Self-Activated Fluorescent Hydroxyapatite Nanoparticles: A Promising

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Self-activated fluorescent hydroxyapatite nanoparticles: a promising agent for bio-imaging and bio-labeling Ketaki Deshmukh, M Monsoor Shaik, Sutapa Roy Ramanan, and Meenal Kowshik ACS Biomater. Sci. Eng., Just Accepted Manuscript • Publication Date (Web): 07 Jul 2016 Downloaded from http://pubs.acs.org on July 11, 2016

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ACS Biomaterials Science & Engineering

Self-activated fluorescent hydroxyapatite nanoparticles: a promising agent for bio-imaging and bio-labeling Ketaki Deshmukha, M. Monsoor Shaika, Sutapa Roy Ramananb*, Meenal Kowshika*. a

Biological Sciences Department, b Chemical Engineering Department.

Birla Institute of Technology and Science Pilani, K K Birla Goa Campus, Goa, India [email protected],

[email protected],

[email protected],

[email protected]. Corresponding authors: Dr.Meenal Kowshik a, Dr.Sutapa Roy Ramananb Present/Permanent Address: Birla Institute of Technology and Science Pilani, K K Birla Goa Campus, Zuarinagar, Goa, India – 403726 Phone No. 0832-2580304, 0832-2580325 Fax no: 0832 - 2517033

ACS Paragon Plus Environment


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Abstract: Bio-imaging has drastically transformed the field of medicine, and made the process of diagnosis easy and fast. Visualization of complete organ to complex biological processes has now become possible. Among the various imaging processes, fluorescence imaging using non-toxic fluorescent nanomaterials is advantageous for several beneficial features including high sensitivity, minimal invasiveness and safe detection limit. In this study, we have synthesized and characterized a new class of non-toxic, self-activated fluorescent hydroxyapatite nanoparticles (fHAp NPs) with different aspect ratios (thin-rods, short-rods, rods) by changing the stabilizing agents (triethyl amine and acetyl acetone) and solvents (water and dimethyl sulfoxide). fHAp NPs showed excellent fluorescence with broad emission spectrum ranging from 350-750nm and maximum at 502nm. Presence of fluorescence was attributed to the electronic transition in the asymmetric structure of fHAp NPs as confirmed by ESR spectroscopy and absence of fluorescence in symmetric HAp NPs. In addition to exceptional fluorescence behavior, these NPs were found to be non-toxic in nature and could be easily internalized in both prokaryotic and eukaryotic systems. We propose that, the fHAps provide a safe and a potential alternative to the current fluorescent materials in use for bio-labelling and bioimaging applications. Keywords: Hydroxyapatite, fluorescence, cell internalization, asymmetric structure, bio-imaging.


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Introduction Since the time of Galileo, imaging technique has become the "eyes of science" rendering an exquisite sense of vision to medical science and research. This technique facilitates the study of biological processes, including the observable changes in receptor kinetics, cellular signaling and the movement of molecules through membranes1-2. Imaging techniques such as magnetic resonance imaging, computed tomography, fluorescence imaging, radio imaging, etc., enable visualization of multidimensional and multi-parameter data3. Compared to other techniques, fluorescence imaging is 4

advantageous as it is highly sensitive, minimally-invasive and quite safe . Fluorescent labeling using usual organic dyes, fluorescent proteins and lanthanide chelates are the preferred methods to 5

observe biological events and processes in both living and non-viable systems . However, they possess inherent drawbacks such as short Stokes shift, poor photochemical stability, susceptibility to photo-bleaching and decomposition under repeated excitation6. In view of the existing limitations, more efficient fluorescent nanomaterials have been developed as alternative bio-imaging agents. Fluorescent nanomaterials including quantum dots, nanoparticles (NPs) doped with lanthanides, upconversion nanoparticles (UpNPs),metal NPs etc., have shown significant progress in diagnostics 6-7

and therapeutic applications, owing to their small size and large surface area . Among these, hydroxyapatite (HAp) NPs doped with lanthanides and organic dyes are being extensively studied as fluorescent probes8-15. Fluorescent lanthanide (Eu3+, Tb3+, Yb3+, Er3+, etc.) doped HAp NPs have been reported to possess narrow emission bandwidths, high photo-chemical stability and long fluorescence lifetimes16. HAp [Ca10(PO4)6(OH)2] is an ideal phase of calcium phosphate for application inside human body due to its excellent bioactivity, biocompatibility, high porosity and osteo-conductivity17. It has been successfully used for applications in the field of biomedical science ranging from bone 18

regeneration to drug delivery . HAp is composed of ions commonly found in physiological environments, which makes it highly biocompatible19. However, HAp NPs doped with lanthanides are toxic, resulting in their limited in-vivo applications10,

