Synthesis, characterization and evaluation of 169Yb-labeled tailored hydroxyapatite nanospheres for potential application in nanobrachytherapy

Synthesis, characterization and evaluation of 169Yb-labeled tailored hydroxyapatite nanospheres for potential application in nanobrachytherapy | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Synthesis, characterization and evaluation of 169Yb-labeled tailored hydroxyapatite nanospheres for potential application in nanobrachytherapy Sourav Patra, Khajan Singh, Partha Sarathi Ghosh, K V Vimalnath, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8117448/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Background The introduction of radiolabeled nanoparticles in the realm of brachytherapy has led to a promising therapeutic strategy for cancer management called ‘nanobrachytherapy’. In the quest of developing a potent radiolabeled inorganic biomaterial for use in nanobrachytherapy, we report the synthesis and evaluation 169 Yb [T 1/2 = 32.02 d]-labeled glucuronic acid (GA) functionalised hydroxyapatite (HA) nanoparticles (GAHAnp) and established its potency in pre-clinical settings. Results GAHAnp having average hydrodynamic diameter of 45 ± 3 nm was synthesized in house and characterized using various analytical methods. Ytterbium-169 was produced with adequate radionuclidic purity required for medical application by direct neutron activation of isotopically enriched (35.8% in 168 Yb) Yb target in research reactor. Radiolabeleing protocol of GAHAnp with 169 Yb to obtain [ 169 Yb]Yb-GAHAnp in high yield and purity was optimized. Adsorption of [ 169 Yb]Yb 3+ on GAHAnp followed Langmuir-Freundlich isotherm and pseudo-second order kinetics. The mechanism of incorporation of [ 169 Yb]Yb + 3 on GAHAnp was investigated using density functional theory (DFT) and experimentally verified by radiotracer investigations and XAFS studies. These investigations suggested replacement of Ca 2+ with Yb 3+ in GAHAnp matrix. The [ 169 Yb]Yb-GAHAnp formulation demonstrated excellent in vitro radiochemical stability in physiological media and cell toxicity in Raaji cells. SPECT/CT imaging and ex vivo biodistribution carried out after intra-tumoral administration of [ 169 Yb]Yb-GAHAnp in tumor bearing mice showed near-complete retention of the formulation in the tumor mass upto 2 weeks. Tumor growth could be significantly arrested after administration of 30 MBq dose of the formulation compared to the control. Conclusion Detailed radiochemical and biological investigations reported in this article demonstrate the potential utility of synthesized [ 169 Yb]Yb-GAHAnp formulation in the treatment of solid tumors through nanobrachytherapy. The formulation exhibited excellent radiochemical stability and significant therapeutic efficacy in pre-clinical models. Nanobrachytherapy Radiolabeled nanoparticles GAHAnp 169Yb chelator-free radiolabeling SPECT/CT tumor regression Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Background Cancer remains one of the most formidable challenges in modern medicine, characterized by uncontrolled cell growth and the potential to invade or spread to other parts of the body (Brown et al., 2023 ). Despite significant advancements in understanding its genesis and prognosis at molecular level, cancer remains a leading cause of mortality worldwide (Siegel et al., 2018 ; Siegel et al., 2023 ; Thun et al., 2010 ). Other than surgery, the current landscape of cancer treatment encompasses a multifaceted approach, including chemotherapy, immunotherapy, and radiation therapy (DeVita Jr and Chu, 2008 ; Gracie et al., 2025 ; Sareen et al., 2025 ). Chemotherapy uses cytotoxic drugs to target rapidly dividing cells, but its non-selective nature can lead to severe side effects, impacting healthy tissues (Pandey et al., 2025 ). Immunotherapy leverages immune system of the body to combat with cancer but its effectiveness can vary depending on the type of cancer and also its immune-related adverse effects can limit its use in cancer management (Zidi et al., 2025 ). In contrast, brachytherapy, which is a form radiation therapy can surmount these limitations by placing a millimeter size sealed sources within or near the tumor surgically. This internal radiation method allows for a higher dose of radiation to be delivered to the tumor with minimal impact on adjacent healthy tissues (Serre et al., 2025 ). Nevertheless, the surgical implementation of millimeter size sealed sources makes this procedure invasive and often leads to uneven distribution of dose within the cancerous tissue, hence minimizing the therapeutic outcome (Seniwal et al., 2021 a). The shortcoming of conventional brachytherapy could be overcome by employing radiolabeled nanosize seeds which can be administered into the tumor mass without surgical intervention. This approach is fast emerging as a promising therapeutic modality and is popularly known as ‘nanobrachytherapy’ (Ghosh et al., 2023 ). The small size of these nanoparticles allows for better penetration and near-homogenous distribution within the tumor microenvironment, thus potentially overcome the limitations of conventional brachytherapy (Ghosh et al., 2024 ). An ideal agent for use in nanobrachytherapy should deliver adequate dose of ionizing radiation to the proliferating cells with minimum radiation induced damage to the surrounding healthy cells or tissue (Seniwal et al., 2021 a). This is achieved by the choice of a suitable radionuclide having particulate emissions of optimum tissue penetration. Apart from this, another major requirement is to minimize the non-target dose to the organs, such as liver, spleen, lymph nodes, due to the leakage of radioactivity from the tumor site (Ghosh et al., 2024 ). The leakage often occurs mainly due the poor in vivo stability of radiolabeled nanoparticles, which can lead to the disintegration of the radioisotope from the formulation. To prevent this leakage, it is essential to engineer the nanoparticles in a manner that securely encapsulates the radioisotope ensuring that it becomes an integral part of the nanoparticle matrix. Further, the nanoparticles require surface conjugation with suitable surfactants, biomolecules etc. to ensure their suspensibility in physiological medium, so that the radiolabeled formulation can be easily administered and distributed in the tumor mass (Huang et al., 2023 ; Kievit and Zhang, 2011 ). Among the inorganic nanoparticles as biomaterials under investigations in the field of nanomedicine in recent years, biomimetic synthetic hydroxyapatite nanoparticles (HAnp) have garnered significant attention (Khajuria et al., 2018 ; Sugimoto et al., 2025 ; Truong et al., 2025 ). Hydroxyapatite is the primary inorganic constituents of mineral phase of bone and are biocompatible and biodegradable in nature. Additionally, HAnp demonstrates a remarkable ability to bind with various radiometals due to its high surface-to-volume ratio, nanoporosity, and the presence of compatible sorption sites (Barroug and Glimcher, 2002 ; Dasgupta et al., 2010 ; Furko, 2025; Grinding, 2025; Patra et al., 2023a ; Patra et al., 2023b ; Patra et al., 2025 ). Despite these excellent attributes, the main drawback of HAnp is that they tend to agglomerate at the microscale, which hinders its use in desired biological applications. This can be overcome by proper surface functionalization which will impart suspensibility to the HAnp in biological medium (Patra et al., 2023b ). It is always advantageous to functionalize HAnp with biocompatible organic molecules containing functional groups such as thiol, amine, carboxyl, hydroxyl, or phosphate, as these can enhance the complexation ability of HAnp with biomolecules, drugs, or cations (Haider et al., 2017 ). Therefore, with an aim to developing a potent radiolabeled inorganic nanoformulation for use in nanobrachytherapy, we have synthesized HAnp with glucuronic acid (GA) surface functionalization as the nanoplatform. GA is a sugar acid derived from oxidation of glucose in biological system and therefore highly biocompatible. Besides the carboxylic group in GA helps in complexation with Ca 2+ present in HAnp, the hydroxyl groups in GA enhance the suspensibility of the nanoparticles in aqueous medium. On the other hand, selection of appropriate radioisotope is a crucial factor that determines the therapeutic effectiveness and safety of a nanobrachytherapy formulation (Ghosh et al., 2023 ; Seniwal et al., 2021 b). It is desirable that the radioisotope used in nanobrachytherpy should not only facilitate targeted and efficient cancer therapy but also enable non-invasive imaging of radioactivity retention at the tumor site using SPECT/PET techniques. Among the various radioisotopes used in brachytherapy, 169 Yb [T 1/2 = 32.02 d, 100% EC] offers distinct advantages as it emits Auger electrons and X-rays (Flynn et al., 2019 ; Ghosh et al., 2024 ; Patel et al., 2001 ; Saxena et al., 2015 ). Moreover, it decays to an excited state of 169 Tm, which subsequently emits several gamma photons with a mean energy of 92.7 keV, making it suitable for SPECT imaging. These favorable nuclear decay properties enhance radiation dose conformity for clinical applications in conventional brachytherapy. The United States Food and Drug Administration (US FDA) has approved use of 169 Yb-based brachytherapy sources (Reynoso et al., 2017 ). Given these advantages, we have chosen 169 Yb to radiolabel GAHAnp in our endeavor to develop the potent nanobrachytherapy formulation based on inorganic nanoplatform. In this article, we report synthesis and characterization of glucuronic acid-functionalized hydroxyapatite nanoparticles (GAHAnp) and the production of 169 Yb via 168 Yb(n,γ) 169 Yb reaction in a medium flux nuclear reactor. Subsequently, a chelator-free radiolabeling protocol was optimized to obtain 169 Yb-labled GAHAnp ([ 169 Yb]Yb-GAHAnp) with high purity. The chelator-free radiolabeling approach rules out the requirement of exogenous chelator for the attachment of the radiometal with the nanoplatform and thus, precludes potential disintegration of the 169 Yb from the chelator. Colloidal and radiochemical stability of [ 169 Yb]Yb-GAHAnp was established in physiological medium and cell toxicity was demonstrated in Raaji cells. In vivo SPECT/CT imaging, ex vivo biodistribution and tumor regression in animal model established the potency of the formulation. Moreover, the sorption mechanism of 169 Yb 3+ onto the matrix of GAHAnp was investigated to prove that the radiometal is an integral part of the GAHA matrix. A schematic of the sequential investigations reported in the manuscript toward synthesis and evaluation of a new radiolabeled biomaterial having promising features for cancer management is presented in Fig. 1 . Figure 1 Schematic presentation of formulation and evaluation of [ 169 Yb]Yb-labeled glucuronic acid functionalized hydroxyapatite nanoparticles for application in nanobrachytherpy Results Synthesis and characterization of GAHAnp Well-dispersed GAHAnp was synthesized at room temperature following a single step process using calcium nitrate and diammonium hydrogen phosphate and GA. The amount of GA required for achieving the smallest particle size of GAHAnp was optimized by varying the amount of GA. It was found that hydrodynamic diameter of GAHAnp decreases with the increment of GA amount and become unaltered when the amount of GA was 100 mg ( Fig. S1 A ). The formation of GAHAnp was confirmed by XRD (Fig. 2 A ) . While the peaks appeared indicated the formation of hydroxyapatite, the broadening of the peaks established the formation of nanocrystalline material. The crystallite size of GAHAnp was determined from (221) peak using the Scherrer formula given bellow Crystallite Size (D) = \(\:\frac{\text{k}{\lambda\:}}{\text{B}\:\text{c}\text{o}\text{s}{\theta\:}}\) (4) Where B is the full-width half maximum, k is the Scherrer constant, D is the crystallite size, and λ is the wavelength of X-ray. The crystallite size of the GAHAnp was found to be 6 ± 1 nm. The XRD of GAHAnp recorded after removal of GA ( Fig. S1 B ) matches well with standard crystalline hydroxyapatite (JCPDS data No. 9-432) and the peaks showed minimal broadening. This could be explained by the fact that GA did not promote the nucleation of HA but instead retarded crystal growth, resulting in GA-functionalized HA nanoparticles. Figure 2 B shows the Raman spectra of GA, HAnp and GAHAnp. The Raman peak observed at ~ 383 cm − 1 is attributed to the bending vibrations in calcium hydroxide (Kozyryev et al., 2019 ). The peaks at 520 and 585 cm − 1 may be assigned to the bending modes of HPO 4 2− and PO 4 3− (Koutsopoulos, 2002 ; Ulian et al., 2013 ). The intense peak recorded at ~ 983 cm − 1 , 1085 cm − 1 and a broad band at 450 cm − 1 could be assigned to the symmetric stretching vibrational modes of phosphate ions (Koutsopoulos, 2002 ). These peaks are present in the Raman spectrum of both bare HAnp and GAHAnp and thereby indicating the formation of HAnp. It is evident that the diagnostic peaks of GA (at 720 cm − 1 and 1365 cm − 1 ) appeared in the GAHAnp giving a clear indication about the functionalization of GA on the surface of HAnp (Mutter et al., 2015 ). The functionalization of GA on the surface of the HAnp was clearly evident in FT-IR spectra as shown in Fig. 2 C. The peak at 3350 cm − 1 was attributed to the OH − ions and the peaks at 1102, 1030, 603, and 563 cm − 1 which are diagnostic peaks of PO 4 3− moiety, were present in both bare HAnp and GAHAnp spectra (Verma et al., 2016 ). The peak observed at 2901 cm − 1 could be assigned to the C-H stretching in both GA and GAHAnp (Ţucureanu et al., 2016 ). The peaks at 1620, 1380, 1085 cm − 1 in GAHAnp could be assigned to the asymmetric stretching of the C = O, symmetric stretching of the C = O and bending of the C-H bond respectively (Tajmir-Riahi, 1984 ). These peaks were not observed in the case of bare HAnp, which indicates the attachment of glucuronic acid on the surface of HAnp thus confirming its functionalization. The shifting of C = O stretching frequency towards the lower frequency (1620 cm − 1 ) in GAHAnp spectra compared to the C = O stretching frequency of 1715 cm − 1 in case of GA is due to the interaction between the C = O of carboxylic group and the Ca 2+ present in the hydroxyapatite. These observations clearly inferred the functionalization of GA on the surface of HAnp. Further, the extent of functionalization of GA on the surface of the HAnp was quantified by comparing the TGA curves of bare HAnp and GAHAnp (Fig. 2 D ) . The weight loss upto 150° C for both samples could be attributed for evaporation of water molecules that were adsorbed physically on their surface. The weight loss observed for GAHAnp in the temperature range of 150°-750° C could be assigned for removal and degradation of the attached glucuronic acid molecules from the surface of HAnp. From the TGA curve it was found that around 25 ± 2 wt % of glucuronic acid was attached to the surface of HAnp. Particle size distribution of the synthesized GAHAnp was obtained from DLS measurements (Fig. 3 A) and the average hydrodynamic diameter of the particles was found to be 45 ± 3 nm. The zeta potential of GAHAnp was determined to be -32.6 ± 2.5 mV at pH ~ 6. The change of hydrodynamic diameter and the zeta potential of a freshly prepared GAHAnp was determined over a period of 15 days ( Fig. S2A and S2B ). It was found that size and zeta potential of GAHAnp were not altered significantly during that time period. To corroborate this observation, magnetic T 2 relaxation was measured over the same time period using Xigo Nanotools. It was observed that T 2 relaxation did not alter significantly with time ( Fig. S2C and S2D ), indicating nanoparticles were not coagulated. Overall, these studies demonstrated robust colloidal stability of the nanoparticles in aqueous medium. One the other hand, the average particle size measured for the bare HAnp ( Fig. S3A ) was found to be 280 ± 5 nm and zeta potential − 2.9 ± 0.8 mV at pH ~ 6. Also, increase of particles size of the bare HAnp was observed over a period of time ( Fig. S3B ), indicating coagulation of the nanoparticles. This indicates that the functionalization of HAnp with GA not only improve the particle size but also it enhances colloidal stability of the nanoparticles. The visual image of GAHAnp and bare HAnp was shown Fig. S4 where the coagulation of HAnp could be clearly seen while aqueous dispersion GAHAnp remained stable upto 14 days from preparation. The TEM image of GAHAnp was acquired to demonstrate the particles shape and size. The TEM image of Fig. 3 B showed formation of spherical shape of GAHAnp. The particle size distribution of GAHAnp obtained from TEM image was shown in Fig. 3 C and their average size was found to be 15.2 ± 1.4 nm. Figure 3 D demonstrate the HRTEM image of GAHAnp and the lattice fringe could also be seen in the image. Using this lattice fringe the interplanar distance was determined to be 0.278 nm which was closely matches with (211) lattice plane (In et al., 2020 ). The SAED pattern ( Inset of Fig. 3 D) demonstrated the polycrystalline nature of GAHAnp and the brightest ring pattern indicated (211) plane of GAHAnp. Production and radiochemical processing of Yb Theoretical calculations were carried out (detail provided in ‘Supporting Information’) to predict the yield and radionuclidic impurities of 169 Yb obtained from 168 Yb(n, γ) 169 Yb reaction using enriched (35.8% in 168 Yb) Yb target at thermal neutron flux of 1.0 × 10 14 n/cm 2 /s for different irradiation time periods. It was found that 21 days of irradiation is required to achieve the maximum radioactivity yield of 169 Yb ( Fig. S5 ). Based on this, enriched (35.8% in 168 Yb) Yb targets were irradiated for 21 days in Dhruva research reactor at Bhabha Atomic Research Centre at the available thermal neutron flux of 1.0 × 10 14 n/cm 2 /s. Post irradiation, targets were radiochemically processed and 169 Yb activity produced was obtained as [ 169 Yb]YbCl 3 solution. Subsequently, gamma ray spectra were recorded to determine the yield and radionuclidic purity of 169 Yb activity produced. A typical gamma ray spectrum is shown in Fig. 4A. The batch yield of 169 Yb along with the co-produced radionuclidic impurities were summarized in Table 1 . It was found that the average yield of 169 Yb was 8.8 ± 1.2 GBq at the end of processing with 175 Yb being the major radionuclidic impurity co-produced by neutron activation of 174 Yb present in the target used ( Table S1 ). The radionuclidic purity of 169 Yb was found to be 97.18 ± 0.02% at the end of irradiation. However, for its utility in medical applications, 169 Yb should be obtained with > 99.9% radionuclidic purity. Based on theoretical calculations, it is found that radionuclidic impurity of 169 Yb would be > 99.9% after 25 days from the end of irradiation for the irradiation protocol followed ( Table S2 ). Gamma ray spectrum of [ 169 Yb]YbCl 3 was recorded after 25 days of decay. The spectrum ( Fig. 4B ) showed no detectable photopeaks