Study of Gallium Oxide Nanoparticles Conjugated with Î²-Cyclodextrin

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Study of Gallium Oxide Nanoparticles Conjugated with #-cyclodextrin -An Application to Combat Cancer Bichitra Nandi Ganguly, Vivek Verma, Debanuj Chatterjee, Biswarup Satpati, Sushanta Debnath, and Partha Saha ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.6b04807 • Publication Date (Web): 22 Jun 2016 Downloaded from http://pubs.acs.org on June 23, 2016

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ACS Applied Materials & Interfaces

Study of Gallium Oxide Nanoparticles Conjugated with β-cyclodextrin -An Application to Combat Cancer Bichitra Nandi Ganguly*1, Vivek Verma2, Debanuj Chatterjee3, Biswarup Satpati1 Sushanta Debnath1 and Partha Saha1 1 Saha Institute of Nuclear Physics, 1/AF Bidhannagar, KOLKATA-700064, INDIA 2 IISER- PUNE, Dr. Homi Bhabha Road, Pashan, Pune-411008 INDIA, 3 IISER- KOLKATA, Mohanpur - 741 246,West Bengal, INDIA

*Corresponding author’s email : [email protected]

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ABSTRACT Bio-active nanomaterials, namely: Gallium oxy-hydroxide GaO(OH), also surfaceconjugated GaO(OH) with a giant sugar molecule: β-cyclodextrin (CD) have been prepared through a simple wet chemical route such that the same could be suitably used in biomedical diagnostics as well as therapeutic applications. Several physical methods were used for their characterization viz: powder X-ray diffraction pattern of GaO(OH) NPs for their grain size determination, optical spectroscopic absorption (UV-Vis and FT-IR) and fluorescence properties of these NPs to ascertain surface conjugation, as also their wide band-gap properties. Besides these, morphological properties of these NPs were studied by transmission electron microscopic (TEM) investigation, justifying the elemental constitution through energy dispersive X-ray analysis(EDX). Further, biological cellular uptake of these nano-particles have been demonstrated on cancerous HeLa cells and reported with total fetal effect after 72 hours, with CD templated GaO(OH) nano-particles, the fact has not been reported so far.

KEYWORDS: Gallium oxide hydroxide/ GaO(OH); β-cyclodextrine ; GaO(OH) conjugated with βcyclodextrine/GaO(OH)-CD ; band gap; size of the nano-particles; TEM, in-vitro cellular studies.


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1. INTRODUCTION Metal oxide nanoparticles have generated a new pathway for biomedical application1 in the form of diagnostic approaches and therapeutic interventions2,3, for selective destruction of tumor cells and holds potential application in drug delivery systems. Nano-scale particles typically posses a larger percentage of atoms at the material’s surface, which can lead to increased surface reactivity4, can maximize their ability to be loaded with surface binding drug molecules to deliver them to target cells. Also, by appropriate choice of surface conjugation/chelation with ligands, these nanomaterials can acquire the ability to selectively target particular types of cells or to pass through physiological barriers and penetrate deep into the sites of rapidly dividing cells. In other words, such surface conjugated nanomaterials have become forefront scaffolds for designing bioactive materials. Gallium oxide nano particles as hydrous oxides5 have been studied, and recently the interest has been revived with modern aspects6,7 in dispersed aqueous solution phase. Interestingly, aqueous chemistry of Ga (III) have considerable resemblance with that of Fe(III) ions, where ample physiological similarities8 have been observed. Gallium(III) ion was chosen primarily because of its similar size and chemical properties with Fe(III) ions. [see Table I]9. The high degree of correspondence in the chemical behaviors of Ga(III) and Fe(III) can be attributed largely to the comparable values for ionic radii and for measures of ionic (electrostatic) versus covalent contributions to bonding. Ga(III) is however virtually irreducible under physiological conditions. Owing to such chemical advantages, Ga(III) compound has been utilized for pharmaceutical and radio-pharmaceutical uses as Ga-68, a



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short lived positron emitter with 68 minutes half –life, for diagnostic studies in positron emission tomography(PET)10-12. Also, since chemical properties of an element are invariant with the change of the isotope of that element, the same nano-particles/or compounds bearing Ga (III) ion could be suitably labeled in aqueous phase13. When gallium ions are mistakenly taken up by living cells, their ability to respire is interfered (as it is incapable to bind with heme)14, the effect is lethal. The mechanism behind this is that iron is redox active, which allows for the transfer of electrons during respiration, but gallium is redox inactive. Thus, although, gallium compounds have shown high bio-availability15 due to its ability to mimic ferric ions (Fe3+), but gallium ions (Ga3+) are anti-proliferative to pathologically proliferating cells, particularly cancer cells and some bacteria. It is known that gallium has a strong affinity for certain tissues, particularly growing or remodeling bone and many tumors16,17 where large amounts of transferrin receptors are expressed18. The property of gallium to selectively accumulate in tumor cells justifies its importance in the designed molecular conjugated compound in the form of nano-particles. Also, semiconductor nanomaterials have been showing considerable potential for cancer treatment recently2,19. The cytotoxic properties of semiconductor nano-particles against cancerous cells is directly related to its size and band gap properties20. Due to their ability to induce reactive oxygen species (ROS) generation, they can lead to cell death when the anti-oxidative capacity of the cell is exceeded2. Thus it is envisaged, apart from diagnostic advantages, the synthesized wide band gap semi-conductor nano materials may also have a therapeutic potential.


