Europium Doped Calcium Deficient Hydroxyapatite ... - ACS Publications


Oct 24, 2017 - Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences & Technology, Poojappura, Thiruvananthapuram 695012, India...
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Europium Doped Calcium Deficient Hydroxyapatite as Theranostic Nanoplatforms : Effect of Structure and Aspect Ratio Sunita Prem Victor, M.G. Gayathri Devi, Willi Paul, Vineeth M. Vijayan, Jayabalan Muthu, and Chandra P. Sharma ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.7b00453 • Publication Date (Web): 24 Oct 2017 Downloaded from http://pubs.acs.org on October 24, 2017

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Europium Doped Calcium Deficient Hydroxyapatite as Theranostic Nanoplatforms: Effect of Structure and Aspect Ratio Sunita Prem Victora,c † , M.G. Gayathri Devia, Willi Paulb,d, Vineeth M. Vijayanc, Jayabalan Muthu c and Chandra P. Sharmab † 1

Division of Polymeric Medical Devices, 2Biosurface Technology, 3Polymer Division, 4Central

Analytical Facility Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences & Technology, Poojappura, Thiruvananthapuram 695012, India



Corresponding Authors - Dr. Chandra P. Sharma, E-mail: [email protected]; Dr. Sunita Prem Victor, E-mail: [email protected]

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Abstract We present the development of theranostic nanoplatforms (NPs) based on europium (Eu3+) doped calcium deficient hydroxyapatite (CDHA) core functionalised with cyclodextrin (βCD) and cucurbitural (CB[7]). The composition, crystalline structure, aspect ratio, surface area, morphology and luminescence property of the NPs were investigated by means of xray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR), energy dispersive x-ray analysis (EDAX), Brunauer–Emmett–Teller (BET) method, transmission electron microscopy (TEM), and fluorescence spectroscopy. The perceivable effects of Eu3+ doping appear in the minor peak shift to larger angles attributed to lower crystallite size and smaller aspect ratios coupled with greater structural strain in the rod shaped theranostic NPs and a shift in their zeta potential towards less negative values. Cell parameter calculations suggest that the doping of Eu3+ would cause the a-axis parameter to decrease slightly as the ionic radius of Eu3+ is smaller than that of Ca2+. Moreover drug release profiles employing 5-fluorouracil (5FU) suggest that these luminescent NPs depict controlled and sustained release profiles. Further the emissive intensities of the NPs in the carrier systems increase with cumulative released amounts of 5FU suggesting that the release of drug can be monitored by changes in luminescent intensity. In addition, native NPs manifest commendable cytocompatibility as demonstrated by MTT and Live/dead protocols whereas the 5FU loaded NPs demonstrated over 80% HeLa cell death signifying their therapeutic potential. We envision that these NPs can serve as effective and practical multifunctional probes for theranostic applications.

Keywords Crytallinity, fluorescence, cytocompatibility, cyclodextrin, cucurbitural

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Introduction “Theranostics” refers to the fusion of therapeutics with diagnostic agents in a collaborative manner to combat specific diseases. They have evolved to encompass nanoplatforms (NPs) that include both therapeutic and imaging components.1 These NPs which possess incredible potential are better than their traditional counterparts owing to their superior capabilities that include image guided therapeutics, synergetic performance, sustained and controlled release, targeted delivery and therapeutic monitoring.2 In this regard, carbon nanomaterials, liposomes, gold nanostars, quantum dots, iron oxide nanoparticles and mesoporous silica nanoparticles have attracted significant attention.3-8 However causes of concerns such as high cost and stability of gold nanoparticles, nonbiodegradability and toxicity of carbon nanotubes, hypersensitivity of polymeric nanoparticles, in vivo biodistribution and toxicity of silica nanoparticles limit their in vivo application potential.2 A plethora of approaches are now underway to develop more efficient and advanced systems that can monitor changes at the cellular level which can be employed for a clinical setting.