for 175 Yb or 177 Lu, indicating near exclusive radionuclidic purity of 169 Yb. Hence, [ 169 Yb]YbCl 3 solution was used for radiolabeling after 25 days from the end of irradiation of target. At that point, the total activity of [ 169 Yb]YbCl 3 available was 5.1 ± 0.7 GBq. Figure 4 Typical gamma ray spectrum of 169 Yb (A) at the end of processing (B) after 25 days of cooling Table 1 Yield of 169 Yb and co-produced impurities from irradiation of 100 µg of 35.8% enriched 168 Yb target for 21 days at thermal neutron flux of 1×10 14 n.cm − 2 .s − 1 Batch No. Activity of 169 Yb* (GBq) Activity of 175 Yb* (GBq) Activity of 177 Lu* (MBq) 1 8.9 0.26 2.3 2 8.7 0.25 2.1 3 8.8 0.25 2.4 (* at the end of irradiation) Radiochemical purity of [ 169 Yb]YbCl 3 as [ 169 Yb]Yb 3+ was 99.1 ± 0.2%, as obtained from radio-TLC. Radiolabeling of GAHAnp with Yb Analysis of the results of the experiment carried out to determine the effect of concentration of GAHAnp on the yield of 169 Yb-radiolabeleing revealed that with increasing the GAHAnp concentration, the radiolabeling yield increases and becomes saturated at the concentration 1 mg/mL (Fig. 5 A). The radiolabeling yield at this concentration was > 99%. On the other hand, radiolebeling yield was found to increases with the increase of pH of the solution between 3–6 (Fig. 5 B) and becomes maximum when pH was ~ 6. Experiments on radiolabeling of GAHAnp with 169 Yb were not carried out beyond pH ~ 7 due to conversion of Yb + 3 to colloidal Yb(OH) 3 in alkaline pH. On the other hand, radiolabeling was not recommended bellow pH ~ 3 due to dissolution of GAHAnp. Overall, thorough mixing of [ 169 Yb]YbCl 3 solution with a suspension of 1 mg of in 1 mL of de-ionized water at pH ~ 6 for 45 min at room temperature was found to be the optimal condition of for formulation of [ 169 Yb]Yb-GAHAnp with > 99% yield and radiochemical purity. Studies on sorption of Yb on the surface of GAHAnp The sorption behaviour of Yb 3+ ion on the surface of GAHAnp could be ascertained from equilibrium sorption isotherm and kinetics. It was found that sorption of Yb 3+ on GAHAnp followed Langmuir-Freundlich isotherm (Fig. 5 C) and pseudo-second order kinetics (Fig. 5 D), indicating that chemisorption of Yb 3+ ion on the surface of GAHAnp and the rate determining step involved sharing or exchange of electrons between GAHAnp and Yb 3+ . The chemisorption process ensures robust binding of Yb 3+ on GAHAnp. The rate of transfer of Yb 3+ on the surface of GAHAnp was determined to be ~ 3.8 × 10 − 4 g/mg/min. Based on the kinetic model, the sorption capacity of Yb 3+ at a concentration of 0.5 mg/mL was calculated from the slope and intercept of the plot in Fig. 5 D and found to be 136 ± 2 mg/g. This value is in close agreement with the results presented in Fig. S6A and S6B . Poor correlation coefficient value (R 2 ) of linear fitting discarded the applicability of Langmuir isotherm ( Fig. S7A ) and Freundlich isotherm ( Fig. S7B ). Similarly, poor R 2 value also discarded the pseudo-first order kinetics model ( Fig. S7C ). The linear fitting formula for the aforementioned isotherm and kinetic models are given in Table S3 . Preparation and characterization of Yb-GHAn The emission spectra of synthesized Yb-GAHAnp and Yb 2 O 3 on excitation with 980 nm are depicted in Fig. 6A . The emission spectra showed intense broad spectral bands around 650 nm to 750 nm, which split into several peaks. Emission spectra of Yb-GAHAnp showed reduced intensity suggesting a chemical interaction between Yb 3+ ions and hydroxyapatite nanoparticles. The fluorescence radiation trapping depends on the concentration of Yb 3+ and occurs even for small doping levels. The decay rates for 2F 5/2 level of Yb 3+ ion exhibit single exponential for all concentrations. The lifetime decreases from 7.296 µs to 1.037 µs for Yb-GAHAnp ( Fig. 6B ), therefore it is inferred that a definite chemical interaction between Yb 3+ and GAHAnp has occurred. Figure 6 (A) Emission spectra of Yb-GAHAnp and free Yb 3+ on excitation with 980 nm (B) fluorescence life time spectra of free Yb 3+ and Yb-GAHAnp In vitro stability of [ 169 Yb]Yb-GAHAnp The radiochemical stability of [ 169 Yb]Yb-GAHAnp was ascertained in PBS and mouse serum media. This study demonstrated that the radiolabeled nanoformulation retained its integrity > 98% in PBS medium and > 97% in mouse serum even after 30 d from the time of formulation ( Fig. S8 ), confirming robustness of binding of Yb 3+ with GAHAnp. In vitro cell toxicity study with [ 169 Yb]Yb-GAHAnp Results of in vitro cell toxicity studies using the radiolabeled nanoparticles in Raji cells by flow cytometry is presented in Fig. 7 . The results showed 92.8 ± 2.1% (Fig. 7 A) and 86.4 ± 3.2% (Fig. 7 B) cell viability in control and vehicle control experiment, respectively. In comparison to these, only 48.1 ± 1.8% (Fig. 7 C) cell viability was observed when the cells were treated with 37 MBq of [ 169 Yb]Yb-GAHAnp formulation. These in vitro experimental data demonstrated the therapeutic potential of the prepared formulation. In vivo SPECT/CT imaging and ex vivo biodistribution Qualitative analysis of whole body SPECT/CT images acquired at different time intervals after intra-tumoral administration of [ 169 Yb]Yb-GAHAnp in B16F10 tumor bearing C57BL6 mice confirmed that the formulation remained at the site of administration (i.e. within the tumor mass) even after two weeks of post-injection upto which period the study was carried out (Fig. 8 A). Also, there was almost no leaching of radioactivity from tumor site as almost no uptake was observed in other organ especially, liver and bone. The near-complete retention of the instilled radioactivity is a favourable attribute toward it use in nanobrachterapy. Quantitative analysis of the distribution of [ 169 Yb]Yb-GAHAnp after intra-tumoral administration in B16F10 tumor bearing C57BL6 mice was carried out from the results of ex vivo biodistribution (BD) studies and the distribution pattern is summarized in Fig. 8 B. The BD pattern showed that the tumor uptake of [ 169 Yb]Yb-GAHAnp was 121.60 ± 10.53% ID/g at 3 h post injection (p.i.) and it was retained to a significant extent (85.36 ± 5.36% ID/g) even after 336 h p.i. A small percentage of the administered formulation cleared through hepatobiliary route, confirmed from 2.61 ± 0.86%ID/g of liver uptake at 3 h p.i. which was reduced to 0.64 ± 0.05%ID/g at 336 h p.i. The uptake of [ 169 Yb]Yb-GAHAnp in any other organ/tissue was negligible, as evident from the BD pattern. On the other hand, the BD pattern of [ 169 Yb]YbCl 3 is given in Fig. 8 C, which shows that although the tumor uptake of was high (112.78 ± 6.7% ID/g) at 3 h p.i., almost entire radioactivity leached out within 24 h. Substantial bone uptake of [ 169 Yb]YbCl 3 was observed at 3 h of p.i (20.5 ± 2.3% ID/g), which increased to 24.6 ± 3.6% ID/g at 24 h p.i. Significant uptake could also be observed in liver and kidney at 24 h p.i. This comparative analysis of the BD patterns of [ 169 Yb]Yb-GAHAnp nanoformulation and [ 169 Yb]Yb 3+ used as control clearly established the utility of the nanoformulation [ 169 Yb]Yb-GAHAnp to ensure the prolonged retention of administered radioactivity in tumor and prevent its accumulation in non-target organs. Assessment of therapeutic efficacy by tumor regression study To assess the therapeutic efficacy of [ 169 Yb]Yb-GAHAnp, four escalating doses, 10, 20, 30, and 40 MBq of the formulation were administered intra-tumorally in four different sets of B16F10 tumor bearing C57BL6 mice (four animals in each set). Saline was injected in another set of tumor bearing animals treated as ‘control’. Tumor growth index (TGI) and body weight index (BWI) were monitored over a 21-day post-treatment period (Fig. 9 ). Notably, tumor progression was effectively suppressed by a single administration of [ 169 Yb]Yb-GAHAnp either 30 MBq or 40 MBq, indicating a clear dose-dependent therapeutic effect (Fig. 9 A). While mice treated with the 30 MBq dose maintained stable body weight throughout the observation period (Fig. 9 B), reduction in BWI was observed in the 40 MBq group—potentially attributable to radiation-induced systemic toxicity. Based on these findings, the 30 MBq dose was identified as the optimal therapeutic window, offering a favorable balance between efficacy and safety. Investigation of sorption mechanism of Yb on the surface of GAHAnp For the understanding of the mechanism of sorption of Yb 3+ on the surface of GAHAnp, a systematic periodic DFT studies were performed. In the HA [Ca 10 (PO 4 ) 6 (OH) 2 ] unit cell, two oxygen atoms have partial occupancies in the P6 3 /m space group. Based on the partial occupation, four configurations can be generated with varying OH group orientations. A unit cell with the opposite orientation of the OH group channels was considered to obtain a unit cell having zero electric polarization (Almora-Barrios and de Leeuw, 2010 ). The (0001) surface of HA was considered for all of the Yb 3+ interaction because the (0001) termination is the most energetically stable(Almora-Barrios and de Leeuw, 2010 ). The DFT optimized (0001) surface structure is shown in Fig. 10 A. In order to decide the nature of interaction (physical/chemical) of Yb 3+ ion with HA surface, a 2×2×1 supercell with 352 atoms are constructed. A vacuum of around 15 Å is taken into consideration along the z-direction to prevent interactions between periodic images in non-periodic directions. To study Yb 3+ adsorption on (0001) surface, the position of Yb 2+ atom on (0001) surface is optimized and shown in Fig. 10 B. The distances of Yb 3+ atom from the nearest O atoms are 2.29, 2.34, 2.35, 2.42, 2.44 Å. To study Yb 3+ chemical interaction on (0001) surface, Yb 3+ ion is exchanged with a surface Ca 2+ ion and the exchanged Ca 2+ ion remain as an adsorbed atom on the surface and the optimized structure is shown in Fig. 10 C. The bond distances of in Yb 3+ O polyhedra are 2.23, 2.24, 2.28, 2.42, 2.44 Å. The distances of Ca 2+ ion from the surface O atoms are 2.36, 2.40, 2.40, 2.41, 2.48, 2.52 Å. Evidently, Yb 3+ ion forms stronger and shorter bonds with O atoms due to substitution at Ca 2+ site rather than adsorption on the (0001) surface. The energetics of these two processes are calculated as follows: HA + Yb 3+ (H 2 O) 9 →Yb 3+ @HA + (H 2 O) 9 ΔE = 2.23 eV HA + Yb 3+ (H 2 O) 9 →Yb 3+ _Ca 2+ @HA + Ca 2+ (H 2 O) 9 ΔE = -1.88 eV where Yb 3+ @HA and Yb 3+ _Ca 2+ @HA represent the Yb 3+ -adsorbed HA surface and Yb 3+ _Ca 2+ -exchanged HA surface, respectively. These processes also considered the energies of Yb 3+ (H 2 O) 9 and Ca 2+ (H 2 O) 9 complexes in the calculation of reaction energies. The positive ΔE value implies that adsorption of Yb 3+ ion on the (0001) surface of HA is energetically unfavourable. On the contrary, chemical substitution of Yb 3+ at Ca 2+ site on the (0001) surface of HA is energetically favourable as implied by negative ΔE value. The theoretical investigations suggest that sorption of Yb 3+ on HA matrix is through ion-exchange mechanism. This observation was corroborated by studying the release of Ca 2+ from intrinsically 45 Ca-labeled GAHAnp due to sorption of Yb 3+ on its surface. It was found that 24.3 ± 1.2% of the 45 Ca was replaced from 45 Ca-labeled GAHAnp matrix when 5 mg Yb was adsorbed on 50 mg GHAnp, which indicated that sorption takes place through ion-exchange mechanism and in this process, Yb ( 169 Yb for radiolabeled nanoformaultion) become integral part of the solid matrix of [ 169 Yb]Yb-GAHAnp formulation. Further, X-ray absorption spectroscopy (XAS) measurements for Yb-GAHAnp along with Yb 2 O 3 and YbCl 3 samples were conducted and measured at Yb L 3 edge. The normalized X-ray near edge structure (XANES) spectra of all these samples was shown in Fig. 11 A which suggest that Yb is loaded on GAHAnp in Yb 3+ form not in Yb 2 O 3 and YbCl 3 as none of these XANES data showed exact match with XANES data of Yb-GAHAnp. The normalized absorption spectra of Yb-GAHAnp at Yb L 3 edge is shown in Fig. 11 B. The fourier transform of k 2 χ(K) vs. k spectra (Fig. 11 C) over the k range of 3.0–11.0 Å -1 was performed to generate versus R plots at Yb L 3 edge (Fig. 11 D). Subsequently, the EXAFS data were fitted in the range of 1.0–4.0 Å in R-space. The scattering paths were obtained from Ca 10 (PO 4 ) 6 (OH) 2 crystal structure, in which some of the Ca atoms were replaced by the Yb atoms. The peak at 1.8 Å appeared from Yb-O coordination shells. The EXAFS peak at 2.75 Å appeared from the Yb-P coordination shell. The small EXAFS peak at 3.20 Å arose from the Yb-O 3 coordination shell. The EXAFS peak around 3.66 Å has contribution from the Yb-Ca coordination shells. In the fitting procedure, the coordination number (N.), the interatomic bond distance between pairs of atoms (R) and the Debye-Waller factor (σ 2 , representing the thermal disorder and mean square fluctuation in atomic bond lengths) were utilized as fitting parameters. The optimal values for these parameters are presented in Table 2 . The goodness of fit was assessed by the value of R factor which is defined as follows where, χ dat and χ th refer to the experimental and theoretical χ(r) values respectively. Im and Re represents the imaginary and real parts of the respective quantities. The results obtained from XAFS study demonstrated shrinkage of bond distance (Table 2 ) after the replacement of Ca by Yb ion. All these investigations conclude that loading of Yb on GAHAnp most likely occurred through ion exchange mechanism, by which Yb becomes an integral part of the GAHAnp matrix. This implies that in the developed 169 Yb-labeled nanoformulation, the radiometal is a part of the solid matrix, which is an essential pre-requisite for a nanoscale brachytherapy agent. Table 2 Bond length, coordination number and disorder factors obtained by EXAFS fitting measured at Yb L 3 edge. Path Parameter Yb-GAHAnp Yb L3 edge Yb-O 1 R (Å) (2.37) 2.27 ± 0.01 N (3) 3.0 ± 0.12 σ 2 0.0064 ± 0.0007 Yb-O 2 R (Å) (2.45) 2.35 ± 0.01 N (3) 3.0 ± 0.12 σ 2 0.0064 ± 0.0007 Yb-P R (Å) (3.20) 3.11 ± 0.01 N (3) 3.0 ± 0.12 σ 2 0.0105 ± 0.002 Yb-O 3 R (Å) (3.41) 3.50 ± 0.02 N (2) 2.0 ± 0.08 σ 2 0.001 ± 0.0005 Yb-Ca R (Å) (3.95) 3.90 ± 0.02 N (6) 6.0 ± 0.24 σ 2 0.0104 ± 0.003 R-factor 0.01 Discussion Well-dispersed GAHAnp was successfully synthesised and extensively characterized by various analytical tools. The inclusion of GA on the surface of HAnp was ascertained by FT-IR (Fig. 2 C), where the shifting of C = O stretching frequency towards the lower frequency (1620 cm -1 ) in GAHAnp spectra compared to the C = O stretching frequency of 1715 cm -1 in GA could be observed. This is due to the interaction between the C = O of carboxylic group and the Ca 2+ present in the hydroxyapatite. These observations clearly inferred the functionalization of GA on the surface of HAnp. TEM image confirmed GAHA nanosphere formation. The optimized chelator free radiolabeling protocol of GAHAnp with 169 Yb yielded [ 169 Yb]Yb-GAHAnp formulation in high yield and radiochemical purity (> 99%). Binding of [ 169 Yb]Yb 3+ with the nanoformulation was found to be through chemisorption from sorption isotherm and kinetics studies (Fig. 5 C and 5 D). This indicates robust binding of [ 169 Yb]Yb 3+ with GAHAnp. Consequently, [ 169 Yb]Yb-GAHAnp formulation demonstrated excellent radiochemical stability in physiological medium ( Fig S8 ). Cytotoxicity of [ 169 Yb]Yb-GAHAnp formulation, demonstrated by in vitro cell toxicity studies in Raji cells (Fig. 7 ), could be attributed to the auger electrons emitted during the decay process of 169 Yb. Auger electrons possess high LET value (1–23 keV/µm) which induce high clustered damage in macromolecular targets within cancer cells, especially DNA and the cell membrane (Ku et al., 2019 ). Serial SPECT/CT images recorded after intra-tumoral injection of [ 169 Yb]Yb-GAHAnp in C57BL6 mice bearing B16F10 tumor (Fig. 8 A) established excellent retention of the nanoformulation in the tumor mass. This investigation also established that there was almost no leakage of the radioalabeled nanoparticles or detachment of the radiometal from the formulation in the biological system. Ex vivo biodistribution studies carried out in the same animal model (Fig. 8 B) further corroborated these observations. Similar studies carried out with [ 169 Yb]YbCl 3 showed extensive leakage of radioactivity from tumor (Fig. 8 C). These observations point toward cellular uptake of the radiolabeled nanoparticles through endocytosis (Sun et al., 2016 ) as well as robust binding of [ 169 Yb]Yb 3+ with HA which remains stable in vivo . In contrast, poor retention of [ 169 Yb]YbCl 3 would be due to its lack of internalization, which leads to leakage into the bloodstream, followed by uptake in various organs /tissues, primarily bone, kidney, and liver (Xu et al., 2024 ). Subsequently, it was shown that administration of 30 MBq dose of the formulation into B16F10 tumor raised in C57bL/6 mice could effectively suppress the tumor progression in the same animal model without any considerable side effect (Fig. 9 ). Overall, these systematic studies established the promising features of developed radiolabeled nanospheres for use in nanobrachytherapy. An essential criterion for any radioactive material to be used for in vivo brachytherapy application is that the radioactive component should be an integral part of the matrix. From this consideration, in the developed [ 169 Yb]Yb-GAHAnp formulation intended for application in nanobrachytherapy as radioactive nanospheres, it is essential that [ 169 Yb]Yb 3+ to be incorporated into the GAHAnp matrix. This was established by elaborate theoretical and experimental investigations. Theoretical studies using DFT shows that replacement of Ca 2+ of the HA matrix by the Yb 3+ is energetically favourable process and hence exchange with Ca 2+ of HA matrix could be the possible mechanism of incorporation of [ 169 Yb]Yb 3+ onto the matrix of HA nanospheres. This observation was experimentally verified XAFS study (Fig. 11 ), where the local geometry around Yb after its adsorption onto GAHAnp matrix was evaluated. The results obtained demonstrated shrinkage of bond distances (Table 2 ) which could only be possible due to the replacement of Ca by Yb ion, providing direct evidence of Yb becoming a part of the solid matrix. This was further corroborated by loading inactive Yb 3+ onto intrinsically 45 Ca-labeled GAHAnp, which resulted in the release of [ 45 Ca]Ca 2+ from the HA matrix, detected by radioactivity measurement. All these investigations conclude that loading of Yb on GAHAnp most likely occurred through ion exchange mechanism, by which Yb becomes an integral part of the GAHAnp matrix. Conclusion In summary, we have accomplished the synthesis of well-dispersed glucuronic acid funtionalized hydroxyapatite nanospheres of 45 ± 3 nm size and demonstrated their chelator free radiolabeling with 169 Yb for potential application of nanobrachtytherapy. The attachment of glucuronic acid with hydroxyapatite nanoparticles was