Calcium Phosphate Nanoparticles Cytocompatibility

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Although calcium phosphate minerals are noted for their cytocompatibility, there are .... the fate of cell exposed to CPNs, morphology, porosity, solubility.

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Calcium Phosphate Nanoparticles A Serendipitous Paradox




Milad Pourbaghi Masouleha*, Vahid Hosseinib*, Masoud Pourhaghgouyc and Martin K. Bakht d,e a

Division of Drug Delivery and Tissue Engineering, School of Pharmacy, Boots Science Building, University of Nottingham , Nottingham NG7 2RD, U.K.; b Department of Health Science and Technology, ETH, Zürich, Switzerland; bNanotechnology and Advanced Materials Department, Materials and Energy Research Center, Karaj, Iran ;cLaboratory of Molecular Imaging and Therapy, Seoul National University College of Medicine, Seoul, Korea; dDepartment of Biological Science, University of Windsor, Windsor, Ontario, Canada

ARTICLE HISTORY Received: January 18, 2017 Accepted: February 14, 2017 DOI: 10.2174/157016381466617032 1115007

Abstract: Due to efficacious characteristics of calcium phosphate nanoparticles (CPNs), they have numerously been employed in nanomedicine, particularly as carrier for therapeutic and diagnostic agents, and also in tissue engineering. Although calcium phosphate minerals are noted for their cytocompatibility, there are outstanding findings from various studies that question whether they are still compatible with cells in nanoscale ranges or not and it leads to the controversial issue of CPNs cytocompatibility versus cytotoxicity. In this regard, it is necessary to know how CPNs could result in cytotoxicity for future studies. Interestingly, most of the researchers have attributed the cytotoxicity to triggering of apoptosis in CPNs-exposed cells. Furthermore, it is reported that CPNs could result in cancer cell demise through induction of apoptosis. According to the findings, not only CPNs are promising for cancer cell drug delivery, but also they have the potential to be employed as therapeutic agents. In this review, firstly the physical and chemical properties of CPNs and their application in medicine are reviewed. Moreover, the interaction between CPNs and different kind of cells are covered. Lastly, employment of CPNs as a therapeutic agent is discussed.

Keywords: Calcium phosphate nanoparticles, nanotoxicity, apoptosis, calcium signaling, mitochondrial-dependent pathway. 1. INTRODUCTION Nanomedicine has enlisted researchers to exploit calcium phosphate nanoparticles (CPNs) for many objectives, owning to their versatile properties. Utilization of CPNs as nanovehicular system for delivery of therapeutics such as nucleic acids (DNA or RNA), proteins and for diagnostic purposes like bioimaging agents or in some cases, combination of these two cargoes as theranostic platform, has deeply been investigated since last decade. Among the advantages of CPNs as nanocarrier, most of the researchers are in agreement with that its cytocompatibility is the prominent privilege in nanomedicine. According to the literature, it seems that cytocompatibility and efficiency of CPNs as carrier is well investigated and the results have shown not only any toxicity, but also appropriate functionality, in vitro [1, 2], and in vivo [3, 4]. It is well notorious that nanomaterials, even when made of inert elements (for example, gold), become extremely active at nanoscales and risks of employment of these materials in human body should be further investigated [3]. Based on what has been mentioned, although one of the prominent advantages of CPNs over other alternatives is their cytocompatibility, it alludes to take the risks of nanotoxicity into further consideration while their utilization matters. In 2003, Liu et al. reported the toxic effect of hydroxyapatite nanoparticles (HANs); a crystalline phase of calcium phosphate, against human hepatoma cells and the results forewarned the future employment of CPNs as one of the alternatives for delivery of therapeutic and diagnostic carriers as the most biocompatible materials ever known. They found that HANs not only *Address correspondence to these authors at Laboratory of Applied Mechanobiology, Department of Health Science and Technology, ETH, Zürich 8093, Switzerland; E-mails: [email protected] and Division of Drug Delivery and Tissue Engineering, School of Pharmacy, Boots Science Building, University of Nottingham, Nottingham NG7 2RD, U.K.; E-mail: [email protected]

