials such as bones, teeth or shells are composed of 120regular hierarchical structure of calcium phosphate and calcium carbonate. These structures 121have much superior mechanical and functional properties compared to the artificial materials.122So it was believed that development of delivery vehicles composed of natural bio-mineral 123would possess an excellent biocompatibility owing to their chemical similarity to human hard 124tissues (bones, teeth) and ions of intracellular signalling pathways. Moreover, they are 125already present in blood at relative high concentration. It was also observed that biologically 126formed CaP and CaCO3 were often nanocrystals (5-20 nm width by 60 nm length) that are 127precipitated under mild conditions and this natural selection as nanostructured materials 128provides them the capability of specific interactions with natural proteins. Hence the 129development of nanosized bioresorbable inorganic materials such as CaP and CaCO3 in 130biomedical research stands to benefit most. In fact, their use in hard tissue engineering and as 131a matrix for localized controlled delivery of therapeutic agents to bones and tissues is very 132well established [3]. But nowadays they are also being explored for systemic delivery of 133therapeutics and this is because of their several attractive features (listed below) over others 134which makes them excellent delivery systems. 135

1363.1. Biocompatibility and biodegradability 137

Ceramic nanoparticles made up of CaP and CaCO3 are completely biocompatible and 138biodegradable compared to any other nanoparticles. Unlike carbon nanotubes, quantum dots, 139magnetic nanoparticles, silica nanoparticles and metallic nanoparticles they are free of severe140toxicity. Metallic nanoparticles like Cr, Cd, Au, Ag, Se, Te,Co,TiO2, CuO and ZnO have141been shown to increase mutation frequency, produce oxidative lesions, decrease cell viability 142and induce damage to DNA. Contrary to this calcium based nanosized carriers belong to the 143

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safest class of materials known so far where by-products like Ca2+, PO43-or CO3

2-are already 144present in the bloodstream within the concentration range of 1-5mM [4]. If fabricated 145appropriately they can possess the same chemistry, size and crystalline structure as the 146targeted tissues which enhance the materials activity and bio-acceptability before releasing 147the drug. Calcium phosphate per se belongs to a category of GRAS (Generally Regarded as 148Safe) as reported by FDA [5]149

1503.2. In-vivo Stability151In-vivo stability refers to the stability of nanoparticles in biological matrix. In this respect 152most of the inorganic nanoparticles bears the superiority over their counterparts and thus are 153relatively much more suitable for in-vivo applications. For example, some of the polymeric 154systems swell or their porosity changes on change of the pH and temperature while these 155nanoparticles do not. Also, they overcome the major limitation of micelle and liposomes 156based carriers, which are subjected to dissipation at below specific critical concentration (a 157major obstacle in in-vivo administration). CaP and CaCO3 nanoparticles are much more 158robust, particularly those with Ca/P ratio equal to hydroxyapatite (HAP, naturally occurring 159calcium phosphate) which is not soluble in blood as blood is itself supersaturated with respect 160to HAP. Also polymers like PLGA and PLLA inhibit rapid or prompt release of the loaded 161drugs and undergo catalytic degradation with time leading to the generation of acidic 162products. These ceramic nanoparticles are not prone to microbial growth, thus its storage 163stability is good.164

1653.3. Ease of preparation and cost effectiveness166The simplicity in the manufacturing process with minimal raw materials (common salts) 167without the use of organic solvents makes them more cost effective and industrially relevant 168compared to other nanoparticulate systems. Convenience in fabrication of these nanoparticles 169in aqueous media provides a suitable platform for the surface functionalization with 170appropriate agents like siRNA, proteins and several polyelectrolytes.171

1723.4. Particles in very small size range173Their synthesis under controlled conditions can result in particles with size range

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animals because of longer biodegradation time, a property which is essential for diffusion 187controlled kinetics.188

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3.6. Suitability as carrier for gene delivery191Escaping the endosome for gene delivery on intracellular uptake is the biggest challenge and 192here calcium based nanoparticles play a significant role. They are the most viable option for 193gene delivery because of the endosomal escape effect. Their complete dissolution or 194disintegration at the low pH of endosome (

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Method of preparation of nanoparticles always play vital role to get the desired properties. 231Therefore, numerous methods have been adapted for synthesis of calcium based232nanoparticles, which are mainly classified as dry or wet synthesis. Dry synthesis includes 233solid state reaction, mechano-chemical reaction and plasma spraying whereas wet synthesis 234includes wet chemical route, solgel technique, hydrothermal reaction, homogeneous 235precipitation, emulsion processing route and recently microemulsions (Table 1). By means of 236these methods various kinds of nanoparticles with varying size, structure and crystal237morphology can be synthesized.238Table 1: Different methods of preparation239

240Method Morphology Advantages Disadvantages References

Co-precipitation

Spherical, needle or rod like

Low cost, easy to synthesize and bio-

macromolecules can control the aggregation

Low crystallinity and rapid aggregation

[9]

Mechano-chemical

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

[12, 13]

HydrothermalNanorods, needle like, spherical and

fibrous Nanocrystals

Highly crystallinity, purity and homogeneity

Time consuming , low yield and need special

devices[14-16]

Emulsion technique

Spherical nanocrystals, needle like, nanorods and

nanoplates

Size and morphology well controlled, low aggregation

and homogenous

Time consuming , use of organic solvents

[17, 18]

Ultrasonic method

Needle like or spherical

Nanocrystals

Low aggregation, narrow size distribution, low

temperature

Special device needed and low yield

[19]

Template method

Nanoplates, nanorods, flower

and layer structure mesoporous and

fibrous nanostructure

Size can be well controlled

Time consuming , low yield, employs template

agents and high sintering temperatures

required

[20, 21]

Microwave processing

Rod, bowknot, needle like and flower structure

High crystalline particles, narrow size distribution , size and morphology can

be well controlled, synthesis is fast

Needs device and large scale synthesis is

difficult[13, 22, 23]

Emulsion hydrothermalcombination

Rod, fibrous, spherical and strap

like structures

Synthesis at low temperatures, narrow size

distribution , low, aggregation and well

controlled size and shape

Needs special devices,reaction system is

complex, time consuming and low

yield

[24]

Microwave hydrothermal combination

Rod, spherical,fibrous and strap

like structures

High crystalline particles, fast synthesis and high temperatures are not

Needs special device, yield is low

[25]

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required

241The main advantage of dry synthesis is that it usually yields well crystallized products with 242precise stoichiometry but at the same time needs long time and high temperature for the243processing. On the other hand wet methods generally require low temperature. Nanosized 244HAP is a particular type of crystalline calcium phosphate nanoparticles (Ca10(PO4)6(OH)2) 245which consist of calcium-to-phosphorus ratio of 1.67 and is the most stable with a least 246solubility out of all calcium orthophosphates. It can be prepared in aqueous solutions by 247mixing exact stoichiometric quantities of calcium and phosphate ions solutions at pH>9, 248followed by boiling at high temperature for several days under a CO2-free atmosphere, 249filtration and drying[26].250Several other methods are continuously being developed like Mechanical alloying [27], ball 251milling [28], liquid-solid solution synthesis [29], laser induced fragmentation [30],252radiofrequency induction plasma [31], radiofrequency magnetron sputtering [32]however 253for all the practical purpose nanoparticles prepared by synthetic and low cost methods are254used. Co-precipitation and microemulsion are the commonly used methods at lab scale level255and are explained in detail (Fig 1.)256

257Co-precipitation is one of the most established techniques for the preparation of CaP and 258CaCO3 nanoparticles and is also called as wet, chemical or aqueous precipitation. This259technique is widely chosen in contrast to another technique because large amount can be 260produced in the absence of organic solvents that too at reasonable cost. The only by-product 261of the reaction is water. The reaction involves the precipitation of nanophase CaP/CaCO3262from an aqueous solution containing calcium and phosphate/carbonate ions using various 263reactants such as calcium hydroxide, calcium nitrate, calcium citrate, calcium chloride, 264sodium carbonate, diammonium phosphate, disodium phosphate, and orthophosphoric etc. 265The reaction was first proposed by Yagai and Akoki where they used calcium hydroxide and 266orthophosphoric acid as starting materials for the preparation of nanoparticles. Later on, the 267same process was modified as the different groups used different starting materials. The 268precipitation process is highly dependent upon various critical process parameters like initial 269concentration of calcium and phosphate/carbonate ions, addition rate, temperature, stirring270rate and final pH of the medium. The major limitation or disadvantage of this technique is 271difficult to control the size by preventing particle aggregation. Though several approaches 272have been tried by using different types of polymers, lipids or molecules that can prevent the 273size of the particles but still the particle aggregation remains the major concern of this 274method. Morphology of the particles is quite sensitive to the presence of additives which also 275control the zeta potential of colloidal particles during precipitation[33].Some researchers 276have carried out the precipitation process in a simulated body fluid to obtain biomimetic 277nanoparticles. Recently advanced techniques like microreactors consisting of microchannels 278are also being used to enable the reaction to be performed in several orders smaller than the 279conventional reactors. Advantages of using these microreactors are high speed mixing, better 280control over the reaction and high yield [34]. Entrapment of molecules can either be carried 281