Calcium phosphates are bequeathed with commendable properties such as high biocompatibility, structural capacity for ionic substitutions, bioactivity, tunable biodegradability and nontoxicity.9,10 Their properties strongly depend on the Ca/P molar ratio which ranges from bioactive hydroxyapatite (HA, Ca/P=1.67) to resorbable tricalcium phosphate (TCP, Ca/P=1.5).11 In particular, the predominant constituent of biological hard tissue is a calcium deficient hydroxyapatite (CDHA, Ca/P ratios ranging from 1.67 to 1.33) which resembles HA stoichiometrically and possesses tunable degradation characteristics.12 The dissolution rate of CDHAs can be contained by adjusting its crystallinity and Ca/P ratio, thereby potentially controlling the release rate of incorporated drug and promoting the development of efficient and controlled delivery systems.13 Moreover the structural capacity of CDHAs to accept lanthanide ionic substitution has already been demonstrated by Graeve et al.14 They observed higher intensity and

broader emission for Europium (Eu2+ /Eu3+) substituted CDHAs when

compared to Eu3+ substituted HA nanopowders. So lanthanide doped CDHAs seem to be a promising material for imaging applications.

Additionally it is imperative that the 3

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synthesized CDHAs possess high crystallinity necessary for greater fluorescence yield and nano size regimes to facilitate efficient cellular internalization properties.

Cyclodextrins (β-CD) and cucurbiturals (CB[7]) are supramolecular systems with unique molecular structures and have been widely recognized as pharmaceutical excipients.15,16 β-CDs are hollow truncated cones and CB[7] are macrocyclic pumpkin shaped hexamers, both comprising of hydrophobic cores which demonstrate superior complexing capabilities with hydrophobic drugs. Several research groups have reported the application of these molecules in enhancing drug stability, drug absorption and altering release patterns from nanoparticles.17-19 From this view point we can assume the presence of β-CD/CB[7] in the engineered CDHA NPs might augment drug loading capabilities and enhance therapeutic efficacy. So the conjunction of CDHAs with Eu3+ and β-CD/CB[7] seems to be an interesting strategy in the development of facile theranostic NPs. The simultaneous presence of CDHA, Eu3+ and β-CD/CB[7] could unravel myriad advantages. The presence of Eu3+ bestows the NPs with fluorescence imaging capabilities. In addition ions such as Eu3+ and gadolinium (Gd3+) are functional mimics of calcium ions and could play a critical role in the bone remodeling cycle.20 Moreover Eu3+ and Gd3+ dual-doped HA nanocrystals have been shown to possess photo luminescent, drug delivery and imaging properties by Xie et al21 and Chen et al.22 The crystalline CDHA core in the NPs further ensures intense fluorescence intensity. In addition the concomitant presence of β-CD/CB[7] should augment the encapsulation efficiency of drugs and assist in a controlled and sustained release profile pattern. Additionally the release of the drug can be controlled by the dissolution of the CDHA core of the NPs that can be fine tuned for optimum efficacy.

Materials and Methods Theranostic nanoplatforms (NPs) were synthesized by an adept co-precipitation method. Pure CDHA having a Ca/P molar ratio of 1.61 was initially synthesized by following previously established protocols. The amount of reactants was calculated based on the Ca/P molar ratio of 1.61. Briefly, 200 ml of calcium chloride (3.672 g) solution was 4 ACS Paragon Plus Environment

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mixed with 20 ml of tri sodium citrate (0.9 g) solution to obtain a solution of pH 7.5. 200 ml of disodium hydrogen phosphate (4.48 g) was then introduced dropwise into the solution mixture. The mixture was continuously stirred at around 400 rpm using an overhead stirrer for 24 h. The CDHA nanoparticles were then filtered, washed, heated to 100˚C and lyophilized to acquire a fine powder. To obtain diverse theranostic NPs varying concentrations of Eu3+ were introduced into the calcium chloride solution and the synthesis procedure modified accordingly. Varying concentrations of Eu3+ (0.5g, 0.75g, 1g) were introduced drop wise in parallel to the addition of disodium hydrogen phosphate to obtain theranostic CDHA NPs. These theranostic CDHA NPs incorporating 0.5g, 0.75g and 1g of Eu3+ are abbreviated as HEu0.5, HEu0.75 and HEu1 respectively and subjected to further characterization studies. To introduce CB[7] or β-CD into these theranostic nanoprobe systems, the preliminary step involving calcium chloride and tri sodium citrate solution mixture was modified to include appropriate 1 ml of CB[7] solution (20 mg in 1ml) or 1 ml of β-CD suspension (20 mg in 1 ml). Subsequently the latter steps in the preparation route remain unchanged. The HEu0.75 theranostic NPs modified using CB [7] or β-CD have been coded as HEu0.75CB or HEu0.75CD respectively Physiochemical Characterization Techniques Average bulk composition and phase identify of the NPs was carried out by x-ray powder diffraction (XRD) technique (Cu kα radiation, reflection mode, Bruker D8, Japan) by comparison with that of standard HA (JCPDS; 09-0432). In addition average crystalline size and cell parameters were calculated employing the Scherrer formula and least square fit method respectively. The Ca/P molar ratio of the synthesized CDHA was obtained by ICP-OES. The chemical composition and functional groups present in the NPs were identified