19

.Therefore, there is clear need for developing

alternative fluorescent nanomaterials for bio-imaging, with biocompatible and tunable photoluminescent properties. In our earlier work, we had reported the synthesis of rod shaped HAp NPs at 100°C using the 20

modified sol-gel method . Herein, we report the synthesis of self-activated fluorescent HAp NPs (fHAp) with different aspect ratios by varying the stabilizing agents and solvents at room temperature.



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These fHAps besides being highly florescent are also non-toxic in nature and are readily internalized by both prokaryotic and eukaryotic systems. Hence, these fHAp NPs can be used as potential materials for bio-imaging and bio-labelling applications.

Materials and Methods: Synthesis of fHAp NPs: HAp NPs were synthesized using modified sol gel method using various protocols as described below. The schematic illustrations for the same are attached as supplementary information (Figure S1). Method1: 2M calcium chloride was dissolved in DMSO and stirred for 30mins followed by drop-wise addition of orthophosphoric acid. Ca:P atomic ratio was maintained at 1.67. Subsequently, the stabilizing agent TEA was added, and the sol obtained was stirred for 1h. The pH was adjusted to 10 using liquid ammonia and stirring was continued till the completion of gel formation. The gel obtained was washed thoroughly with ethanol and dialyzed (using dialysis bag, Bangalore genie 110) against de-ionized water for 12h with frequent change of water for active removal of adsorbed ions. The dialyzed samples were dried at room temperature. Method2: In this procedure all the steps followed were similar to method 1 except ACA was added in place of TEA. Method3: These NPs were synthesized using our previous protocol20. Only change was that these samples were dried at room temperature instead of 100⁰C. 1M phosphoric acid in TEA was added drop-wise to 2M calcium chloride solution in de-ionized water under constant stirring. Rest of the protocol was same as method 1. Method4: ACA was used as a stabilizing agent in place of TEA in method 3. Method5: 2M calcium chloride was dissolved in deionized water and orthophosphoric acid was added drop-wise to it. In this procedure no stabilizing agent was added. The mixture was heated at 80°C for 1h followed by dialysis and the remaining steps were followed as mentioned in method 1. Characterization of the HAp NPs: The morphology of the synthesized HAp was studied using TEM (Phillips CM 200) and particle size was analyzed by dynamic light scattering (DLS) using a particle size analyzer (Delsa Nano S, Beckman Coulter, USA). Zeta potential of HAp NPs was determined using Zeta Plus, zeta potential analyzer (Delsa Nano S, Beckman Coulter, USA) at room-temperature.


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XRD studies of the powdered samples were carried out for phase identification using X-Ray Diffractometer (Miniflex II Rigaku) with monochromatic Cu Kα radiation (λ = 1.5405Å) and a scan range of 2θ = 20° to 80°. The functional groups present in the synthesized compounds were ascertained by FTIR (8201 PC Shimadzu), over the region 4000– 450cm-1. The pellets for FTIR analysis were obtained by mixing 1mg of the powdered sample with spectroscopic grade KBr (Merck). Fluorescence excitation spectra of the samples were determined using spectrofluorometer (Shimadzu RF 5301), at an emission wavelength of 502 nm. The samples were suspended in water (1mg/ml) and sonicated for 15mins before analysis. The fluorescence spectra of the sample were recorded using a He-Cd laser at temperature ranging from 300-15K with an excitation wavelength of 325nm. The fluorescence of the sample was also observed under epi-fluorescence microscope (Olympus BX41) using FITC and TRITC filters. The fluorescence spectra of HAp NPs in liquid media were recorded using Spectrofluorimeter (JascoFP-6300) by dispersing in DMEM and PBS at pH 7. ESR spectra were recorded using JEOL ESR spectrometer (JES-FA200). All the measurements were performed at room temperature.