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out during the precipitation process (pre-loading) or after the preparation of nanoparticles282(post-loading). Conjugation of proteins, polymers, peptides, cell penetrating moieties or other 283functional ligands onto the surface of CaP nanoparticles is usually expected to proceed non-284covalently that is via adsorption which is governed by the hydrophobic or vanderwaals 285forces.286

287Another approach although less explored is microemulsion or reverse micelles technique,288where inverse micelles are used as nanosized vectors. The advantage of this technique is their 289capability to produce the particles of uniform shape, controlled size, narrow size distribution, 290low degree of agglomeration and high chemical purity. Reverse micelle or microemulsions 291are transparent isotropic liquid with nanosized droplets dispersed in continuous phase of oil 292and stabilized by a layer of surfactant/polymer at their interface. These water cavities 293stabilized by surfactant act like a cage which control the nucleation and growth resulting in 294generally very fine and monodisperse particles. Like co-precipitation it also depends upon the 295several parameters like ion concentration (Ca2+, PO4

2-,CO32-),ionic strength, pH, temperature 296

and concentration of surfactants etc. Microemulsion is also currently the most commonly 297used technique for the preparation of nanoparticles at laboratory scale with a wide range of 298surfactants. Hexadecyl(cetyl) trimethyl ammonium bromide (CTAB) surfactant has been 299frequently used to synthesize these nanoparticles where on altering concentrations of CTAB300nanoparticles with different diameter and morphology has been obtained [35]. Co-surfactants 301are also sometimes used in these systems to enhance the stability as well as solubilizing 302capacity of microemulsions. Example Co-surfactant like butanol is known to reduce the 303rigidity of CTAB/Toulene/water interface systems [36]. However, this method also suffers 304from some limitation, unlike co-precipitation which is extremely simple process, this 305technique is fairly complicated and involves the removal of organic solvents. Also the yield 306of particles is less. 307

308309310311312313314315

316317318319320321

Fig 1: Commonly used method for preparation of CaP/CaCO3nanoparticles322323

5. Biomedical applications:324

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Biomedical applications of CaP/CaCO3 can broadly be classified into localized delivery or 325systemic delivery. Earlier main applications of these nanoparticles were limited to localized 326tissue engineering however their excellent properties make them attractive for other purpose 327as well (Fig 2.). The following sections discuss their applications in wide areas. 328

329330

Fig 2: A) Various Different biomedical applications of CaP and CaCO3 nanoparticles B) Functionalization of CaP/ 331CaCO3 nanoparticles with different molecules332

3335.1. Hard tissue engineering334

3355.1.1. Bone repair336In hard tissue engineering the material with higher mechanical strength such as metals, alloys 337or their composites are always preferred. However, most of these materials are not 338bioresorbable and need surgical removal after the implantation. Also, most of them have the 339tendency to elicit an unnatural response which leads to the formation of capsule round the 340implant. The ideal material for the hard tissue engineering should be the one possessing 341sufficient mechanical strength and bioactivity to stimulate active tissue regeneration.342Though there are number of other ceramic materials which also bear these properties but 343particularly calcium based ceramic has gained more attention due to the excellent 344cytocompatibility, biodegradability and osteoinductive properties in physiological 345environment. CaCO3 has also been employed as scaffolds for bone repair and osteo-346regenerative applications use of CaP is much more extensive than its carbonate counterparts.347This might be due to the major occurrence of phosphate ions in human body than the 348carbonate ions. CaCO3 composites with CaP have broader range of applications than the 349calcium carbonates alone. In fact in natural systems CO3

2- is the most common dopant of350hydroxyapatite (HAP) and is known to improve bioactivity compared to pure HAP [37].351Earlier traditional ceramics as implants with grain size > 1micron were only used in 352orthopedic and dentistry as filler materials, structural forms and surface coatings but there 353biological properties were limited to a great extent because of large grain size which resulted354in variety of implant failures due to insufficient osteo-integration, osteolysis as well as 355implant wears. These conventional implants also lack the property of evoking natural cellular 356responses to regenerate tissue. The limitations of conventional implants as well as the fact 357

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that naturally occurring CaP also exist in nanocrystalline form led the necessity for 358development of new generation implants comprising of nanophase CaP that can lengthen the 359service time of implants and can dramatically reduce the patient pain [38]. They are known to 360overcome many limitations of conventional scaffolds and simultaneously exhibits enhanced 361bioactivity, resorbability and mechanical strength. This combination of nanotechnology and 362bioceramics has resulted in effective biological interactions as bioactivity of selected proteins 363is altered on these nanomaterials (important for cell recognition) because of their surface 364grain size, pore size, wettability, etc. which is closer to natural bone. Basically nanophase 365ceramics show these properties in tissue engineering because of the enhanced adhesion of 366osteoblasts compared to the conventional ceramics (Fig 3) [11, 39]. Nanocrystalline HAP is 367the most popular choice out of all calcium phosphate categories to be used in tissue 368engineering because of its high stability and structural similarity as a major inorganic 369component of biological systems [40]. CaP or CaCO3 nanoparticles in its 3D nanocomposites 370form with other metals, minerals or polymers are the major developments in hard tissue 371engineering. These 3D nanocomposites are composed of CaP or CaCO3 nanoparticles 372incorporated into a biodegradable polymer matrix having interconnected porous structure 373which serve as support and reinforce tissue regeneration or replacement in a natural way. 374These composites improve the mechanical properties as well as incorporate nanotopographic 375features that mimic the structure of natural bone. In Injectable hydrogels CaP or CaCO3376nanoparticles mainly serves as tool to control the release kinetics of bioactives by reducing377the fast degradation of hydrogels without affecting their self -healing behaviour of 378nanoparticles[41]. In 2014 Thi Phuong Nguyen et al developed the enzyme assisted injectable 379chitosan hydrogels consisting of biphasic calcium phosphate nanoparticles and HAP for bone 380regeneration and found that incorporation of nanoparticles increased both the mechanical 381strength as well as binding to mesenchymal stem cells (MSC) on the hydrogel composite382[42]. Now days synthetic polymers like poly (lactic acid), poly (glycolic acid), poly (lactide-383co-glycolide) are more widely used in scaffolds than the natural polymers or proteins as they 384avoid the risk of virus infection and their mechanical properties are better which can be 385controlled during synthesis. CaP also provides the advantage to buffer the acidic degradation 386product of some polymers like PLGA and other lactides and glycolides [43].The application 387of nanophase CaP/CaCO3 in localized delivery can be divided in two parts. First in tissue 388engineering, they can directly act as bone substitutes or regenerate the bone because of 389osteoinductive action. Secondly, they can be loaded with specific growth factors (e.g. growth 390hormones, bone morphogenetic protein, transforming growth factor-beta and insulin growth 391factor),genes or drugs for the local and controlled release specifically useful in the treatment 392of bone regeneration, bone tumour, osteoporosis or osteomyelitis [44]. NanoOss(bone void 393filler, US FDA approved) and Ostim (injectable paste, CE approved) are approved 394nanotechnology based medical devices as bone substitutes. Several groups have worked on395localized delivery of drugs, gene, growth factors etc. by means of CaP nanoparticles396composites. Kreb et al. reported that BMP-3 encoded plasmid DNA loaded in CaP 397Nanoparticles and embedded into injectable alginate hydrogel were more efficacious in bone 398regeneration applications[45]. They have gained specific attention in bone diseases like 399osteoporosis and osteomyelitis because of their capability to augment the osteogenic response 400of osteoblasts [46, 47]. Fibres loaded with pDNA-CaP Nanoparticles encoding basic 401