by

Fourier

transform

infrared

(FTIR)

spectra

(Jasco,

FT/IR-4200

spectrophotometer) method. The aspect ratio, nanoprobe size and morphology were characterized by transmission electron microscopy (TEM, Philips CM12 STEM, Netherlands). Samples were drop cast on carbon coated copper grids, dried and visualized using TEM. Specific surface areas of the NPs were calculated by the Brunauer–Emmett– Teller (BET) method. The zeta potential of the NPs was calculated using a zetasizer (Malvern nanoseries, Worcestershire, U.K) at pH 7.4 (PBS). We also calculated the 5 ACS Paragon Plus Environment

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amounts of Eu3+ in the NPs by energy dispersive X-ray analysis (EDX). An anticancer drug 5 Fluorouracil (5FU) was employed to test the efficacy of these NPs for theranostic applications. The dissolution studies were carried out by dispersing the samples in sodium acetate buffer having pH 5.2 at 37˚C. 0.5 ml of buffer were withdrawn at regular intervals and the amount of calcium released was analyzed at 578 nm with a spectrophotometer (Cary 50, Varian) employing a calcium assay kit (Enzyme Technologies Pvt. Ltd., Baroda). Drug loading was quantitatively optimized by varying the parameters mentioned in the equation given below.

Drug loading = Mbound/Wparticle

(1)

where Mbound is the amount of drug (mg) eluted from the particles in PBS (pH~7.4) in 24 h and Wparticle is the amount of particle (g) utilized for drug loading. In a typical loading process, 25 mg of HEu0.75 and 400 µg of 5FU drug resulted in optimum loading. Briefly, 25 mg of HEu0.75 was dispersed in 1ml PBS. 400 ug of 5FU was then added and the solution was left overnight with constant stirring. The excess PBS was removed and the resulting HEu0.75-5FU mixture was lyophilized. To calculate Mbound the lyophilized mixture was suspended in PBS for 24 h and 5FU content in HEu0.75 was calculated. Similar procedure was repeated for all the engineered NPs. The amount of 5FU released from these NP was quantitatively measured using a UV/Vis spectrophotometer (Cary 50, Varian) and quantified by referencing it against a 5FU standard calibration plot (correlation coefficient R2 = 0.992). Briefly, 10 mg of HEu0.755FU mixture was dispersed in 10 ml Sodium acetate buffer (pH= 5.2) and the vial placed inside an orbital shaker maintained at RPM 200 and temperature 37˚C. At predetermined time intervals, aliquots (2ml) were withdrawn; and the release determined by measuring absorbance at 266 nm and quantified by comparing to previously constructed standard plot. The buffer was replaced after each withdrawal to maintain constant volume conditions. All release experiments were completed in triplicate and have been reported with standard deviation