Biocompatibility studies Cytotoxicity was measured by MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) 3

4

method . Exponentially growing HeLa cells were inoculated with 5 × 10 cells/well supplemented with complete media. The cells were exposed to various concentrations (10, 30, 50, 100, 500 and 1000 µg/ml) of the HAp NPs dispersed in DMEM, and incubated for 24h. MTT (20µl of 5mg/ml) was added to the test samples, and the plates were incubated at 37ºC for 4h. The media was pipetted out carefully without disturbing the cells after 4h. For dissolving the formazan crystals, 150µl of DMSO was added, and the absorbance was measured at 570nm using UV-visible spectrophotometeter (UV2450). All measurements were done in triplicates. The relative cell viability (%) was calculated as: ([At] / [Ac]) × 100, Where, [At] is the absorbance of the test sample and [Ac] is the absorbance of the cells without treatment. Lactate dehydrogenase assay was performed using a commercial LDH kit (Coral Clinical Systems, India). LDH is a soluble enzyme located in the cytosol. The enzyme is released into the surrounding culture medium upon cell damage or cell lysis, processes that occur during both apoptosis and necrosis. LDH activity, therefore, can be used as an indicator of cell membrane integrity, and a



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measurement of cytotoxicity21. Growth medium obtained from cells (HeLa) incubated in complete media with varying concentrations (10, 30, 50, 100, 500 and 1000 µg/ml) of HAp NPs for 24 h was used for LDH analysis. The protocol followed was as specified by the manufacturer (Coral Clinical Systems) for the LDH kit. Absorbance was measured at 340 nm (Thermofisher scientific Multiskan™ GO Microplate Spectrophotometer). Intracellular ROS generation was determined using 2’, 7’- dichlorofluorescein diacetate (DCFH-DA), a fluorescent probe that readily diffuses through the cell membrane into the cellular cytoplasm, and is enzymatically hydrolyzed to non-fluorescent DCFH. The oxidation of DCFH to highly fluorescent 2’, 7’dichlorofluorescein provides a quantitative estimate of ROS formation22-23. The ROS studies were 4

carried out using HeLa cell line. Exponentially growing cells (5 x 10 cells/ well), were seeded in 24well plates supplemented with complete media. After 24 hrs of growth the cells were exposed to varying concentrations of fHAp NPs (10, 50, 100, 500 and 1000 µg/ml) dispersed in DMEM, and incubated for 24 hrs. Following exposure, DCFH- DA (20 µM) was added to all the wells, and the plate was incubated in dark at 37°C for 30mins. The fluorescence was measured using a spectrofluorometer (JASCO FP-6300), with an excitation wavelength of 485 nm, and an emission wavelength of 530 nm. 1 µM hydrogen peroxide (H2O2) was used as the positive control. All the experiments were performed in triplicates. The hemolytic activity of the samples was investigated according to the method described by Dobrovolskaia et al

24

. 4ml blood was diluted with 5mL of sterile saline solution and 20µL of it was

added to 500 and 1000 µg/ml of fHAp NPs. The suspensions were incubated for 60min in water bath shaker at 37ºC and centrifuged at 700 g for 10min. The amount of free hemoglobin was determined by measuring the absorbance of the supernatant at 540nm (UV Spectrophotometer Shimadzu UV 2450). Sterile saline solution and distilled water were used as the negative and positive controls, respectively. The hemolysis rate (HR) was calculated using25: =

− 100%, −

Where Dt, Dnc and Dpc are the absorbance of the sample, the negative control and the positive control, respectively. All the experiments were carried out in triplicates.

Uptake of HAp NPs by eukaryotic and prokaryotic cells:


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HeLa cells were maintained in DMEM supplemented with 10% fetal bovine serum with antibiotics. Cells were maintained at 37ºC in a humidified atmosphere with 5% CO2 and sub-cultivated according to standard cell culture protocols26. Exponentially growing cells were inoculated at a concentration of 4

5 x 10 cells/well onto 2×2cm glass slide in a 6 well plate supplemented with 1 ml complete media and were grown for 12h. The cells were incubated with all the HAp NPs [30µg/ml] for 4h and the glass slides were washed twice with PBS,(pH 7), to remove un–internalized particles and debris. These slides were observed using epi-fluorescence microscope under TRITC and FITC filters. Candida albicans (MTCC 183) as a representative of yeast culture was used to study the internalization of NPs. Culture was grown in Sabouraud dextrose media (SB) at 37°C in an incubator. 6

Exponentially growing cells were inoculated at a concentration of 10 cells/well onto 2×2cm glass slide in a 6 well plate supplemented with 2ml SB media for 48h. The cells were then incubated with the HAp NPs [70µg/ml] for 4h and the glass slides were washed twice with PBS (pH 7) to remove un– internalized particles and debris. These slides were observed using epi-fluorescence microscope under TRITC and FITC filters.