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fibroblast growth factors (pbFGF) and vascular endothelial growth factor (pVEGF) led to 402drastically higher density of mature blood vessels than those containing plain plasmid and 403also particles showed higher cell viability as well as comparatively less inflammation 404compared to PEI-pDNA complex[48]. Localized delivery of pDNA, siRNA or shRNA by405multishell calcium phosphate nanoparticles embedded into different layers of multilayer films 406for gene silencing of osteopontin and osteocalcin is a new recent strategy in tissue 407engineering to control the bone formation[49]. 4085.1.2. Enamel and tooth repair409Further nanocrystalline CaP/CaCO3 has also shown excellent results in enamel and dentin 410erosion, gingivitis, dentin hypersensitivity and marginal periodontitis. Like bones, 411nanostructured CaP is also analogous to the basic building block of enamel which is needle 412like apatite crystals with diameter between 20-40nm. Thus their remineralizing nature, 413capability to neutralize lactic acid and their mechanical strength make them better tools414compared to the commercially available dental composites [50, 51]. This was clearly visible415when eroded sample of tooth were treated with both nanophase HAP as well as traditional 416HAP crystals where in-vitro repair effects were much effective with nanophase HAP 417compared to conventional crystals. MD et al reported enhanced remineralization under nano-418composites of CaP after the demineralization/remineralization treatment as compared to the 419commercial fluoride releasing composites[52]. Dental fillers with nanophase CaP have also 420shown substantially reduced caries formation and mineral loss from enamel near the margins 421of nanocomposite compared to the control composites, which may be due to the release of 422calcium and phosphate ions from the composites [53].The efficacy of these composites in 423caries or periodontitis can further be improved by encapsulating nanoparticles with molecules 424like antibacterial agents chlorhexidine[54] or using them along with Ag nanoparticles having 425inherent antibacterial properties[55]. Most of the results show that nanophase CaP ceramics426have enhanced in-vivo bioactivity and thus have prospective application in tissue engineering.427

4285.1.3. Coating material for other material implants429Nanostructured CaP as coating material for biomedical metals is another application in hard 430tissue engineering. Elements such as cobalt chromium alloy, titanium and its alloy and 431stainless steel are coated with nano-CaP to improve their biological attributes. Their coating 432on biomedical metals accelerates growth of natural cells and thus enhances their fixation 433making them useful for high load sites such as femoral and tibial bones. This approach is 434basically used to improve the bonding between the implants and surrounding bones by 435enhancing the interaction between the proteins, cells and tissue on the implant surface [56]436[57-59]. Hamdan S Alghamdi et al reported that titanium implants coated with 437bisphosphonate (BP)-loaded CaP nanoparticles when implanted for 4 weeks, bone volume 438and bone area was significantly increased compared to control implants in healthy and 439osteoporotic conditions. They concluded that simultaneous targeting of bone resorption (by 440BP) and bone formation (nano-CaP) can be an effective strategy in bone implants for 441osteoporotic conditions [60]. 442

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Fig 3: Osteoblast adhesion on conventional implants and nano-calcium based implants. The number of cells 448adhered to nanoparticles based implants are more compared to conventional implants449

450451

5.2. Gene and siRNA delivery452Gene therapy is the impending treatment option for some incurable and genetic disorders 453(such as cancer, diabetes, hemophilia etc.) which works by substituting the defective gene or 454by silencing the undesired mRNA. However, the delivery of these molecules inside the cell 455has always been challenging task because of their inherent properties like large size, negative 456charge and rapid degradation by enzymes like RNAse and nuclease which inhibit their 457penetration in most of the cell membrane. Though viral vectors investigated initially appear 458to be the most effective as they have natural tendency to infect cells but these carriers grieve459from some shortcomings like difficulties in pharmaceutical processing, immune response 460against the transfection system, low capacity to incorporate exogenous genes, difficulties in 461scale up and the possibility of reversion of an engineered virus to the wild type[61]. Despite 462low transfection efficiency non viral vectors are favoured alternative to viral vectors owing to 463their low cost of preparation procedures and absence of any immunogenicity. However most 464of the preferred non viral vectors or delivery systems usually contains cationic polymers like 465polyethyleneimine (PEI) or cationic lipids like 1,2-dioleyltrimethylammoniumpropane466(DOTAP) which are not satisfactory to use because of their polycationic nature making them 467significantly toxic to normal cells. At present different approaches used in non-viral delivery 468systems are aimed at reducing the toxicity and simultaneously increasing the transfection 469efficacy. CaP/CaCO3 nanoparticles are attractive option among various non-viral gene 470delivery methods because of their good binding capacity with nucleic acids, low toxicity and 471endosome disruption effect. The calcium ion (Ca2+) of the carrier provides the binding site for472the phosphate backbone of antisense oligonucleotides. As previously mentioned these473nanoparticles also have endosome disrupting properties which makes them desirable carriers 474specifically for DNA /miRNA/siRNA delivery. Calcium phosphate is in use for in vitro475transfection for more than 30 years. Although their ability to encapsulate negatively charged 476molecules during precipitation and protection from enzymatic attack make them desirable 477systems for the delivery of oligonucleotides but their rapid aggregation precludes its clinical 478usage. After the initial mixing of the calcium ion and other inorganic anions the growth of the 479precipitates is uncontrollable which results in large size particles triggering unfavourable480effects on cellular internalization which is a main cause for the steep drop in transfection 481efficacy. This also causes difficulties in handling and reproduction. However, this limitation 482

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of Calcium based nanoparticles has been overcome to some extent by the use of hybrid 483nanospheres [62]. Hybrid nanospheres are inorganic nanoparticles coated or stabilized with a 484layer of organic molecules like polymer or lipid. The outer coating of polymer or lipid not 485only protects the active constituent but also control the size, thermodynamically stabilize as 486well as provide the site where a targeting moiety can be attached thus enhancing the delivery 487efficiency[63]. The same techniques have also been extended for the delivery of other 488immunoactive oligonucleotides like DNAzymes. Some of the groups have reported that small 489molecules like PEGylated chelating agents bisphosphonates or inositolpentakisphosphate[64], 490adenosine 5-triphosphate disodium salt (ATP)[65] can also be used as potential stabilizers for 491CaP nanoparticles[66]. 492Out of the various approaches used to control particle size and aggregation of CaP and 493CaCO3 nanoparticle lipid coated (LCP) are the ones which are most extensively explored. 494They consist of a nanosized CaP core coated with a layer of cationic lipid (DOTAP, ()-495N,N,N- trimethyl-2,3-bis(z-octadec-9-ene-oyloxy)-1-propanaminium chloride ) and pegylated 496lipid (DSPE-PEG). The CaP core gets dissolved at low pH in the endosome releasing the 497entrapped siRNA whereas the PEG coating at the surface shield the genetic material from 498nuclease attack leading to improved gene silencing in-vitro as well as in-vivo. The potential 499of LCP in gene delivery can be adjudged from the fact that siRNA loaded LCP were efficient 500in reducing 78% B16F10 lung metastasis in C57BL/6 mice that too at a very low dose [67].501Even though the technique is elegant the only concerns is that it involves the use of organic 502solvents[68]. 503Just like lipids, potential of several polymers as stabilizers have also been established. 504Conventionally used block co-polymers like Pluronic 127 are the ones that have been used to 505some extent [64]. However, these simple classical block co-polymers are not sufficient 506enough to produce small size nanoparticles because of which several research groups are 507focussing on development of specially designed polymers with improved properties to make 508the nanoparticles stable as well as more effective. A Japan based research group developed a 509new block-copolymer poly (ethylene glycol)-block-poly (aspartic acid) (PEG-PAA) and used 510that for coating of nanoparticles during precipitation technique only to inhibit the crystal 511growth. Even though this organic-inorganic hybrid was found to have good colloidal stability 512as well as high siRNA loading but the behaviour of these particles in physiological relevant 513media needs to be established [69].514PEG modified bisphosphonates are also potential stabilisers due to their strong affinity for 515naturally occurring crystalline calcium phosphate (nanophase HAP). These bisphosphonates 516form a tight bond with the calcium present in the CaP and thus inhibit aggregation in in-vitro517systems. However, the use of bisphosphonates as stabilizers can raise the question of toxicity.518However, bisphosphonates being itself a therapeutic drug and moreover it can stabilize the 519system in micromolar concentration the toxicity issue can be ruled out or can be assessed in 520detail. Also the extreme simplicity of the process makes it a safe and attractive option [70].521Some of the groups have specially used positively charged molecules that serve the dual 522purpose. They condense with pDNA/siRNA as well as stabilize the particles leading to 523improved transfection efficiency. Semi-synthetic polymer DOPA-chitosan conjugate is an 524example of it that has been used to prepare stabilised nano-CaP/CaCO3which adsorb at the 525surface of growing particles to act as both gene carrier as well as stabilizer [71]. Synthetic 526