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It is within this perspective, we have chosen to study gallium oxide hydroxide nano particles which could be prepared through a simple wet-chemical route (different from references 5 and 6) under physiological conditions and could be suitably conjugated to a non-reducing cyclic saccharide, a giant sugar molecule namely β-cyclodextrin21, with a hydrophilic outer surface and a lipophilic central cavity. Cyclodextrins have mainly been used as complexing agent to increase aqueous solubility of poorly soluble drugs, and to increase their bio-availability and stability22. Since this giant polysaccharide molecule possesses –OH polar groups projected outwards, it renders the compound water soluble, keeping the hydrophobic moiety intact. This type of conjugation also enhanced the water solubility of the encapsulated drug, so that it could be easily taken up by the malignant cells (those are rapidly dividing), and would be appropriate for the renal clearance of the same after its administration and investigation time duration in molecular imaging processes ( such as in PET), which is very important and hence qualifies as a drug carrier23, 24. In order to have a complete data of GaO(OH) and its CD conjugate, developed under physiological conditions through a soft chemical route25 within the energy regime of hydrogen bonding or dipole-dipole type interactions, their physical characterization is quite pertinent. In this article, we report the physical aspects of these nano-particles and their surface conjugation, so that the nano-structured materials suitably finds their use in biomedical applications. Their properties both as in dry nano particle state as well as in aqueous dispersive phase have been discussed in this article, studying their microscopic morphological properties as well as spectroscopic properties. Further, in-vitro uptake of these inorganic nano particles within the HeLa cell line26 have been investigated and reported following the consequent lethal effect, not known so far.



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2. EXPERIMENTAL SECTION 2.1. Chemical Method : a) GaO(OH) nanoparticles: Gallium (III) nitrate hydrated (Ga(NO3)3.xH2O) Merck, triple distilled water (TDW), Ammonia solution (Merck India) in TDW (1:1/vol), β-cyclodextrin Merck, were utilized for chemical synthesis of pure gallium oxide hydroxide (GaO(OH)) nanoparticles, by the sol-gel technique from gallium (III) nitrate hydrated (Ga(NO3)3.xH2O). A 2mM solution of this gallium salt was prepared in triple distilled water (TDW) and (1:1/vol) ammonia solution was added to an aliquot portion of this solution, drop by drop, maintaining pH~7.5; initially gallium was precipitated as hydroxide. After centrifugation, the precipitate has been collected and re-dispersed several times into triple distilled water (TDW) for removing excess of ammonia and other ions (the supernatant liquid was tested for pH). Finally, the precipitate was recollected and dried at 60oC for two days in vacuum oven (~133 Pa) to get GaO(OH), the same was further stored under vacuum (Figure1a). b) GaO(OH) grown under β -Cyclodextrin template: β-Cyclodextrin, CD (M.F.: C42H70O35, procured from Sigma), was dissolved in mildly warm TDW(35-40°C) to prepare several concentrations of the aqueous solution, within a range of 0.05 to 0.5mM. Desired concentration of CD solution was added to aliquot portions of gallium nitrate solution(2mM) and the final pH was adjusted to ~7.5. After precipitation and centrifugation, the precipitate was again collected and re-dispersed into TDW and checked for the removal of excess of ammonia and other ions. Finally, the precipitate was recollected and dried at 60oC for two days in vacuum oven(~133 Pa) and


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also stored under vacuum for future use(Figure 1b). The prepared samples have been characterized by various physical techniques as given in the following classified sections. c) Biological cell culture and cell incubation with nano particles HeLa cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM, HiMedia, India) supplemented with 10% (v/v) fetal bovine serum (Gibco, Life Technologies, USA) and penicillin-streptomycin (HiMedia India) (0.5 U/ml of penicillin and 0.5 µg/ml streptomycin) in 35 mm dishes at 37 °C in an atmosphere of air with 5% CO2 and constant humidity. Cells were seeded on a TEM grid (pasted on a micro slide) submerged in complete media, to near confluence a day before incubation with the nano-particles. After the cells were grown on the grids, gallium nano-particle suspension was added to the medium to final nano-particle concentrations of 4-10 mg.L-1 for a microscopic assay. After 24hrs, the micro-slides containing the grids were collected and the cells were fixed using 4% para-formaldehyde according to standard protocol. The fixed cells on the TEM grid were studied under Transmission electron microscope (TEM). d) MTT assay for cell viability : Cytotoxicity of GaO-CD on HeLa cells was determined by conventional MTT [MTT= (3(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide, a yellow tetrazole] assay27. Exponentially growing HeLa cells were seeded in 96-well flat-bottom culture plates at a density of 6 x 104 cells per well in 100 µl DMEM medium (Himedia, India) containing 10% FBS (Gibco, USA). The cells were allowed to grow for 24h at 37 ºC in a CO2 incubator (New Brunswick Scientific, U.S.A.), and then the medium was replaced with 100 µl fresh medium containing various concentrations of GaO-CD (0 to 100 mg.L-1). The assay was performed in quadruplet for each concentration. The cells were then incubated 7 ACS Paragon Plus Environment