Similar studies were done with bacterial cultures, using Escherichia coli (NCIM 2345) and Staphylococcus aureus (MTCC 737) as the Gram-negative and Gram-positive culture, respectively. Both the cultures were grown in Luria Bertani (LB) medium at 37°C in a shaker incubator. Exponentially growing cells were inoculated at a concentration of 106 cells/well onto 2×2cm glass slide in a 6 well plate supplemented with 2 ml LB media for 6h. The cells were then incubated with the HAp NPs [50µg/ml] for 4h after which the glass slides were washed twice with PBS (pH 7) to remove un–internalized particles and debris. These slides were observed using epi-fluorescence microscope under TRITC and FITC filters. Results and Discussion: Synthesis and characterization of HAp NPs Currently, HAp NPs with different morphologies are being synthesized using various methods such as precipitation, hydrothermal processing, wet-chemical process, sol-gel synthesis27-30 etc. The process of nucleation and growth of the NPs primarily depends on the corresponding monomers’ 31

concentration and solubility . The growth, and thereby the morphology, of the NPs is further controlled by the addition of stabilizing/capping agents such as ethylenediamine tetraacetic acid (EDTA), hexadecyl trimethyl-ammonium bromide, sodium dodecyl sulfate (SDS), etc.,18, 31.



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In the current work, we have synthesized a new class of HAp NPs at room temperature using triethyl amine (TEA) or acetyl acetone (ACA) as stabilizing agents with either water or dimethyl sulfoxide (DMSO) as the solvent. Water and DMSO were selected as solvents based on the differences in their polarity, which would eventually influence the interactions among the calcium ions resulting in varied morphologies. Besides, DMSO being less polar and aprotic in nature, facilitates the formation of smaller particles32. To study the effect of basic and acidic stabilizing agents on the shape and size of HAp NPs, TEA (as basic) and ACA (as acidic) were used. The formation of the HAp NPs was confirmed by carrying out XRD and FTIR analysis after the completion of the synthesis. The XRD spectra (Figure S2A), of all the three NPs and commercially purchased HAp, corresponded well with the hexagonal HAp crystal (ICDD 09-0432). All the synthesized HAp NPs exhibited the characteristic signature HAp bands in the FTIR spectra (Figure S2B)20.Depending on the conditions used in the reaction mixture (refer to the materials and methods), the newly formed nanoparticles exhibited different morphologies (Table 1 and Figure S3). It was observed that NPs synthesized using water as a solvent and TEA as a stabilizing agent, were rod shaped with average length 92.8nm and average diameter 23.2nm (Figure S3a). The nanostructure changed to short rods with an average length 35nm and an average diameter 7nm when DMSO was used as a solvent, keeping TEA as the stabilizing agent (Figure S3b). Thin rods of average length 62.5nm and diameter 6.2nm were obtained when the stabilizing agent was changed to ACA while maintaining DMSO as the solvent (Figure S3c). When water was used as the solvent and ACA as the stabilizing agent, particles obtained were irregular with mixed morphologies (Figure S3d). In the absence of either of the stabilizing agents, HAp microparticles of rod shape with an average length 1.42µm and diameter 0.29µm were formed (Figure S4a). This demonstrated the role of stabilizing agent in controlling nucleation and growth, which resulted in different sizes of rods. The details of size of fHAp NPs determined by TEM, DLS and the surface charge determined by zeta potential analysis are summarized in Table 1. To the best of our knowledge, this is the first study to report the effect of TEA and ACA as stabilizing agents with water and DMSO as solvents, on the morphology of HAp NPs.


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Solvent

Stabilizing agent

Size and shape using TEM Morphology

Average length (A) (nm)

Tri-ethyl Rod 92.8 amine Tri-ethyl DMSO Short-rod 35 amine Acetyl DMSO Thin-rod 62.5 acetone Acetyl Mixed and Water 30-90 acetone irregular a ratio of average length and average diameter Water

*

Hydrodynamic size using DLS (nm)

Zeta potential value (mv)

Average diameter (B) (nm)

*Aspect ratio (A/B)

23.2

1:4

85±10

3.85

7

1:5

28±10

13.25

6.2

1:10

57±10

2.12

6-25

NA*

85±10

6.923

Not applicable due to its mixed morphology

Table 1: Different morphologies of HAp NPs synthesized using various solvent/stabilizing agent combinations.