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polymers like protamine, polyallylamine hydrochloride and PEI have been reported to show 527better transfection efficiency compared to natural polymers (alginate or chitosan) because of 528their self endosomal escape property [72, 73]. Babak Mostaghaci et al reported one step 529synthesis of stable amino functionalized calcium phosphate nanoparticles for DNA delivery. 530They used novel N-(2-aminoethyl)-3-aminopropyltrimethoxysilane as stabilizing and 531modifying agent and reported that different pH values can generate different crystalline forms 532like HAP and Brushite. Whereas because of amine functionalization, the zeta potential of the 533particles were positively charged leading to enhanced physical stability and pDNA 534condensation capability [74, 75].535Another approach that has been used to prevent the aggregation and protection of the loaded 536gene is layer-by-layer coating of CaP nanoparticles. In-vitro experiments of multiple shell 537CaP nanoparticles have shown enhanced transfection efficiency when loaded with DNA or 538siRNA in comparison to traditionally used single shell nanoparticles on different cancer cell 539lines [76]. Even though polyelectrolytes like PEI are known to enhance transfection 540efficiency of CaP nanoparticles their use in gene delivery should be limited because of 541toxicity. Further important consideration must be given to the fact that coating the particles 542with addition shells of either polymer or CaP itself may lead to significant increase in size of 543the particle which can alter the bio-distribution as well as activity of the particles [77]. To 544avoid this toxicity as well as to maintain the high transfection efficiency a group has 545developed siRNA loaded hybrid micelles consisting of nanophase CaP and poly (ethylene 546glycol)-block-charge conversion polymer. They observed 90% inhibition of luciferase 547expression at 200nM compared to control micelles with sequence of siRNA [78]. Sangmok 548Jang et al developed a phosphate backbone co-polymer PEG-b-PMOEP (poly (ethylene 549glycol)-b-poly(2-methacryloyloxyethyl phosphate) which strongly interacts with calcium ions 550of pDNA loaded nanoparticles and prevent aggregation or uncontrolled growth of 551nanoparticles. The zeta potential of the particles was close to zero compared to plain CaP 552nanoparticles which showed ZP of +33mV at normal pH. The advantage of using either 553charge conversion polymer or the polymers which gives almost neutral zeta potential at blood554pH 7.4 is to have reduced unwanted interaction which leads to enhanced potential for in-vivo555applications compared to positively charged system [79]. Also the ease of surface 556modification of these nanoparticles makes them acceptable for targeted delivery. Attachment 557of molecules like Galactose can result in increased targeting capability of nanoparticle558[80].Treatment of antibody conjugated CaP nanoparticles to dendritic cells in in-vitro mixture 559of primary cells resulted in more specific activation of immune system suggesting the 560possibility of in-vivo targeting [81]. CaP Nanoparticles are efficient carries for almost all the 561types of nucleic based drug which can be inference from the report that DNAzymes delivery 562by arginine modified HAP nanoparticles showed much more biological activity in in-vivo563compared to the Fugene HD [82]. 564CaP nanoparticles are definitely very promising carriers but in recent years researchers have 565also shown interest towards calcium carbonate particles for the gene delivery which might be 566due to the fact of simplicity in manufacturing process of CaCO3/DNA co-precipitates with an 567advantage that no buffer solution is required to adjust the pH compared to approach used for 568the preparation CaP/DNA co-precipitates[83]. But the aggregation phenomenon during 569preparation is rapid in both a CaP and CaCO3 nanoparticles which results in large increase in 570

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size. To overcome this issue just like CaP several strategies have been used for calcium 571carbonate. Semi-synthetics and water soluble polymers like Carboxylmethyl cellulose 572carboxymethyl chitosan, alginate, protamine or lipids[84] have been utilized to form hybrid 573particles with CaCO3 with controlled sizes for gene and/or drug delivery. The implication of 574calcium carbonate nanoparticles in siRNA delivery can be seen from the fact that these 575nanoparticles results in 65 % transfection efficiency without any cytotoxicity in cell culture 576experiments or mortality in whole animals unlike lipofectin which although have high 577transfection efficiency (approx 70%) but also cause large proportion of cell death [85].CaCO3578nanoparticles loaded with siRNA exhibited significantly reduced vascular endothelial growth 579factor-C (VEGF-C) expression in SGC-7901 cells .When activity was further checked in in-580vivo models with subcutaneous xenografts tumor lymphangiogenesis, tumor growth and 581regional lymph-node metastasis was dramatically suppressed [85]. Most of the experimental 582results that have been published till now prove that nanosized CaP or CaCO3 possess higher 583penetration rate in cells and transfection efficiency than conventional particles. 584

5855.3. Protein and antigen delivery586Protein therapy is the most direct and safe approach for treating disease. Proteins have 587enormous biological and medical applications including cancers, vaccination, genetic 588diseases and imaging. But their optimal performance depends upon the delivery vehicles 589which protect their degradation until it reaches the target site. Proteins cannot be delivered 590without the help of carrier system. 5915.3.1. Vaccine delivery592Vaccines are also kind of antigenic proteins that when administered into the body stimulates 593the production of antibodies[86]. Vaccines given with adjuvants have much more efficacy 594than soluble antigens. Alum is one of the most regularly used adjuvant in licensed vaccines 595for human use but it suffers from several disadvantages like severity of local tissue irritation, 596minimal induction of cell mediated immunity, longer duration of inflammation and elicitation 597of undesirable IgE response. Furthermore, the major objective behind the development of 598new adjuvants especially against virus vaccine is fact that alum based vaccines are frequently 599ineffective for the induction of antiviral immunity. CaP nanoparticles are the new generation 600and suitable alternative to alum based adjuvant (especially for viral antigen). They are simple 601having good immunogen adsorption efficiency as well as several outstanding immunological 602features. Further nanoparticles itself bear the advantage of having relatively higher cellular 603uptake. A research group in BioSante Pharmaceuticals, Georgia developed CaP604nanoparticles as adjuvant and compared them with alum for their ability to induce immunity 605against herpes simplex virus type 2 (HSV-2) and EBV infection on intraperitoneal 606administration. They observed that CaP nanoparticles were more potent as adjuvants, induced 607very little inflammation at the site of entry and facilitated high percentage of protection by 608inducing high titer of IgG2 antibody and neutralizing antibody. They proposed that CaP 609nanoparticles has the potential to reduce the antigen dose by sustaining the release over 610extended period of time[87]. The same group in 2002 demonstrated that CaP nanoparticles 611can also be effective mucosal adjuvant[88]. Even in immunized mice against influenza virus 612infection CaP nanoparticles loaded with immunoactive TLR9 ligand (CpG) combined with a 613viral antigen from the influenza A virus hemagglutinin showed efficient uptake by dendritic 614

