Radiolabelled nanohydroxyapatite with 99mTc: perspectives to nanoradiopharmaceuticals construction

Abstract

The use of nanoparticles is under intense investigation. The possible advantages proposed by these systems are very impressive and the results may be quite schemer. In this scenario, the association of nanoparticles with radioactive materials (radionuclide) may be the most important step since the discovery of radioactive for nuclear medicine and radiopharmacy, especially for cancer targeting and therapy. In this study, we developed radiolabelled nanoparticles of hydroxyapatite with technetium 99m for bone cancer imaging. The results demonstrated that it is possible to label nanoparticles of hydroxyapatite, and due to its physicochemical properties is possible to develop nano-radiopharmaceutical for bone imaging.

Keywords::

• Bone cancer
• bioceramics
• bone imaging
• nanoparticles

Introduction

The last decade has brought major advances in cancer treatment and imaging including the development of conformal radiation treatments, robotic and endoscopic surgery and the use of nanoparticles as a drug delivery system. These techniques rely on accurate target delineation and visualization of tumor targets, and require accuracy on the millimeter scale.

Several new therapies and diagnostic kits are in development using innovative methods of delivery besides the current practice of direct introduction of drugs and devices into the tumor. Nanobiotechnology, particularly nanoparticles, is making a significant contribution to the improvement of drug delivery in cancer. The application of nanotechnology to drug delivery is widely expected to change the landscape of pharmaceutical and biotechnology industries for the foreseeable future. The development of nanotechnology products may play an important role in adding a new armamentarium of therapeutics to cancer fighting. Using nanotechnology, it may be possible to achieve (1) improved delivery of poorly water-soluble drugs; (2) targeted delivery of drugs in a cell- or tissue-specific manner; (3) transcytosis of drugs across tight epithelial and endothelial barriers; (4) delivery of large macromolecule drugs to intracellular sites of action; (5) co-delivery of two or more drugs or therapeutic modality for combination therapy; (6) visualization of sites of drug delivery by combining therapeutic agents with imaging modalities; and (7) real-time read on the in vivo efficacy of a therapeutic agent. Recently, various types of nanoprobes have been developed as blood pool CT contrast agents, such as gold nanoprobes and nanotags, iodine-based emulsions and tantalum oxide nanoparticles (Reuvenie et al. 2011, Farokhzad and Langer 2009, Jain 2005, Jain 2007, Sa et al. 2012).

The inorganic biomaterials based on calcium orthophosphate have their wide range of applications in medicine. Among them, synthetic hydroxyapatite (HAP, Ca₁₀(PO₄)₆(OH)₂) is the most promising because of its biocompatibility, bioactivity and osteoconductivity. The calcium apatite phase forming the main mineral part of bones and teeth (Morelli et al. 2011) contains several ions in different amounts substituting calcium and phosphorus in the HA lattice Hydroxyapatite has been used to fill a wide range of bony defects in orthopedic and maxillofacial surgeries and dentistry. Whereas the synthesized hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂, HA) is a pure phase that is a well-established bone replacement material in orthopedics and dentistry. These substitutions provoke changes in the HA surface structure and charge, raising its solubility and increasing the ability of synthetic HA to be involved in a natural bone remodeling process.Thus the importance of silicon on bone formation and calcification was confirmed in different studies. In them, it was shown that a reduction in Si in bone results in a decrease in the number of osteoblasts, osseomatriceal collagen and glycosaminoglycans. Nanophase hydroxyapatite is chemically and structurally similar to natural bone minerals and is biodegradable. Additionally, increased new bone formation has been observed as early as 2 weeks on nano-hydroxyapatite-coated tantalum scaffolds compared with micron-sized hydroxyapatite-coated scaffolds and uncoated scaffolds when implanted into rat calvarial bone. Also, nanoparticles with different material composition, inorganic nanoparticles composed of calcium phosphate have numerous advantages including ease of synthesis, control of physicochemical properties, strong interactions with their payload and biocompatibility. Moreover, hydroxyapatite nanoparticles (HAP) are low crystalline with highly active surfaces and used as carriers in drug delivery systems as well as for protein separation as an absorptive material (Yan-Zhong et al. 2011, Lock and Liu 2011, Ibrahim et al. 2011, Ciobanu et al. 2011, Zhun et al. 2011).

Materials and methods

Hydroxyapatite

Hydroxyapatite was precipitated by dropwise addition of (NH₄)₂HPO₄ aqueous solution containing NH₄OH to a Ca(NO₃)₂ solution at 37°C, and pH equal to 11. The precipitate was separated by filtration, repeatedly washed with deionized boiling water and dried at 100°C for 24 h. The dried powder was manually grounded and the < 210 μm particles were separated by sieving.

Calcium and phosphorous concentrations (Ca/P = 1.66) were determined by X-ray fluorescence spectroscopy. Sample mineral phase and crystallinity were evaluated by X-ray diffractometry (XRD) with CuK radiation at 40 kV and 40 mA. Phosphate species and OH – groups in apatite structure were identified by Fourier transform infrared spectrophotometry (FTIR) in transmission mode from 400 to 4000 cm ⁻¹. Crystallite mean size (τ) along hydroxyapatite (002) and (300) directions was determined by Debye–Sherrer formula, τ = Kλ/β_1/2cosθ, where β_1/2 is the peak line width (values in radians) of the reflection and K = 0.9.