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for 24h, after which the culture medium was removed aseptically, and 100 µl of 3 mg/ml MTT reagent in DMEM (without FBS) was added to each well. Thereafter, it was incubated for 4h; during this period active mitochondria of viable cells reduce MTT to purple formazan. Unreduced MTT were then discarded and “MTT solubilization solution” [10% Triton-X-100 in acidic (0.1N HCl) iso-propanol] (100 µl) was added into each well to dissolve the formazan precipitate, which was then measured spectrophotometrically using a microplate reader at 570 nm. The cytotoxic effect of each treatment was expressed as percentage of cell viability relative to the untreated control cells. The simplest estimate of IC50 is a plot : concentration of GaO(OH)-CD (X) vs percentage of cell viability (Y), the data has been fitted with a straight line (linear regression). IC50 value is then estimated using the fitted line, i.e., Y = a * X + b, and IC50 = (0.5 - b)/a.

2.2. PHYSICAL METHODS OF CHARACTERIZATION: a) X-ray diffraction (XRD) measurements: The phase structures of the nano-particle samples were identified by X-ray diffraction technique using Rigaku Goniometer TTRAX3 diffractometer with CuKα radiation (wavelength of the radiation, λ = 1.54 Å). The data have been collected in the range (2θ) 4o –80o with a step size of 0.02o. Si has been used as external standard to deconvolute the contribution of instrumental broadening

28

. The XRD pattern has been

shown in Figure 2. The grain sizes of the synthesized samples have been calculated using Scherrer formula28: here the diffraction peak corresponding to 110 plane of the GaO(OH) nano-crystals was considered. The width of the diffraction curve increases as the thickness of the crystal 8 ACS Paragon Plus Environment

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decreases. The width βP is measured in radians as full width at half of the maximum intensity(FWHM). Dhkl = Kλ/βP cosθ

…………… (1)

where, Dhkl is the average grain size, K the shape factor (taken as 0.9), λ is the X-ray wavelength, (here 110 peak of the GaO(OH) spectrum was fitted with a Gaussian, for precision measurement) and the Bragg angle has been 21.4850.

b) Transmission Electron Microscopic (TEM) study: Transmission electron microscopy (TEM) investigations were carried out using FEI, Tecnai G2 F30-ST microscope operated at 300 kV. High-angle annular dark field scanning transmission electron microscopy (STEM-HAADF) was used here using the same microscope, which was equipped with a scanning unit and a HAADF detector from Fischione (model 3000). The compositional analysis was performed using energy dispersive X-ray spectroscopy (EDS, EDAX Inc.) attachment on the Tecnai G2 F30. Energy-filtered TEM (EFTEM) measurements were carried out using a GIF Quantum SE (model 963). For this analysis, the GaO(OH) sample and the CD templated sample have been dispersed in isopropyl alcohol through a bath sonicator; a drop of the same was placed onto a carbon coated copper grid and dried at room temperature. Furthermore, selected area electron diffraction (SAED) patterns have been recorded to determine the growth orientation of the synthesized GaO(OH).

c) Spectroscopic Measurements: i) Fourier transmission infrared (FT-IR) spectra:



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Fourier transmission infrared (FT-IR) spectra of the samples in

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powder form

(finally as pellets in KBr, without moisture) were recorded using a Fourier transform infrared spectrometer (Perkin Elmer FTIR system; Spectrum GX) in the range of 400-6000 cm-1 with a resolution of 0.2 cm-1. ii) UV –Vis Spectroscopic measurements: The optical absorption spectra were measured in the range of 190–1000 nm using a UVVIS spectrophotometer Jasco V-630 (wave length accuracy ±0.2nm) after the dispersion of nano –particles in aqueous solution under physiological pH, at room temperature. iii) Flouresence measurement : The spectrofluorimetry was performed using Jasco FP-8500 Spectrofluorimeter (excitation wavelength: 220nm) with the aqueous dispersion of

GaO(OH), and GaO(OH)-CD

complex, the emission spectrum has been recorded at room temperature.