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cells in-vivo and elicit sufficient immune response with high production of effector T-cells615[89-91].Vladimir M Temchura et al very recently reported that how antigen coated CaP 616nanoparticles can be efficient carriers to induce humoral immunity. They observed that in in-617vitro conditions Hen Egg Lysozyme (HEL) antigen functionalized nanoparticles were 618preferentially internalized by HEL-specific B-cells and were specifically able to increase the 619surface expression of B cell activation markers. Nanoparticles were 100 folds more efficient620in activation of B cells compared to soluble antigen [92]. Flagellin, an immune activator, 621generate more pronounced immunostimulation when loaded in CaP nanoparticles producing 622higher levels of IL8 after intraperitoneal administration in mice compared to free flagellin 623[93]. University of Washington researchers worked on on demand vaccines. They used an 624engineered protein which mimics the antigen and can bind to CaP nanoparticles. After 8 625months of immunization in mice there was 3 fold increase in the levels of protective killer T 626cells compared to plain protein. This approach has potential application specifically in 627developing countries against diseases which are hard to vaccinate [94]. HAP nanoparticles 628loaded with malarial merozoite surface protein-1(19) (MSP-1(19)) was able to induce 629vigorous immunoglobulin G (IgG) response, with higher IgG2a than IgG1 titre. Considerable 630amount of IFN-g and IL-2 was observed in spleen cells of mice immunized with 631nanoparticles compared to plain antigen or with alum[95]. To completely explore the 632potential of CaP nanoparticles in antigen delivery several other animal models have been 633tested. In a fish model Labeo rohita H the protein adsorbed on CaP particles elicited both 634innate and adaptive immune responses. This parentral immunization persisted for 63 days and 635also gave cross protection [96]. Results of vaccine delivery by CaP nanoparticles through 636oculo-nasal route was found better than commercial available vaccines in chickens against637Newcastle Disease Virus (NDV)[97].6385.3.2. Insulin and other therapeutic protein delivery639Apart from the antigenic properties the other protein which has been extensively explored in 640nanotechnology is Insulin. Insulin delivery other than the subcutaneous injection has always 641been an elusive goal for many investigators. Even though the two FDA approved dosage 642forms (buccal delivery through Oral-lyn and pulmonary delivery by Exubera) have shown 643much improvement in diabetic patients but still the oral delivery of either proteins or drugs is 644always the preferred choice. Exubera though being potential, later on withdrawn from the 645market because of poor sales. So, the cost of the product is always important for market 646applicability. Oral delivery of insulin also provides the advantage that oral absorption occurs 647through the portal circulation mimicking the natural physiological secretion. Number of 648polymeric carriers have been developed however none of them were successful enough to be 649used commercially but the many beneficial properties of CaP nanoparticles makes them 650potential carriers that can make it reach to the market. Rukmani Ramachandran et al 651synthesized CaP nanoparticles core with PEG coating for the oral delivery of insulin. The 652particles were able to sustain the release at physiological pH of intestine without any 653degradation and conformational change in secondary structure of protein. The nontoxic 654nature of the particle was also established by MTT assay and found to be non-toxic [98]. 655Later on lauric acid conjugated and alginate coated CaP nanoparticles were prepared with the 656hypothesis that conjugating lauric acid will improve the intestinal uptake across the intestinal 657epithelium [99]. Transdermal delivery of insulin is another non-invasive technique to attain 658

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sustained physiological levels of basal insulin. CaCO3 nanoparticles system has shown 659successful delivery of insulin transdermally by significantly sustaining the decrease in blood 660glucose level of normal mice as well as diabetic mice supporting the feasibility of developing 661transdermal systems for human application. Efficacy of these nanoparticles in delivery of 662antibodies has also been tested by in-vitro as well as in-vivo assays[100]. Lipid coated CaCO3663nanoparticles loaded with a therapeutic peptide EEEEpYFELV (EV) and conjugated with a 664targeting ligand for lung cancer cells provoked higher apoptosis in H460 lung cancer cell line 665as well as significant retardation in tumour growth on in-vivo administration with minimal 666uptake by other organs[101]. Xia Cao et al. used the CaP nanoparticles as the delivery vector 667of Yamanaka factors into HUMSCs which was capable of establishing virus free human 668iPSCs [102].669

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Fig 4: (A) Fate of CaP/CaCO3 nanoparticles after parenteral administration. The image represents how 674CaP/CaCO3 nanoparticles behave on coming in contact with different pH conditions of blood, tumor extracellular 675fluid and tumour intracellular compartment. (B) The Stability of the pH sensitive calcium carbonate core studied at 6765, 10, 15, 20, 30 and 45 min exposure to different pH conditions (pH 5.5, 6.5 or 7.4). The disruption of the 677formation of calcium cores was measured using dynamic light scattering (average mean value of detected 678particle intensity per second (%)) Reproduced with permission from Elsevier [101](C) Kinetics of calcium 679dissolution from mineralized micelles under pH control Reproduced with permission from Elsevier [103].680

6815.4. Small molecules delivery682Nanotechnology is spreading widely in drug delivery. Many drugs are being investigated for 683therapeutic efficacy using nanoparticles for several diseases especially cancer. Use of 684nanoparticles in cancer have specific advantage is that they can access to tumor site 685selectively because of small size and modifiability [104]. Therefore just like gene and protein 686delivery CaP and CaCO3 nanoparticles can be potential carriers for small molecules as well.687In contrast to inorganic nanoparticles like mesoporous silica, which are known to have porous 688structure and thus higher loading, little work has been done on these alternative nanoparticles.689These nanoparticles also possess the advantage that they can be made sufficiently porous by 690enhancing the drug loading which is one of the most important parameters for drug delivery691systems. In fact CaCO3 nanoparticles are very good carrier for drugs because of their porous 692

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nature and high surface area. These nanoparticles compared to mesoporous silica 693nanoparticles have superior biocompatibility, biodegradability and also offers pH dependant 694solubility. In our lab itself multilayered CaCO3 microparticle had been used for the oral 695delivery of kaempferol in osteoporosis [105] [106]. These particles resulted in improved 696outcomes like enhanced bone mineralization, bone mineral density and anabolic effects. 697

6985.4.1. Anticancer drugs delivery699In comparison to other diseases their use in cancer as nanoparticulate forms provides the 700advantage of having stimuli based responsive targeted delivery where the encapsulated drug 701is released only in the acidic environment of tumours (Fig 4). The small size, easy adsorption702of anticancer drugs on the surface through electrostatic interactions and pH tunable properties703makes them versatile nanoparticulate carriers for the targeted delivery of anticancer drugs. 704CaP nanoparticles have been explored for the delivery of various anticancer drugs like 705Cisplatin [107, 108], Methotrexate [109], docetaxel[110] and Gemcitabine triphosphate [111]706and in almost all the cases better efficacy observed with nanoparticles. In case of cisplatin 707loaded CaP nanoparticles almost equivalent anti-tumor effectiveness was observed with 708respect to plain drug solution but the main advantage was reduction in side effects where 709weight loss and kidney damage was minimized. [107, 108]. The cytotoxicity studies 710performed on A2780cis human ovarian cancer cell line indicated that conjugation procedure 711did not adversely affect cisplatin structure and release [107]. CaP nanoparticles also provide 712the advantage of intracellular delivery which can be implicated from the reports where 713Methotrexate loaded nanoparticles prepared using reverse micelles approach showed more 714than 90% release at endosomal pH and enhanced uptake of dextran FITC loaded particle 715The particles were also found stable for the period of 90 days at 2-8 C but significant 716agglomeration occurred at room temperature[109]. For cancer targeted delivery systems 717reduce macrophage uptake is primary requirement and though CaP itself bear the advantage 718of reduce macrophage uptake several groups are working on development of PEGylated 719systems which can further prolong the circulation time in body. A research group has 720developed PEGylated phospholipids (DSPE-PEG2000-COOH) stabilized and oleic acid 721coated HAP nanoparticles as a vehicle for the delivery of docetaxel in hormone refractory 722prostate cancer (HRPC) with the aim of enhancing the encapsulation efficiency. The in-vitro723activity studies conducted on PC3 and DU cell lines showed enhanced cytotoxicity as 724compared to the free docetaxel (DTX)[112]. Later on another group developed multilayered 725DTX loaded CaP nanoparticles prepared by precipitation using oppositely charged 726polyelectrolytes ((poly(diallyldimethylammonium chloride) (PDADMAC) and poly(acrylate 727sodium) (PAS)) as dual templates) and found much higher anticancer efficacy of 728nanoparticles [113] .pH dependant profile of DTX was also reported by Chao Yang et al.729where they observed that only 20% of the adsorbed drug was released in 120h at pH 6.8 in 730comparison to pH 5 (endosome pH) at which release was increased up to 90% in same 731duration. Increase in cytotoxicity to A549 cells was also observed at 24 h by nanoparticles 732which was almost 29.37% compared to free DTX [114]. Earlier reported LCP for siRNA 733delivery have also been recently used for the delivery of anticancer drugs like gemcitabine 734triphosphate (GTP, a bioactive form of gemcitabine & a nucleoside analog) and acyclovir 735monophosphate (ACVP) for the treatment of non-small cell lung cancer and pancreatic 736