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hinder proliferation of the cells but also induce apoptosis [5]. In this regard, very recently, Meena et al. reported that the higher dose of HANs induces oxidative stress to the human breast cancer cells, leading to apoptotic-like conditions [6]. It was supposed that this finding restricts employment of CPNs or at least HANs as nanovehicular system due to the apoptosis induction in cells; in contrast, they have variously been utilized without any proper attention toward their nanotoxicity referring to previous studies [7]. It appears rather paradoxical whether CPNs are cytocompatible or cytotoxic while it shows good functionality not only as carrier but also as apoptogenic agents. At present, there is an issue of deficiency of more clarified correlation between employments of CPNs cytocompatibility and cytotoxicity in various studies. The bridge that connects these two simultaneous responses toward cells (cytocompatibility and cytotoxicity) could disentangle the fact about this serendipitous paradox for future developments of CPNs. First and foremost, it is requisite to clarify how it interacts with cells that lead to toxicity. On the other hand, understanding their nanotoxicity might result in enhancement of the nanomedicine where these nanoparticles (CPNs) could exactly serve the needs. The focus of this review is to highlight this bridge. In this regard, firstly, the chemical and physical properties of CPNs are briefly explained. Afterwards, the interaction between cell and CPNs is mentioned. In this division, the biochemical mechanisms of interaction between CPNs and intracellular environment are considered to demonstrate how apoptosis gets activated. Meanwhile, the studies which investigated the impact of HANs on cells and apoptosis induction are reviewed. Lastly, the correlation between nanotoxicity resulting from CPNs and therapy utilizing these nanoparticles (NPs) is discussed. Utilizing the understanding resulting from the previous subdivisions; an induction that CPNs has the potential to be employed as therapeutic agent will be made.

© 2017 Bentham Science Publishers

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2. CALCIUM PHOSPHATE NANOPARTICLES Considering the employment of CPNs in nanomedicine, an enormous variety of required characteristics for divers applications are important. For tackling this issue, different kind of CPNs with various chemical and physical properties are synthesized. The desirable characteristics could be reached through the experimented synthesizing methods such as precipitation methods, sol-gel, hydrothermal and other conventional techniques which were thoroughly reviewed elsewhere [8]. On the condition that the synthesis process is meticulously controlled, the products of these methods could be the NPs with the predicted characteristics. Although CPNs are mostly made from two sources in which one provides Ca and the other one provides P, due to their degree of crystallinity, chemical and physical properties vary from one phase to another phase. This variance between the phases leads to difference in values that matter in biological dissolution of NPs such as biodegradability, stochiometric ratio and morphology. In order to well-understand the impact of CPNs in cell life, it is mandatory to notice their physicochemical properties. It should be noted that there could be an undesirable belief among the toxicologist or related researchers that all the calcium phosphate materials refer to hydroxyapatite (HA) and the difference between various crystalline phases of calcium phosphates should be further considered. 2.1. Chemical and Physical Properties Although CPNs have various characteristics for being mentioned, the chemical and physical properties that play key role in cell-NPs interaction could be satisfied with the properties including biodegradability and morphology [9]. Due to the fact that these characteristics could be affected by the microscopic arrangement of atoms inside the NPs, scrutiny of the periodic arrangement of atoms, or in other words, crystallinity is very important. As it is seen in Fig. 1, degree of crystallinity could result in the variation in morphology of CPNs. Specific surface area (SSA) is the characteristic that directly correlates to the morphology of the NPs. On the condi-