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Biocompatibility Evaluation Protein adsorption behaviour of the NPs was assessed by Lowry method by following previously established procedures. Briefly, calibration curves for albumin, fibrinogen and globulin were initially plotted from different known concentrations of protein solutions.23 The quantification of the particular protein was then carried out by immersing 50 mg of the sample in 10 ml of 25% protein solution. At time intervals of 5, 15, 30 and 60 min, solutions are withdrawn and the amount of protein in the solution was obtained by Lowry method by measuring the absorbance values at 750nm using UV/Vis spectrophotometer (Cary 50, Varian). The concentration of the protein adsorbed was subsequently calculated by using the respective calibration curve obtained for pure protein. All experiments were carried out in triplicate and the values reported with standard deviation The in vitro biocompatibility of the engineered NPs was determined by the proliferation of HeLa cells using MTT assay (ISO/EN10993-5). Briefly HeLa cells (1x105 cells/well) were seeded on a 96-well tissue culture plate and incubated in 5% CO2 and 95% humidity with DMEM medium (containing 10% FBS) for 24 h. The medium was then removed and replaced with different concentrations (10 mg/ml, 15 mg/ml, and 20 mg/ml) of the NPs and incubated for 24 h. 20 µl MTT (5 mg/ml ) solution was then added, followed by incubation for another 4 h. Thereafter 200 µl DMSO was added in each well and absorbance measured at 570 nm (Plate reader, Tecan, Infinite M200, Switzerland). Further; to determine the therapeutic potential of these NPs, varying concentrations of 5FU loaded NPs were incubated with HeLa cells for 24 h and the viability determined as discussed above. Live/dead staining was also performed alongside employing a Calcein/Ethidium homodimer-1 kit. Evaluation of Luminescence properties The aptitude of these NPs to function as imaging agents was assessed by recording their excitation and emission spectra (JASCO FP-8200 spectrophotometer). Measurements were carried out on suspensions of NPs in PBS (10 mg/1.5 ml) at pH 7.4. Results and Discussion Composition, morphology, physicochemical and spectral properties of NPs

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A series of theranostic nanomaterials based on CDHA core were systematically synthesized. The Ca/P molar ratio of pristine CDHA samples obtained by ICP-OES was 1.61 ± 0.19. The crystalline structure and phase purity of the engineered theranostic NPs were evaluated using XRD. Figure 1 represents the XRD pattern of pristine CDHA, the theranostic NPs HEu0.5, HEu0.75 and HEu1 with different Eu3+ concentrations and the supramolecular NPs HEu0.75CB and HEu0.75CD respectively.

Figure 1. XRD micrographs of CDHA, HEu0.5, HEu0.75, HEu1, HEu0.75CB and HEu0.75CD nanoplatforms.

It was observed that the prepared theranostic NPs could be indexed to pristine HA hexagonal phase (JCPDS; 09-432). This reaffirms the difficulty of differentiating CDHA

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from HA using XRD method. The patterns were found to be similar for the HEu0.5, HEu0.75 and HEu1 NPs with no additional peaks being detected, suggesting that Eu3+ has been successfully doped into the crystal lattice confirming the purity of the synthesized NPs. The perceivable effect of Eu3+ doping appears in the minor peak shift to larger angles as observed in the XRD patterns of HEu0.5, HEu0.75 and HEu1 NPs. This slight shift to larger angles is attributed to lower crystallite23 size coupled with greater structural strain in the theranostic NPs. In a study involving dual doped HA nanoparticles, effects of Fe3+/Eu3+

doping, manifested changes in peak position, intensity and broadening.24

However in our present study the ionic radius of Eu3+ which is around 108 pm closely matches that of Ca2+ ensuring minimal lattice effects and no changes in observed XRD intensity patterns.

On the other hand the diffractograms of the HEu0.75CB and

HEu0.75CD (Figure 1 inset) supramolecular NPs displayed all reflections observed for the CDHA phase in addition to the characteristic peaks of CB[7] and β-CD respectively. CB[7] in its crystalline form exhibits peaks at 27°, 30°, 31°, 32°, 36°, 38°, 39° and 45°. Similarly crystalline β-CD exhibits main diffraction peaks at 4.75°, 12.7°, 15°, 19.7°, 21.1°, 22.8°, 29° and 34° respectively. The diffractogram of HEu0.75CB depict the characteristic peaks of CB[7] at 27°, 31° and 32° in addition to the signature peaks of CDHA. Similarly the HEu0.75CD diffractogram reveal peaks corresponding to β-CD at 29° and 34° together with the characteristic peaks of CDHA. Overall the NPs reveal good crytallinity as indicated by the narrow XRD diffraction peaks recorded in the XRD spectra. This increased crystallinity of the apatite could induce increase in luminescence signal intensity of the fluorescent ion ensuring optimum fluorescence.25 Moreover it has been reported that the luminescence associated with lanthanide doped apatites is brought about by the substitution of Ca2+ ions by that of lanthanide in the apatite lattice.25

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Sample

Aspect ratio

Zeta Potential (mV)

Crystal Size (nm)

Surface area (m2/g)