ascertained by FT-IR and Raman spectroscopy while the amount of content of GA in GAHAnp was determined using TGA study. The high colloidal stability of the functionalized hydroxyapatite nanoparticles over the its bare counterpart was determined by measuring particles size and T 2 relaxation time at different time intervals. The prolonged in vitro stability of [ 169 Yb]Yb-GAHAnp in physiological medium illustrated the robustness of binding of the radiometal with the nanoplatform. The chemical interaction between Yb 3+ and GAHAnp was studied by DFT calculations, sorption studies, radiotracer studies and XAFS. These studies ascertained that in the synthesized Yb-GAHAnp nanoformulation ( 169 Yb in case of radiolabeled formulation), Yb becomes an integral part of GAHAnp matrix which fulfils the criteria for nanoscale brachytherapy. In order to demonstrate the potency of [ 169 Yb]Yb-GAHAnp in biological system, SPECT/CT images were acquired after the intra-tumor administration in the melanoma tumor bearing C57BL/6 mice which revealed prolonged retention in tumor and almost no leaching of radioactivity in any other organ. This result was compared with ex vivo biodistribution pattern of the formulation after intra-tumoral injection in same animal model. Further, treatment with 30 MBq dose of the formulation could effectively suppress the tumor progression in the same animal model without any considerable side effect. In light of the detailed studies reported herein, it could be concluded that [ 169 Yb]Yb-GAHAnp nanoformulation could be a promising candidate for nanobracthytherapy. Experimental Materials Isotopically enriched ytterbium oxide (35.8% in 168 Yb, > 99.99% chemical purity) was purchased from Isoflex Corporation, Russian Federation. Ytterbium oxide of natural isotopic composition (spectroscopic grade, > 99.99% pure) was procured from American Potash Inc., USA. Calcium nitrate tetrahydrate (≥ 99 99% pure), ammonium dihydrogen phosphate (99.99% pure) and ammonia solution (AR grade), hydrochloric acid (Suprapur ® ), glucuronic acid (99.99% pure) were purchased from Merck, Germany. All other materials used were of AR grade and were obtained from reputed manufacturers unless mentioned otherwise. Synthesis of GAHAnp One pot synthesis of GAHAnp was carried out at room temperature using calcium nitrate and diammonium hydrogen phosphate and GA as calcium precursor, phosphate precursor and surface functionality, respectively. The amount of the precursor is chosen in such a way so that Ca/P ratio becomes 1.67. Briefly, 100 mg of GA was dissolved in 10 mL of 1 mM calcium nitrate tetrahydrate (solution A). Subsequently, equivolume of 0.6 mM diammonium hydrogen phosphate was added dropwise into solution A under stirring at room temperature. The pH of the solution was adjusted to ~ 9 by adding ammonia solution and incubated for 4 h at room temperature while continuing the gentle stirring. At end of incubation time, the dispersed nanoparticles were transferred to a dialysis tube (MW cut-off 10 kDa) and dialyzed to eliminate excess GA not associated with HA. Subsequently, the dispersed nanopartilces were lyophilized at -50°C and 1 mbar pressure using a lyophilizer (Martin Christ lyophilizer). Thus, the synthesised GAHAnp was obtained as white powder and subjected to physiochemical characterization. For comparison, bare HAnp was synthesized under the same experimental conditions in the absence of GA. Characterization of GAHAnp Structural characterization of synthesized GAHAnp was carried out by powder X-ray diffraction (XRD), utilizing monochromatized Cu-Kα radiation and conducted on a PANalytical X-ray diffractometer (X’pert PRO). The XRD of GAHAnp was again recorded after removing of GA from its surface by heating at 900 ºC inside a furnace. Raman spectra were recorded using a micro-Raman spectrometer (STR-300, SEKI Technotron, Japan) where a diode-pumped solid-state laser (DPSS, gem532, Laser Quantum) with a wavelength of 532 nm was employed as an excitation source. Calibration of the spectrograph was performed using the 520.5 cm − 1 line from a silicon wafer. The encapsulation of GA on the surface of HAnp was ascertained by analysing fourier transform infrared (FT-IR) spectra recorded for GA, bare HAnp and GAHAnp in the range of 4500 cm − 1 to 500 cm − 1 . The hydrodynamic diameter of GAHAnp was determined by dynamic light scattering (DLS) using Microtrac Series MN420 – Nanotrac Wave II particle analyzer and using the same instrument zeta potential of the functionalized nanoparticles was determined. The average particles size of GAHAnp was obtained from high-resolution transmission electron microscope (HRTEM) using Philips CM 200 TEM system. In order to determine the GA content on the surface of GAHAnp, thermogravimetric analysis (TGA) of the bare HAnp and GAHAnp were carried out using Mettler Toledo TG/DSC stare system in which a certain amount of sample (~ 22.14 mg) was placed in an alumina sample holder and analyses were executed under nitrogen gas flow (rate ~ 50 mL/min) from room temperature to 900° C at the heating rate of 10° C/min. The colloidal stability and relaxation time measurement of GAHAnp were determined using Xigo Nanotools (Acorn Area instrument). Production and radiochemical processing of 169 Yb Measured amount (1.0 mg) of isotopically enriched (35.8% in 168 Yb) Yb 2 O 3 was dissolved in minimum volume of ultrapure 0.1 M HNO 3 by gently heating. The resulting solution was evaporated to near dryness and reconstituted in 1 mL of deionized water. An aliquot of 100 µL was transferred into a clean quartz ampoule and gently heated to facilitate the deposition of a thin film of [¹⁶⁸Yb]Yb(NO₃)₃ on the inner surface. The ampoule was then flame sealed and placed inside a standard aluminium irradiation container. The container was which was sealed by cold-wielding and subsequently irradiated in the Dhruva reactor. At the end of irradiation, the target was cooled for 4 h and subsequently dissolved in ultrapure 0.1 M HCl (5 mL) by gentle warming inside a 100 mm lead-shielded radiochemical processing cell. The resultant solution was evaporated to near-dryness and reconstituted in 5 mL of deionized water to obtain 169 Yb in the form of [ 169 Yb]YbCl 3 solution. The radioactivity produced was determined by γ-ray spectrometry using a pre-calibrated HPGe detector coupled with 4K Multi Channel Analyser system (Canberra, Eurisys). For this, an aliquot of [ 169 Yb]YbCl 3 solution was appropriately diluted and counted for 1 h in HPGe detector ensuring that the dead time of the detector < 2%. Radionuclidic purity of the formulation was also determined from the gamma ray spectra recorded. The radiochemical purity of [ 169 Yb]YbCl 3 formulation was determined by radio-TLC developed in 0.9% NaCl in 0.02 M HCl as the eluting solvent. Further detail on production of 169 Yb is provided in the ‘Supporting Information’. Radiolabeling of GAHAnp with 169 Yb A chelator free radiolabeling protocol was optimized for formulation of 169 Yb-labeled GAHAnp. For this, 0.1 mL (~ 370 MBq) of 169 Yb activity as [ 169 Yb]YbCl 3 solution was added to each of the reaction vials containing suspension of GAHAnp (1.0 mL) of various concentration (0.25 -2 mg/mL) in de-ionized water. After thorough mixing, the suspensions were incubated at room temperature under continuous stirring for 45 min. The pH of reaction mixtures was maintained at ~ 6. Concurrently, a reference solution was prepared by mixing 0.1 mL of [ 169 Yb]YbCl 3 solution with 1.0 mL of de-ionized water. Upon completion of incubation time, the reaction mixture was vortexed thoroughly and centrifuged at 5000 rpm for 20 min. Subsequently, an aliquot (0.1 mL) of the supernatant solution was carefully withdrawn from the reaction mixture and radioactivity was measured (R) using NaI(Tl) scintillation detector (Mucha, Raytest GmbH). Aliquot of same volume was also withdrawn from the reference solution and radioactivity was measured (B) using same detector. Radiolabeling yield was determined using following formula Radiolabeling yield (%) = (1− \(\:\:\frac{R}{B}\) ) × 100 (1) The radiolabeling yield of GAHAnp was also determined by varying the pH of the GAHAnp suspension (pH ~ 3–7) while keeping the concentration of GAHAnp fixed (1 mg/mL). Finally, the radiolabeling of GAHAnp with 169 Yb was carried out using optimal reaction parameters obtained from previous experiments for the in vitro and in vivo assessment of 169 Yb-labeled GAHAnp. Studies on sorption of Yb 3+ on the surface of GAHAnp Sorption of Yb + 3 on the surface of GAHAnp was assessed using the same method described in previous reports (Joshi et al., 2022 ; Patra et al., 2025 ). Briefly, 5 mL of YbCl 3 solution with various concentrations of Yb (0.25-2 mg/mL) were spiked with [ 169 Yb]YbCl 3 (~ 15.6 MBq, 50 µL) and incubated with 5.0 mg of GAHAnp at room temperature for 1 h. The pH of the solutions was adjusted to ~ 6. Concurrently, a reference solution of same volume (5 mL) was prepared without addition of GAHAnp nanoparticles. After 1 h of incubation, the solutions were centrifuged at 5000 rpm for 20 min. An aliquot (50 µL) of supernatants were withdrawn from each solution and the activity associated with each aliquot was measured using same NaI(Tl) detector. At the same time, activity of 50 µL reference solution was measured and then equilibrium sorption capacity (q e ) of GAHAnp at each concentration of Yb 3+ was obtained using the following formula Where A o and A e were the radioactivity of supernatant solution before and after equilibration, respectively, C o (mg/mL) is total Yb content in the solution before sorption, V (mL) is volume of solution and m was the mass (g) of GAHAnp. Data were obtained in triplicate. In order to determine the rate of sorption of Yb 3+ on the surface of GAHAnp, radiolabeling study was conducted as a function of time. For this sorption capacity of GAHAnp for a particular concentration (0.5 mg/mL) was determined at various time interval at room temperature. Preparation and characterization of Yb-GHAnp Yb-GAHAnp was prepared following the protocol optimized for the formulation of [ 169 Yb]Yb-GAHAnp (Section 2.5), only using YbCl 3 solution instead of 169 Yb[YbCl 3 ] radiochemical formulation. Photoluminescence (PL) spectra of Yb-GAHAnp formulation were recorded using an excitation wavelength of 980 nm in FLS1000 fluorescence spectrometer (Edinburgh Instruments, UK). For comparison, the PL spectra of free Yb³⁺ ions were also recorded using YbCl₃ solution. In vitro stability of [ 169 Yb]Yb-GAHAnp The radiolabeled formulation prepared using optimized radiolabeling protocol was centrifuged at 5000 rpm for 20 min. Then the supernatant was discarded and the radiolabeled particulates were dispersed with 1.0 mL phosphate buffered saline (PBS). The radiochemical purity of [ 169 Yb]Yb-GAHAnp was determined over a periods 30 d following the procedure described in Section 2.5 (Eq. 1). In a similar way radiochemical purity of [ 169 Yb]Yb-GAHAnp was determined in mouse serum over the same time period. In vitro cell toxicity study with [ 169 Yb]Yb-GAHAnp Therapeutic potential of [ 169 Yb]Yb-GAHAnp was evaluated in vitro by cell toxicity studies using Raji cells. Raji cells were cultured in RPMI medium with 10% fetal calf serum (FCS) and 1% antibiotic/antimycotic solution. Approximately one million cells were seeded in 6-well plates, and 100 µL (~ 37 MBq) of [ 169 Yb]Yb-GAHAnp formulations were added into it. For comparison, the same procedure was carried out using an equivalent amount of cold Yb-GAHAnp (vehicle control) and without any treatment (control). Then, the cells were incubated for 48 hours in a humidified 5% CO 2 atmosphere at 37°C. After the incubation, the cells were washed, trypsinized, and mixed with Guava ViaCount reagents (Luminex Corp., USA). After 5 min incubation, the cells were analyzed using a Guava flow cytometer (Luminex Corp., USA) to assess cell toxicity. In vivo SPECT/CT imaging and ex vivo biodistribution Biological evaluation of [ 169 Yb]Yb-GAHAnp was carried out in C57BL/6 mice bearing melanoma tumor. All animal experiments were carried out following relevant guidelines and regulations approved by the institutional Animal Ethics Committee of Bhabha Atomic Research Centre (Reference: BAEC/12/2024). The animals were bred and reared in a dedicated animal house facility, BARC. A total of ~ 2 × 10 6 B16F10 melanoma cells dispersed in PBS solution were injected subcutaneously in the shoulder region of C57BL/6 mice (5 weeks older) weighing 20–25 g each. On 15 d after the injection of B16F10 melanoma cells, the tumor diameters reached 6–9 mm. Subsequently, ~ 50 µL (~ 20 MBq) of [ 169 Yb]Yb-GAHAnp radiolabeled nanoparticles was injected intratumorally after diluting in PBS medium. Then, whole body SPECT/CT imaging of the animals were performed at different time points post-injection (p.i.) over a period of two weeks. Further, the melanoma tumor has grown in eighteen C57BL/6 mice using the same protocol. The tumor bearing animals were randomized into six groups of 3 mice each and 10 MBq (~ 25 µL) [ 169 Yb]Yb-GAHAnp was administered into each mice by intra-tumoral injection. At the end of six different time points post-injection (3, 24, 48, 72, 168 and 336 h), one group of mice was sacrificed by carbon dioxide asphyxiation. Subsequently, the radioactivity and the weight associated with each organ and blood samples were measured and presented as percentage injected dose per gram (%ID/g). As control experiment, ex vivo biodistribution study was performed in melanoma tumor bearing C57BL/6 mice by intra-tumoral administration of 10 MBq (~ 25 µL) [ 169 Yb]YbCl 3 solution (pH ~ 5.0) following same protocol described for [ 169 Yb]Yb-GAHAnp formulation. Assessment of therapeutic efficacy by tumor regression study Tumor regression study was conducted using C57BL/6 mice bearing melanoma tumors with an average initial tumor volume of approximately 150 mm³. The mice were randomly assigned into five groups (n = 3 per group). One group of mice received a single intra-tumoral injection of normal saline (control), while the remaining four groups were administered a single dose of [¹⁶⁹Yb]Yb-GAHAnp at activity levels of 10, 20, 30 and 40 MBq, respectively. Tumor progression and body weight were monitored over a 21 days period post treatment. Tumor volume was estimated using the formula: (length × width²) / 2. The tumor growth index (TGI) at each time point was determined by normalizing tumor volume to its baseline value (day 0). Similarly, the body weight index (BWI) was calculated by dividing the body weight at each time point by the initial body weight. Investigation of sorption mechanism of Yb 3+ on the surface of GAHAnp Utility of [ 169 Yb]Yb-GAHAnp in nanoscale brachytherapy necessitates that 169 Yb becomes an integral part of HA matrix in the radiolabeled formulation. To establish this, it is imperative to study the sorption mechanism of Yb 3+ on GAHAnp. In this regard, a theoretical investigation was carried out considering unfunctionalized HAnp as sorption matrix and assuming that GA does not influence the sorption of Yb 3+ on GAHAnp matrix. To understand the basic interaction between hydroxyapatite surface and Yb 3+ at the molecular level, plane-wave based spin polarized density functional theory (DFT) calculations are performed using Vienna ab initio simulation package (VASP) (Kresse and Furthmüller, 1996 ; Kresse and Joubert, 1999 ). The interaction between ions and electrons are described using the projector augmented wave (PAW) potentials (Blöchl, 1994 ). The exchange correlation energy is described using the generalised gradient approximation (GGA) as parameterized by Perdew-Burke-Ernzerhof (PBE) (Perdew et al., 1996 ). A 550 eV is taken into consideration as the kinetic energy cut-off for the electronic self-consistent field iterations. Additionally, every structure underwent optimisation until the force tolerance is less than 0.01 eV/Å and the difference value of the total energy is less than 10 − 5 eV. The Brillouin-zone integrations are performed using a 2×2×1 Monkhorst-Pack k-point mesh (Monkhorst and Pack, 1976 ). The dispersion corrections to the total energies are implemented using Grimme's D3 semiempirical approach (PBE-D3) with Becke-Jonson damping (Grimme et al., 2011 ). To corroborate the theoretical finding, radiotracer investigations were carried out to establish if the sorption of Yb 3+ on GAHAnp matrix results in the replacement of Ca 2+ using 45 Ca-labeled GAHAnp. The experimental details of this study are provided in the ‘Supporting Information’. Further, the local geometry of Yb after sorption on GAHAnp was investigated by X-ray absorption fine structure (XAFS) spectroscopy. The detail of this study is provided in the ‘Supporting Information’. Abbreviations GA Glucuronic acid HA Hydroxyapatite GAHAnp Glucuronic acid functionalized hydroxyapatite nanoparticles DFT Density functional theory SPECT Single photon emission computed tomography CT Computed tomography XAFS X-ray absorption fine structure XRD X-ray diffraction FT-IR Fourier transform infrared TGA Thermogravimetric analysis DLS Dynamic light scattering TEM Transmittance electron microscope HRTEM High resolution transmittance electron microscope PL Photoluminescence Declarations Ethics approval and consent to participate All animal experiments were carried out following relevant guidelines and regulations approved by the institutional Animal Ethics Committee of Bhabha Atomic Research Centre (Reference: BAEC/12/2024). Consent for publication Not applicable. Conflicts of interest There are no conflicts of interest to declare. Funding Open access funding provided by Department of Atomic Energy (DAE), Government of India. This research did not receive any specific grant from funding agencies. Author contributions SP : Data curation, Formal analysis, Writing – original draft. KS : Data curation. PSG : Data curation, Formal analysis. KVV : Data curation. PB : Data curation. RK : Data curation, Formal analysis. CK : Data curation, Formal analysis. AC : Data curation, Formal analysis. SG : Data curation, Formal analysis. DB : Formal analysis. RC : Formal analysis, Writing – review & editing. SC : Conceptualization, Formal analysis, Supervision, Writing – review & editing. Acknowledgment The authors are grateful to Dr. Tapas Das, Head, Radiopharmaceuticals Division, Dr. Sandip Basu, Head, Radiation Medicine Centre (Medical) and Dr. N.S. Baghel, Radiation Medicine Centre (General), Bhabha Atomic Research Centre (BARC) for their support. The authors also acknowledge the valuable support of the staff of Radiopaharmceuricals Division, BARC who has facilitated irradiation targets in the reactor. 