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cancer. The phosphorylated analogs of the corresponding drugs were develops to enhance the 737drug loading in the CaP core with encapsulation efficiency over 60 %. Moreover use of GTP738or ACVP avoids the activation required in cell. [111]. With both the drugs encapsulation in 739CaP nanoparticles resulted in enhanced efficacy against inhibiting tumor cell proliferation 740and cell cycle progression[115].CaP nanoparticles itself are also potential apoptogenic agents 741against cancer cells and this is because either they elevate the levels of Ca2+ in cytosol or may 742be due to their nuclear localization on intracellular uptake which results in growth inhibition 743or induction of apoptosis [116, 117]. HAP nanoparticles have been found toxic to several 744cancer cell lines MGC80-3 > HepG2 > HeLa but had no toxicity on normal hepatic cell line 745(L-02)[118]. Just like CaP nanoparticles CaCO3 nanoparticles are also potential candidates 746for drug delivery particularly due to their porous nature. Several modifications have been 747done to improve their usage and applicability for in vitro and in vivo purpose. Carboxy 748Methyl Chitosan (CMC) modified CaCO3 micro and nanoparticles showed 60 % 749encapsulation efficiency of doxorubicin (DOX) because of its porous structure and 750electrostatic interaction between negatively charged CMC and positively charged DOX [62]. 751Low molecular weight drug betamethasone or bioactive protein loaded CaCO3 nanoparticles 752showed enhanced chemical stability and sustained release on subcutaneous injection [122]. 753The nanoparticles also show potential application as calcium supplement where enhanced 754serum calcium concentrations has been achieved than conventional formulations [123].7555.4.2. Ocular hypotensive agents delivery756Apart from the delivery of anticancer drugs the other major area where CaP nanoparticles 757have been explored are in delivery of ocular hypotensive agents like timolol [119], 7-758hydroxy-2-dipropyl-aminotetralin [120] and methazolamide [121] with the major aim is to759sustain the drug levels in anterior tissue for a period of few hours and reduce the dosing 760frequency as compared to the solution. Unlike CaP nanoparticles CaCO3 nanoparticles have 761not been investigated much for systemic delivery. 762

7635.5. In-vitro/ in-vivo imaging and photodynamic therapy764Diagnosis of particular disease is very essential and for that several imaging methods have 765been in practice. The broad spectrum profile, photochemical instability and low bleaching 766threshold restricts the direct use of organic dyes and lanthanide chelates for bio-sensing and 767imaging purposes[124]. Quantum dots which are stable and produce bright fluorescence are 768frequently accepted as an alternative but their heavy metal toxicity limits their in-vivo use. 769Nanocarriers which can encapsulate fluorescent molecules in their rigid matrix of colloidal 770carrier can be a better substitute to shields interaction of dyes with solvent molecules as well 771as to improve photostability and in vivo stability [125]. The use of surface decorated 772particulate carriers for diagnostic imaging gives the additional advantage of cellular targeting 773and therapeutic activity. The encapsulation of fluorophores in CaP/CaCO3 nanoparticulate 774systems retains the stability associated with silica and polymeric nanoparticles but 775simultaneously eliminates the problems associated with other systems and therefore results in 776bright and stable fluorescent particles like quantum dots. Several reports are available CaP777nanoparticles have been used as carriers for fluorescent probes either by doping with 778lanthanides or by surface functionalizing the organic dyes. CaP nanoparticles doped with 779lanthanide were prepared by precipitation method at low temperature and stabilised with 780

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DNA to give a stable colloid. These particles were tested on in vitro HUVEC cell where781much enhanced uptake of particles were observed by confocal laser scanning microscopy. In 782this study though the results were good but some morphological changes also observed which 783raises the concern regarding future use of lanthanide doped particles on in-vivo use due to 784potential toxicity[126, 127]. In another study conducted by Erhan I. Altnog lu et al 785Indocyanine green (ICG, NIR emitting fluorophore) doped PEGylated CaP nanoparticles786prepared for in vivo studies of breast adenocarcinoma tumors. The dye loaded particles 787showed 2-fold higher quantum efficiency per molecule and photostability which was 4.7-fold 788longer relative to free molecule making these ICG doped particles an attractive fluoroprobe 789for sensitive diagnostic imaging applications[128]. Number of other dyes such as rhodamine790WT, fluorscein, Cy3 loaded particles have also been developed along with drugs for 791simultaneous imaging and drug delivery. Just like encapsulation surface functionalization is 792also an approach for loading of actives in CaP nanoparticles than involves either 793incorporation in coating layers or stabilisation by the actives only. In 2008 Kathirvel Ganesan 794et al prepared CaP nanoparticles surface functionalized with water soluble p-TPPP 795(5,10,15,20-tetrakis(4-phosphonooxyphenyl) porphine dye for photodynamic therapy in 796cancer. Photodynamic therapy is a technique useful in the treatment of tumours and bacterial 797biofilms in which light-sensitive dye is brought into the malignant tissue and irradiated with a 798laser source. The excited dye destroys the malignant cells or bacteria by forming singlet 799oxygen species. The goal of the study was to demonstrate that surface functionalization of800organic dyes on CaP can also be a biocompatible and harmless technique for the delivery of 801fluorescing molecules[129]. They showed enhanced uptake of particles into NIH 3T3 802fibroblast cells after a few hours of administration. Organic dyes can also be loaded on to 803polymeric shell decorated nanoparticles. Epple and co-workers did the experimental analysis 804by loading m-THPP (porphyrin dye) dye and methylene blue dye into the polymeric shell 805functionalized CaP nanoparticles. The final charge of the particle was adjusted by selecting 806an appropriate polymer. The efficiency of these positively charged nanoparticles were 807compared to free dye on HT- 22(human colon adenocarcinoma cells), HIG-82 (synoviocytes 808from rabbits) and J774A.1 cells (murine macrophages). The different activity profile was809obtained depending upon the cell line, like for J774 A the particles were toxic even in dark 810and moderate activity was observed for HT29 epithelial cells but contrastingly particles 811showed a good performance with HIG-82 synoviocytes[130]. Thus though they were able to 812prepare water-dispersible system for dye by avoiding the alcoholic solution, the final 813efficiency/activity was dependent upon the number of other parameters, e.g. particle charge, 814kind of polymer, types of cell and cell culture medium (e.g. the presence of proteins) which 815needs further studies for complete justification[131]. Incorporation of a pH sensitive dye 816SNARF-1 and MRI probes in CaP nanoparticles has further widened the application of CaP 817nanoparticles in imaging[132]. Peng Mi et al observed higher amount of contrast agent at 818tumor position after intravenous injection of diethylenetriaminepentaacetic acid 819gadolinium(III) Gd-DTPA (an MRI probe) encapsulated CaP nanoparticles than free Gd-820DTPA which was due to EPR effect leading to higher tumor accumulation as well as higher 821molecular relaxivity of Gd-DTPA than the free Gd-DTPA [133]. Recently, Yu-Cheng Tseng822et al reported how lymphotropism can be achieved by LCP with particle size ~25nm. 823Intravenous administration of Indium (111In) loaded LCP by SPECT/CT imaging showed 824

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~70% ID/g accumulation in lymph nodes compared to ~25% ID/g accumulation in liver and 825spleen. However they also found that particles with size >67nm were less lymphotropic. 826These particles were sufficiently able to visualize 4T1 breast cancer lymph node metastasis 827model [134]. Potential of CaP nanoparticles have also been shown through multi-modal 828imaging where combined delivery of nuclear (99m-Technetium-methylene diphosphonate 82999mTc-MDP), magnetic (Gadolinium Gd3+) and near infrared imaging (indocyanine green 830ICG) in in-vivo models without showing any toxicity on healthy human mononuclear cells, 831red-blood cells and platelets [135].832