Labelling

The labelling process was done using 150 μL of hydroxyapatite incubated with stannous chloride (SnCl₂) solutions (80 μL/mL) (Sigma-Aldrich) for 20 min at room temperature. This solution was then incubated with 100 μCi (approximately 300 μL) of technetium-99 m for another 10 min in order to label their structures with Tc-99m.

In order to characterize the labelled nanoparticles, thin layer chromatography (TLC) was made using Whatman paper No. 1. The TLC was performed using 2 μl of each labelled sample in acetone (Proquimios) as mobile phase. The radioactivity of the strips was verified in a gamma counter (Packard, Cobra II) as described in Table I.

Table I. Labelling stability of nanoparticles of hydroxyapatite with 99mTc.

	Time in hours
	0	1	3	5	24
Labelling test	99.3
Test I	98.4	98.3	98.1	97.4	93.9
Test II	99.0	97.4	97.3	95.9	91.2
Test III	98.0	96.4	94.6	95.7	91.0
Final average	98.5	97.4	96.7	96.3	92.0

Biodistribution

Biodistribution studies were done using 3 mice for the nanoparticle-labelled sample. The Institutional Review Board and the Animal Ethics Committee approved the study protocol. The labelled samples (3.7 MBq/0.2 mL) were administered by intraocular administration. Counts were acquired for 5 min in a 15% window centred at 140 KeV. The animals were subsequently sacrificed and their organs removed, weighed and the radioactivity uptake was counted in a gamma counter (Packard-Cobra II). The results were expressed as percentage of injected dose per gram of tissue.

Scanning Electron Microscopy

The morphology of hydroxyapatite nanoparticles was examined by Scanning electron microscopy (SEM) (JEOL LSM 5800). The samples were sputter coated with a layer of gold for observation at 10 kV.

Transmission Electron Microscopy

Micrographs were recorded using a JEOL transmission electron microscope (TEM) model JEM-2010 with a LaB6 filament as the electron source, operated at 200 kV. Material samples were mounted on a microgrid carbon polymer, supported on a copper grid, by placing a few droplets of a suspension of the sample in water followed by drying at ambient temperature (Figure 5).

Results and discussion

Before its association with technetium-99m, the nanostructured powder presented an X-ray diffraction pattern of a pure and crystalline hydroxyapatite as shown in Figure 1. The FTIR spectrum, as shown in Figure 2, has typical hydroxyapatite vibration bands of OH⁻ (3571 cm⁻¹ and 630 cm⁻¹) and PO₄³⁻ (1088, 1025 and 963 cm⁻¹) groups.

Figure 1. X-ray diffraction pattern of nanostructured hydroxyapatite.

Figure 2. FTIR spectrum of hydroxyapatite sample showing OH⁻ and PO₄³⁻ bands.

The labelling process showed a percentage of 99.3% of ligation with the 99mTc. This is quite excellent and supports the use of nanohydroxyapatite as nanoradiopharmaceuticals.

The stability of the labelling process using 99mTc was tested for 24 h. This time was chosen considering the necessary time for transportation and subsequent clinical application. The results are expressed in Table I. All the tests were made in triplicate.

The great stability of the nanohydroxyapatite with 99mTc supports its use for clinical application, especially in bone imaging. Further tests are required for bone therapy. However, this primary result showed that it is possible to develop nanoradiopharmaceuticals for bone using nanohydroxyapatite.

The biodistribution (Figures 3 and 4) showed interesting results and can confirm the use of the nanohydroxyapatite as a drug delivery system for bone. First the nanoparticle demonstrated a low hepatic metabolism with a great value in blood pool confirming the high affinity for blood proteins, especially albumin. Second, the nanoparticle showed a great attraction for the kidney and not in terms of clearance, since it is reabsorbed and is still circulating, as can be seen from the percentage in heart.

Figure 3. Biodistribution % dose per organ of radioactivity.

Figure 4. Biodistribution % gram per tissue of radioactivity.

Figure 5. a) Transmission Electron Microscopy (TEM) image of the HA37 sample showing agglomerates of nanoparticles, b) High resolution TEM image (HRTEM) of the framed area with its respective FFT along the hydroxyapatite [1101] zone axis.

The percentage of absorption in lungs is normal and may mean that this use can be widespread for lung cancer targeting. The nanoparticle crossed the brain barrier but in a negligible amount. The nanoparticle also demonstrated low reticulum endothelial attraction which can be confirmed by the low concentration in the intestine. These data together with the data from the low hepatic metabolism showed that the nanoparticle has a sustainable release and is not recognized by the lymphatic system and probably does not activate the immunologic system. We do believe that more studies are required to understand the whole pharmacokinetic behavior of the nanoparticle. However, these findings alone are very interesting and encouraging for the use this nanoparticle has in the design of targeted therapeutic and imaging agents.

Conclusion

The results although preliminary are very interesting. The use of nanohydroxyapatite as nanoradiopharmaceuticals can change the global scenario of oncology and nuclear medicine drastically, especially for bone cancer imaging and treatment.

Declaration of interest

The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.