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tion of same morphology, although lower particle size is negligently supposed to result in higher SSA, porosity in NPs which affects the dissolution of NPs with biological milieu should be evaluated. In addition to the characteristics mentioned, stochiometric ratio of NPs would affect their biological responses. It was demonstrated that the Ca/P ratio in CPNs could affect the exposed cells [10]. In this regard, for introducing the parameters that could affect the fate of cell exposed to CPNs, morphology, porosity, solubility and stochiometric ratio of CPNs are briefly discussed in the following categories, although part of these subjects are well-reviewed before [8, 11]. 2.1.1. Crystalline Phase The common crystalline phases of calcium phosphate mineral family includes monocalcium phosphate (MCP), dicalcium phosphate (DCP), octacalcium phosphate (OCP), tricalcium phosphate (TCP), calcium-deficient hydroxyapatite (CDHA), HA, fluorapatite (FA) and tetracalcium phosphate (TTCP). Each crystalline phase possesses specific Ca/P ratio and the overall ratio varies from 0.5 to 2. Degree of crystallinity in CPNs is crucial for prediction of their probable usage. According to the literature, the common morphology of synthesized CPNs could be sphere-like, plate-like, and rodlike or needle-like (Fig. 1: B to E). The desired morphology should be controlled while the experiments are being designed through employing precise circumstances such as method of synthesis, pH and temperature. The ratio of Ca/P should also be controlled due to the fact that it could change the desired phase. Taking the synthesis of CPNs with various Ca/P ratios into account, Liu et al [10] demonstrated that the chemistry, crystalline phases, grain size, dissolution rate, porosity and pore size were all connected to just the Ca/P ratio and consequently, it could control the CPNs dissolution in biological milieus. 2.1.2. Amorphous Phase Unlike each crystalline phase of calcium phosphate that has a specific Ca/P ratio, the Ca/P ratio of amorphous phase of calcium

Fig. (1). Synthesized calcium phosphates morphology and crystallinity. (A) X-ray diffractometry ‎of different synthesized calcium phosphate shows crystalline, semi-crystalline and amorphous phases. Nanoparticles morphology: (B) sphere-like, (C) plate-like, (D) rod-like and (E) needle-like. Reprinted with permission from ref. 14, S. V. Dorozhkin, Acta Biomater 6 (12), 4457 (2010). Copyright @ Elsevier. B.V.

Calcium Phosphate Nanoparticles Cytocompatibility versus Cytotoxicity

phosphate varies from 1.2 to 2.2. It was reported that solubility of amorphous calcium phosphate (ACP) is far higher than crystalline phases of this mineral. Concerning the solubility of calcium phosphate’s different phases, Dorozhkin suggested a comparison as following; ACP >> TCP> CDHA >> HA > FA. The high rate of solubility for ACP could be attributed to its disordered atomic arrangement and like other amorphous phases, its rate of solubility increases [12]. Contrary to the four morphologies commonly reported for crystalline CPNs, the only morphology that has been reported for ACPNs is sphere-like which is observed as disc-shaped in transmission electron microscopy (TEM) (Fig. 1: B). 2.2. Applications in Nanomedicine Due to the favorable characteristics of CPNs such as appropriate biocompatibility and cheap methods of synthesis, employment of these NPs is more promising than before. Not only CPNs have been used as nanovehicular system for cellular delivery of therapeutic (i.e. gene, and drug) and/or diagnostic agents, but also these have been utilized in fabrication of tissue engineering scaffolds, specifically for bone regeneration [13]. Attaching and encapsulation are the most conventional tactics for loading the drug on cytocompatible particulate carriers. Considering the high SSA and controlled size of NPs, the efficacy of drug loading has been enhanced since the advent of nanotechnology and consequently NPs synthesis. In addition to cytocompatibility, CPNs have shown favorable characteristics such as high rate of dissolution in low pH of lysosome after uptake [14]. Various drugs such as doxorubicin [15], insulin [16], cisplatin [17], or ceramide [18] as therapeutic cargos are loaded on CPNs. On the other hand, CPNs were also employed as carrier for diagnostic cargoes such as photoluminescent [19] or fluorescence materials [20]. Employment of CPNs as the structural body of theranostic platforms which contain both therapeutic and diagnostic agents was also reported [18]. Likewise drug delivery, delivery of genes has also confronted some obstacles. In addition to what has been mentioned about the facilities that nanovehicular system could provide for drug delivery, in gene delivery, favorable NPs are supposed to possess nucleophilic affinity. Based on fundamentals of gene delivery, the cargo is aimed to be delivered to the nucleus by the nanovehicular system which should be meticulously designed to protect the cargo in case any damage might happen during the transfer. Apparently, because of intracellular trafficking and the difference between intracellular and extracellular pH, employment of appropriate carriers such as pH sensitive NPs are desirable. Gene delivery through carriers is subdivided into viral and non-viral methods. Attributing the risk of virus employment to viral delivery, employment of nanovehicular systems as non-viral method is appealed to researchers [21, 22]. Among the utilized nanocarriers for this purpose, CPNs have recently grasped the opportunity to posit the potential as a safe and efficacious nanostructure for transferring the gene to nucleus [1, 23] due to their nucleophilic nature [24]. Among the inorganic carriers studied up to now, calcium phosphate is still the most widely used and investigated biomaterial in this purpose because it can transfect a wide variety of mammalian cells in vitro [25], also avoiding issues such as immune responses [26] and high toxicity associated with other techniques [27]. Calcium phosphate is promising in this respect because of its unequivocal biocompatibility and biodegradability, and also because it is not subjected to microbiological degradation like organic or polymeric carrier systems. It is worthy to mention that several reviews have exhaustively summarized recent advances on employment of CPNs as carrier for therapeutic or diagnostic cargoes [28-32]. Not only CPNs are employed for cellular delivering of desirable cargoes, but also they have been used in fabrication of scaffolds that could provide the microenvironment like extracellular matrix where the cells could proliferate and consequently tissues could be