3. RESULTS 3.1. Phase and morphology : XRD analysis The samples synthesized via a simple wet chemical route under normal temperature ~25°C, under physiological conditions, first without the addition of any capping agent/macromolecular surface active agent. After drying of the precipitate at 60°C nearly for two days under vacuum, the sample has been characterized by X-ray diffraction method. Figure 2a. shows the XRD patterns of samples characterized28, with their respective main lattice planes, the diffraction peaks could be assigned to orthorhombic GaO(OH) shape (JCPDS card No 01-071-2778), the sharp diffraction pattern indicated high crystallinity of the samples. A simple grain size calculation of the powdered nano –sample by Scherrer 10 ACS Paragon Plus Environment

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method was found to be : ~ 380 – 400 nm with reproducible results for different samples prepared. A Rietveld refinement analysis performed with the same samples Figure 2b with lattice parameters : a= 4.58Å, b=9.81Å and c=2.97Å, space group: Fddd, corroborates with the data obtained in reference 6, for a orthorhombic spheroid structure, where the sample was prepared at pH-7, by a soft chemical route. However, when the giant sugar molecule namely β-cyclodextrin(CD) is used as a conjugating agent, in small concentrations (0.05mM to 0.5mM) in the preparative soft chemical route the crystallinity parameters are lost, the yield of the product (in the form of the precipitate) depends on the concentration of the added ligand, which renders it more soluble in the aqueous solution. It appears from the observation that the surface polar –OH groups of the sugar moiety links with gallium oxide hydroxide from all side and serves as a template25,29,30, the crystal diffraction patterns have been diffused/masked and lost. 3.2. Transmission electron microscopy(TEM): Transmission electron microscopic images along with the electron diffraction patterns enables us to elucidate the detailed morphology of the prepared nano-particles vis-a-vis a perception of analytical inference of the shape, size and elemental purity. Figure 3. shows the TEM images of the GaO(OH) nano –particle synthesized. The inset of Figure 3(a) presents magnified image of GaO(OH) nono-disc like structure as shown in Figure 3(b). The selected area electron diffraction (SAED) pattern, from a region marked by a dotted circle is shown in Figure 3(c), which indicates the single crystal nature of the GaO(OH) nano-disc. The HRTEM image in inset of Figure 3(a)

clearly show lattice fringes

indicating the crystalline phase and measured d-spacings 2.82 and 2.51 Å are very close to



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the (202) and (400) inter-planar spacing of orthorhombic GaO(OH) (JCPDS # 73-1028), respectively. To investigate the morphology and chemical composition of these nano-discs, we have performed EFTEM and STEM-HAADF-EDS analysis. The obtained relative thickness (thickness/mean free path) map using EFTEM technique and the corresponding area profile taken from this map for a nano-disc is plotted in the inset of Figure 4(a). This indicates flattop morphology of this nano-disc. Figure 4(b) shows the STEM-HAADF image. The chemical composition of the GaO(OH) was determined by EDS analysis (Figure 4c), confirming the presence of Ga and O which is from the rectangular area 1 marked in Figure 4(b). The observed Cu and C signal is due to the C-coated Cu grid. The EDX line profile along the line 2 in Figure 4(b) further confirm the flat-top morphology of this nano-disc. For a detailed distribution of atomic contents in the materials, elemental mapping was performed using STEM-HAADF-EDS technique (Figure 4d). From TEM imaging and elemental mapping, it can be concluded that GaO(OH) nanoparticles are crystalline, twodimensional and having uniform elemental distribution. There has been no presence of impurity elements within these nano-materials. The shape GaO(OH) NPs which were otherwise referred hitherto as alike to nano –disc like structure (as bare hydrous oxide), have shown a remarkable change in morphological shape and structure, when surface conjugated with β-cyclodextrin. Figure 5a is a representative TEM image where such shape change can be seen. This effect also confirms the observations of an earlier study by Yeh et al.7. Dipole-Dipole interaction of polar –OH group moiety of the giant sugar molecule with the negatively charged surfaces of the nascent nano-particle7 and a slow process of growth along the surface of CD could be


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responsible for such a distinct change in shape of the CD capped NPs (see Figure. 5b), although the detailed physico-chemical mechanism remains to be solved. It is worthwhile to report that surface coroneted nano-particle clusters have been shown to present a different morphological effect when a certain concentration level of the surface active molecule has been attained through aqueous sol-gel route, as shown in case of the synthesis of ZnO NPs31. The chemical composition of the CD capped GaO(OH) was also determined by EDS analysis, confirming the presence of C, Ga and O which is from the rectangular area marked in Figure 5(c). For a detailed distribution of atomic contents, elemental mapping was performed, the composite map in Figure 5d clearly shows the capping of CD. 3.3. Spectroscopic studies: a) FT-IR investigation : The structural properties of the GaO(OH) nano particles as grown and stored under vacuum for two days, has been studied by the FT-IR spectroscopy32-34 and presented in Figure 6. The broad region of stretching frequencies of -OH group is elaborately shown in Figure 6b., for GaO(OH), at a higher energy 3440cm-1 (peak position of the red curve) depicting a greater force constant through the expression: ν = 1/2πc [K/µ]1/2, where ‘K’ is the force constant, µ is the reduced mass of the oscillating dipole and ‘c’ is the velocity of light, (also see Table -II and III). The same absorption due to –OH from CD (black curve) appears at a lower frequency at 3409 cm-1(with a broader FWHM), along with another absorption peak ~2923 cm-1due to free terminal –OH groups in the molecule, attached to the sugar moiety. The latter frequency has been practically insignificant when the conjugating ligand has been loaded by the nano-particles (Figure 6b, in rest of the colored spectra) under different concentrations of CD and in case of pure GaO(OH) nano particles(red curve). The other modes of frequencies are explained in 13 ACS Paragon Plus Environment