CDHA

65.7±7.25

-26.6±0.72

62

83.39

57.3±7.56

-24.1±0.32

53

84.21

HEu0.75

40.5±4.95

-22.5±0.63

39

86.56

HEu1

35.9±4.06

-19.4±0.56

36

88.96

36.4±7.01

-18.8±0.51

38

92.45

35.6±4.88

-20.2±0.32

37

96.38

HEu0.5

HEu0.75 CD HEu0.75 CB

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Cell Parameter (nm)

Pore volume cm3/g

a = b = 0.9413, c = 0.6879 a = b = 0.9412, c = 0.6878 a = b = 0.9412, c = 0.6878 a = b = 0.9411, c = 0.6878 a = b = 0.9412, c = 0.6878 a = b = 0.9412 c= 0.6878

0.26 0.27 0.30 0.32 0.37 0.37

Table 1 Characterization of aspect ratio, zeta potential, crystalline size, surface area, cell parameter and pore volume of pristine CDHA, HEu0.5, HEu0.75, HEu1, HEu0.75CB and HEu0.75CD nanoprobes. The peak broadening of the diffraction peak at 25.9◦ assigned to (001) Miller plane was selected for the calculation of crystallite size using Scherer’s formula. We notice that the doping of Eu3+ led to smaller crystallite size as expressed in Table 1. As the concentration of Eu3+ increases there is a corresponding decrease in the size of the theranostic NPs HEu0.5, HEu0.75 and HEu1 when compared to pristine CDHA. Martin et al

26

postulated

charge compensation mechanism for occupancy of Eu3+ into Ca2+ sites. Since the electric charge of Eu3+ is different from that of Ca2+, this could influence crystal growth resulting in the observed smaller size. However in the case of the supramolecular NPs HEu0.75CB and HEu0.75CD we do not discern any obvious change in crystallite size.

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Cell parameters evaluated by least square fit method (Table 1) indicate that the values obtained for the theranostic NPs are in good agreement with that of CDHA. The results suggest that the doping of Eu3+ would cause the a-axis parameter to decrease slightly as the ionic radius of Eu3+ is smaller than that of Ca2+. Moreover we do not notice any significant decrease in c-axis since there is a possibility of carbonate ions substituting for phosphate ions thereby countering the reduction of c-axis parameter. Similar trends have been observed by Chen et al in Fe3+/Eu3+ co-doped HA nanoparticle systems.24 The amounts of Eu3+ in the NPs calculated by EDX were found to be 6.38±0.57 wt%, 11.94±0.78 wt% and 13.8±0.23 wt% for HEu0.5, HEu0.75 and HEu1 NPs respectively.

Accordingly the FTIR spectrum of native CDHA (Supplementary Fig 1) reveals characteristic bands for phosphate bending at 548 cm−1 and 601 cm−1 and phosphate stretching at 962 cm−1, 1044 cm−1 and 1090 cm−1. The pertinent bands of hydroxyl, carbonate and water appeared at 3400 cm−1, 1392 cm−1 and 1568 cm−1 respectively. Additionally the characteristic HPO42- band appears at 878 cm−1 confirming the presence of CDHA phase. The second derivatives of the FTIR spectra of theranostic particles and the Raman data have been added in Supplementary Figure 3 and 4. The respective adsorption isotherms of the NPs exhibit type IV isotherms (Supplementary Figure 3) related to the fundamental mesoporous structure of CDHA. Typical surface areas and average pore volumes of the engineered samples are listed in Table 1. Pure CDHA has BET surface area of 83.39 m2/g, and average pore volume of 0.26 cm3/g. Similarly the HEu0.75 nanoprobe presented values of 86.56 m2/g and 0.30 cm3/g for BET surface area and average pore volume respectively. However the HEu0.75CB NPs display a pronounced increase in BET surface area (96.38 m2/g) coupled with increase in pore volume (0.37 cm3/g). The results affirm that the surface areas of the Eu3+ doped NPs are larger than that of CDHA nanoparticles. In retrospect the greater surface area and higher accessible pore volume could be advantageous for the encapsulation of biological molecules emanating in improved cargo loading characteristics.