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08:09:26","extension":"html","order_by":37,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":162163,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-8117448/v1/d181a52f7a4b0c9edf77a6dc.html"},{"id":97124104,"identity":"e9fb1231-27ef-479d-a1e8-f806582ba563","added_by":"auto","created_at":"2025-12-01 08:09:35","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":96262,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic presentation of formulation and evaluation of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-labeled glucuronic acid functionalized hydroxyapatite nanoparticles for application in nanobrachytherpy\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8117448/v1/bc50ec97e1c00b344d6bff69.jpg"},{"id":97124088,"identity":"fcacda70-abc4-42a0-b9c7-6c2cde6053eb","added_by":"auto","created_at":"2025-12-01 08:09:31","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":121643,"visible":true,"origin":"","legend":"\u003cp\u003e(A) XRD pattern of GAHAnp (B) Raman spectra of bare HAnp, GA and GAHAnp (C) FT-IR spectra of bare HAnp, GA and GAHAnp (D) TGA curve of bare HAnp and GAHAnp showing their weight loss pattern with increased temperature\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8117448/v1/fa603ba1511860e1251eeb56.jpg"},{"id":97124085,"identity":"c689ca7c-1cd1-4e97-bb63-18c1dfc7fb35","added_by":"auto","created_at":"2025-12-01 08:09:30","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":182683,"visible":true,"origin":"","legend":"\u003cp\u003e(A) DLS size distribution plot of GAHAnp (B) TEM image of GAHAnp (C) Size distribution of GAHAnp obtained from TEM image (D) HRTEM image illustrating lattice fringes (Inset: SAED pattern of the nanoparticle)\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8117448/v1/3fd612d9114d44ca5279dff6.jpg"},{"id":97124105,"identity":"7dd30ecb-6204-4a51-ad3e-45a13d81de82","added_by":"auto","created_at":"2025-12-01 08:09:35","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":92076,"visible":true,"origin":"","legend":"\u003cp\u003eTypical gamma ray spectrum of \u003csup\u003e169\u003c/sup\u003eYb (A) at the end of processing (B) after 25 days of cooling\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8117448/v1/17b8f527cb3c43b09eb37e6f.jpg"},{"id":97124001,"identity":"59f49393-a3d7-46b1-abba-1220cbed6b6e","added_by":"auto","created_at":"2025-12-01 08:09:23","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":84191,"visible":true,"origin":"","legend":"\u003cp\u003eVariation of radiolabeling yield of GAHAnp with \u003csup\u003e169\u003c/sup\u003eYb as a function of (A) concentration of GAHAnp and (B) pH of the medium. (C) Linear fitting of sorption isotherm with Langmuir-Freundlich model. (D) Linear fitting of sorption kinetics with pseudo-second order model\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8117448/v1/670fa53256abe867f824ead6.jpg"},{"id":97141232,"identity":"35146824-51f3-4857-ae90-09926f4f2f05","added_by":"auto","created_at":"2025-12-01 10:06:27","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":95814,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Emission spectra of Yb-GAHAnp and free Yb\u003csup\u003e3+\u003c/sup\u003e on excitation with 980 nm (B) fluorescence life time spectra of free Yb\u003csup\u003e3+\u003c/sup\u003e and Yb-GAHAnp\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8117448/v1/705ff041abe18e9349e63983.jpg"},{"id":97124098,"identity":"8e21ab2c-574a-4c82-ad3b-98dbf20999de","added_by":"auto","created_at":"2025-12-01 08:09:33","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":114194,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eIn vitro \u003c/em\u003ecell toxicity of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp formulation by cell viability study using flow cytometer (A) Control sample (B) vehicle control and (C) treated sample\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8117448/v1/3561852ff73b63aa15f48c09.jpg"},{"id":97124038,"identity":"dd3e76ca-b588-4f3f-85d2-8ce561809948","added_by":"auto","created_at":"2025-12-01 08:09:25","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":96060,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Whole body SPECT/CT images of C57BL6 mice bearing B16F10 tumor at different time point of points post-injection of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp. (B) and (C) Results of \u003cem\u003eex vivo\u003c/em\u003e biodistribution of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp and [\u003csup\u003e169\u003c/sup\u003eYb]YbCl\u003csub\u003e3\u003c/sub\u003e in C57BL6 mice bearing B16F10 tumor at different points after intra-tumoral administration of the formulations\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8117448/v1/5dec25a80168097f4597dcf5.jpg"},{"id":97124040,"identity":"989d7964-ed5d-44ed-9843-186138739a51","added_by":"auto","created_at":"2025-12-01 08:09:25","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":129711,"visible":true,"origin":"","legend":"\u003cp\u003eTumor regression studies with [\u003csup\u003e169\u003c/sup\u003eYb]Yb\u003csub\u003e-\u003c/sub\u003eGAHAnp (A) Tumor growth index and (B) body weight index plot of C57BL/6 mice bearing melanoma tumor after intra-tumoral injection of saline (control), and 10, 20, 30, 40 MBq of [\u003csup\u003e169\u003c/sup\u003eYb]Yb\u003csub\u003e-\u003c/sub\u003eGAHAnp.\u003c/p\u003e","description":"","filename":"9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8117448/v1/6f66f253d4e8cec7ce0b3682.jpg"},{"id":97124039,"identity":"7d2f787b-1729-4694-a57b-730a14795f08","added_by":"auto","created_at":"2025-12-01 08:09:25","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":191749,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Slab model of HA (0001) surface (B) Yb\u003csup\u003e3+\u003c/sup\u003e atom adsorbed (0001) surface and (C) Yb\u003csup\u003e3+\u003c/sup\u003e atom substituted at Ca\u003csup\u003e2+\u003c/sup\u003e ion site on the (0001) surface and the exchanged Ca\u003csup\u003e2+\u003c/sup\u003e ion remain as an adsorbed atom. Blue, Green and red atoms represent Ca, Yb and O atoms, respectively. The PO\u003csub\u003e4 \u003c/sub\u003eunits are shown as light pink polyhedra.\u003c/p\u003e","description":"","filename":"10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8117448/v1/ad4bd759f4a1b36569d6d911.jpg"},{"id":97124056,"identity":"1e39c0ff-7127-413f-a4ae-92192b95e078","added_by":"auto","created_at":"2025-12-01 08:09:28","extension":"jpg","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":108299,"visible":true,"origin":"","legend":"\u003cp\u003e(A) Normalized XANES spectra of Yb-GAHAnp along with YbCl\u003csub\u003e3\u003c/sub\u003e and Yb\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e measured at Yb L\u003csub\u003e3\u003c/sub\u003e edge (B) normalized XAS spectra of Yb-GAHAnp measured at Yb L\u003csub\u003e3\u003c/sub\u003eedge. (C) EXAFS (\u003cem\u003ek\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003ec(k) Vs k) of Yb-GAHAnp measured at Yb L\u003csub\u003e3\u003c/sub\u003e edge (D) fourier transformed EXAFS of Yb-GAHAnp at Yb L\u003csub\u003e3\u003c/sub\u003e edge. The experimental spectra are represented by scatter points and theoretical fits are represented by solid lines.\u003c/p\u003e","description":"","filename":"11.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8117448/v1/f40448cab5be15f8f9ba6219.jpg"},{"id":97145255,"identity":"7faf1740-49cc-42cb-85e7-e50ea66d5c9a","added_by":"auto","created_at":"2025-12-01 10:13:31","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2808075,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8117448/v1/2fd89f73-a612-4d9a-82df-c0b5516377d7.pdf"},{"id":97124051,"identity":"5fadfa86-58aa-41fc-ab1d-640e40ba68d1","added_by":"auto","created_at":"2025-12-01 08:09:27","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":4511056,"visible":true,"origin":"","legend":"","description":"","filename":"SIPatraetal221125.docx","url":"https://assets-eu.researchsquare.com/files/rs-8117448/v1/ed786ed402e0b73d8bb3f40f.docx"}],"financialInterests":"","formattedTitle":"Synthesis, characterization and evaluation of 169Yb-labeled tailored hydroxyapatite nanospheres for potential application in nanobrachytherapy","fulltext":[{"header":"Background","content":"\u003cp\u003eCancer remains one of the most formidable challenges in modern medicine, characterized by uncontrolled cell growth and the potential to invade or spread to other parts of the body (Brown et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Despite significant advancements in understanding its genesis and prognosis at molecular level, cancer remains a leading cause of mortality worldwide (Siegel et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Siegel et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Thun et al., \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Other than surgery, the current landscape of cancer treatment encompasses a multifaceted approach, including chemotherapy, immunotherapy, and radiation therapy (DeVita Jr and Chu, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2008\u003c/span\u003e; Gracie et al., \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Sareen et al., \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Chemotherapy uses cytotoxic drugs to target rapidly dividing cells, but its non-selective nature can lead to severe side effects, impacting healthy tissues (Pandey et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Immunotherapy leverages immune system of the body to combat with cancer but its effectiveness can vary depending on the type of cancer and also its immune-related adverse effects can limit its use in cancer management (Zidi et al., \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). In contrast, brachytherapy, which is a form radiation therapy can surmount these limitations by placing a millimeter size sealed sources within or near the tumor surgically. This internal radiation method allows for a higher dose of radiation to be delivered to the tumor with minimal impact on adjacent healthy tissues (Serre et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Nevertheless, the surgical implementation of millimeter size sealed sources makes this procedure invasive and often leads to uneven distribution of dose within the cancerous tissue, hence minimizing the therapeutic outcome (Seniwal et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003eThe shortcoming of conventional brachytherapy could be overcome by employing radiolabeled nanosize seeds which can be administered into the tumor mass without surgical intervention. This approach is fast emerging as a promising therapeutic modality and is popularly known as \u0026lsquo;nanobrachytherapy\u0026rsquo; (Ghosh et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The small size of these nanoparticles allows for better penetration and near-homogenous distribution within the tumor microenvironment, thus potentially overcome the limitations of conventional brachytherapy (Ghosh et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). An ideal agent for use in nanobrachytherapy should deliver adequate dose of ionizing radiation to the proliferating cells with minimum radiation induced damage to the surrounding healthy cells or tissue (Seniwal et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003ea). This is achieved by the choice of a suitable radionuclide having particulate emissions of optimum tissue penetration. Apart from this, another major requirement is to minimize the non-target dose to the organs, such as liver, spleen, lymph nodes, due to the leakage of radioactivity from the tumor site (Ghosh et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). The leakage often occurs mainly due the poor \u003cem\u003ein vivo\u003c/em\u003e stability of radiolabeled nanoparticles, which can lead to the disintegration of the radioisotope from the formulation. To prevent this leakage, it is essential to engineer the nanoparticles in a manner that securely encapsulates the radioisotope ensuring that it becomes an integral part of the nanoparticle matrix. Further, the nanoparticles require surface conjugation with suitable surfactants, biomolecules etc. to ensure their suspensibility in physiological medium, so that the radiolabeled formulation can be easily administered and distributed in the tumor mass (Huang et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Kievit and Zhang, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eAmong the inorganic nanoparticles as biomaterials under investigations in the field of nanomedicine in recent years, biomimetic synthetic hydroxyapatite nanoparticles (HAnp) have garnered significant attention (Khajuria et al., \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Sugimoto et al., \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2025\u003c/span\u003e; Truong et al., \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Hydroxyapatite is the primary inorganic constituents of mineral phase of bone and are biocompatible and biodegradable in nature. Additionally, HAnp demonstrates a remarkable ability to bind with various radiometals due to its high surface-to-volume ratio, nanoporosity, and the presence of compatible sorption sites (Barroug and Glimcher, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Dasgupta et al., \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2010\u003c/span\u003e; Furko, 2025; Grinding, 2025; Patra et al., \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2023a\u003c/span\u003e; Patra et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e; Patra et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Despite these excellent attributes, the main drawback of HAnp is that they tend to agglomerate at the microscale, which hinders its use in desired biological applications. This can be overcome by proper surface functionalization which will impart suspensibility to the HAnp in biological medium (Patra et al., \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023b\u003c/span\u003e). It is always advantageous to functionalize HAnp with biocompatible organic molecules containing functional groups such as thiol, amine, carboxyl, hydroxyl, or phosphate, as these can enhance the complexation ability of HAnp with biomolecules, drugs, or cations (Haider et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Therefore, with an aim to developing a potent radiolabeled inorganic nanoformulation for use in nanobrachytherapy, we have synthesized HAnp with glucuronic acid (GA) surface functionalization as the nanoplatform. GA is a sugar acid derived from oxidation of glucose in biological system and therefore highly biocompatible. Besides the carboxylic group in GA helps in complexation with Ca\u003csup\u003e2+\u003c/sup\u003e present in HAnp, the hydroxyl groups in GA enhance the suspensibility of the nanoparticles in aqueous medium.\u003c/p\u003e\u003cp\u003eOn the other hand, selection of appropriate radioisotope is a crucial factor that determines the therapeutic effectiveness and safety of a nanobrachytherapy formulation (Ghosh et al., \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e; Seniwal et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2021\u003c/span\u003eb). It is desirable that the radioisotope used in nanobrachytherpy should not only facilitate targeted and efficient cancer therapy but also enable non-invasive imaging of radioactivity retention at the tumor site using SPECT/PET techniques. Among the various radioisotopes used in brachytherapy, \u003csup\u003e169\u003c/sup\u003eYb [T\u003csub\u003e1/2\u003c/sub\u003e = 32.02 d, 100% EC] offers distinct advantages as it emits Auger electrons and X-rays (Flynn et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Ghosh et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2024\u003c/span\u003e; Patel et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2001\u003c/span\u003e; Saxena et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). Moreover, it decays to an excited state of \u003csup\u003e169\u003c/sup\u003eTm, which subsequently emits several gamma photons with a mean energy of 92.7 keV, making it suitable for SPECT imaging. These favorable nuclear decay properties enhance radiation dose conformity for clinical applications in conventional brachytherapy. The United States Food and Drug Administration (US FDA) has approved use of \u003csup\u003e169\u003c/sup\u003eYb-based brachytherapy sources (Reynoso et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Given these advantages, we have chosen \u003csup\u003e169\u003c/sup\u003eYb to radiolabel GAHAnp in our endeavor to develop the potent nanobrachytherapy formulation based on inorganic nanoplatform.\u003c/p\u003e\u003cp\u003eIn this article, we report synthesis and characterization of glucuronic acid-functionalized hydroxyapatite nanoparticles (GAHAnp) and the production of \u003csup\u003e169\u003c/sup\u003eYb via \u003csup\u003e168\u003c/sup\u003eYb(n,γ)\u003csup\u003e169\u003c/sup\u003eYb reaction in a medium flux nuclear reactor. Subsequently, a chelator-free radiolabeling protocol was optimized to obtain \u003csup\u003e169\u003c/sup\u003eYb-labled GAHAnp ([\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp) with high purity. The chelator-free radiolabeling approach rules out the requirement of exogenous chelator for the attachment of the radiometal with the nanoplatform and thus, precludes potential disintegration of the \u003csup\u003e169\u003c/sup\u003eYb from the chelator. Colloidal and radiochemical stability of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp was established in physiological medium and cell toxicity was demonstrated in Raaji cells. \u003cem\u003eIn vivo\u003c/em\u003e SPECT/CT imaging, \u003cem\u003eex vivo\u003c/em\u003e biodistribution and tumor regression in animal model established the potency of the formulation. Moreover, the sorption mechanism of \u003csup\u003e169\u003c/sup\u003eYb\u003csup\u003e3+\u003c/sup\u003e onto the matrix of GAHAnp was investigated to prove that the radiometal is an integral part of the GAHA matrix.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eA schematic of the sequential investigations reported in the manuscript toward synthesis and evaluation of a new radiolabeled biomaterial having promising features for cancer management is presented in \u003cb\u003eFig.