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Table II. It has been observed that the bending modes of –OH and their overtones are distinct and clear in CD, these are however shifted in frequency, diffused and diminished in intensity in the templated GaO(OH)-CD due to surface conjugation and involvement of the said moiety with the gallium oxide hydroxide nano-particles.

b) UV-Vis spectrophotometric investigations: The result of the absorption studies are shown in Figure 7, with the absorption peaks in the UV region ~200 -300nm. The absorption peak of the GaO(OH) nano-particle dispersions showed a spectrum with wider (FWHM), indicative of a distribution in size, with its absorption edge (red curve) at 235 nm, corresponding to 5.28 eV, where as that of CD templated GaO(OH) (black curve) showed a narrower distribution with its absorption edge at 230nm corresponding to 5.37 eV. It can be remarked that the synthesized nano-particles show a wide band gap properties. This difference in energy level is due to surface conjugation of GaO(OH) nano particles through a dipole-dipole interaction, which could further show up in either charge transfer35 or energy transfer36 mechanism in the emission spectrum. The absorption spectrum of CD has been found only in UV and near UV-Vis borderline region.

c) Fluorescence : The results of the emission spectrum are given in Figure 7, it shows two emission peaks in the UV region, at 238nm (corresponding to 5.22 eV) and 274 nm(corresponding to 4.52eV), for both GaO(OH) and the conjugated GaO(OH)-CD nano particles, when exited by 220 nm photons. The intensity of the latter is quenched when GaO(OH) nanoparticles are grown on the template CD. When the concentration of CD in the conjugated samples has been increased (0.1mM to 0.5mM), the intensity of the same


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peak is decreased and blue shifted (~ 10 nm) which means emission from a higher energy domain, owing to the change in the chemical environment due to the template. The quenched intensity is attributed to transfer of the exited state energy from the donor GaO(OH) to β-cyclodextrin molecule (CD as an acceptor) through a dipole-dipole interaction37. Thus, suggesting a surface orientation of the dipoles of nanoparticles on to the template –OH groups on CD projecting outwards to the polar region. Moreover, comparing the absorption spectra of β-Cyclodextrin and the emission spectra of GaO(OH) we find a significant overlap [see Figure 7 c] between the two. This bolsters the fact that the quenching has occurred via exited state energy transfer from the donor fluorophore to the acceptor. So a part of the energy emitted by GaO(OH) corresponding to 274nm, is taken up by β-cyclodextrin(CD) and hence the net fluorescence intensity is reduced in the conjugated nano particle. This energy transfer mechanism has been feasible since the donor and acceptor are within the relevant distance and following the calculation of Föster resonance energy transfer (FRET) efficiency, this distance is found to be ~11.5 Å, which in turn suggests GaO(OH) has been conjugated to CD. It is important to note that with increase in CD concentration to (Figure 7 c, 0.5mM, blue curve), there is a strong quenching effect and also prominent blue shift is observed, which depicts a shift in chemical environment due to a strong ensconcement of giant sugar moiety. However, from Figure 7 d, we see that the fluorescence peak of GaO(OH) at 240 nm also shows a significant overlap with the absorption spectrum of β-cyclodextrin, but still it does not undergo quenching. This can be explained by the fact that FRET efficiency also depends on the relative orientation of the donor and acceptor dipoles and is extremely dependent on the donor–acceptor distance, R, falling off at a rate of 1/R6. So, a part of the



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improper orientation of dipoles (out of phase) may not lead to quenching interaction in that wavelength region. In such instances, those interactions that result in a fluorescent donor molecule coming in close proximity will only result in loss of fluorescence intensity38. One may conclude from these observations that the dipoles of GaO(OH) are oriented in a particular fashion on the surface of CD, as a result an elongated spindle shape growth of the nanoparticles are obtained7(compare Figures 5 and 7 and TEM results) as the end product due to ligand encroachment.

d) Biological uptake and interaction:

Biological up take of GaO(OH) and CD coated nanoparticles in the HeLa cells were studied through TEM observation (Figure 8.) along with the control maintained exactly under the same condition. Since the surface of the HeLa cells contain high affinity transferrin receptors, which are the principal vehicles by which iron is brought in to the cells39,40, it could also play the same role for gallium because of the similarity in the ionic radii as well as tri-valent state of the two elements. In this case it is possible that transferrin (associated to its receptor molecule at the cell surface) instead of binding to iron, the same is also capable of binding to Ga (III) in the nano –particles41 which are then internalized and formed clusters within the cytoplasmic compartment areas as seen in the micrograph (Figure 8). Our findings suggest that all the bare gallium nano-particles dose given to the cell line (varied from 4 to 10 mg L-1) have been ingested in to the cells. Such nano – particles are usually negatively charged7 at the surface, likely to be surface coroneted by proteins42 and are localized in specialized compartments37,39. However, the CD wrapped nano-particles (spindle shaped structure) have a larger structure, although appears to have