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regenerated. Owning to cells’ responds accumulated from in vivo and in vitro studies of various biocompatible materials, calcium phosphate minerals have shown promising potential as candidate for restoration of defects arisen in bones and they are counted as reputable pioneer biomaterials in reconstruction of bone defects. The reason for this achievement could be through accordance of calcium phosphate with modified form of nano-HA (carbonated nano-HA) that more than half of bone by weight is made up of it which is known as bone mineral [33]. Hence, fabrication of scaffolds with CPNs or composites of these NPs in order to regenerate the damaged bone tissues has flourished [34-36]. 3. CELL-NANOPARTICLES INTERACTION When the cell membrane and receptor molecules come into contact with the NPs, interaction of cell-NPs happens and the NPs are internalized into the cell. The internalization takes place by an organelle called endosome which is a membrane-bound compartment within eukaryotic cells. The uptake of NPs can occur via different mechanisms including endocytotic, phagocytotic and pinocytotic pathways [37]. These pathways are performed by formation of vesicles around the NPs and based on the size of the NPs, the pathway varies which is excellently reviewed elsewhere [38]. Since the time that NPs enter into the cell, the interaction between cell and NPs is initiated and the impact of NPs on cell has consequences. Programmed cell death through NPs is one of the major consequences that has attracted toxicologists’ attention. Due to the fact that one of the aims in this chapter is to clarify how the CPNs induce apoptosis, the fundamental aspects of apoptosis and calcium signaling which are both of great concerns are taken into account. 3.1. Intrinsic Induction of Apoptosis Mediated by Calcium Cell demise caused through apoptosis is defined as programmed cell death which is attributed to mediation of intracellular program [39]. Different stimuli result in cell apoptosis and during this process, cell dies in a controlled and regulated fashion. This characteristic makes the apoptosis distinct from necrosis that is another form of cell death in an uncontrolled fashion. Necrosis leads to lysis of cell and inflammatory responses that serious health problems could be the consequences of these malfunctions [40],[41]. However, apoptosis is defined as a process where cell has a pivotal role in its own death; cell contraction, DNA fragmentation, chromatin condensation, and plasma membrane blobbing are the series of significant morphological and biochemical changes to the cell which make it easy to be distinguished from necrosis [42]. The cradle of cellular apoptosis is referred to many stimuli and reactive oxygen species (ROS) production plays a prominent role in this process. Ephemeral diffusible entities like hydroxyl (-OH), alkoxyl (RO-), peroxyl (ROO-), superoxide (O2-) or nitroxyl radical (NO-) are termed as ROS. It also includes the non-radicals hydrogen peroxide (H2O2), organic hydroperoxides (ROOH) and hypochlorous acid (HOCl). ROS are produced by aerobic metabolism induction, are part of the defense mechanism against bacteria, due to which they have destructive actions on both DNA and proteins by inducing oxidative stress [43]. The cellular antioxidant machinery can control the intracellular ROS concentration in a physiological range but perturbation in prooxidant/antioxidant balance, which could be a consequence of induced oxidative stress, induces cell damage and apoptosis [44]. This induction is partially due to the fact that ROS are able to interact with cell signalling pathways by way of modifications of thiol groups (SH groups) on proteins that possess regulatory functions, including Ca2+ channel forming proteins and transporters [45]. Ca2+ is known as the most protean intracellular messenger due to the fact that it is involved in the regulation of almost all known cellular functions and reactions [46]. Malfunctions in homeostatic control of Ca2+ signals could result in triggering the apoptotic process in cell [47]. On the whole, it is reported that ROS can act in the way that augment the intracellular Ca2+ ion concentration ([Ca2+]i) of various types of cells, including renal tubular