Self-activated fluorescent properties of HAp NPs: Ideally, HAp is not a fluorescent material; however, in this study all the HAp NPs synthesized by solgel method at room temperature using TEA/ACA as stabilizing agents and water/DMSO as solvents exhibited green and red fluorescence under epi-fluorescence microscope with FITC and TRITC filters (Figure1A). Mechanism of the fluorescence of the newly synthesized HAp NPs is not very clear at this stage, because, neither Ca2+ nor PO43- are known to show any fluorescing property; hence this behavior could be attributed to self-activated fluorescence of these particles. Since this new class of HAp NPs exhibit self-activated fluorescence properties, we name them as “fluorescent HAp NPs” (fHAps). Very few reports have described the synthesis of self-activated fluorescence in HAp NPs wherein fluorescence has been attributed to the presence of CO

˙2

radical in the crystal lattice,

introduced through the precursor materials33-37. All these studies have shown blue emission with maxima between 400-460nm. In the present work, all fHAps prepared by the low temperature sol gel method exhibit excitation at 325nm (shown as inset in Fig 1B), and broad emission spectrum ranging from 350-750nm with maximum at 502nm and additional peaks at 528nm, 567nm and 609nm when excited at 325nm (Figure 1B). The peak at ~ 442nm is characteristic peak of He-Cd laser. Commercially purchased spherical HAp NPs neither showed any fluorescence under microscope nor any excitation/ emission spectra. The emission spectra of all three fHAps synthesized were similar, but with significant changes in the intensity of fluorescence signal, which was in the order thin-rod >



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short-rod >rod. Since the thin rods have shown the maximum intensity, we have mainly focused on these fHAps to further explore their properties.

Figure1. A) Epi-fluorescence microscope images of fHAp NPs using FITC (green) and TRITC (red) filters a) thin-rods b) short-rods c) rods, synthesized at room temperature by sol gel method. All the fHAps exhibit both green and red fluorescence. Commercially obtained spherical HAp NPs that do not show any fluorescence are shown for comparison in (d). B) Fluorescence spectra of fHAp NPs at an excitation wavelength of 325nm; inset shows the excitation spectra at an emission wavelength of 502nm. fHAps exhibit a broad emission spectrum ranging from 350-750nm with maximum at 502nm and additional peaks at 528nm, 567nm and 609nm. The fluorescence spectrum of commercially obtained spherical HAp NPs (green) is shown for comparison. Although, the emission spectra of fHAp NPs were similar, significant changes in the intensity of fluorescence signal of the order of thin-rods (black line) > short-rods (red) >rods (blue) were observed.

Presence of artifacts in the NPs, can be traced by observing changes in the fluorescence signals as a function of temperature. At low temperatures, sharpening of the fluorescence peaks, which are otherwise hidden, is observed due to presence of shallow defects in the material, whereas an increase in the emitted signal intensity is observed due to the suppression of phonon assisted nonradiative transitions38-41. To check for defects in fHAps, we determined the fluorescence spectra at different temperatures ranging from 300K to 15K. Only an increase in the intensity of the emitted


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fluorescence signal, without any additional peaks, was observed at low temperatures along with a shift of maximum from 502nm to 528nm (Figure 2A). This clearly indicates that there are no artifacts present in the fHAps and the fluorescence could be attributed to their asymmetric nature. To confirm this assumption, we further carried out similar experiments on commercially available symmetric spherical HAp NPs (Figure S4B). As assumed, these spherical NPs did not show any fluorescence (Figure 1A, 1B), indicating a role of asymmetry in the fluorescence of fHAps. ESR spectra of fHAps were obtained to confirm the presence of free electrons having been confined in the asymmetric geometry. These fHAps exhibited ESR signals with three hyperfine splittings and g values indicative of presence of free electrons (Figure 2Ba). These free electrons, due to their confinement in an asymmetric potential created by the elongated structure of the fHAps, have discrete energy levels which are optically active, leading to fluorescence phenomena. On the other hand, the commercially obtained symmetric spherical HAps did not show any ESR signal (Figure 2Bb). This observation further underlines the importance of asymmetry for optical activity. Further experiments are underway to elucidate the detailed mechanism of fluorescence in fHAps and will be reported elsewhere.

a)

b)

Figure 2. A) Fluorescence spectra of thin rod fHAPs at different temperatures ranging from 300 to 15K.The emission signals across the spectra are shown with a connected line for clarity. Only an



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increase in the intensity of the emitted fluorescence signal, without any additional peaks was observed at low temperatures. B) ESR spectra of a) thin-rods fHAps with three hyperfine splittings and g values (2.0082, 2.0069 and 2.0046) indicating the presence of free electrons owing to the asymmetric nature of these NPs. For comparison, the ESR spectrum of the commercially obtained spherical HAp NPs, which are symmetric, is shown in (b). Notice the absence of hyperfine splitting in the case of spherical HAps.