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penetrated the cellular medium with difficulty but have been found distributed though out the cytoplasm in a cell (Figure 8). This observed phenomenon can be due to either i) the process of uptake is different, owing to the sugar moiety or ii) because of the fact that once they gained entry in to the cell, the sugar phase can interact through dissolution and diffusion to different regions in the cell. Thus, for identical materials of nanoparticles and cells, the biological outcomes are determined by the combined properties of nanoparticles and their conjugated surface42. Even though, given the high surface energy of nanoscaled objects, it is unlikely that the cellular machinery would interact with the pristine surface of the nanoparticles in vitro (as per the observation), this comparison helps to further clarify the importance of the layer of protein and biomolecules adsorbed on the nanoparticles in mediating the interactions of nanomaterials with cells. A systematic dose dependent cell viability assay, following a standard protocol44 was also performed on the HeLa cell line through standard MTT assay25, which shows (Figure 9) results for IC50 value at a dose of 51.65 mgL-1GaO(OH)-CD. This is however an average effect on the cell line. Finally, in order to assay the effect of administered NPs in the HeLa cells, the TEM observation was minutely followed for about 3 days under the specified conditions as given in section 2.c, along with the control. As observed by the Figure 10, the entry of spindle shaped GaO(OH)-CD NPs distributed throughout the cells, dose: 9 mg L-1onwards, show a total lethal effect on the cell line after 72 hours.

4. DISCUSSION:



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Growing of nano crystalline GaO(OH) nano particles through a wet chemical process under physiological conditions enables one to visualize the physical aspects of the particles, namely the size and morphology, the wide band gap properties and structural changes through XRD and spectroscopic investigations. Initially the NPs show orthorhombic crystalline structure (more specifically nano disc like structure by TEM). But the shape of these NPs after being conjugated with large β-Cyclodextrin molecule, have transformed to spindle shaped structure. The spectroscopic investigations also confirm the changes due to surface conjugation of the GaO(OH)-CD nano particles. It is possible that the surface polar –OH groups of the sugar moiety links with gallium oxide hydroxide from all side and serves as a template30, therefore the growth of the nanoparticles are oriented according to the surface area and attractive potential provided by the sugar moiety. As a result the crystal diffraction patterns have been diffused/masked and lost. In the slow process of crystallization, shape of the nano grains have been also changed due to the conjugating ligand, as evidenced by the TEM figures, it also affects the emission properties in the fluorescence spectra as described through FRET process. Dipole-Dipole interaction of polar –OH group moiety of the giant sugar molecule with the negatively charged surfaces of the nascent nano-particle7and a slow process of growth along the surface of CD could be responsible for such a distinct change of shape of the CD capped NPs. Application prospect in the diagnostic field, for PET imaging, is apparently clear through the cellular uptake of the GaO(OH) nano-particles within malignant HeLa cell line. Our findings suggest that all the bare gallium nano-particles dose given have been ingested in to the cells. Such nano –particles are usually negatively charged7 at the surface, likely to be


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surface coroneted by proteins42 and are localized in specialized compartments39,42 . However, the CD wrapped nano-particles (spindle shaped structure) have a larger structure, although appears to have penetrated the cellular medium with difficulty but have been found distributed though out the cytoplasm in a cell. This observed phenomenon can be due to either i) the process of uptake is different, owing to the sugar moiety or ii) because of the fact that once they gained entry in to the cell, the sugar phase can interact through dissolution and diffusion to different regions in the cell43. Thus, for identical materials of nanoparticles and cells, the biological outcomes are determined by the combined properties of nanoparticles and their conjugated surface. Even though, given the high surface energy of nanoscaled objects, it is unlikely that the cellular machinery would interact with the pristine surface of the nanoparticles in vitro (as per the observation), this comparison helps to further clarify the importance of the layer of protein and biomolecules adsorbed on the nanoparticles in mediating the interactions of nanomaterials with cells. A major mechanism suggested is for gallium’s anti-proliferative activity is its ability to mimic, compete with and substitute for Fe3+45. Since gallium is known to accumulate mostly via transferrin receptor46, generally highly over expressed by HeLa cells, a compelling rationale exists for exploring the potential utility of aqueous dispersed gallium ion in some form (e.g. NPs) in treating the same. The strategy of treating cancerous cells by interfering with cellular iron uptake and metabolism is further supported by the significant observed suppression and regression of HeLa cell growth in-vitro due to possible iron deprivation, which ultimately inhibits cell division, leading to programmed cell death. In another report however, the ability of gallium compounds to promote apoptosis was also demonstrated by generation of reactive oxygen species47is not ruled out.


The main


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consequences of administration of gallium oxide –hydroxide NPs conjugated with CD have been aptly pointed out for diagnostic and drug delivery purposes and noticed to produce a total lethal effect after 72 hours when applied even at low dose of GaO(OH)-CD nanoparticles (as observed through TEM).