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cells [48], smooth muscle cells [49, 50], skeletal muscle cells [51], mesangial cells [52], blood mononuclear cells [53], pancreatic betacells [54], neurons [55] and cardiomyocytes [56]. As mentioned in literature, apoptosis can be triggered via two paramount signaling pathways: (I) extrinsic pathway which is activated through extracellular stimuli such as withdrawal of growth factors that incite the engagement of transmembrane death receptors [57-59] and, (II) intrinsic pathway which is directly related to mitochondrial membrane permeabilization (MMP) (mitochondrial apoptotic pathway) [60, 61]. Unexpected changes of [Ca2+]i ubiquitously cause increase in Ca2+ entry and Ca2+ release from endoplasmic reticulum (ER) and mitochondria [62, 63]. It was reported that depletion in Ca2+ in ER calcium storages could result in apoptosis [64]. In the other study, it was found that capacitive Ca2+ influx through Ca2+ release-activated Ca2+ channels is apoptogenic [65]. These evidences imply that mitochondria promote apoptosis through different ways utilizing Ca2+ signaling. Hence, they are indispensable organelle in regulation of alterations in Ca2+ which could act as a relevant amplification loop of the death signal [66]. Release of Ca2+ from ER not only increases the [Ca2+]i, but also intensifies the Ca2+ concentration of mitochondria ([Ca2+]m). The cells maintain a 20000-fold gradient of Ca2+ between extracellular free Ca2+ (~1.2 mM) and resting cytoplasmic free Ca2+ (~100 nM). Depending upon the stimulus, [Ca2+]i can exceed more than 1 µM [66] and also Ca2+ is regulated to 400–600 µM within lysosomes [67]. Based on these facts, it is signified that slight changes in intracellular calcium concentration can cause profound effects on cellular metabolism. For instance, the intensification in [Ca2+]m could open the permeability transition pore or generation of ROS, which leads to mitochondrial dysfunction and as the consequence, some apoptogenic proteins release into cytosole to trigger apoptosis in the cell [68, 69]. Moreover, fluctuations in [Ca2+]i also affect multiple enzymes, including Ca2+- activated proteases, calcineurin, endonucleases, phospholipases, nitric oxide synthase and transglutaminases. These enzymes control the breakdown of various cellular constituents, some of which are directly associated with apoptosis [70]. Generally, mitochondrial apoptotic pathway could also be subdivided into (I) caspase-dependent [71] and, (II) caspaseindependent pathway [72]. Caspases are a family of cysteine proteases that play an important role in apoptosis process of the cell [73]. When the cell is exposed to pro-apoptotic stimuli, cytochrome c (Cyt c) located in mitochondria releases into cytosole [74] and formation of a large complex comprising Cyt c, apoptosis-activating factor 1 (Apaf-1) and pro-caspase-9, that consist of a long prodomain, happens. Caspases-9 will then go on to activate caspases 3 and 7, which lead to apoptosis of the cell [75]. Particularly, Caspase-3 possesses the most effect in the DNA fragmentation process and other morphological changes are associated with apoptosis [76, 77]. In addition to the mentioned proteins, investigation of the apoptosis in cells necessitates examination of the changes in some protein expression of apoptosis-related genes. In the mitochondrial pathway, the Bcl-2 (B-cell lymphoma 2) family proteins are perhaps the most important regulators. These proteins are compromise of both anti-apoptosis and pro-apoptosis members while in caspase-dependent pathway, the anti-apoptosis members are responsible for inhibiting the commencement of the cell death program [78]. On the other hand, it was observed that in presence of caspase inhibitors, there is a process which ensures cellular death while displaying morphology similar to apoptosis [79]. It means that there are stimuli which employ caspase-independent pathways to induce apoptosis. One of the stimuli is apoptosis inducing factor (AIF) existing in mitochondrial inter-membrane proteins space which releases when it cleaves by calpain protease (a calciumdependent cysteine proteases) [80]. Notwithstanding abovementioned explanations which offer a succinct structure of apoptosis that is provided in a way to cover the