Applications of fHAp NPs for bio-imaging and bio-labelling: Bio-labelling and bio-imaging are important techniques for detection and diagnosis of diseases. To use any material for such purposes, its cytotoxicity/biocompatibility needs to be studied. In recent years, use of lanthanide doped HAp NPs has successfully resulted in a series of multifunctional fluorescent materials for bio-imaging and bio-labeling5, 10. However, rare-earth elements are toxic and have adverse effects due to their accumulation in the body10,

42

. In the current study, we have

synthesized a new class of fHAps with self-activated fluorescence properties. Since these fHAps do not contain any toxic dopants, they may be used as safe alternatives for bio-imaging. The cytotoxicity of fHAps was tested using dose dependent MTT and LDH assays in HeLa cell lines. MTT assay is mainly based on enzymatic reduction of MTT dye whereas LDH assay is based on the release of lactose dehydrogenase enzyme after cell membrane damage. Figure 3A shows result of cell viability measured using MTT assay. Cell growth was reduced by ~3-5% at concentrations above 500µg/ml of fHAp NPs. There was no significant difference between the cell viability of untreated cells and cells treated with fHAps, indicating non-toxicity of fHAp NPs (Figure 3A). Similar results were obtained with LDH assay further confirming the non-toxic nature of fHAp NPs (Figure 3B). ROS assay was carried out, to determine ROS generation in HeLa cells exposed to fHAp NPs. ROS damages cells by protein denaturation, DNA damage and altering macromolecules like polyunsaturated fatty acids in membrane lipids. No significant ROS generation was observed even at concentrations of 500 and 1000 µg/ml of fHAp NPs (Figure 3C). Several reports have shown that HAp is biocompatible and its internalization, toxicity, ROS generation depends on its size, shape, charge, concentration, method of synthesis, etc.,

18, 22-23, 43-45

.

In vitro assessment of nanoparticles with the blood component is a necessary factor to classify NPs as biocompatible. Hemolytic assay was performed to examine the interaction of various


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concentrations of fHAp NPs with red blood cell membranes by measuring the release of hemoglobin. Hemolytic rate of fHAp NPs was 0.78% and 0.98% at concentrations of 500 and 1000µg/ml respectively. According to ASTM F 756-00, samples with hemolysis rate less than 2% can be 24

considered non-hemolytic, indicating that the fHAp NPs are non-hemolytic .

Figure3. A) MTT, B) LDH, C) ROS generation data showing biocompatibility of thin-rod fHAp NPs on HeLa cell lines after 24 h of incubation at various concentrations ranging from 10-1000 µg/ml. D) Fluorescence spectra of thin-rod fHAp NPs in biological media DMEM (red) and PBS(pH 7) (black). No change in fluorescence spectra in both the conditions indicating that fluorescence of fHAp NPs is not dependent on the surrounding environment, and is rather an intrinsic property of the nanoparticles.

In order to be a good bio-imaging/labeling agent, the fHAp NPs need to retain their fluorescing nature in biological environment. To check this, we placed fHAp NPs in phosphate buffered saline (PBS, pH



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7), to mimic the extracellular environment, and in Dulbecco’s modified Eagle’s medium (DMEM), to mimic in-vitro conditions. No change in fluorescence spectra was observed in all these conditions indicating that fluorescence of fHAp is not dependent on the environment, and is rather an intrinsic property of the nanoparticles (Figure 3D).

Figure 4. Bright intracellular fluorescence with green and red emissions was observed inside the cells under epi-fluorescence microscope, using bright field, FITC filter, and TRITC filter indicating that thinrods HAp NPs were internalized by a) HeLa cells b) Candida albicans c) Staphylococcus aureus d) Escherichia coli.