8335.6. Multifunctional nanoparticles834A recent advancement in nanomedicine-mediated therapy is the development of 835multifunctional carriers which involves combined delivery of two or more active molecules836to serve the dual purpose. In fact these multifunctional nanoparticles have become a hot 837topic in recent times for biomedical applications. So far several promising systems for co-838delivery have been developed based on liposomes, polymeric/lipidic and silica-based 839nanoparticles[83]. Advantage of co-delivery of a gene and drug is that it can overcome the 840multidrug resistance (MDR) to main line drug and thus can increase its therapeutic efficacy. 841Just like other nanosized CaP and CaCO3 has also been investigated for simultaneous 842delivery of multiple molecules and as expected in this arena also they had proven themselves 843as potential candidates. On in vivo administration Alginate and KALA peptide modified 844CaCO3 nanoparticles showed enhanced tumor cell apoptosis on simultaneous incorporation of 845Doxorubicin (DOX) and gene plasmid (pGL3Luc) compared to unmodified nanoparticles. 846The observed enhanced efficacy with Alginate modified CaCO3 nanoparticles was because of 847decreased size and improved physical stability [136] whereas better activity observed with 848KALA modified particle was because of endosomolytic and fusogenic property of the peptide 849[137, 138]. Another example of such includes the combined incorporation of cytotoxic drug 850(PTX, DOX) and fluorophores like Rhodamine-WT (Rh-WT)/fluorescein for theranostic 851purpose. A research group in The Pennsylvania State University used microemulsion 852approach to encapsulate the Cer6 and fluorescein in CaP nanoparticles. The nanoparticles 853induced 80% growth inhibition in human vascular smooth muscle cells at 25 fold lower 854concentration than ceramide solution in DMSO. The same research group also prepared 855nanoparticles loaded with Cer10 and WT rhodamine and performed in vitro studies in UACC 856903 melanoma cells. Results revealed 5% lower survival of melanoma cells as compared to 857free drug dissolved in DMSO at a concentration of 5M. Also no morphological changes 858were observed in control wells (particle without dye and ceramide) in both cases implying859that blank particle themselves were not toxic. The Cer10 doped particles were also found to 860be significantly effective in sensitive as well as resistant MCF-7 breast cancer cell lines [139, 861140]. Feng Chen et al developed dual doped Eu3+ (NIR probe) and Gd3+ (MRI probe) CaP 862vesicle like nanospheres in the presence of PLA-mPEG amphiphillic block copolymer as 863multifunctional delivery system for in-vivo bio-imaging and therapeutic activity. The 864particles were found non-toxic in in-vitro. Further the particle when loaded with drug 865(ibuprofen as a model drug) they observed that release of drug was sustained for a very long 866time with more than 80 days [141].Gemcitabine monophosphate and c-myc siRNA co-867delivery by anisamide conjugated LCP nanoparticles triggered apoptosis in ~28% of H460 868

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subcutaneous and A549 orthotopic xenografts whereas only drug and only siRNA loaded 869LCP triggered apoptosis in ~ 23 % and ~ 3 % tumor cells respectively. Tumour volume was 870also found to be significantly less compared to the control on treatment with co-loaded 871nanoparticles [142].872

8735.7. As inert templates for polyelectrolyte capsule874The calcium based inorganic microparticles or nanoparticles can also be used as inert 875templates for the preparation of drug loaded hollow nanocapsules in place of particles like 876polystyrene latex and gold which are not biocompatible and biodegradable [106] [105]. 877Basically in this technique CaP or CaCO3 nanoparticles can serve as core template which 878carries the bioactive molecule and over which alternate layers of oppositely charged polymers 879are deposited as per the necessity followed by the removal of core in the presence of acidic 880condition (Fig 5)[143]. The formed nanocapsules serve as containers for drug, enzymes or 881other bioactive molecules. Since they are biodegradable they have an edge over other 882frequently used non-biodegradable templates (like polystyrene nanoparticles) as it has been 883reported that for some microcapsule systems core cannot be removed completely. These 884systems have also been used as template for the synthesis of porous silica nanoparticles[144]885.886

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Fig 5: Layer by layer deposition of oppositely charged polyelectrolyted on CaP/CaCO3nanoparticles followed by 890the core removal891

8925.8. Mineralization 893

894Mineralization is deposition of minerals like calcium phosphate or calcium carbonate over 895other organic particles to develop robust and pH sensitive carrier systems. This kind of 896deposition of inorganic material over organic material has widened the choice of new 897functional nanoparticles. In the field of drug delivery stability of liposomes and micelles has 898always been a great concern. They suffer the limitation of low structural ability and therefore 899disassemble upon injection into blood stream and thus cannot be used for the preferential 900targeting the drug to a site or for sustained effect. Chemical cross linkers used to enhance 901their stability are mostly organic for which their toxicities are not well defined. Moreover the902process of crosslinking requires complex chemistry and also acts as permanent barriers which903reduce the drug release even at the target site. Earlier in inorganic deposition, silicication 904was the only choice to enhance the stability of micelles against disrupting chemical agents. 905

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But recently CaP or CaCO3 has found to be better alternative showing superiority in respect 906of biocompatibility [145].Apart from enhancing the stability, coating of the CaP and CaCO3907over other nanoparticles also makes them to show pH dependent dissolution behavior and 908thus increasing their application in targeted or intracellular delivery without compromising 909the net payload (Fig 6). This kind of mineralization at the surface of organic nanoparticles 910does not change their structure since the process of deposition occurs without any chemical 911reaction. There are several publications reporting the deposition of CaP over micelles 912containing especially anticancer drugs loaded. It was observed that the release of doxorubicin 913from polymeric micelles of poly(ethylene glycol)-b-poly(l-aspartic acid)-b-poly(l-914phenylalanine) (PEG-PAsp-PPhe) was almost negligible in extracellular condition and 915significantly enhanced in the intracellular acidic conditions. Similarly, in another study 916release of doxorubicin was sustained at normal blood pH-7.4 but rate was much higher at 917mildly acidic conditions from mineralized PEGylated hyaluronic acid nanoparticles. The size 918of the mineralized particle (153.7 4.5 nm) was also less than bare nanoparticles919(265.1 9.5 nm), which means that mineralization allows the formation of compact 920nanoparticles[146].921

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Fig 6: Schematic representation of mineralization of nanoparticles showing sustained release at pH 7.4 but burst 926release at endosomal pH.927

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In another study mineralized chitosan-grafted-p(ethylene glycol)-dodecylamine (CMC-g-930PEG-DDA) micelles were developed which exhibited much enhanced serum stability as well 931pH dependant release at endosomal pH (

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blocking species have crucial drawbacks for clinical applications like poor applicability in 941aqueous solutions and their body toxicity which has not been well defined. The 942biocompatibility and pH dissolution behaviour of calcium phosphate makes it an exciting 943pore blocking agent for mesoporous silica nanoparticles [149, 150]. It increases the 944structural rigidity to otherwise fragile systems in addition to providing tailorable diffusive 945barrier. The application of these mineralized nanoparticles has also been extended to protein 946delivery. Cyclodextrin nanoparticles having size 310 nm releases the protein in 3 days on the 947other hand mineralized cyclodextrin nanoparticles were much smaller in size (121nm) and 948release the drug upto 21 days in sustained fashion due to the strong barrier of calcium 949phosphate which inhibit the drug release. So another advantage that can be derived from this 950data is sustaining the release at blood pH [151, 152]. Mineralization has also been used to 951enhance the stability of nanogels in blood stream. Nanogels are drug delivery systems 952composed of hydrophobically modified polysaccharides which form self assembled structures 953in aqueous environment. These nanogels can trap drugs, proteins or semiconductor 954nanocrystals or adsorbed liposomes but suffers from the stability issues in blood stream. 955Hybridization of nanogels like liposomes adsorbed nanogels with inorganic compounds may 956result in the fabrication of new hybrid and stable pH responsive drug carrier [153]. Definitely 957controlled mineralization of polymeric carriers is a rational choice for successful 958development of biocompatible, biodegradable, robust intracellular delivery systems. These 959hybrid systems enhance the cytocompatability and biomedical application of other inorganic 960nanoparticles. The approach has been used for Au and iron oxide nanoparticles where the 961theranostic hybrid nanoparticles showed selective release of drug after cellular uptake [154].962Zhaomin Tang et al developed multifunctional drug and DNA encapsulated CaP coated 963SPIONS (also called as magnetite nanoparticles SPIONs@PEI-CPs) for the magnetic guided 964delivery to the tumor area. The particles displayed pH dependent property as well as high 965transfection efficiency in A549 and HepG2 cells under external magnetic field [155].When 966cellular viability of CaP coated amphiphilic self assembled gelatin iron oxide nanoparticles967(AGIO@CaP-DOX) was compared to bare nanoparticles it was found that HeLa cells were 968viable over wide range of nanoparticles concentration for those with calcium phosphate 969shell[156]. In tissue engineering this coating provides excellent cell attachment and possibly970induces growth of new bone. These kinds of delivery systems would provide more efficient 971local delivery of higher drug payloads to the site of infection and in parallel minimize the risk 972of systemic toxicity. Limitations like low encapsulation efficiency and poor controlled 973degradation makes the use of mineralization favourable over directly used CaP/CaCO3974nanoparticles. Coating would provide all their advantages with the expense of their 975limitations [157]. Many biodegradable polymers such as collagen, chitosan, proteins, 976polyhydroxyalkanoates are considered as best choices for the mineralization because of their 977biological speciality[158, 159]. This process of mineralization can be modified or greatly 978enhanced depending upon the functional groups of the polymers which may act as nucleating 979site for the deposition[160].980