The morphologies and aspect ratios of pristine CDHA, HEu0.5, HEu0.75, HEu1, HEu0.75CB and HEu0.75CD samples were investigated with TEM, as depicted in Figure 11 ACS Paragon Plus Environment

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2(a-f). All the NPs display a homogenous particle size distribution and possessed rod shaped morphology suggesting that the presence of Eu3+ did not result in any perceivable change in morphology of the CDHA nanoparticles. The size distribution of the NPs depicted as histogram is provided as supplementary figure 5. The aspect ratios (Table 1) calculated from TEM software varied from 66 for CDHA to about 36 for the supramolecular theranostic NPs HEu0.75CB and HEu0.75CD. Qui et al investigated the dependence of cellular uptake on aspect ratios of gold nanorods and observed that rod shaped nanoparticles with smaller aspect ratios are internalized faster with effectively nominal membrane disruption.27 In particular, longer rods tend to form large aggregates which are difficult to be internalized as more energy needs to be expended for successful internalization.27 So the smaller aspect ratios obtained for the synthesized NPs HEu0.75, HEu0.75CB and HEu0.75CD could be favorable for effective internalization. The zeta potential and poly dispersity index of the NPs are summarized in Table 1. The zeta potential of pristine CDHA was measured to be about -26.6 ± 0.42 mV. The presence of Eu3+ induces a shift in the zeta potential towards less negative values (Table 1).

Figure 2. TEM micrographs of (a) CDHA, (b) HEu0.5, (c) HEu0.75, (d) HEu1, (e) HEu0.75CB and (f) HEu0.75CD nanoplatforms 12 ACS Paragon Plus Environment

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. A similar trend in zeta potential values has been noticed for Eu3+ and Gd3+ doped mesoporous silica nanoparticles wherein the measured zeta potential of silica nanoparticles doped with Eu3+ and Gd3+ shift to less negative values when compared to native silica nanoparticles.28

The suitability of these theranostic NPs as fluorescence agents for imaging has been investigated by PL studies. A detailed analysis of the luminescent variations in Eu3+ doped HA and CDHA report excitation wavelengths of 305, 337 and 359 nm for promoting Eu3+ emissions.14 Tagaya et al reported an excitation wavelength of 394 which showed two prominent emission peaks at 592 and 615 nm in Eu3+ containing nanoporous silica particles. These two pertinent peaks arise from 5D0-7F1 and 5D0-7F2 transitions of Eu3+.29 Figure 3 represents the emission spectra of the samples at an excitation wavelength of 397 nm i.e. at the maximum of excitation spectrum. The emission spectra revealed two strong emissions at 597 nm and 620 nm corresponding to the 5D0-7F1 and 5

D0-7F2 transitions of Eu3+ respectively.

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Figure 3. PL emission intensity of (a) CDHA, (b) HEu0.5, (c) HEu0.75, (d) HEu1, (e) HEu0.75CB and (f) HEu0.75CD nanoplatforms. As can be discerned the concentration of Eu3+ affects the luminescent intensity of the NPs with intensity decreasing in the order HEu1> HEu0.75> HEu0.5. The supramolecular NPs HEu0.75CB and HEu0.75CD also exhibit strong emissions indicating that the crystallinity of the samples help enhance luminescent properties. Enhancement of luminescence has also been reported by Xie et al in Eu3+/Gd3+ co-doped HA nanocrytals based on energy transfer mechanism.21 In addition, heat treatment of apatites has also been suggested as a route to optimize crystallinity and improve luminescent behavior.30 The overall results obtained suggest that the NPs in the present study possess promising properties that include good crytallinity, optimal aspect ratios, rod shaped morphology, optimum surface area and luminescence potential. The HEu0.75, HEu0.75CB and HEu0.75CD NPs with smaller aspect ratios, optimal crystallinity, suitable morphology and effective luminescence behavior have been selected for further biological evaluation.

Drug adsorption and release properties Drug loading in calcium phosphate based nanoparticle systems are governed by factors including drug concentration, density, surface area, surface charge and porosity of the nanoparticles. The respective loading degrees of selected NPs HEu0.75, HEu0.75CB and HEu0.75CD were 37.5 mg/g, 49.6 mg/g and 44 mg/g respectively when compared to pristine CDHA which had a loading degree of 33.5 mg/g. The amount of drug loading has been found to depend on the surface area values of the samples and loading was significantly greater for HEu0.75CB when compared to HEu0.75 and HEu0.75CD. The HEu0.75CB NPs possessing highest surface area among the evaluated systems display augmented drug loading capabilities. Iafisco et al