Cellular internalization of fluorescent NPs is essential to study cellular/molecular events in-situ. We explored the application of fHAp NPs as in-situ fluorescent probe by examining their potential of cellular internalization in both prokaryotic and eukaryotic systems. HeLa, Candida albicans, Staphylococcus aureus and Escherichia coli were used as the representatives of eukaryotic and prokaryotic cells. Bright intracellular fluorescence with green and red emissions (Figure 4) was


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observed inside the cells under epi-fluorescence microscope, indicating that the fHAp NPs were internalized by both the cell types. Internalization of HAp and lanthanide doped HAp NPs by cells such as fibroblasts, epithelial cells, monocytes, macrophages, human hepatoma etc.,

11, 19, 43-44, 46

have been extensively studied by many

groups. NPs such as quantum dots, carbon dots, lanthanide-based fluorophores, UpNPs, etc. are being studied as multimodal, multifunctional fluorescent probes for in vitro and in vivo bio-imaging. However, cytotoxicity of these particles is of great concern, limiting their in vivo applications10, 19, 47. This limitation can easily be overcome by using non-toxic and self-activated fluorescence of HAp. To the best of our knowledge, this is the first report on uptake and fluorescence of undoped HAp NPs in yeast, bacterial and animal cells. We believe that these fHAps provide a safe and equally dependable alternative for the current fluorescent materials. By tagging these fHAps with specific antibodies, RNA, aptamers or proteins; they can further be modified to specifically recognize certain types of pathogens in blood or environmental samples making on-site diagnosis a possibility.

Conclusion: A new class of fHAps with varied aspect ratios was synthesized at room temperature by sol-gel method. Solvents and stabilizing agents played an important role in obtaining fHAp NPs with different morphologies (rods, thin-rods, short-rods). The intense fluorescence observed is most probably associated with the electronic transitions arising due to asymmetric structure of the NPs as all of them have elongated morphology. Another interesting property exhibited by the fHAp NPs was the internalization by both prokaryotic and eukaryotic systems without causing any cytotoxicity. These multifunctional NPs provide exciting opportunities in the field of bio-medical sciences, making it possible to carry out both Imaging and treatment in conjunction.

Supporting Information Available The following files are available free of charge. Figure S1. Schematic illustration of various synthesis of HAp NPs by using DMSO (A) and water (B) as solvent while TEA/ACA as reducing agents. Figure S2. contains the XRD patterns and FT-IR spectra of (i) sphere (control), (ii) rods (water/TEA), (iii) short-rods (DMSO/TEA), and (iv) thin-rods (DMSO/ACA) shaped HAp NPs.



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Figure S3. contains the TEM micrographs of HAp NPs synthesized by using water/DMSO as solvent in the presence of ACA (acidic) and TEA (alkaline) stabilizing agents. Figure S4. contains TEM micrographs of HAp in the absence of stabilizing agents along with the commercially available HAp from Sigma-Aldrich.

Acknowledgements The authors are grateful to Dr. Teny T. John and Ms. P. R Chithira, Department of physics, BITS Pilani Goa campus, for kind help with photoluminescence studies. Special thanks to Dr. Ravi Prasad Aduri for critically evaluating the manuscript and his valuable suggestions. Authors extend their thanks to Dr. Amrita Chatterjee, for the spectrofluorometric studies and Dr. N. N. Ghosh for the DLS analysis. Authors would also thank, E. S. Kannan for help with the interpretation of photoluminescence and the ESR data. We would like to thank Sophisticated Analysis Instrumentation Facility, Indian Institute of Technology, Bombay for their help with TEM and ESR studies.

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Table of Contents Graphic for Self-activated fluorescent hydroxyapatite nanoparticles: a promising agent for bio-imaging and bio-labeling a

a

b*

a*

Ketaki Deshmukh , M. Monsoor Shaik , Sutapa Roy Ramanan , Meenal Kowshik . a

b

Biological Sciences Department, Chemical Engineering Department.

Birla Institute of Technology and Science Pilani, K K Birla Goa Campus, Goa, India [email protected],

[email protected],

[email protected],

[email protected]

pilani.ac.in. Corresponding authors: Dr.Meenal Kowshik a, Dr.Sutapa Roy Ramananb Present/Permanent Address: Birla Institute of Technology and Science Pilani, K K Birla Goa Campus, Zuarinagar, Goa, India – 403726 Phone No. 0832-2580304, 0832-2580325 Fax no: 0832 - 2517033


Self-Activated Fluorescent Hydroxyapatite Nanoparticles: A Promising

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