(578f) Nanoparticles for the Treatment of Osteoporosis

I.  INTRODUCTION

Although osteoporosis has been studied for a number of years, no current effective prevention and treatment methods exist for this disease.  There are several major barriers that exist for the use of any pharmaceutical agents to stimulate new bone formation. First, the agents can cause non-specific bone formation in areas not desirable. This is because these agents are often delivered in non-specific ways (such as through the mouth, directly into the blood stream, etc.). Second, if delivered locally to the tissue around the area of low bone density, they rapidly diffuse to adjacent tissues which limit their potential to promote prolonged bone formation in targeted areas of weak osteoporotic bone. It is because of these limitations that even the best strategies to sufficiently increase bone mass (although, to date, still unproven) require at least one year to see any change; a time period not acceptable especially for the elderly. For these reasons, we will use nanotechnology (or the design of materials with 10^-9 m dimensions) to develop novel drug-carrying systems that will specifically attach to osteoporotic (not healthy) bone.  Moreover, some of these novel drug carrying systems will then distribute pharmaceutical agents locally to quickly increase bone mass.

On the other hand, based on their unique mesoscopic physical, chemical, thermal, and mechanical properties, nanoparticles offer a high potential for several biomedical applications, including bioanalysis and bioseparation, tissue specific drug therapeutic applications, gene and radionuclide delivery [1]. In order to be used effectively in fighting diseases, specific surface chemistry of the nanoparticles need to be tailored for their desired biomedical applications. Magnetic nanoparticles are also of interest. Specifically, the main interest for the use of magnetic nanoparticles in biomedical applications is that an inhomogeneous external magnetic field exerts a force on them, and thus they can be manipulated or transported to a specific diseased tissue by a magnetic field gradient. They also have controllable sizes, so that their dimensions can match either that of a virus (20?500 nm), of a protein (5?50 nm) or of a gene (2nm wide and 10?100 nm long).  In addition, magnetic particles are of interest because they do not retain any magnetism after removal of the magnetic field.

Specifically in this study, efforts will focus on the prolonged release of bioactive agents to efficiently regenerate enough bone for a patient to return to a normal active lifestyle.  Specifically, inorganic biodegradable nanoparticles (including ceramics like hydroxyapatite) will be functionalized with bioactive chemicals such as bone morphogenetic protein-2 (BMP-2) that bond to bone of low mass. Such bioactive groups will be placed on the outer surface of the magnetic nanoparticle systems using various techniques (such as covalent chemical attachment). After bonding specifically to osteoporotic bone and not healthy bone, magnetic nanoparticle systems will deliver bioactive compounds to locally increase bone mass.  Lastly, the outer coating of the embedded nanoparticle systems will be created to have different biodegradation rates for the release of bone-building agents over various time spans; this will allow for not only quick bone formation but also long-term sustained bone regeneration.  One potential advantage of formulating HA magnetic nanoparticles is that as the magnetic particles accumulate, e.g., in bone tissue, they can play an important role in detection through MRI to locate, monitor and control drug activities.

II. EXPERIMENTAL METHODS

HA is chemically similar to the mineral component of bones and hard tissues in mammals. It is one of few materials that are classified as bioactive, meaning that it will support bone ingrowth and osseointegration when used in orthopaedic, dental and maxillofacial applications.  This is because bone itself is composed of hydroxyapatite and other calcium phosphates.  When loaded with bioactive compounds, such systems can also release the drug at therapeutic concentrations directly to the needed area.  The implant of this type is capable of releasing the bioactive agents over the entire period of resorption. To achieve a fast resorption rate, amorphous calcium phosphate and nanocrystalline HA drug delivery carriers are excellent candidates.

A. Chemical Synthesis of Hydroxyapatite (HA) with Different Particle Morphologies  

HA powders can be synthesized via numerous production routes, using a range of different reactants.  Some processing techniques include: wet chemical methods (precipitation), hydrothermal techniques, sol-gel, and hydrolysis of other calcium phosphates.  We synthesized HA nanoparticles by a wet chemical process followed by hydrothermal treatment [2].  This method used for producing HA involves calcium nitrate, diammonium hydrogen phosphate and ammonium hydroxide. This method results in a faster production rate, with ammonium hydroxide being added to maintain a constant pH.  This precipitated HA is then processed hydrothermally at 200°C for 20 h.  High crystallization is achieved at relatively low temperatures but under a higher pressure than atmospheric.   As a result, nano-sized HA can be obtained.  