\u0026nbsp;1\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure\u0026nbsp;1\u003c/b\u003e Schematic presentation of formulation and evaluation of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-labeled glucuronic acid functionalized hydroxyapatite nanoparticles for application in nanobrachytherpy\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eSynthesis and characterization of GAHAnp\u003c/h2\u003e\u003cp\u003eWell-dispersed GAHAnp was synthesized at room temperature following a single step process using calcium nitrate and diammonium hydrogen phosphate and GA. The amount of GA required for achieving the smallest particle size of GAHAnp was optimized by varying the amount of GA. It was found that hydrodynamic diameter of GAHAnp decreases with the increment of GA amount and become unaltered when the amount of GA was 100 mg (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA\u003c/b\u003e). The formation of GAHAnp was confirmed by XRD (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eA\u003cb\u003e)\u003c/b\u003e. While the peaks appeared indicated the formation of hydroxyapatite, the broadening of the peaks established the formation of nanocrystalline material. The crystallite size of GAHAnp was determined from (221) peak using the Scherrer formula given bellow\u003c/p\u003e\u003cp\u003eCrystallite Size (D) = \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\frac{\\text{k}{\\lambda\\:}}{\\text{B}\\:\\text{c}\\text{o}\\text{s}{\\theta\\:}}\\)\u003c/span\u003e\u003c/span\u003e (4)\u003c/p\u003e\u003cp\u003eWhere B is the full-width half maximum, k is the Scherrer constant, D is the crystallite size, and λ is the wavelength of X-ray. The crystallite size of the GAHAnp was found to be 6\u0026thinsp;\u0026plusmn;\u0026thinsp;1 nm. The XRD of GAHAnp recorded after removal of GA (\u003cb\u003eFig. \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eB\u003c/b\u003e) matches well with standard crystalline hydroxyapatite (JCPDS data No. 9-432) and the peaks showed minimal broadening. This could be explained by the fact that GA did not promote the nucleation of HA but instead retarded crystal growth, resulting in GA-functionalized HA nanoparticles. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eB shows the Raman spectra of GA, HAnp and GAHAnp. The Raman peak observed at ~\u0026thinsp;383 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e is attributed to the bending vibrations in calcium hydroxide (Kozyryev et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). The peaks at 520 and 585 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e may be assigned to the bending modes of HPO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e2\u0026minus;\u003c/sup\u003e and PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e (Koutsopoulos, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2002\u003c/span\u003e; Ulian et al., \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). The intense peak recorded at ~\u0026thinsp;983 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e, 1085 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and a broad band at 450 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e could be assigned to the symmetric stretching vibrational modes of phosphate ions (Koutsopoulos, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). These peaks are present in the Raman spectrum of both bare HAnp and GAHAnp and thereby indicating the formation of HAnp. It is evident that the diagnostic peaks of GA (at 720 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e and 1365 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) appeared in the GAHAnp giving a clear indication about the functionalization of GA on the surface of HAnp (Mutter et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). The functionalization of GA on the surface of the HAnp was clearly evident in FT-IR spectra as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eC. The peak at 3350 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e was attributed to the OH\u003csup\u003e\u0026minus;\u003c/sup\u003e ions and the peaks at 1102, 1030, 603, and 563 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e which are diagnostic peaks of PO\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e3\u0026minus;\u003c/sup\u003e moiety, were present in both bare HAnp and GAHAnp spectra (Verma et al., \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The peak observed at 2901 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e could be assigned to the C-H stretching in both GA and GAHAnp (Ţucureanu et al., \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). The peaks at 1620, 1380, 1085 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in GAHAnp could be assigned to the asymmetric stretching of the C\u0026thinsp;=\u0026thinsp;O, symmetric stretching of the C\u0026thinsp;=\u0026thinsp;O and bending of the C-H bond respectively (Tajmir-Riahi, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e1984\u003c/span\u003e). These peaks were not observed in the case of bare HAnp, which indicates the attachment of glucuronic acid on the surface of HAnp thus confirming its functionalization. The shifting of C\u0026thinsp;=\u0026thinsp;O stretching frequency towards the lower frequency (1620 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e) in GAHAnp spectra compared to the C\u0026thinsp;=\u0026thinsp;O stretching frequency of 1715 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e in case of GA is due to the interaction between the C\u0026thinsp;=\u0026thinsp;O of carboxylic group and the Ca\u003csup\u003e2+\u003c/sup\u003e present in the hydroxyapatite. These observations clearly inferred the functionalization of GA on the surface of HAnp. Further, the extent of functionalization of GA on the surface of the HAnp was quantified by comparing the TGA curves of bare HAnp and GAHAnp (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eD\u003cb\u003e)\u003c/b\u003e. The weight loss upto 150\u0026deg; C for both samples could be attributed for evaporation of water molecules that were adsorbed physically on their surface. The weight loss observed for GAHAnp in the temperature range of 150\u0026deg;-750\u0026deg; C could be assigned for removal and degradation of the attached glucuronic acid molecules from the surface of HAnp. From the TGA curve it was found that around 25\u0026thinsp;\u0026plusmn;\u0026thinsp;2 wt % of glucuronic acid was attached to the surface of HAnp.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eParticle size distribution of the synthesized GAHAnp was obtained from DLS measurements (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eA) and the average hydrodynamic diameter of the particles was found to be 45\u0026thinsp;\u0026plusmn;\u0026thinsp;3 nm. The zeta potential of GAHAnp was determined to be -32.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2.5 mV at pH\u0026thinsp;~\u0026thinsp;6. The change of hydrodynamic diameter and the zeta potential of a freshly prepared GAHAnp was determined over a period of 15 days (\u003cb\u003eFig. S2A and S2B\u003c/b\u003e). It was found that size and zeta potential of GAHAnp were not altered significantly during that time period. To corroborate this observation, magnetic T\u003csub\u003e2\u003c/sub\u003e relaxation was measured over the same time period using Xigo Nanotools. It was observed that T\u003csub\u003e2\u003c/sub\u003e relaxation did not alter significantly with time (\u003cb\u003eFig. S2C and S2D\u003c/b\u003e), indicating nanoparticles were not coagulated. Overall, these studies demonstrated robust colloidal stability of the nanoparticles in aqueous medium. One the other hand, the average particle size measured for the bare HAnp (\u003cb\u003eFig. S3A\u003c/b\u003e) was found to be 280\u0026thinsp;\u0026plusmn;\u0026thinsp;5 nm and zeta potential \u0026minus;\u0026thinsp;2.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.8 mV at pH\u0026thinsp;~\u0026thinsp;6. Also, increase of particles size of the bare HAnp was observed over a period of time (\u003cb\u003eFig. S3B\u003c/b\u003e), indicating coagulation of the nanoparticles. This indicates that the functionalization of HAnp with GA not only improve the particle size but also it enhances colloidal stability of the nanoparticles. The visual image of GAHAnp and bare HAnp was shown \u003cb\u003eFig. S4\u003c/b\u003e where the coagulation of HAnp could be clearly seen while aqueous dispersion GAHAnp remained stable upto 14 days from preparation. The TEM image of GAHAnp was acquired to demonstrate the particles shape and size. The TEM image of Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eB showed formation of spherical shape of GAHAnp. The particle size distribution of GAHAnp obtained from TEM image was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and their average size was found to be 15.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1.4 nm. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eD demonstrate the HRTEM image of GAHAnp and the lattice fringe could also be seen in the image. Using this lattice fringe the interplanar distance was determined to be 0.278 nm which was closely matches with (211) lattice plane (In et al., \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The SAED pattern (\u003cb\u003eInset of\u003c/b\u003e Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e3\u003c/span\u003eD) demonstrated the polycrystalline nature of GAHAnp and the brightest ring pattern indicated (211) plane of GAHAnp.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eProduction and radiochemical processing of Yb\u003c/h3\u003e\n\u003cp\u003eTheoretical calculations were carried out (detail provided in \u0026lsquo;Supporting Information\u0026rsquo;) to predict the yield and radionuclidic impurities of \u003csup\u003e169\u003c/sup\u003eYb obtained from \u003csup\u003e168\u003c/sup\u003eYb(n, γ)\u003csup\u003e169\u003c/sup\u003eYb reaction using enriched (35.8% in\u003csup\u003e168\u003c/sup\u003eYb) Yb target at thermal neutron flux of 1.0 \u0026times; 10\u003csup\u003e14\u003c/sup\u003e n/cm\u003csup\u003e2\u003c/sup\u003e/s for different irradiation time periods. It was found that 21 days of irradiation is required to achieve the maximum radioactivity yield of \u003csup\u003e169\u003c/sup\u003eYb (\u003cb\u003eFig. S5\u003c/b\u003e). Based on this, enriched (35.8% in \u003csup\u003e168\u003c/sup\u003eYb) Yb targets were irradiated for 21 days in Dhruva research reactor at Bhabha Atomic Research Centre at the available thermal neutron flux of 1.0 \u0026times; 10\u003csup\u003e14\u003c/sup\u003e n/cm\u003csup\u003e2\u003c/sup\u003e/s. Post irradiation, targets were radiochemically processed and \u003csup\u003e169\u003c/sup\u003eYb activity produced was obtained as [\u003csup\u003e169\u003c/sup\u003eYb]YbCl\u003csub\u003e3\u003c/sub\u003e solution. Subsequently, gamma ray spectra were recorded to determine the yield and radionuclidic purity of \u003csup\u003e169\u003c/sup\u003eYb activity produced. A typical gamma ray spectrum is shown in \u003cb\u003eFig.\u0026nbsp;4A.\u003c/b\u003e The batch yield of \u003csup\u003e169\u003c/sup\u003eYb along with the co-produced radionuclidic impurities were summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. It was found that the average yield of \u003csup\u003e169\u003c/sup\u003eYb was 8.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2 GBq at the end of processing with \u003csup\u003e175\u003c/sup\u003eYb being the major radionuclidic impurity co-produced by neutron activation of \u003csup\u003e174\u003c/sup\u003eYb present in the target used (\u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e\u003c/b\u003e). The radionuclidic purity of \u003csup\u003e169\u003c/sup\u003eYb was found to be 97.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02% at the end of irradiation. However, for its utility in medical applications, \u003csup\u003e169\u003c/sup\u003eYb should be obtained with \u0026gt;\u0026thinsp;99.9% radionuclidic purity. Based on theoretical calculations, it is found that radionuclidic impurity of \u003csup\u003e169\u003c/sup\u003eYb would be \u0026gt;\u0026thinsp;99.9% after 25 days from the end of irradiation for the irradiation protocol followed (\u003cb\u003eTable S2\u003c/b\u003e). Gamma ray spectrum of [\u003csup\u003e169\u003c/sup\u003eYb]YbCl\u003csub\u003e3\u003c/sub\u003e was recorded after 25 days of decay. The spectrum (\u003cb\u003eFig.\u0026nbsp;4B\u003c/b\u003e) showed no detectable photopeaks for \u003csup\u003e175\u003c/sup\u003eYb or \u003csup\u003e177\u003c/sup\u003eLu, indicating near exclusive radionuclidic purity of \u003csup\u003e169\u003c/sup\u003eYb. Hence, [\u003csup\u003e169\u003c/sup\u003eYb]YbCl\u003csub\u003e3\u003c/sub\u003e solution was used for radiolabeling after 25 days from the end of irradiation of target. At that point, the total activity of [\u003csup\u003e169\u003c/sup\u003eYb]YbCl\u003csub\u003e3\u003c/sub\u003e available was 5.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.7 GBq.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure\u0026nbsp;4\u003c/b\u003e Typical gamma ray spectrum of \u003csup\u003e169\u003c/sup\u003eYb (A) at the end of processing (B) after 25 days of cooling\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eYield of \u003csup\u003e169\u003c/sup\u003eYb and co-produced impurities from irradiation of 100 \u0026micro;g of 35.8% enriched \u003csup\u003e168\u003c/sup\u003eYb target for 21 days at thermal neutron flux of 1\u0026times;10\u003csup\u003e14\u003c/sup\u003e n.cm\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e.s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eBatch No.\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eActivity of \u003csup\u003e169\u003c/sup\u003eYb*\u003c/p\u003e\u003cp\u003e(GBq)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eActivity of \u003csup\u003e175\u003c/sup\u003eYb*\u003c/p\u003e\u003cp\u003e(GBq)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eActivity of \u003csup\u003e177\u003c/sup\u003eLu*\u003c/p\u003e\u003cp\u003e(MBq)\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e8.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"4\"\u003e(* at the end of irradiation)\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eRadiochemical purity of [\u003csup\u003e169\u003c/sup\u003eYb]YbCl\u003csub\u003e3\u003c/sub\u003e as [\u003csup\u003e169\u003c/sup\u003eYb]Yb\u003csup\u003e3+\u003c/sup\u003e was 99.1\u0026thinsp;\u0026plusmn;\u0026thinsp;0.2%, as obtained from\u003c/p\u003e\u003cp\u003eradio-TLC.\u003c/p\u003e\n\u003ch3\u003eRadiolabeling of GAHAnp with Yb\u003c/h3\u003e\n\u003cp\u003eAnalysis of the results of the experiment carried out to determine the effect of concentration of GAHAnp on the yield of \u003csup\u003e169\u003c/sup\u003eYb-radiolabeleing revealed that with increasing the GAHAnp concentration, the radiolabeling yield increases and becomes saturated at the concentration 1 mg/mL (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eA). The radiolabeling yield at this concentration was \u0026gt;\u0026thinsp;99%. On the other hand, radiolebeling yield was found to increases with the increase of pH of the solution between 3\u0026ndash;6 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eB) and becomes maximum when pH was ~\u0026thinsp;6. Experiments on radiolabeling of GAHAnp with \u003csup\u003e169\u003c/sup\u003eYb were not carried out beyond pH\u0026thinsp;~\u0026thinsp;7 due to conversion of Yb\u003csup\u003e+\u0026thinsp;3\u003c/sup\u003e to colloidal Yb(OH)\u003csub\u003e3\u003c/sub\u003e in alkaline pH. On the other hand, radiolabeling was not recommended bellow pH\u0026thinsp;~\u0026thinsp;3 due to dissolution of GAHAnp. Overall, thorough mixing of [\u003csup\u003e169\u003c/sup\u003eYb]YbCl\u003csub\u003e3\u003c/sub\u003e solution with a suspension of 1 mg of in 1 mL of de-ionized water at pH\u0026thinsp;~\u0026thinsp;6 for 45 min at room temperature was found to be the optimal condition of for formulation of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp with \u0026gt;\u0026thinsp;99% yield and radiochemical purity.\u003c/p\u003e\n\u003ch3\u003eStudies on sorption of Yb on the surface of GAHAnp\u003c/h3\u003e\n\u003cp\u003eThe sorption behaviour of Yb\u003csup\u003e3+\u003c/sup\u003e ion on the surface of GAHAnp could be ascertained from equilibrium sorption isotherm and kinetics. It was found that sorption of Yb\u003csup\u003e3+\u003c/sup\u003eon GAHAnp followed Langmuir-Freundlich isotherm (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eC) and pseudo-second order kinetics (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eD), indicating that chemisorption of Yb\u003csup\u003e3+\u003c/sup\u003eion on the surface of GAHAnp and the rate determining step involved sharing or exchange of electrons between GAHAnp and Yb\u003csup\u003e3+\u003c/sup\u003e. The chemisorption process ensures robust binding of Yb\u003csup\u003e3+\u003c/sup\u003e on GAHAnp. The rate of transfer of Yb\u003csup\u003e3+\u003c/sup\u003e on the surface of GAHAnp was determined to be ~\u0026thinsp;3.8 \u0026times; 10\u003csup\u003e\u0026minus;\u0026thinsp;4\u003c/sup\u003e g/mg/min. Based on the kinetic model, the sorption capacity of Yb\u003csup\u003e3+\u003c/sup\u003e at a concentration of 0.5 mg/mL was calculated from the slope and intercept of the plot in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and found to be 136\u0026thinsp;\u0026plusmn;\u0026thinsp;2 mg/g. This value is in close agreement with the results presented in \u003cb\u003eFig. S6A\u003c/b\u003e and \u003cb\u003eS6B\u003c/b\u003e.\u003c/p\u003e\u003cp\u003ePoor correlation coefficient value (R\u003csup\u003e2\u003c/sup\u003e) of linear fitting discarded the applicability of Langmuir isotherm (\u003cb\u003eFig. S7A\u003c/b\u003e) and Freundlich isotherm (\u003cb\u003eFig. S7B\u003c/b\u003e). Similarly, poor R\u003csup\u003e2\u003c/sup\u003e value also discarded the pseudo-first order kinetics model (\u003cb\u003eFig. S7C\u003c/b\u003e). The linear fitting formula for the aforementioned isotherm and kinetic models are given in \u003cb\u003eTable S3\u003c/b\u003e.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\n\u003ch3\u003ePreparation and characterization of Yb-GHAn\u003c/h3\u003e\n\u003cp\u003eThe emission spectra of synthesized Yb-GAHAnp and Yb\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e on excitation with 980 nm are depicted in \u003cb\u003eFig.\u0026nbsp;6A\u003c/b\u003e. The emission spectra showed intense broad spectral bands around 650 nm to 750 nm, which split into several peaks. Emission spectra of Yb-GAHAnp showed reduced intensity suggesting a chemical interaction between Yb\u003csup\u003e3+\u003c/sup\u003e ions and hydroxyapatite nanoparticles. The fluorescence radiation trapping depends on the concentration of Yb\u003csup\u003e3+\u003c/sup\u003e and occurs even for small doping levels. The decay rates for 2F\u003csub\u003e5/2\u003c/sub\u003e level of Yb\u003csup\u003e3+\u003c/sup\u003e ion exhibit single exponential for all concentrations. The lifetime decreases from 7.296 \u0026micro;s to 1.037 \u0026micro;s for Yb-GAHAnp (\u003cb\u003eFig.\u0026nbsp;6B\u003c/b\u003e), therefore it is inferred that a definite chemical interaction between Yb\u003csup\u003e3+\u003c/sup\u003e and GAHAnp has occurred.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFigure\u0026nbsp;6\u003c/b\u003e (A) Emission spectra of Yb-GAHAnp and free Yb\u003csup\u003e3+\u003c/sup\u003e on excitation with 980 nm (B) fluorescence life time spectra of free Yb\u003csup\u003e3+\u003c/sup\u003e and Yb-GAHAnp\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003estability of [\u003c/b\u003e\u003csup\u003e\u003cb\u003e169\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eYb]Yb-GAHAnp\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe radiochemical stability of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp was ascertained in PBS and mouse serum media. This study demonstrated that the radiolabeled nanoformulation retained its integrity\u0026thinsp;\u0026gt;\u0026thinsp;98% in PBS medium and \u0026gt;\u0026thinsp;97% in mouse serum even after 30 d from the time of formulation (\u003cb\u003eFig. S8\u003c/b\u003e), confirming robustness of binding of Yb\u003csup\u003e3+\u003c/sup\u003e with GAHAnp.