9815.9. Nanocomposite with other metals 982Substitution of some of the ions in a matrix of CaP nanoparticles is an approach generally 983used by material scientist to overcome some of its limitations. In case of CaP nanoparticles 984

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usually lower levels of transfection are observed because of low amount of DNA 985condensation and poor endosomal escape and this is basically due to its physicochemical 986properties. Apart from using as an efficient stabilizer, this property can also be altered with 987the substitution of ions which are naturally present in the body. These ions for substitution 988can be either cation or anion where cations include Mn2+, Mg2+, Zn2+, Na+,Sr2+ whereas 989anions include F-,CO3

2-,HPO42-. Significantly enhanced transfection efficiency was reported 990

by A. Hanifi et al when Ca2+ ions were substituted with Sr2+ ions. As reported by them this 991enhancement could either due to decrease in particle size and crystallinity or increase in 992cationic strength which was leading to higher binding of DNA as well as improved 993endosomal escape [161]. Other than these ions silver is also common dopant for CaP 994nanoparticles. Silver in ionic form or particulate form is a proven bactericidal agent and 995usually used in medicine by coating it on catheter, bandages or as silver containing implant 996coatings. Alexander Peetscha, et al developed silver doped CaP nanoparticles and reported997that this kind of doping can be biocompatible approach for the delivery of silver ions and can 998be easily be incorporated in bone cements, paste like formulations or to coat the implant 999surface[162]. Silica doped CaP nanoparticles loaded with cisplatin in polymeric scaffolds 1000showed approximately 50% reduction in tumor volume (Hepatocellular carcinoma) after five 1001days of implantation [163].1002Well-tailored gold nanostructures show Surface Plasmon Resonance (SPR) effects (means 1003that they can strongly absorb the NIR and destroy the cancer cells by localized heating 1004without affecting the normal tissues). Jinyoung Kang, et al developed the biocompatible CaP1005gold nanocomposites conjugated with antibody Erbitux as a bimodal treatment which can 1006kill the cancer cells by inhibiting the EGFR signalling as well by localized photothermal 1007effect by the NIR laser. However in-vivo studies of these particles have not been done which 1008are essential for the complete evaluation of their clinical potential[164].1009

10105.10. Other applications in medicine 1011Apart from their primary application these delivery systems are also known to be useful in 1012many other ways. For example, hollow structured CaP nanospheres activated by ultrasound 1013can be used for delivery of actives as they are known to collapse and transform into pin-1014shaped crystallites under ultrasonic treatment to release the encapsulated drug [165]. Further 1015Calcium based nanoparticles have also proven their benefit in wound healing process after 1016intravenous administration by providing ionized calcium ions. Although intravenous 1017administered calcium chloride also enhance healing process but this cause significant 1018elevation in the total serum calcium level causing calcium related side effects[166]. Coating 1019of CaP or CaCO3 over other systems like emulsion or micelles can be useful artificial oxygen 1020carriers as they would combine the strength of polymersomes with permeability characteristic 1021of liposomes. Hydroxyapatite nanoparticles are used as particulate emulsifiers specifically for 1022oils containing ester groups. These emulsions are called as pickering emulsions stabilised by 1023solid particles at the interface [167].1024

10256. Drawbacks and challenges1026Certainly CaP and CaCO3 nanoparticles possess many advantages and are attractive delivery 1027systems in biomedicine but they also suffer from some major drawbacks or limitation which 1028

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constricts their wide utilization in clinical systems. The present section deals with major 1029issues associated with theses nanoparticles as well as discuss that how they can be overcome. 1030

10316.1. Low drug loading1032Loading or amount of encapsulation of therapeutic molecules is one of the most essential 1033parameters in a delivery system and therefore should be the primary focus while developing 1034the same for particular drug. Here comes one of the major limitations of these biologically 1035accepted ceramic nanoparticles. In comparison to other polymeric or lipidic delivery systems 1036they possess low drug loading capacity. In this case drug is basically adsorbed on particles 1037and this is further dependent upon many factors like the method of preparation used, 1038conditions at the time of preparation, size and morphology of the particles. Although 1039preloading of drug in particles is generally expected better than post loading but still it is very 1040less. CaCO3 nanoparticles are little better in this aspect to CaP nanoparticles as they possess1041more porous structure thereby providing more space for drug binding. While several1042techniques has been tried by researchers which includes making the amorphous or porous 1043particles to improve the drug loading still they have not reached to the point where the drug 1044loading in these particle become comparable to other soft nanoparticles.1045

10466.2. Rapid Aggregation1047Rapid aggregation is another serious draw back in the fabrication process of these 1048nanoparticles. Co-precipitation is the most commonly employed technique but it bears 1049uncontrollable reaction rate which results in the formation of large sized particles, producing1050unfavourable effects on cellular internalization and transfection efficiency. This crystal 1051growth also causes difficulties in handling and reproduction during the pharmaceutical scale 1052up. Various modification techniques like the use of synthesized polymers or lipids to control 1053the reaction rate have been tried but still researchers lag in approach which can control the 1054reaction as per the requirement. Though other commonly used techniques i.e. micro-emulsion 1055is much better with respect in controlling size and polydispersity, but the use of organic 1056solvents like cyclohexane which are difficult to remove completely limits the widespread use 1057of this technique.1058

10596.3. Toxicity1060Biomedical advantages of nanoparticles are widely known but knowledge of their potential 1061threats or risk are just emerging. For examples, particles with size

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the calcium based nanoparticles did not produce any cytotoxicity. In fact HAP nanoparticles 1073are known to inhibit proliferation of osteosarcoma cells but stimulate the proliferation of 1074bone marrow mesenchymal stem cells. Cao et al observed that even very high dose 1075(0.56mg/ml) of HAP nanoparticles was non-toxic to normal hepatic cells [169]. In contrast, 1076CaP nanoparticles showed toxicity at concentration more than 250g/ml in human 1077monocytes-derived macrophages (HMMs) implying that intervention with intracellular 1078[Ca2+] homeostasis would be the prime reason of nanoparticles toxicity. Slow degradation of 1079CaP nanoparticle at normal pH is also expected to raise concerns on in vivo application. For 1080example if used in implants it is possible that broken particles may migrate to other tissues1081and may alter the gene expression of the cell. During the toxicity studies we must consider 1082this fact that any kind of toxicity associated with nanoparticles is very much dependent upon 1083the size as well as dose and therefore needs to be established for complete understanding of 1084biological effects of nano-sized particles [11].1085

10866.4. Difficult to predict release kinetics1087Release kinetic involves the prediction of release of encapsulated active molecule from a 1088delivery system. pH dependent accelerated release of drug from these nanoparticles is well 1089known and this also makes it suitable for delivery of anticancer drugs and gene but yet1090release at normal pH from them has not been assessed in detail. It has also been observed that 1091even though the particles are suitable for encapsulation of both water soluble and insoluble 1092drugs, sometimes it becomes difficult to control the rapid release at normal pH especially of 1093water soluble drugs.1094

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7. Summary of applications in biomedicine:

Applications Modifications Remarks References

Gene delivery

p-DNA No modification Particles show strong aggregation behaviour in aqueous dispe

an insight

Documents

functionalized calcium

accepted date

calcium phosphatecap

accepted manuscript1highlights

accepted manuscripttitle

calcium carbonate caco3

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b venkatesh teja1