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essentials, there are plenty of reviews which thoroughly cover different parts of apoptosis and its correlation with calcium signaling [42, 46, 47, 66, 81-84]. Considering better understanding of apoptosis induction in cells, normal or cancerous, requisite verities concerning molecular interactions between cell and NPs should be elucidated. In this regard, thorough knowledge of apoptosis mechanisms and the stimuli that induce apoptosis lead to meticulous postulation. On the other hand, with regard to start of the interaction at molecular scale, biodegradation of NPs carries an outstanding amount of weigh in this process. Collectively, these basic facts could coalesce into a whole idea for further consideration of apoptosis induction. 3.2. Crystalline Calcium Phosphate Nanoparticles and Apoptosis Induction Various crystalline CPNs with different Ca/P ratio have already been synthesized. Based on their characteristics, they have been utilized in number of applications as mentioned above. Among these crystalline phases, HA has more attracted researchers’ attention. More recently, HANs have variously been used as carrier for different cargoes such as DNA [85], siRNA [86], anticancer drugs [87], diagnostic agents [88] and so forth [89]. They have been employed in scaffold production for tissue engineering as well [90]. Based on great interaction between HANs and cells in nanomedicne, it necessitates meticulous considerations over its cytotoxicity [91]. Although the subject is significant enough to draw researchers’ attention, it is approached lightly and the number of studies is not enough considerably. Nevertheless, the studies on HANs cytotoxicity are adequately informative and the facts and ideas are worthy of mention and taken into consideration. 3.2.1. HANs Cytotoxicity: Role of Size, Morphology and Cell Type Owing to the fact that hydroxyapatite is the most allocated substitute material to bone regeneration, employing nanotechnology has developed the HANs in order to be exploited to perfection. As each undertaken research about HANs has its own positive and negative aspects, rather than outstanding osteocompatibility of hydroxyapatite [92], there are some reports that have shown some inflammatory responses of HANs [93, 94]. According to the reported inflammatory potential, irrational employment of HANs for bone regeneration materials appears more ostentatious rather than brilliant [95, 96]. Shi et al. studied the effect of synthesized HANs on human osteoblast-like cells in vitro [97]. They reported that smaller size of HANs results in less toxicity than the other sizes. Loss of contact with surrounding cells, condensation of the cells, swollen mitochondria, deformation in nuclei and compressed chromatin were the changes observed in the cells interacting with larger particles which were signs for the process of programmed cell death; apoptosis [98]. They found that higher apoptosis percentage is caused by larger HANs and the toxicity of lager NPs is because of this fact, although it could not be generalized to all sizes of HANs [99]. Notwithstanding impact of HANs size on apoptosis induction, any valid reason regarding effect of morphology on cytotoxicity was not reported. Furthermore, while NPs are being studied, morphology of NPs and their SSA signify the overall results. Based on mentioned hallmarks of NPs, Xu et al. examined the impact of morphology and SSA of HANs in addition to their size, on cultured osteoblast [100]. They account for inhibition of osteoblast proliferation in a dose-dependent manner of HANs and cell proliferation was decreased along with scaling up in the concentration of HANs in culture dish. Through staining with Annexin V conjugated to fluorescein isothiocyanate (FITC-AV) (only binds to apoptotic cells), they found that most cytotoxicity is due to apoptosis in comparison to necrosis (staining with propidium iodide (PI) which only binds to necrotic cells) which is in agreement to what Shi et al. [97] attributed the toxicity to. Moreover, it was found that most apoptosis percentage, among various types of HANs morphologies, was for the needle-like HANs (N-HANs) (more details of experiments