B. Synthesis of Magnetic Nanoparticles (Magnetite) Dispersion (Fe₃O₄)  

The wet chemical routes to synthesize magnetite nanoparticles are simpler, more tractable and more efficient with appreciable control over size, composition and sometimes even the shape of the nanoparticles.  The control of size, shape and composition of nanoparticles depends on the type of salts used (e.g. chlorides, sulphates, nitrates, perchlorates, etc.), Fe²⁺ and Fe³⁺ ratio, pH and ionic strength of the media.  Conventionally, magnetite is prepared by adding a base to an aqueous mixture of Fe²⁺ and Fe³⁺ chloride at a 1:2 molar ratio. Variation in reaction conditions critically affects the physical and chemical properties of the nanosized magnetite.  To control the reaction kinetics, which is strongly related with the oxidation speed of iron species, the synthesis of particles must be done in an oxygen-free environment by passing N₂ gas. For the HA coated magnetic nanoparticles, the pH of magnetite (Fe₃O₄) nanoparticle dispersions was adjusted to 10 by adding NH₄OH followed by the addition of diammonium hydrogen phosphate and calcium nitrate.

C. Functionalization of Aminophase Molecules on Nanomaterials

For the attachment of bioactive compounds, we will use silane chemistry [3].  Alkoxysilanes were chosen in this study because of their ability to graft to hydroxyl terminated surfaces of HA.  It is expected that the surface energy of biomaterials can be controlled using the attachment site (e.g., silane), spacer (e.g., alkane), and end group (e.g., amine).  It is important to note that although aminophase chemistry has been used for glass, little to no reports are in the literature for its use on HA. 

III. EXPERIMENTAL RESULTS

A. HA coated Magnetic Nanomaterial Synthesis

Material properties of the nano sized crystalline HA produced were determined by X-ray diffraction, inductively coupled plasma-atomic emission spectroscopy (ICP-AES) to measure Ca/P ratio, particle size analyzer to measure mean particle size, BET to measure grain size and TEM to analyze HA particle morphology.  We have also synthesized amorphous calcium phosphate of increased degradability with nano particulate sizes.  By using highly degradable amorphous calcium phosphate and slowly degradable crystalline hydroxyapatite, we have materials that can provide for a wide range of drug release profiles. We have also successfully prepared magnetic HA nanoparticles.  

B. Aminophase Functionalization of HA Coated Magnetic Nanoparticles and Characterization Using Novel CBQCA Test

HA-aminophase functionalization was characterized by a novel CBQCA method [4].  Inherently CBQCA (3-(4-Carboxybenzoyl)quinoline-2-carboxaldehyde) is a non-fluorescence molecule and upon reaction with amine groups in the presence of cyanide or thiols, it fluoresces well. This observation is highly precise based on our earlier experimental observations with similar experiments. Through this technique we have shown that all crystalline HA and amorphous calcium phosphate nanoparticles were functionalized with various bioactive groups that can rebuild osteoporotic bone. 

IV. CONCLUSIONS

Overall the results of this study indicate our ability: (i) to synthesize HA coated magnetic nanoparticles and calcium phosphate nanoparticles, (ii) to further chemically modify these particles using aminosilane chemistry, (iii) and to attach drug molecules, particularly BMP-2, that will allow these nanoparticles once directed to osteoporotic bone to rebuild bone mass. Next steps in this research are to determine attachment efficiency to osteoporotic compared to healthy bone, imbed bone building agents into the nanoparticles, determine release profiles once attached to weak bone, and determine bone regeneration.

V. ACKNOWLEDGMENTS

The authors acknowledge support for this work provided by the Showalter grant as well as the assistance of Dr. Alex Wei and Ms. Jie Liu (Department of Chemistry, Purdue University).

VI. REFERENCES

[1] A.K.Gupta, M.Gupta, ?Synthesis and surface engineering of iron oxide nanoparticles for biomedical applications,? Biomaterials, vol.26, pp.3995-4021, 2005.

[2] T.J Webster, R.W Siegel, R.Bizios, ?Enhanced functions of osteoblasts on nanophase ceramics,? Biomaterials, vol.21, pp.1803-1810, 2000.

[3] R.B Danczyk, A.Krieder, T.J.Webster, H HogenEsch, A Rundell, ?Comparison of antibody functionality using different immobilization methods,? Biotechnology and Bioengineering, vol.84, pp.215-223, 2003.

 [4] J.Liu, O.Shirota, M.Novotny, ?Capillary electrophoresis of amino sugars with laser-induced fluorescence detection,? Anal.Chem., vol.63, pp.414-417, 1991.