31

reports loading amounts of 475µg of

DOX/mg of HA and 449 µg of DOX/mg of FeHA corresponding to the maximum drug loading capacity of HA. Likewise Zhang et al 32 reports an ibuprofen loading of 32.9 wt% on luminescent strontium /HA nanorods wherein the driving force behind drug loading was associated to the hydroxyl groups present on the surface of apatite molecules; which

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facilitate hydrogen bonding with the respective carboxyl groups present on ibuprofen drug moieties. To further understand the interaction mechanism of NPs with the drug 5FU we measured the zeta potential of HEu0.75, HEu0.75CB and HEu0.75CD after incorporation of 5FU. The coupling of HEu0.75, HEu0.75CB and HEu0.75CD with 5FU caused a shift of zeta potential towards less negative values (-15.3 ± 0.34, -13.6 ± 0.46 and -11 ± 0.41mV for HEu0.75, HEu0.75CB and HEu0.75CD respectively). This can be attributed to a possible interaction of the positively charged -NH3+ groups of the 5FU31 with the negatively charged surface groups present on the NP. Similar shift in zeta potential towards less negative values has been reported for Fe3+ doped HA nanoparticles and was explained by the possible interaction between -NH3+ groups on DOX with surface groups on HA and FeHA nanoparticles.31 This surface uptake of DOX resulted in inducing less negative surface charge leading to decreased interparticle repulsion between the nanoparticles. Thus possible electrostatic interactions also play an active role in assisting and improving drug loading efficiency in the engineered NPs.

Efficient degradation behavior of the carrier is crucial for drug delivery applications. The degradation behavior of CDHA was initially evaluated and compared with the other samples. We notice that more than thirty percent of calcium was dissolved in 12 hours for the CDHA sample. This trend in dissolution has been attributed to its nonstoichiometric behavior where the removal of calcium precedes that of phosphates.32 Similarly all the samples exhibit similar trend in dissolution with around 30-35% of calcium being removed within 24 hours. The cumulative 5FU release profiles of the samples as a function of release time is shown in Figure 4.

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Figure 4. Release profile of 5FU from (a) CDHA, (b) HEu0.5, (c) HEu0.75, (d) HEu1, (e) HEu0.75CB and (f) HEu0.75CD nanoplatforms.

During the release process the solvent gradually enters the matrix phase resulting in a slow diffusion of 5FU from the system through solvent filled capillary channels. 33 Native CDHA nanoparticles depict a single phase release while the theranostic NPs represent a two-phase release profile. The initial drug burst observed in all the theranostic samples can be associated to the 5FU molecules adsorbed on the surface of the nanoparticles. However in the case of the HEu0.75CB and HEu0.75CD samples this initial burst phase (1 - 6 h) is then succeeded by a slow and sustained release presumably due to the presence of CB[7] and β-CD molecules which assist subsequent sustained release for over 100 hrs. Similar release profiles have been observed in HA/Eu systems33 and HA/Sr systems30, though around 94% IBU drug was released around 14 h. So the sustained release patterns observed in the HEu0.75CB and HEu0.75CD samples can be attributed primarily due to

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greater hydrophobic interactions of 5FU with CB[7] and β-CD coupled with enhanced electrostatic interactions between 5FU molecules and the NPs.

It has been established that the presence of hydroxyl groups in drug molecules lower the emissive property of drug loaded luminescent materials. So it is imperative that we evaluate the PL of HEu0.75CB and HEu0.75CD NPs after drug loading. As can be discerned from Figure 5a the drug loaded NPs have lower intensities when compared to those without 5FU. However it is striking to note that the characteristic emission line at 620 nm appears in the emission spectrum and can be monitored by PL studies. Furthermore the PL emission intensities of these NPs are found to increase with a gradual release in entrapped 5FU (Figure 5b).

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Figure 5. (a) PL emission intensity of HEu0.75CB and HEu0.75CD nanoplatforms with and without 5FU and (b) Cumulative 5FU release from HEu0.75CB nanoplatforms as a function of release time.

The slow release of 5FU will impair quenching effects, resulting in the observed increase in emissive intensities.

This relationship between PL intensity and cumulative drug

release is concurrent with reports in literature30,33 and could be effectively utilized as a probe to track release of drug.