Calcium Phosphate Nanoparticles Cytocompatibility versus Cytotoxicity

in Table 1). According to the cell proliferation assay, they reported that short rod-like HANs (R-HANs) induced more apoptosis in comparison with the other HANs and ascribe this higher amount of apoptosis to higher particle-cell interaction which causes mechanical stresses on the cell surface. They also observed that increases in SSA of HANs led to rise in the apoptotic rate (cytotoxicity) which conforms with the other studies [101-104]. Nevertheless, they mentioned that sintering temperature might change the range of toxicity; the study lacks phase characterization of HANs which could directly affect induction of toxicity. Zhao et al. also studied the effect of HANs morphology on human bronchial epithelial and mouse macrophage cell line [105]. Four different shapes of HANs were synthesized (Table 1) and their cytotoxicity induction was evaluated through measuring the amount of double-stranded DNA by Picogreen assay. The assay showed evident differences in cell viability between two cell lines. According to the results, no toxicity was observed in mouse macrophage cell line; in contrast, HANs dose-dependent cytotoxicity was observed in human bronchial epithelial cell line where N-HANs induced the highest cytotoxicity which is in agreement with other studies in this regard [100, 106] and plate-like HANs (P-HANs) have the lowest rate of cytotoxicity, as demonstrated before [107] (Fig. 2: A-1 and A-2). Flowcytometry (FCM) results for measurement of cell necrosis/apoptosis in cultured human bronchial epithelial cell mediated to a constant concentration of HANs showed that dominant cytotoxicity was because of necrosis rather than apoptosis (Fig. 2: A-3). Comparing the amount of cellar uptake between N-HANs and R-HANs, it was found that internalization of R-HANs is more than N-HANs although the cytotoxicity caused by N-HANs is higher than the other (Fig. 2: B). Hence, the study supports the conclusion that resulted cytotoxicity not only is HAN shape-dependent but also directly related to the type of cell which HANs interacts with. HANs has shown selective effects on bone related cells as very recently investigated by Qing et al. [108]. They studied the effect of different concentrations of HANs which interacted with two type of bone related cells; osteosarcoma cells (OCs) and osteoblasts (Table 1). Employing in vitro staining technique, no dead cell was found in any osteoblasts exposed to HANs; in contrast, many dead cells were observed in OCs culture dish. According to experimental data obtained via TEM, it was suggested that the toxicity which resulted in cell death is attributed to exhibition of typical characteristics of initial apoptotic cells in a dose-dependent manner. During the mediation of OCs to HAN, in minimum dosage, accumulation of NPs in lysosomes was observed while some NPs were spotted in cytoplasm and no deformation was seen in OCs morphology. This occurrence was, nevertheless, detected in OCs mediated to higher dosages of HANs showing chromatin margination and in maximum dosage exposure, complete DNA fragmentation was evident (Fig. 3: A). The reason for apoptosis induction in OCs was reported due to outburst of so-called myelinosome (lamellated ultrastructural inclusions) structures (Fig. 3: B-1) which is described elsewhere [109, 110]. The entrance of HANs into nucleus (Fig. 3: C) resulted in some classical morphology change which is related to apoptosis process (Fig. 3: B-2 and B-3). It was reported that increase in Ca2+ ion [Ca2+] and PO43+ ion [PO43-] within lysosomes caused the rise of their pH, thus leading to the interference of intracellular homeostasis and eventually causing the cell apoptosis, although Motskin et al. [111] research results on reasons of apoptosis induction caused by HANs are not in accordance with above-mentioned explanations; neither outburst of myelinosome structures nor interference of high-pH lysosomes with intracellular homeostasis. They reported that apoptosis induction happened because of high increase in [Ca2+]i. In this study, the interaction of hydroxyapatite nanoand microparticles (HAMs) with human monocytes’-derived macrophages (HMMs) and the resulted toxicity were studied. Both HANs and HAMs uptake were due to phagocytosis. Consequently, HAN accumulated not only in phagososmes and lysosomes after appropriate period of incubation, but also in smooth endoplasmic