5. CONCLUSION From the experimental evidences, we conclude that GaO(OH) grains as prepared under physiological conditions are nano-crystalline orthorhombic spheroid structures and morphologically as nano-disc shaped particles. These nano-particles were successfully conjugated with β-Cyclodextrin, as a drug carrier, where in the structural morphology changes. We have made a detailed spectroscopic study of these NPs on material aspects. Our initial investigation and application of the conjugated nanoparticles when injected in cancerous HeLa cell line (in-vitro) showed, that those were taken up by the malignant cells and thus these NPs hold potential to be used for imaging / as a diagnostic tool (may be in PET if properly tagged with a short lived radiotracer 68Ga) and probably also lead to a new therapeutic route since the sugar coated nano-particles resulted in total lethal effect of the HeLa cell line. For a detailed biological aspect, more investigation using CLSM and FCM analysis would be necessary as also the mechanistic study regarding endocytosis path and apoptosis could be a separate issue.

5. ACKNOWLEDGEMENT : Mr. Anish Karmahapatra is acknowledged for X-ray data taking, Dr. Jens Röder for the analysis and Ms. Soma Roy for all her technical help in the laboratory.


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(17) Nelson B; Hayes RL; Edwards CL; Kniseley RM; Andrews GA . Distribution of Gallium in Human Tissues after Intravenous Administration . J Nucl Med 1972, 13, 92– 100. 22 ACS Paragon Plus Environment

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(44) http://www.atcc.org/~/media/DA5285A1F52C414E864C966FD78C9A79.ashx (45) Chua Mei –Sze; Bernstein Lawrence R.; Li Rui and So Samuel K.S. Gallium Maltolate is a Promising Chemotherapeutic Agent for the Treatment of Hepato cellular Carcinoma Anticancer Research 2006, 26, 1739-1744.

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Tables : Table I. Some chemical parameters for Ga+3 and Fe+3 (reference 9). Parameter

Unit

Ga+3

Fe+3 high spin

Ionic radius (octrahedral)

Å

0.620

0.645

Ionic radius (tetrahedral)

Å

0.47

0.49

Ionization potential (4th ionization potential) Electron affinity (3rd ionization potential) Absolute hardness (pearson)

eV

64

54.8

eV

30.71

30.65

eV

17

12.08

Electronegetivity (Pauling)

Pauling 1.81 units KJ mol- 353.5

Metal-oxygen bond dissociation energy First metal –hydroxide formation constant K1= [MOH2-]/ [M3+][OH-] Tendency to ionic bonding (HA)

1.83 390.4

1

logK1

11.4

11.81

none

7.69

7.22


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Table II: The characteristic frequencies due to FT-IR investigation of GaO(OH) nano particle, CD and conjugated GaO(OH)CD.

Characteristic groups

Peak position -1

(frequency, cm )

Modes

of

vibrations

3440

O-H due to GaO(OH)

stretching

3409

O-H from CD

stretching

2923

Terminal O-H from CD stretching

1627

O-H

from

all bending

compounds 1415-1250

-OH from CD

bending

1390

-OH from GaO(OH)

bending

1154

-O-H from CD

bending

1024, 1024, 935

-O-H

500-600

Ga-O

bending and their overtones from CD and GaO(OH) Stretching(red curve)

Table III: Change in the O-H stretching frequencies and their force constant due to conjugation of GaO(OH) to β-cyclodextrin.

Sample Name

CD GaO(OH) GaOCD1 GaOCD2 GaOCD3

IR frequencies (cm-1) Force Constant (dyne/cm)x10-11µ peak position (for O-H bond) 3409 4.132 3440 4.200 3437 4.185 3436 4.190 3435 4.192



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Figures:

Gallium nitrate + tripledistilled water (10ml)

Add 1:4 Ammonia water by vol. pH~7-7.5

Separate the white precipitate, dry in vacuum oven at 60°C for 48 hrs. to obtain GaO(OH)

Precipitation of Gallium hydroxide Ga(OH)3 (gelatinous)

Centrifuge (300 rpm) for 20mins., each time removing the supernatant liquid and washing with water to remove excess of ammonia

a)

Gallium nitrate in triple distilled water (10ml)

add to

β-cyclodextrin dissolved in warm water (40°C)

mix well

Add 1:4 Ammonia water by vol. pH~7-7.5

Off-white color precipitate appears, Centrifuge (300 rpm) for 20mins., each time removing the supernatant and washing with water to remove excess of ammonia

Separate the precipitate, dry in vacuum oven at 60°C for 48 hrs. to obtain GaO(OH) capped in β-cyclodextrin

b) Figure 1. Process diagram for synthesis of a) GaO(OH) and b) GaO(OH)-CD.


GaO(OH) dried

(140)

(151)

(221)

(111) (121)

(130)

(120)

100000

(021)

200000

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


(110)

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0

20

40

60

80

Bragg angle 2θ

Fig.2a) Fig.2b)

Figure 2. a) X-ray diffraction pattern (from experimental data) of synthesized GaO(OH) b) Rietveld refinement analysis of the data, with points in red, fitted curve in green, peak positions are shown in black dots and the residual intensity in pink. 29 ACS Paragon Plus Environment


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Figure 3 (a) Low magnification TEM images of GaO(OH). Width and length of these flat top hexagonal particles and HRTEM image showing lattice spacings of these particles are shown in the inset. (b) High magnification TEM images of one GaO(OH) particle and single crystal SAD pattern from this particle is shown in (c).


