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 This 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 proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Highlights of the present review

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 Focus is more on systemic applications than localized delivery

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 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: [email protected] [email protected]

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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 material’s 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 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 20 Page 21 of 47

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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˙. Altınogˇ 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 (pGL3–Luc) 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 5µM. 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, silicification was the only choice to enhance the stability of micelles against disrupting chemical agents. 23 Page 24 of 47

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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 ( 3 PO< sub> 4 solution with Ca (OH)< sub> 2, Ultrasonics Sonochemistry, 8 (2001) 8588. [20] Z. Yang, Y. Huang, S.-T. Chen, Y.-Q. Zhao, H.-L. Li, Z.-A. Hu, Template synthesis of highly ordered hydroxyapatite nanowire arrays, Journal of materials science, 40 (2005) 1121-1125. [21] Q. He, Z. Huang, Y. Liu, W. Chen, T. Xu, Template-directed one-step synthesis of flowerlike porous carbonated hydroxyapatite spheres, Materials Letters, 61 (2007) 141-143. [22] J. Liu, K. Li, H. Wang, M. Zhu, H. Yan, Rapid formation of hydroxyapatite nanostructures by microwave irradiation, Chemical physics letters, 396 (2004) 429-432. [23] A. Escudero, M.E. Calvo, S. Rivera-Fernandez, J.M. de la Fuente, M. Ocana, Microwaveassisted synthesis of biocompatible europium-doped calcium hydroxyapatite and fluoroapatite luminescent nanospindles functionalized with poly(acrylic acid), Langmuir, 29 (2013) 1985-1994. [24] K. Lin, J. Chang, R. Cheng, M. Ruan, Hydrothermal microemulsion synthesis of stoichiometric single crystal hydroxyapatite nanorods with mono-dispersion and narrow-size distribution, Materials Letters, 61 (2007) 1683-1687. [25] J.-K. Han, H.-Y. Song, F. Saito, B.-T. Lee, Synthesis of high purity nano-sized hydroxyapatite powder by microwave-hydrothermal method, Materials chemistry and physics, 99 (2006) 235-239. [26] L.-y. Cao, C.-b. Zhang, J.-f. Huang, Synthesis of hydroxyapatite nanoparticles in ultrasonic precipitation, Ceramics International, 31 (2005) 1041-1044. [27] M. Fathi, E.M. Zahrani, Fabrication and characterization of fluoridated hydroxyapatite nanopowders via mechanical alloying, Journal of Alloys and Compounds, 475 (2009) 408414. [28] C.C. Silva, M.P.F. Graça, M.A. Valente, A.S.B. Sombra, Crystallite size study of nanocrystalline hydroxyapatite and ceramic system with titanium oxide obtained by dry ball milling, J Mater Sci, 42 (2007) 3851-3855. [29] X. Wang, J. Zhuang, Q. Peng, Y.D. Li, Liquid–Solid–Solution Synthesis of Biomedical Hydroxyapatite Nanorods, Advanced Materials, 18 (2006) 2031-2034.

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Graphical abstract

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