Protein adsorption, cytoxicity and cellular uptake Nanoparticles are rapidly covered by a dense layer of protein when they come in contact with a biological fluid. This adherence of proteins to form a “corona” is strongly influenced by parameters including protein diffusion, protein affinity, type of protein and physiochemical properties of involved protein and the material surface.34 This “corona” then decides the type of response to the nanoparticles which influence circulation time, internalization and biodistribution. So an accurate knowledge of protein adsorption is necessary to understand nanotoxicolgical challenges. Lowry method was used to corroborate protein adsorption by the CDHA, HEu0.75, HEu0.75CB and HEu0.75CD samples. Figure 6 displays the percentage of protein adsorbed on the samples in isolated protein solution. All the samples depict a preferential adsorption of albumin when compared to fibrinogen and gamma globulin. The HEu0.75CB and HEu0.75CD NPs exhibit around 25-30% increase in albumin adsorption when compared to HEu0.75 nanoprobe. This augmented adsorption has been corroborated to the enhanced hydrophobicity associated with the presence of CB[7] and β-CD; which demonstrate innate capabilities to stabilize proteins by sequestration and protect against denaturing and degradation.35

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Figure 6. Percentage of protein adsorbed on the nanoplatforms in isolated protein solution using Lowry protocol.

We evaluated the cellular toxicity of CDHA, HEu0.5, HEu0.75, HEu1, HEu0.75CB and HEu0.75CD samples at different concentrations (10 mg/ml, 15 mg/ml, and 20 mg/ml) on HeLa cells by MTT assay. The corresponding results in Fig 7a demonstrate that all the tested samples depict greater than 80% viability indicating negligible cytotoxicity of the synthesized NPs. Figure 7b shows the viability of HeLa cells when incubated with 5FU loaded CDHA, HEu0.5, HEu0.75, HEu1, HEu0.75CB and HEu0.75CD samples for 24h. The drug loaded NPs presented a prominent cytotoxic effect and revealed low cell viability when compared to NPs without drug and plain 5FU (as standard) even at very low concentrations (10 mg/ml).

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Figure 7. (a) Cellular toxicity of the blank nanoplatforms and (b) Cellular toxicity of 5FU loaded nanoplatforms by MTT assay respectively.

This observed decrease in cell viability is attributed to the release of the anticancer drug from the drug loaded NPs. These results emphasize the good therapeutic potential of the theranostic NPs. In parallel, live/dead assay studies further substantiate the results obtained by MTT assay. Representative images of the response of HeLa cells to HEu0.75CB NPs shown in Fig 8a-8c indicate green fluorescence signaling that the samples did not induce necrosis and preserved cell membrane integrity. In addition, the Live/dead images of 5FU loaded HEu0.75CB NPs (Fig 8d-8f) supplement the observed cytotoxicity to HeLa cells. This observed reduction in cell viability is attributed to the release of anticancer drug 5FU from the NPs suggesting excellent therapeutic potential.

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Figure 8.

(a-c) Live/Dead images of HeLa cells incubated with blank HEu0.75CB

nanoplatforms and (d-f) Live/Dead images of HeLa cells incubated with 5FU loaded HEu0.75CB nanoplatforms (Live cells are represented in green and dead cells in orange). Scale bar denotes 100 micron. Conclusion In conclusion, we report the synthesis of theranostic NPs based on CDHA core. The concomitant presence of Eu3+ and β-CD or CB[7] endows these NPs with fluorescence potential and augmented cargo loading capabilities respectively. Series of characterization techniques confirm the successful synthesis of NPs which possess encouraging properties that include good crytallinity, smaller aspect ratios, rod shaped morphology, optimum surface area and luminescence potential. These NPs further manifested the advantages of the presence of β-CD or CB[7] with respect to 5FU loading, encapsulation efficacy and release kinetics. In addition, native NPs manifest commendable cytocompatibility whereas the 5FU loaded NPs demonstrated over 80% HeLa cell death signifying their therapeutic potential.

Acknowledgment We express our sincere thanks to the Director and Head of the BMT Wing, SCTIMST for the facilities provided. This work was supported by the Innovative Young Biotechnologist Award

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(BT/07/IYBA; 2013-2017), awarded to Dr. Sunita Prem Victor by the Department of Biotechnology, Govt. of India. Funder Author Sunita Prem Victor received funding from IYBA award (BT/07/IYBA; 2013-2017). Supporting Information FTIR of the NPs; Raman spectrum of the NPs, adsorption isotherms and size distribution histograms have been included as supporting information.

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