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Figure 4. (a) Thickness map and corresponding average thickness profile from a region marked by dotted box in the inset indicating the presence of nano-disc with flat top morphology. (b) STEM-HAADF image of GaO(OH). (c) EDX spectrum from a region marked by area 1 in (d). EDX line profile along a line 2 on a single particle. (e) STEMHAADF-EDX images taken from the area marked by an alloy orange box 3 indicating the locations of different atoms across the structure.



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Figure 5. (a) Low magnification TEM images of GaO(OH)-CD. SAD pattern and EDX spectrum of these particles are shown in the inset.( b) High magnification TEM images of one GaO(OH)-CD particle. (c) indicating the locations of different atoms across the structure. (d) STEM-HAADF-EDX images taken from the area marked by an alloy orange box in (c).


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100

a) Absorbance

80

60 CD GaO GaOCD1 GaOCD2 GaOCD3

40

20 3500 3000 2500 2000 1500 1000

500

-1

Wavenumber(cm )

100

b)

enlarged portion of -OH stretching frequencies

80

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


60 CD GaO GaOCD1 GaOCD2 GaOCD3

40

20 3500 3000 -1 Wavenumber(cm )

2500

Figure 6 : Fourier transformed Infra-red (FT-IR) spectroscopy of a) GaO(OH) (red), CD (black) and the conjugated samples of GaO(OH) with CD at different concentrations: GaOCD1:prepared from 0.5mM CD , GaO-CD2 :prepared in 0.1mMCD , and GaO-CD3 : prepared in 0.05mM CD, as the capping agent in different colours, b) a part of the spectra, 2500-3700cm-1 are shown as the enlarged portion.



a)

2.0 1.8 1.6

GaO(OH) conjugated with β-cyclodextrine

1.4

Absorbance

1.2 1.0 0.8

β-cyclodextrine

GaO(OH)

0.6 0.4 0.2 0.0 -0.2 200

300

400

500

600

700

800

900

1000

Wavelength (nm)

b)

2.0 1.8 1.6

GaO(OH) conjugated with β−cyclodextrine

1.4

Absorbance

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1.2 1.0

GaO(OH)

0.8 0.6 0.4 0.2 0.0 -0.2 190

P

R Q

200

210

220

230

240

250

260

270

280

290

300

Wavelength (nm)

Figure 7. a and b


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c)

7000 6000 5000

Intensity

GaO(OH)

4000

GaO-CD med

3000

GaO-CD high

2000 1000 0 220

230

240

250

260

270

280

290

300

Wavelength (nm)

Figure 7 c).

d)

1.0

Absorption spectrum of β-cyclodextrine 0.8

Emission spectrum of GaO(OH) 0.6

Intensity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


0.4

0.2

Overlap region 0.0 200

220

240

260

280

300

Wavelength (nm)

Figure 7 d) Figure 7 . a) Absorption spectroscopic data : Obtained plots for β-cyclodextrin (in blue), GaO(OH)-CD (in black), GaO(OH) (in red)., b) enlarged portion of the curves where absorption edges are marked for the respective curves (black:Q=230 nm, red :R=235nm), and the peak, ‘P’ ~ λmax is at 200nm. c) Observed fluorescence spectra of GaO(OH) in red and GaO(OH)CD, with increase in concentration of CD (0.05mM for black curve, 0.5mM for blue curve). d) Overlap of the absorption spectra of β-Cyclodextrin(in blue) and the emission spectra of GaO(OH)(in red). 35 ACS Paragon Plus Environment


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Figure 8. a) TEM image showing HeLa cells after 24 hrs cell culture, b) HeLa cells treated with GaO(OH) particles for 24 hrs. as pointed by the arrows, (c) Magnified TEM image from cell regions showing clustering of nanoparticles, (d) HeLa cells treated with CD coated GaO(OH) particles for 24 hrs., (e) entry of CD coated GaO(OH) NPs (f) Magnified TEM images from cell regions showing distributed NPs throughout the cytoplasm in a cell.


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100

Percentage of viability

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


80

60

40

20

0 0

20

40

60

80

100

GaO(OH)-CD conc. (mg.L-1)

Figure 9: Results for dose dependent cell viability as determined through MTT assay, showing cytotoxic effect on HeLa cells using GaO(OH)-CD nanoparticles.



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Figure 10. a) TEM image showing grown Hela cells treated with CD coated GaO(OH) particles for 48 hrs, b) Magnified image from (a) showing clearly particle penetration inside the cell, (c) TEM image showing grown Hela cells treated with CD coated GaO(OH) particles for 72 hrs. (d) Magnified TEM image from same sample in (c) showing cell death, (e) Hela cells in controlled atmosphere after 72 hrs, (f) Magnified TEM from (e) clearly showing the active nature of the cell.


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For Table of Content Use Only Title: Study of Gallium Oxide Nanoparticles Conjugated with β-cyclodextrin -An Application to Combat Cancer Authors: Bichitra Nandi Ganguly*, Vivek Verma, Debanuj Chatterjee, Biswarup Satpati Sushanta Debnath and Partha Saha


Study of Gallium Oxide Nanoparticles Conjugated with Î²-Cyclodextrin

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