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003ecell toxicity study with [\u003c/b\u003e\u003csup\u003e\u003cb\u003e169\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eYb]Yb-GAHAnp\u003c/b\u003e\u003c/p\u003e\u003cp\u003eResults of \u003cem\u003ein vitro\u003c/em\u003e cell toxicity studies using the radiolabeled nanoparticles in Raji cells by flow cytometry is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003e. The results showed 92.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2.1% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003eA) and 86.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.2% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003eB) cell viability in control and vehicle control experiment, respectively. In comparison to these, only 48.1\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003eC) cell viability was observed when the cells were treated with 37 MBq of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp formulation. These \u003cem\u003ein vitro\u003c/em\u003e experimental data demonstrated the therapeutic potential of the prepared formulation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eSPECT/CT imaging and\u003c/b\u003e \u003cb\u003eex vivo\u003c/b\u003e \u003cb\u003ebiodistribution\u003c/b\u003e\u003c/p\u003e\u003cp\u003eQualitative analysis of whole body SPECT/CT images acquired at different time intervals after intra-tumoral administration of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp in B16F10 tumor bearing C57BL6 mice confirmed that the formulation remained at the site of administration (i.e. within the tumor mass) even after two weeks of post-injection upto which period the study was carried out (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e8\u003c/span\u003eA). Also, there was almost no leaching of radioactivity from tumor site as almost no uptake was observed in other organ especially, liver and bone. The near-complete retention of the instilled radioactivity is a favourable attribute toward it use in nanobrachterapy.\u003c/p\u003e\u003cp\u003eQuantitative analysis of the distribution of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp after intra-tumoral administration in B16F10 tumor bearing C57BL6 mice was carried out from the results of \u003cem\u003eex vivo\u003c/em\u003e biodistribution (BD) studies and the distribution pattern is summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e8\u003c/span\u003eB. The BD pattern showed that the tumor uptake of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp was 121.60\u0026thinsp;\u0026plusmn;\u0026thinsp;10.53% ID/g at 3 h post injection (p.i.) and it was retained to a significant extent (85.36\u0026thinsp;\u0026plusmn;\u0026thinsp;5.36% ID/g) even after 336 h p.i. A small percentage of the administered formulation cleared through hepatobiliary route, confirmed from 2.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.86%ID/g of liver uptake at 3 h p.i. which was reduced to 0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05%ID/g at 336 h p.i. The uptake of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp in any other organ/tissue was negligible, as evident from the BD pattern. On the other hand, the BD pattern of [\u003csup\u003e169\u003c/sup\u003eYb]YbCl\u003csub\u003e3\u003c/sub\u003e is given in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e8\u003c/span\u003eC, which shows that although the tumor uptake of was high (112.78\u0026thinsp;\u0026plusmn;\u0026thinsp;6.7% ID/g) at 3 h p.i., almost entire radioactivity leached out within 24 h. Substantial bone uptake of [\u003csup\u003e169\u003c/sup\u003eYb]YbCl\u003csub\u003e3\u003c/sub\u003e was observed at 3 h of p.i (20.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3% ID/g), which increased to 24.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6% ID/g at 24 h p.i. Significant uptake could also be observed in liver and kidney at 24 h p.i. This comparative analysis of the BD patterns of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp nanoformulation and [\u003csup\u003e169\u003c/sup\u003eYb]Yb\u003csup\u003e3+\u003c/sup\u003e used as control clearly established the utility of the nanoformulation [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp to ensure the prolonged retention of administered radioactivity in tumor and prevent its accumulation in non-target organs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eAssessment of therapeutic efficacy by tumor regression study\u003c/h2\u003e\u003cp\u003eTo assess the therapeutic efficacy of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp, four escalating doses, 10, 20, 30, and 40 MBq of the formulation were administered intra-tumorally in four different sets of B16F10 tumor bearing C57BL6 mice (four animals in each set). Saline was injected in another set of tumor bearing animals treated as \u0026lsquo;control\u0026rsquo;. Tumor growth index (TGI) and body weight index (BWI) were monitored over a 21-day post-treatment period (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Notably, tumor progression was effectively suppressed by a single administration of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp either 30 MBq or 40 MBq, indicating a clear dose-dependent therapeutic effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). While mice treated with the 30 MBq dose maintained stable body weight throughout the observation period (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e9\u003c/span\u003eB), reduction in BWI was observed in the 40 MBq group\u0026mdash;potentially attributable to radiation-induced systemic toxicity. Based on these findings, the 30 MBq dose was identified as the optimal therapeutic window, offering a favorable balance between efficacy and safety.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eInvestigation of sorption mechanism of Yb on the surface of GAHAnp\u003c/h3\u003e\n\u003cp\u003eFor the understanding of the mechanism of sorption of Yb\u003csup\u003e3+\u003c/sup\u003e on the surface of GAHAnp, a systematic periodic DFT studies were performed. In the HA [Ca\u003csub\u003e10\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e6\u003c/sub\u003e(OH)\u003csub\u003e2\u003c/sub\u003e] unit cell, two oxygen atoms have partial occupancies in the P6\u003csub\u003e3\u003c/sub\u003e/m space group. Based on the partial occupation, four configurations can be generated with varying OH group orientations. A unit cell with the opposite orientation of the OH group channels was considered to obtain a unit cell having zero electric polarization (Almora-Barrios and de Leeuw, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The (0001) surface of HA was considered for all of the Yb\u003csup\u003e3+\u003c/sup\u003e interaction because the (0001) termination is the most energetically stable(Almora-Barrios and de Leeuw, \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). The DFT optimized (0001) surface structure is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e10\u003c/span\u003eA. In order to decide the nature of interaction (physical/chemical) of Yb\u003csup\u003e3+\u003c/sup\u003e ion with HA surface, a 2\u0026times;2\u0026times;1 supercell with 352 atoms are constructed. A vacuum of around 15 \u0026Aring; is taken into consideration along the z-direction to prevent interactions between periodic images in non-periodic directions. To study Yb\u003csup\u003e3+\u003c/sup\u003e adsorption on (0001) surface, the position of Yb\u003csup\u003e2+\u003c/sup\u003e atom on (0001) surface is optimized and shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e10\u003c/span\u003eB. The distances of Yb\u003csup\u003e3+\u003c/sup\u003e atom from the nearest O atoms are 2.29, 2.34, 2.35, 2.42, 2.44 \u0026Aring;. To study Yb\u003csup\u003e3+\u003c/sup\u003e chemical interaction on (0001) surface, Yb\u003csup\u003e3+\u003c/sup\u003e ion is exchanged with a surface Ca\u003csup\u003e2+\u003c/sup\u003e ion and the exchanged Ca\u003csup\u003e2+\u003c/sup\u003e ion remain as an adsorbed atom on the surface and the optimized structure is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e10\u003c/span\u003eC. The bond distances of in Yb\u003csup\u003e3+\u003c/sup\u003eO polyhedra are 2.23, 2.24, 2.28, 2.42, 2.44 \u0026Aring;. The distances of Ca\u003csup\u003e2+\u003c/sup\u003e ion from the surface O atoms are 2.36, 2.40, 2.40, 2.41, 2.48, 2.52 \u0026Aring;. Evidently, Yb\u003csup\u003e3+\u003c/sup\u003e ion forms stronger and shorter bonds with O atoms due to substitution at Ca\u003csup\u003e2+\u003c/sup\u003e site rather than adsorption on the (0001) surface.\u003c/p\u003e\u003cp\u003eThe energetics of these two processes are calculated as follows:\u003c/p\u003e\u003cp\u003eHA\u0026thinsp;+\u0026thinsp;Yb\u003csup\u003e3+\u003c/sup\u003e(H\u003csub\u003e2\u003c/sub\u003eO)\u003csub\u003e9\u003c/sub\u003e \u0026rarr;Yb\u003csup\u003e3+\u003c/sup\u003e@HA + (H\u003csub\u003e2\u003c/sub\u003eO)\u003csub\u003e9\u003c/sub\u003e ΔE\u0026thinsp;=\u0026thinsp;2.23 eV\u003c/p\u003e\u003cp\u003eHA\u0026thinsp;+\u0026thinsp;Yb\u003csup\u003e3+\u003c/sup\u003e(H\u003csub\u003e2\u003c/sub\u003eO)\u003csub\u003e9\u003c/sub\u003e \u0026rarr;Yb\u003csup\u003e3+\u003c/sup\u003e_Ca\u003csup\u003e2+\u003c/sup\u003e@HA\u0026thinsp;+\u0026thinsp;Ca\u003csup\u003e2+\u003c/sup\u003e(H\u003csub\u003e2\u003c/sub\u003eO)\u003csub\u003e9\u003c/sub\u003e ΔE = -1.88 eV\u003c/p\u003e\u003cp\u003ewhere Yb\u003csup\u003e3+\u003c/sup\u003e@HA and Yb\u003csup\u003e3+\u003c/sup\u003e_Ca\u003csup\u003e2+\u003c/sup\u003e@HA represent the Yb\u003csup\u003e3+\u003c/sup\u003e-adsorbed HA surface and Yb\u003csup\u003e3+\u003c/sup\u003e_Ca\u003csup\u003e2+\u003c/sup\u003e-exchanged HA surface, respectively. These processes also considered the energies of Yb\u003csup\u003e3+\u003c/sup\u003e(H\u003csub\u003e2\u003c/sub\u003eO)\u003csub\u003e9\u003c/sub\u003e and Ca\u003csup\u003e2+\u003c/sup\u003e(H\u003csub\u003e2\u003c/sub\u003eO)\u003csub\u003e9\u003c/sub\u003e complexes in the calculation of reaction energies. The positive ΔE value implies that adsorption of Yb\u003csup\u003e3+\u003c/sup\u003e ion on the (0001) surface of HA is energetically unfavourable. On the contrary, chemical substitution of Yb\u003csup\u003e3+\u003c/sup\u003e at Ca\u003csup\u003e2+\u003c/sup\u003e site on the (0001) surface of HA is energetically favourable as implied by negative ΔE value.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe theoretical investigations suggest that sorption of Yb\u003csup\u003e3+\u003c/sup\u003e on HA matrix is through ion-exchange mechanism. This observation was corroborated by studying the release of Ca\u003csup\u003e2+\u003c/sup\u003e from intrinsically \u003csup\u003e45\u003c/sup\u003eCa-labeled GAHAnp due to sorption of Yb\u003csup\u003e3+\u003c/sup\u003e on its surface. It was found that 24.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.2% of the \u003csup\u003e45\u003c/sup\u003eCa was replaced from \u003csup\u003e45\u003c/sup\u003eCa-labeled GAHAnp matrix when 5 mg Yb was adsorbed on 50 mg GHAnp, which indicated that sorption takes place through ion-exchange mechanism and in this process, Yb (\u003csup\u003e169\u003c/sup\u003eYb for radiolabeled nanoformaultion) become integral part of the solid matrix of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp formulation. Further, X-ray absorption spectroscopy (XAS) measurements for Yb-GAHAnp along with Yb\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e and YbCl\u003csub\u003e3\u003c/sub\u003e samples were conducted and measured at Yb L\u003csub\u003e3\u003c/sub\u003e edge. The normalized X-ray near edge structure (XANES) spectra of all these samples was shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e11\u003c/span\u003eA which suggest that Yb is loaded on GAHAnp in Yb\u003csup\u003e3+\u003c/sup\u003e form not in Yb\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e and YbCl\u003csub\u003e3\u003c/sub\u003e as none of these XANES data showed exact match with XANES data of Yb-GAHAnp. The normalized absorption spectra of Yb-GAHAnp at Yb L\u003csub\u003e3\u003c/sub\u003e edge is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e11\u003c/span\u003eB. The fourier transform of \u003cem\u003ek\u003c/em\u003e\u003csup\u003e\u003cem\u003e2\u003c/em\u003e\u003c/sup\u003e\u003cem\u003eχ(K)\u003c/em\u003e vs. \u003cem\u003ek\u003c/em\u003e spectra (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e11\u003c/span\u003eC) over the \u003cem\u003ek\u003c/em\u003e range of 3.0\u0026ndash;11.0 \u0026Aring;\u003csup\u003e-1\u003c/sup\u003e was performed to generate \u003cspan class=\"InlineEquation\"\u003e\u003c/span\u003eversus \u003cem\u003eR\u003c/em\u003e plots at Yb L\u003csub\u003e3\u003c/sub\u003e edge (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e11\u003c/span\u003eD). Subsequently, the EXAFS data were fitted in the range of 1.0\u0026ndash;4.0 \u0026Aring; in R-space. The scattering paths were obtained from Ca\u003csub\u003e10\u003c/sub\u003e(PO\u003csub\u003e4\u003c/sub\u003e)\u003csub\u003e6\u003c/sub\u003e(OH)\u003csub\u003e2\u003c/sub\u003e crystal structure, in which some of the Ca atoms were replaced by the Yb atoms. The peak at 1.8 \u0026Aring; appeared from Yb-O coordination shells. The EXAFS peak at 2.75 \u0026Aring; appeared from the Yb-P coordination shell. The small EXAFS peak at 3.20 \u0026Aring; arose from the Yb-O\u003csub\u003e3\u003c/sub\u003e coordination shell. The EXAFS peak around 3.66 \u0026Aring; has contribution from the Yb-Ca coordination shells. In the fitting procedure, the coordination number (N.), the interatomic bond distance between pairs of atoms (R) and the Debye-Waller factor (σ\u003csup\u003e2\u003c/sup\u003e, representing the thermal disorder and mean square fluctuation in atomic bond lengths) were utilized as fitting parameters. The optimal values for these parameters are presented in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. The goodness of fit was assessed by the value of R\u003csub\u003efactor\u003c/sub\u003e which is defined as follows\u003c/p\u003e\u003cp\u003e\u003cimg 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style=\"width: 412px; height: 68.3216px;\" width=\"412\" height=\"68.3216\"\u003e\u003c/p\u003e\u003cp\u003ewhere, χ\u003csub\u003edat\u003c/sub\u003e and χ\u003csub\u003eth\u003c/sub\u003e refer to the experimental and theoretical χ(r) values respectively. Im and Re represents the imaginary and real parts of the respective quantities. The results obtained from XAFS study demonstrated shrinkage of bond distance (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) after the replacement of Ca by Yb ion. All these investigations conclude that loading of Yb on GAHAnp most likely occurred through ion exchange mechanism, by which Yb becomes an integral part of the GAHAnp matrix. This implies that in the developed \u003csup\u003e169\u003c/sup\u003eYb-labeled nanoformulation, the radiometal is a part of the solid matrix, which is an essential pre-requisite for a nanoscale brachytherapy agent.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eBond length, coordination number and disorder factors obtained by EXAFS fitting measured at Yb L\u003csub\u003e3\u003c/sub\u003eedge.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"3\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003ePath\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eParameter\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eYb-GAHAnp\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"3\" nameend=\"c3\" namest=\"c1\"\u003e\u003cp\u003eYb L3 edge\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eYb-O\u003c/b\u003e\u003csub\u003e\u003cb\u003e1\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR (\u0026Aring;) (2.37)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.27\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN (3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eσ\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0064\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0007\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eYb-O\u003c/b\u003e\u003csub\u003e\u003cb\u003e2\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR (\u0026Aring;) (2.45)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.35\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN (3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eσ\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0064\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0007\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eYb-P\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR (\u0026Aring;) (3.20)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.11\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN (3)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eσ\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0105\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eYb-O\u003c/b\u003e\u003csub\u003e\u003cb\u003e3\u003c/b\u003e\u003c/sub\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR (\u0026Aring;) (3.41)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN (2)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e2.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eσ\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.001\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0005\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e\u003cp\u003e\u003cb\u003eYb-Ca\u003c/b\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR (\u0026Aring;) (3.95)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e3.90\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eN (6)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e6.