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reticula. Apparently, some HANs escaped from the phagocytic pathway and after passing through intracellular traffic, they reached to a pore in nuclear envelope. It is reported that elevation of [Ca2+]i leads phagocytosed agents to escape from the pathway [112, 113] and causes localization in the nuclear owning to their nucleophilic attribute (Fig. 4: A) [24, 114]. Motskin et al. suggested HAMs as outstanding candidates for vaccine delivery as they are internalized intensively by HMMs and they show toxicity only at high concentration. The detached nanoparticles from HAMs which occasion the [Ca2+]i elevation, is implicated in the cytotoxicity (Fig. 4: B). It was notified that increase in [Ca2+] and [PO43-] within lysosomes will cause rise in pH [67, 115] and supersaturation of lysosomes with [Ca2+] is controlled by gradual dissolution of HAN in a rather long period of time (Fig. 4: C) and this could be the reason for longterm presence of HAN in lysosomes [111], in spite of the fact that pH rise in lysosome could cause apoptosis in cells [108]. The in vivo study also supports that HANs could induce apoptosis in macrophages in a dose-dependent manner [116]. 3.2.2. HANs Interaction with Cancer Cells Cancer therapy through employment of nanomedicine has distinguished the prominence of nanocarriers-cancer cells interaction. In targeted drug or gene delivery to cancer cells, many factors matter and the most important one is the sustained release of the cargo [117]. With regard to the cancer therapy efficiency through delivery, if the carrier induces toxicity to cancer cell in addition to control the release, it will result in more effective therapy. Considering the studies conducted on utilization of HANs in cancer treatment, appropriate functionality of nanocarriers were the most provoked responses from cancer cells. According to abovementioned details, study of HANs toxicity in cancer cells was appealed to researchers in order to attain a more promising nanocarrier. Initially, in 2003, Liu et al. examined the effect of HANs on human hepatoma cell line in vitro [5]. They reported that HANs could significantly inhibit the proliferation of cultured cells and cytochemical staining revealed that inhibition is due to induction of apoptosis. The results showed that HANs influence the apoptosis rate in a dose-dependent manner although the mechanism of apoptosis induction was not discovered. Similarly, Venkatasubbu et al. also investigated HANs interaction with human hepatoma cells [118]. Although the study shows no data of nanoparticles size, it was observed that augmentation of toxicity is in a rather direct relation to increase in exerted concentration and, significantly, decrease in grain size of HANs. Through morphological changes of the cultured cells, they attributed the cytotoxicity to apoptosis. The cells were treated with various concentrations of HANs for a whole day and G2/M phase arrest was observed in a dose-dependent manner by FCM. The induced apoptosis was related to HANs penetration into cell membrane and nuclear trafficking which led to the greatest changes in the apoptotic proteins. They mentioned that the HANs with greater grain size could not penetrate into cellular nucleus to induce more apoptotic induction while the idea appears somehow irrational and the study possesses a lack of clear mechanisms for apoptotic induction. In another study, HeLa cells were exposed to HANs by Cao et al. and anti-proliferative interaction of the nanoparticles was observed [119]. It was found that HANs induce toxicity toward HeLa cells in a time- and dose-dependent manner. The data obtained from fluorescence observation of stained HeLa cells clarified that induced toxicity in the cells was due to increase in nuclear fragmentation which attributed to apoptosis. The amount of apoptotic cells was augmented along with increase in HANs concentration and longer treatment time. According to data from FCM, it was reported that the most percentage of dead cells was due to apoptosis and necrosis held a minor share (further data could be found in Table 1) and it was concluded that HANs could have therapeutic effect on HeLa cells but no apoptotic mechanism for apoptosis induction was suggested (Fig. 5).

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1% HAN suspen-



sion - 120 h

> 200

R-HAN ~ 47



Expressed protein

~ 6.68% Normal

~ 13.61%



~ 23.13%

~ 23 2

Apoptotic rate (%)

and time



Cell type


~ 20 ~ 80


System of cultured

Average size (nm)

HAN characteristics versus induced apoptosis percentage and related expressed genes.



Table 1.

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1.65% Normal


200 µg/ml -



120 h


The expressed proteins are Collagen I,

in support of bone regenera-


tion, although no apoptotic


gene was detected. N-HAN S-HAN 3

10-20 (db) /

10-30 20-40 (d) /



30-50 (lc)

Normal cell

Rat osteoblast

100 mg/L – 72 h

200-400 (l)


20-30<  l