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.24\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eσ\u003csup\u003e2\u003c/sup\u003e\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.0104\u0026thinsp;\u0026plusmn;\u0026thinsp;0.003\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eR-factor\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.01\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWell-dispersed GAHAnp was successfully synthesised and extensively characterized by various analytical tools. The inclusion of GA on the surface of HAnp was ascertained by FT-IR (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e2\u003c/span\u003eC), where the shifting of C\u0026thinsp;=\u0026thinsp;O stretching frequency towards the lower frequency (1620 cm\u003csup\u003e-1\u003c/sup\u003e) in GAHAnp spectra compared to the C\u0026thinsp;=\u0026thinsp;O stretching frequency of 1715 cm\u003csup\u003e-1\u003c/sup\u003e in GA could be observed. This is due to the interaction between the C\u0026thinsp;=\u0026thinsp;O of carboxylic group and the Ca\u003csup\u003e2+\u003c/sup\u003e present in the hydroxyapatite. These observations clearly inferred the functionalization of GA on the surface of HAnp. TEM image confirmed GAHA nanosphere formation. The optimized chelator free radiolabeling protocol of GAHAnp with \u003csup\u003e169\u003c/sup\u003eYb yielded [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp formulation in high yield and radiochemical purity (\u0026gt;\u0026thinsp;99%). Binding of [\u003csup\u003e169\u003c/sup\u003eYb]Yb\u003csup\u003e3+\u003c/sup\u003e with the nanoformulation was found to be through chemisorption from sorption isotherm and kinetics studies (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). This indicates robust binding of [\u003csup\u003e169\u003c/sup\u003eYb]Yb\u003csup\u003e3+\u003c/sup\u003e with GAHAnp. Consequently, [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp formulation demonstrated excellent radiochemical stability in physiological medium (\u003cb\u003eFig S8\u003c/b\u003e). Cytotoxicity of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp formulation, demonstrated by \u003cem\u003ein vitro\u003c/em\u003e cell toxicity studies in Raji cells (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e7\u003c/span\u003e), could be attributed to the auger electrons emitted during the decay process of \u003csup\u003e169\u003c/sup\u003eYb. Auger electrons possess high LET value (1\u0026ndash;23 keV/\u0026micro;m) which induce high clustered damage in macromolecular targets within cancer cells, especially DNA and the cell membrane (Ku et al., \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Serial SPECT/CT images recorded after intra-tumoral injection of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp in C57BL6 mice bearing B16F10 tumor (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e8\u003c/span\u003eA) established excellent retention of the nanoformulation in the tumor mass. This investigation also established that there was almost no leakage of the radioalabeled nanoparticles or detachment of the radiometal from the formulation in the biological system. \u003cem\u003eEx vivo\u003c/em\u003e biodistribution studies carried out in the same animal model (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e8\u003c/span\u003eB) further corroborated these observations. Similar studies carried out with [\u003csup\u003e169\u003c/sup\u003eYb]YbCl\u003csub\u003e3\u003c/sub\u003e showed extensive leakage of radioactivity from tumor (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e8\u003c/span\u003eC). These observations point toward cellular uptake of the radiolabeled nanoparticles through endocytosis (Sun et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2016\u003c/span\u003e) as well as robust binding of [\u003csup\u003e169\u003c/sup\u003eYb]Yb\u003csup\u003e3+\u003c/sup\u003e with HA which remains stable \u003cem\u003ein vivo\u003c/em\u003e. In contrast, poor retention of [\u003csup\u003e169\u003c/sup\u003eYb]YbCl\u003csub\u003e3\u003c/sub\u003e would be due to its lack of internalization, which leads to leakage into the bloodstream, followed by uptake in various organs /tissues, primarily bone, kidney, and liver (Xu et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e2024\u003c/span\u003e). Subsequently, it was shown that administration of 30 MBq dose of the formulation into B16F10 tumor raised in C57bL/6 mice could effectively suppress the tumor progression in the same animal model without any considerable side effect (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Overall, these systematic studies established the promising features of developed radiolabeled nanospheres for use in nanobrachytherapy.\u003c/p\u003e\u003cp\u003eAn essential criterion for any radioactive material to be used for \u003cem\u003ein vivo\u003c/em\u003e brachytherapy application is that the radioactive component should be an integral part of the matrix. From this consideration, in the developed [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp formulation intended for application in nanobrachytherapy as radioactive nanospheres, it is essential that [\u003csup\u003e169\u003c/sup\u003eYb]Yb\u003csup\u003e3+\u003c/sup\u003e to be incorporated into the GAHAnp matrix. This was established by elaborate theoretical and experimental investigations. Theoretical studies using DFT shows that replacement of Ca\u003csup\u003e2+\u003c/sup\u003e of the HA matrix by the Yb\u003csup\u003e3+\u003c/sup\u003e is energetically favourable process and hence exchange with Ca\u003csup\u003e2+\u003c/sup\u003e of HA matrix could be the possible mechanism of incorporation of [\u003csup\u003e169\u003c/sup\u003eYb]Yb\u003csup\u003e3+\u003c/sup\u003e onto the matrix of HA nanospheres. This observation was experimentally verified XAFS study (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e11\u003c/span\u003e), where the local geometry around Yb after its adsorption onto GAHAnp matrix was evaluated. The results obtained demonstrated shrinkage of bond distances (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e) which could only be possible due to the replacement of Ca by Yb ion, providing direct evidence of Yb becoming a part of the solid matrix. This was further corroborated by loading inactive Yb\u003csup\u003e3+\u003c/sup\u003e onto intrinsically \u003csup\u003e45\u003c/sup\u003eCa-labeled GAHAnp, which resulted in the release of [\u003csup\u003e45\u003c/sup\u003eCa]Ca\u003csup\u003e2+\u003c/sup\u003e from the HA matrix, detected by radioactivity measurement. All these investigations conclude that loading of Yb on GAHAnp most likely occurred through ion exchange mechanism, by which Yb becomes an integral part of the GAHAnp matrix.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn summary, we have accomplished the synthesis of well-dispersed glucuronic acid funtionalized hydroxyapatite nanospheres of 45\u0026thinsp;\u0026plusmn;\u0026thinsp;3 nm size and demonstrated their chelator free radiolabeling with \u003csup\u003e169\u003c/sup\u003eYb for potential application of nanobrachtytherapy. The attachment of glucuronic acid with hydroxyapatite nanoparticles was ascertained by FT-IR and Raman spectroscopy while the amount of content of GA in GAHAnp was determined using TGA study. The high colloidal stability of the functionalized hydroxyapatite nanoparticles over the its bare counterpart was determined by measuring particles size and T\u003csub\u003e2\u003c/sub\u003e relaxation time at different time intervals. The prolonged \u003cem\u003ein vitro\u003c/em\u003e stability of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp in physiological medium illustrated the robustness of binding of the radiometal with the nanoplatform. The chemical interaction between Yb\u003csup\u003e3+\u003c/sup\u003e and GAHAnp was studied by DFT calculations, sorption studies, radiotracer studies and XAFS. These studies ascertained that in the synthesized Yb-GAHAnp nanoformulation (\u003csup\u003e169\u003c/sup\u003eYb in case of radiolabeled formulation), Yb becomes an integral part of GAHAnp matrix which fulfils the criteria for nanoscale brachytherapy. In order to demonstrate the potency of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp in biological system, SPECT/CT images were acquired after the intra-tumor administration in the melanoma tumor bearing C57BL/6 mice which revealed prolonged retention in tumor and almost no leaching of radioactivity in any other organ. This result was compared with \u003cem\u003eex vivo\u003c/em\u003e biodistribution pattern of the formulation after intra-tumoral injection in same animal model. Further, treatment with 30 MBq dose of the formulation could effectively suppress the tumor progression in the same animal model without any considerable side effect. In light of the detailed studies reported herein, it could be concluded that [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp nanoformulation could be a promising candidate for nanobracthytherapy.\u003c/p\u003e\u003cdiv id=\"Sec12\" class=\"Section2\"\u003e\u003ch2\u003eExperimental\u003c/h2\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003eMaterials\u003c/h2\u003e\u003cp\u003eIsotopically enriched ytterbium oxide (35.8% in \u003csup\u003e168\u003c/sup\u003eYb, \u0026gt;\u0026thinsp;99.99% chemical purity) was purchased from Isoflex Corporation, Russian Federation. Ytterbium oxide of natural isotopic composition (spectroscopic grade, \u0026gt;\u0026thinsp;99.99% pure) was procured from American Potash Inc., USA. Calcium nitrate tetrahydrate (\u0026ge;\u0026thinsp;99 99% pure), ammonium dihydrogen phosphate (99.99% pure) and ammonia solution (AR grade), hydrochloric acid (Suprapur\u003csup\u003e\u0026reg;\u003c/sup\u003e), glucuronic acid (99.99% pure) were purchased from Merck, Germany. All other materials used were of AR grade and were obtained from reputed manufacturers unless mentioned otherwise.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eSynthesis of GAHAnp\u003c/h2\u003e\u003cp\u003eOne pot synthesis of GAHAnp was carried out at room temperature using calcium nitrate and diammonium hydrogen phosphate and GA as calcium precursor, phosphate precursor and surface functionality, respectively. The amount of the precursor is chosen in such a way so that Ca/P ratio becomes 1.67. Briefly, 100 mg of GA was dissolved in 10 mL of 1 mM calcium nitrate tetrahydrate (solution A). Subsequently, equivolume of 0.6 mM diammonium hydrogen phosphate was added dropwise into solution A under stirring at room temperature. The pH of the solution was adjusted to ~\u0026thinsp;9 by adding ammonia solution and incubated for 4 h at room temperature while continuing the gentle stirring. At end of incubation time, the dispersed nanoparticles were transferred to a dialysis tube (MW cut-off 10 kDa) and dialyzed to eliminate excess GA not associated with HA. Subsequently, the dispersed nanopartilces were lyophilized at -50\u0026deg;C and 1 mbar pressure using a lyophilizer (Martin Christ lyophilizer). Thus, the synthesised GAHAnp was obtained as white powder and subjected to physiochemical characterization. For comparison, bare HAnp was synthesized under the same experimental conditions in the absence of GA.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003eCharacterization of GAHAnp\u003c/h2\u003e\u003cp\u003eStructural characterization of synthesized GAHAnp was carried out by powder X-ray diffraction (XRD), utilizing monochromatized Cu-Kα radiation and conducted on a PANalytical X-ray diffractometer (X\u0026rsquo;pert PRO). The XRD of GAHAnp was again recorded after removing of GA from its surface by heating at 900 \u0026ordm;C inside a furnace. Raman spectra were recorded using a micro-Raman spectrometer (STR-300, SEKI Technotron, Japan) where a diode-pumped solid-state laser (DPSS, gem532, Laser Quantum) with a wavelength of 532 nm was employed as an excitation source. Calibration of the spectrograph was performed using the 520.5 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e line from a silicon wafer. The encapsulation of GA on the surface of HAnp was ascertained by analysing fourier transform infrared (FT-IR) spectra recorded for GA, bare HAnp and GAHAnp in the range of 4500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e to 500 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e. The hydrodynamic diameter of GAHAnp was determined by dynamic light scattering (DLS) using Microtrac Series MN420 \u0026ndash; Nanotrac Wave II particle analyzer and using the same instrument zeta potential of the functionalized nanoparticles was determined. The average particles size of GAHAnp was obtained from high-resolution transmission electron microscope (HRTEM) using Philips CM 200 TEM system. In order to determine the GA content on the surface of GAHAnp, thermogravimetric analysis (TGA) of the bare HAnp and GAHAnp were carried out using Mettler Toledo TG/DSC stare system in which a certain amount of sample (~\u0026thinsp;22.14 mg) was placed in an alumina sample holder and analyses were executed under nitrogen gas flow (rate\u0026thinsp;~\u0026thinsp;50 mL/min) from room temperature to 900\u0026deg; C at the heating rate of 10\u0026deg; C/min. The colloidal stability and relaxation time measurement of GAHAnp were determined using Xigo Nanotools (Acorn Area instrument).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003eProduction and radiochemical processing of \u003csup\u003e169\u003c/sup\u003eYb\u003c/h2\u003e\u003cp\u003eMeasured amount (1.0 mg) of isotopically enriched (35.8% in \u003csup\u003e168\u003c/sup\u003eYb) Yb\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e3\u003c/sub\u003e was dissolved in minimum volume of ultrapure 0.1 M HNO\u003csub\u003e3\u003c/sub\u003e by gently heating. The resulting solution was evaporated to near dryness and reconstituted in 1 mL of deionized water. An aliquot of 100 \u0026micro;L was transferred into a clean quartz ampoule and gently heated to facilitate the deposition of a thin film of [\u0026sup1;⁶⁸Yb]Yb(NO₃)₃ on the inner surface. The ampoule was then flame sealed and placed inside a standard aluminium irradiation container. The container was which was sealed by cold-wielding and subsequently irradiated in the Dhruva reactor.\u003c/p\u003e\u003cp\u003eAt the end of irradiation, the target was cooled for 4 h and subsequently dissolved in ultrapure 0.1 M HCl (5 mL) by gentle warming inside a 100 mm lead-shielded radiochemical processing cell. The resultant solution was evaporated to near-dryness and reconstituted in 5 mL of deionized water to obtain \u003csup\u003e169\u003c/sup\u003eYb in the form of [\u003csup\u003e169\u003c/sup\u003eYb]YbCl\u003csub\u003e3\u003c/sub\u003e solution. The radioactivity produced was determined by γ-ray spectrometry using a pre-calibrated HPGe detector coupled with 4K Multi Channel Analyser system (Canberra, Eurisys). For this, an aliquot of [\u003csup\u003e169\u003c/sup\u003eYb]YbCl\u003csub\u003e3\u003c/sub\u003e solution was appropriately diluted and counted for 1 h in HPGe detector ensuring that the dead time of the detector\u0026thinsp;\u0026lt;\u0026thinsp;2%. Radionuclidic purity of the formulation was also determined from the gamma ray spectra recorded. The radiochemical purity of [\u003csup\u003e169\u003c/sup\u003eYb]YbCl\u003csub\u003e3\u003c/sub\u003e formulation was determined by radio-TLC developed in 0.9% NaCl in 0.02 M HCl as the eluting solvent.\u003c/p\u003e\u003cp\u003eFurther detail on production of \u003csup\u003e169\u003c/sup\u003eYb is provided in the \u0026lsquo;Supporting Information\u0026rsquo;.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e\u003ch2\u003eRadiolabeling of GAHAnp with \u003csup\u003e169\u003c/sup\u003eYb\u003c/h2\u003e\u003cp\u003eA chelator free radiolabeling protocol was optimized for formulation of \u003csup\u003e169\u003c/sup\u003eYb-labeled GAHAnp. For this, 0.1 mL (~\u0026thinsp;370 MBq) of \u003csup\u003e169\u003c/sup\u003eYb activity as [\u003csup\u003e169\u003c/sup\u003eYb]YbCl\u003csub\u003e3\u003c/sub\u003e solution was added to each of the reaction vials containing suspension of GAHAnp (1.0 mL) of various concentration (0.25 -2 mg/mL) in de-ionized water. After thorough mixing, the suspensions were incubated at room temperature under continuous stirring for 45 min. The pH of reaction mixtures was maintained at ~\u0026thinsp;6. Concurrently, a reference solution was prepared by mixing 0.1 mL of [\u003csup\u003e169\u003c/sup\u003eYb]YbCl\u003csub\u003e3\u003c/sub\u003e solution with 1.0 mL of de-ionized water. Upon completion of incubation time, the reaction mixture was vortexed thoroughly and centrifuged at 5000 rpm for 20 min. Subsequently, an aliquot (0.1 mL) of the supernatant solution was carefully withdrawn from the reaction mixture and radioactivity was measured (R) using NaI(Tl) scintillation detector (Mucha, Raytest GmbH). Aliquot of same volume was also withdrawn from the reference solution and radioactivity was measured (B) using same detector. Radiolabeling yield was determined using following formula\u003cdiv class=\"BlockQuote\"\u003e\u003cp\u003eRadiolabeling yield (%) = (1\u0026minus;\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\:\\frac{R}{B}\\)\u003c/span\u003e\u003c/span\u003e) \u0026times; 100 (1)\u003c/p\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe radiolabeling yield of GAHAnp was also determined by varying the pH of the GAHAnp suspension (pH\u0026thinsp;~\u0026thinsp;3\u0026ndash;7) while keeping the concentration of GAHAnp fixed (1 mg/mL). Finally, the radiolabeling of GAHAnp with \u003csup\u003e169\u003c/sup\u003eYb was carried out using optimal reaction parameters obtained from previous experiments for the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e assessment of \u003csup\u003e169\u003c/sup\u003eYb-labeled GAHAnp.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e\u003ch2\u003eStudies on sorption of Yb\u003csup\u003e3+\u003c/sup\u003e on the surface of GAHAnp\u003c/h2\u003e\u003cp\u003eSorption of Yb\u003csup\u003e+\u0026thinsp;3\u003c/sup\u003e on the surface of GAHAnp was assessed using the same method described in previous reports (Joshi et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e2022\u003c/span\u003e; Patra et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2025\u003c/span\u003e). Briefly, 5 mL of YbCl\u003csub\u003e3\u003c/sub\u003e solution with various concentrations of Yb (0.25-2 mg/mL) were spiked with [\u003csup\u003e169\u003c/sup\u003eYb]YbCl\u003csub\u003e3\u003c/sub\u003e (~\u0026thinsp;15.6 MBq, 50 \u0026micro;L) and incubated with 5.0 mg of GAHAnp at room temperature for 1 h. The pH of the solutions was adjusted to ~\u0026thinsp;6. Concurrently, a reference solution of same volume (5 mL) was prepared without addition of GAHAnp nanoparticles. After 1 h of incubation, the solutions were centrifuged at 5000 rpm for 20 min. An aliquot (50 \u0026micro;L) of supernatants were withdrawn from each solution and the activity associated with each aliquot was measured using same NaI(Tl) detector. At the same time, activity of 50 \u0026micro;L reference solution was measured and then equilibrium sorption capacity (q\u003csub\u003ee\u003c/sub\u003e) of GAHAnp at each concentration of Yb\u003csup\u003e3+\u003c/sup\u003e was obtained using the following formula\u003c/p\u003e\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\" style=\"width: 442px; height: 61.3697px;\" width=\"442\" height=\"61.3697\"\u003e\u003c/p\u003e\u003cp\u003eWhere A\u003csub\u003eo\u003c/sub\u003e and A\u003csub\u003ee\u003c/sub\u003e were the radioactivity of supernatant solution before and after equilibration, respectively, C\u003csub\u003eo\u003c/sub\u003e (mg/mL) is total Yb content in the solution before sorption, V (mL) is volume of solution and m was the mass (g) of GAHAnp. Data were obtained in triplicate.\u003c/p\u003e\u003cp\u003eIn order to determine the rate of sorption of Yb\u003csup\u003e3+\u003c/sup\u003e on the surface of GAHAnp, radiolabeling study was conducted as a function of time. For this sorption capacity of GAHAnp for a particular concentration (0.5 mg/mL) was determined at various time interval at room temperature.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e\u003ch2\u003ePreparation and characterization of Yb-GHAnp\u003c/h2\u003e\u003cp\u003eYb-GAHAnp was prepared following the protocol optimized for the formulation of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp (Section 2.5), only using YbCl\u003csub\u003e3\u003c/sub\u003e solution instead of \u003csup\u003e169\u003c/sup\u003eYb[YbCl\u003csub\u003e3\u003c/sub\u003e] radiochemical formulation. Photoluminescence (PL) spectra of Yb-GAHAnp formulation were recorded using an excitation wavelength of 980 nm in FLS1000 fluorescence spectrometer (Edinburgh Instruments, UK). For comparison, the PL spectra of free Yb\u0026sup3;⁺ ions were also recorded using YbCl₃ solution.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003estability of [\u003c/b\u003e\u003csup\u003e\u003cb\u003e169\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eYb]Yb-GAHAnp\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe radiolabeled formulation prepared using optimized radiolabeling protocol was centrifuged at 5000 rpm for 20 min. Then the supernatant was discarded and the radiolabeled particulates were dispersed with 1.0 mL phosphate buffered saline (PBS). The radiochemical purity of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp was determined over a periods 30 d following the procedure described in Section 2.5 (Eq.\u0026nbsp;1). In a similar way radiochemical purity of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp was determined in mouse serum over the same time period.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vitro\u003c/b\u003e \u003cb\u003ecell toxicity study with [\u003c/b\u003e\u003csup\u003e\u003cb\u003e169\u003c/b\u003e\u003c/sup\u003e\u003cb\u003eYb]Yb-GAHAnp\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTherapeutic potential of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp was evaluated \u003cem\u003ein vitro\u003c/em\u003e by cell toxicity studies using Raji cells. Raji cells were cultured in RPMI medium with 10% fetal calf serum (FCS) and 1% antibiotic/antimycotic solution. Approximately one million cells were seeded in 6-well plates, and 100 \u0026micro;L (~\u0026thinsp;37 MBq) of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp formulations were added into it. For comparison, the same procedure was carried out using an equivalent amount of cold Yb-GAHAnp (vehicle control) and without any treatment (control). Then, the cells were incubated for 48 hours in a humidified 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere at 37\u0026deg;C. After the incubation, the cells were washed, trypsinized, and mixed with Guava ViaCount reagents (Luminex Corp., USA). After 5 min incubation, the cells were analyzed using a Guava flow cytometer (Luminex Corp., USA) to assess cell toxicity.\u003c/p\u003e\u003cp\u003e\u003cb\u003eIn vivo\u003c/b\u003e \u003cb\u003eSPECT/CT imaging and\u003c/b\u003e \u003cb\u003eex vivo\u003c/b\u003e \u003cb\u003ebiodistribution\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBiological evaluation of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp was carried out in C57BL/6 mice bearing melanoma tumor. All animal experiments were carried out following relevant guidelines and regulations approved by the institutional Animal Ethics Committee of Bhabha Atomic Research Centre (Reference: BAEC/12/2024). The animals were bred and reared in a dedicated animal house facility, BARC. A total of ~\u0026thinsp;2 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e B16F10 melanoma cells dispersed in PBS solution were injected subcutaneously in the shoulder region of C57BL/6 mice (5 weeks older) weighing 20\u0026ndash;25 g each. On 15 d after the injection of B16F10 melanoma cells, the tumor diameters reached 6\u0026ndash;9 mm. Subsequently, ~ 50 \u0026micro;L (~\u0026thinsp;20 MBq) of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp radiolabeled nanoparticles was injected intratumorally after diluting in PBS medium. Then, whole body SPECT/CT imaging of the animals were performed at different time points post-injection (p.i.) over a period of two weeks.\u003c/p\u003e\u003cp\u003eFurther, the melanoma tumor has grown in eighteen C57BL/6 mice using the same protocol. The tumor bearing animals were randomized into six groups of 3 mice each and 10 MBq (~\u0026thinsp;25 \u0026micro;L) [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp was administered into each mice by intra-tumoral injection. At the end of six different time points post-injection (3, 24, 48, 72, 168 and 336 h), one group of mice was sacrificed by carbon dioxide asphyxiation. Subsequently, the radioactivity and the weight associated with each organ and blood samples were measured and presented as percentage injected dose per gram (%ID/g). As control experiment, \u003cem\u003eex vivo\u003c/em\u003e biodistribution study was performed in melanoma tumor bearing C57BL/6 mice by intra-tumoral administration of 10 MBq (~\u0026thinsp;25 \u0026micro;L) [\u003csup\u003e169\u003c/sup\u003eYb]YbCl\u003csub\u003e3\u003c/sub\u003e solution (pH\u0026thinsp;~\u0026thinsp;5.0) following same protocol described for [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp formulation.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003eAssessment of therapeutic efficacy by tumor regression study\u003c/h2\u003e\u003cp\u003eTumor regression study was conducted using C57BL/6 mice bearing melanoma tumors with an average initial tumor volume of approximately 150 mm\u0026sup3;. The mice were randomly assigned into five groups (n\u0026thinsp;=\u0026thinsp;3 per group). One group of mice received a single intra-tumoral injection of normal saline (control), while the remaining four groups were administered a single dose of [\u0026sup1;⁶⁹Yb]Yb-GAHAnp at activity levels of 10, 20, 30 and 40 MBq, respectively. Tumor progression and body weight were monitored over a 21 days period post treatment. Tumor volume was estimated using the formula: (length \u0026times; width\u0026sup2;) / 2. The tumor growth index (TGI) at each time point was determined by normalizing tumor volume to its baseline value (day 0). Similarly, the body weight index (BWI) was calculated by dividing the body weight at each time point by the initial body weight.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec21\" class=\"Section2\"\u003e\u003ch2\u003eInvestigation of sorption mechanism of Yb\u003csup\u003e3+\u003c/sup\u003e on the surface of GAHAnp\u003c/h2\u003e\u003cp\u003eUtility of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp in nanoscale brachytherapy necessitates that \u003csup\u003e169\u003c/sup\u003eYb becomes an integral part of HA matrix in the radiolabeled formulation. To establish this, it is imperative to study the sorption mechanism of Yb\u003csup\u003e3+\u003c/sup\u003e on GAHAnp. In this regard, a theoretical investigation was carried out considering unfunctionalized HAnp as sorption matrix and assuming that GA does not influence the sorption of Yb\u003csup\u003e3+\u003c/sup\u003e on GAHAnp matrix. To understand the basic interaction between hydroxyapatite surface and Yb\u003csup\u003e3+\u003c/sup\u003e at the molecular level, plane-wave based spin polarized density functional theory (DFT) calculations are performed using Vienna ab initio simulation package (VASP) (Kresse and Furthm\u0026uuml;ller, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e1996\u003c/span\u003e; Kresse and Joubert, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). The interaction between ions and electrons are described using the projector augmented wave (PAW) potentials (Bl\u0026ouml;chl, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e1994\u003c/span\u003e). The exchange correlation energy is described using the generalised gradient approximation (GGA) as parameterized by Perdew-Burke-Ernzerhof (PBE) (Perdew et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). A 550 eV is taken into consideration as the kinetic energy cut-off for the electronic self-consistent field iterations. Additionally, every structure underwent optimisation until the force tolerance is less than 0.01 eV/\u0026Aring; and the difference value of the total energy is less than 10\u003csup\u003e\u0026minus;\u0026thinsp;5\u003c/sup\u003e eV. The Brillouin-zone integrations are performed using a 2\u0026times;2\u0026times;1 Monkhorst-Pack k-point mesh (Monkhorst and Pack, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e1976\u003c/span\u003e). The dispersion corrections to the total energies are implemented using Grimme's D3 semiempirical approach (PBE-D3) with Becke-Jonson damping (Grimme et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2011\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eTo corroborate the theoretical finding, radiotracer investigations were carried out to establish if the sorption of Yb\u003csup\u003e3+\u003c/sup\u003e on GAHAnp matrix results in the replacement of Ca\u003csup\u003e2+\u003c/sup\u003e using \u003csup\u003e45\u003c/sup\u003eCa-labeled GAHAnp. The experimental details of this study are provided in the \u0026lsquo;Supporting Information\u0026rsquo;. Further, the local geometry of Yb after sorption on GAHAnp was investigated by X-ray absorption fine structure (XAFS) spectroscopy. The detail of this study is provided in the \u0026lsquo;Supporting Information\u0026rsquo;.\u003c/p\u003e\u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGlucuronic acid\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHydroxyapatite\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eGAHAnp\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eGlucuronic acid functionalized hydroxyapatite nanoparticles\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDFT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDensity functional theory\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eSPECT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eSingle photon emission computed tomography\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eCT\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eComputed tomography\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eXAFS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eX-ray absorption fine structure\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eXRD\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eX-ray diffraction\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eFT-IR\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eFourier transform infrared\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTGA\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eThermogravimetric analysis\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eDLS\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eDynamic light scattering\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eTEM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eTransmittance electron microscope\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003eHRTEM\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003eHigh resolution transmittance electron microscope\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv class=\"DefinitionListEntry\"\u003e\u003cdiv class=\"Term\"\u003ePL\u003c/div\u003e\u003cdiv class=\"Description\"\u003e\u003cp\u003ePhotoluminescence\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003cp\u003eAll animal experiments were carried out following relevant guidelines and regulations approved by the institutional Animal Ethics Committee of Bhabha Atomic Research Centre (Reference: BAEC/12/2024).\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003cp\u003eNot applicable.\u003c/p\u003e\u003c/p\u003e\u003cp\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003cp\u003eThere are no conflicts of interest to declare.\u003c/p\u003e\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eOpen access funding provided by Department of Atomic Energy (DAE), Government of India. This research did not receive any specific grant from funding agencies.\u003c/p\u003e\u003ch2\u003eAuthor contributions\u003c/h2\u003e\u003cp\u003e\u003cb\u003eSP\u003c/b\u003e: Data curation, Formal analysis, Writing \u0026ndash; original draft. \u003cb\u003eKS\u003c/b\u003e: Data curation. \u003cb\u003ePSG\u003c/b\u003e: Data curation, Formal analysis. \u003cb\u003eKVV\u003c/b\u003e: Data curation. \u003cb\u003ePB\u003c/b\u003e: Data curation. \u003cb\u003eRK\u003c/b\u003e: Data curation, Formal analysis. \u003cb\u003eCK\u003c/b\u003e: Data curation, Formal analysis. \u003cb\u003eAC\u003c/b\u003e: Data curation, Formal analysis. \u003cb\u003eSG\u003c/b\u003e: Data curation, Formal analysis. \u003cb\u003eDB\u003c/b\u003e: Formal analysis. \u003cb\u003eRC\u003c/b\u003e: Formal analysis, Writing \u0026ndash; review \u0026amp; editing. \u003cb\u003eSC\u003c/b\u003e: Conceptualization, Formal analysis, Supervision, Writing \u0026ndash; review \u0026amp; editing.\u003c/p\u003e\u003ch2\u003eAcknowledgment\u003c/h2\u003e\u003cp\u003eThe authors are grateful to Dr. Tapas Das, Head, Radiopharmaceuticals Division, Dr. Sandip Basu, Head, Radiation Medicine Centre (Medical) and Dr. N.S. Baghel, Radiation Medicine Centre (General), Bhabha Atomic Research Centre (BARC) for their support. The authors also acknowledge the valuable support of the staff of Radiopaharmceuricals Division, BARC who has facilitated irradiation targets in the reactor. The Sophisticated Analytical Instrumentation Facility (SAIF) of Indian Institute of Technology Bombay, Mumbai is acknowledged for the TEM images.\u003c/p\u003e\u003ch2\u003eData availability\u003c/h2\u003e\u003cp\u003eAll data are available upon reasonable request from corresponding author ([email protected])\u003c/p\u003e"},{"header":"References","content":"\u003cp\u003eAlmora-Barrios, N., de Leeuw, N.H., 2010. A density functional theory study of the interaction of collagen peptides with hydroxyapatite surfaces. Langmuir 26, 14535-14542.\u003c/p\u003e\n\u003cp\u003eBarroug, A., Glimcher, M.J., 2002. 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Front Immunol 15, 1537956.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-radiopharmacy-and-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"erpc","sideBox":"Learn more about [EJNMMI Radiopharmacy and Chemistry](http://ejnmmipharmchem.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/erpc/default.aspx","title":"EJNMMI Radiopharmacy and Chemistry","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Nanobrachytherapy, Radiolabeled nanoparticles, GAHAnp, 169Yb, chelator-free radiolabeling, SPECT/CT, tumor regression","lastPublishedDoi":"10.21203/rs.3.rs-8117448/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8117448/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eThe introduction of radiolabeled nanoparticles in the realm of brachytherapy has led to a promising therapeutic strategy for cancer management called \u0026lsquo;nanobrachytherapy\u0026rsquo;. In the quest of developing a potent radiolabeled inorganic biomaterial for use in nanobrachytherapy, we report the synthesis and evaluation \u003csup\u003e169\u003c/sup\u003eYb [T\u003csub\u003e1/2\u003c/sub\u003e = 32.02 d]-labeled glucuronic acid (GA) functionalised hydroxyapatite (HA) nanoparticles (GAHAnp) and established its potency in pre-clinical settings.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003eGAHAnp having average hydrodynamic diameter of 45\u0026thinsp;\u0026plusmn;\u0026thinsp;3 nm was synthesized in house and characterized using various analytical methods. Ytterbium-169 was produced with adequate radionuclidic purity required for medical application by direct neutron activation of isotopically enriched (35.8% in \u003csup\u003e168\u003c/sup\u003eYb) Yb target in research reactor. Radiolabeleing protocol of GAHAnp with \u003csup\u003e169\u003c/sup\u003eYb to obtain [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp in high yield and purity was optimized. Adsorption of [\u003csup\u003e169\u003c/sup\u003eYb]Yb\u003csup\u003e3+\u003c/sup\u003e on GAHAnp followed Langmuir-Freundlich isotherm and pseudo-second order kinetics. The mechanism of incorporation of [\u003csup\u003e169\u003c/sup\u003eYb]Yb\u003csup\u003e+\u0026thinsp;3\u003c/sup\u003e on GAHAnp was investigated using density functional theory (DFT) and experimentally verified by radiotracer investigations and XAFS studies. These investigations suggested replacement of Ca\u003csup\u003e2+\u003c/sup\u003e with Yb\u003csup\u003e3+\u003c/sup\u003e in GAHAnp matrix. The [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp formulation demonstrated excellent \u003cem\u003ein vitro\u003c/em\u003e radiochemical stability in physiological media and cell toxicity in Raaji cells. SPECT/CT imaging and \u003cem\u003eex vivo\u003c/em\u003e biodistribution carried out after intra-tumoral administration of [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp in tumor bearing mice showed near-complete retention of the formulation in the tumor mass upto 2 weeks. Tumor growth could be significantly arrested after administration of 30 MBq dose of the formulation compared to the control.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e\u003cp\u003eDetailed radiochemical and biological investigations reported in this article demonstrate the potential utility of synthesized [\u003csup\u003e169\u003c/sup\u003eYb]Yb-GAHAnp formulation in the treatment of solid tumors through nanobrachytherapy. The formulation exhibited excellent radiochemical stability and significant therapeutic efficacy in pre-clinical models.\u003c/p\u003e","manuscriptTitle":"Synthesis, characterization and evaluation of 169Yb-labeled tailored hydroxyapatite nanospheres for potential application in nanobrachytherapy","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-12-01 08:08:37","doi":"10.21203/rs.3.rs-8117448/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2025-11-24T12:43:49+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-11-24T12:23:56+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-11-24T04:49:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"EJNMMI Radiopharmacy and Chemistry","date":"2025-11-22T12:00:05+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"ejnmmi-radiopharmacy-and-chemistry","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"erpc","sideBox":"Learn more about [EJNMMI Radiopharmacy and Chemistry](http://ejnmmipharmchem.springeropen.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/erpc/default.aspx","title":"EJNMMI Radiopharmacy and Chemistry","twitterHandle":"@officialEANM","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"5f3b0943-922a-4578-a516-d7f6915014d5","owner":[],"postedDate":"December 1st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-12-18T09:32:17+00:00","versionOfRecord":[],"versionCreatedAt":"2025-12-01 08:08:37","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8117448","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8117448","identity":"rs-8117448","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}