Bifunctional bisphosphonates for delivering PTH (1-34) to bone mineral with enhanced bioactivity.

The objective of this work was to demonstrate the bioactivity of parathyroid hormone (1-34) (PTH) delivered through a single molecule of bisphosphonate to improve tissue/cell interactions. Bifunctional hydrazine-bisphosphonates (HBPs) with varying length and lipophilicity were used as a drug delivery vehicle. PTH was oxidized with periodate treatment to obtain an N-terminal aldehyde that was then conjugated to HBPs. The toxicity and apoptotic properties of HBPs and HBP-PTH conjugates were studied with macrophages (RAW 264.7). It was found that one of the HBPs had significant apoptotic characteristics similar to alendronate, which is a widely prescribed drug in the treatment of osteoporosis. The improved binding affinity of PTH following conjugation to HBP was determined using a hydroxyapatite binding assay. The amount of PTH delivered to bone through HBPs was not affected by the length or lipophilicity of the HBPs. Furthermore, the improved bioactivity of PTH delivered to bone through HBPs, in comparison to adsorbed PTH, was demonstrated by quantifying the cAMP produced by pre-osteoblastic (MC3T3-E1) cells in response to PTH. The delivery of bioactive PTH to bone tissue by HBP conjugation demonstrates the potential use of HBPs in delivering therapeutic macromolecules to bone for the treatment of several skeletal diseases.

Oriented immobilization of proteins on hydroxyapatite surface using bifunctional bisphosphonates as linkers.

Oriented immobilization of proteins is an important step in creating protein-based functional materials. In this study, a method was developed to orient proteins on hydroxyapatite (HA) surfaces, a widely used bone implant material, to improve protein bioactivity by employing enhanced green fluorescent protein (EGFP) and ß-lactamase as model proteins. These proteins have a serine or threonine at their N-terminus that was oxidized with periodate to obtain a single aldehyde group at the same location, which can be used for the site-specific immobilization of the protein. The HA surface was modified with bifunctional hydrazine bisphosphonates (HBPs) of various length and lipophilicity. The number of functional groups on the HBP-modified HA surface, determined by a 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay, was found to be 2.8 × 10(-5) mol/mg of HA and unaffected by the length of HBPs. The oxidized proteins were immobilized on the HBP-modified HA surface in an oriented manner through formation of a hydrazone bond. The relative protein immobilization amounts through various HBPs were determined by fluorescence and bicinchoninic acid (BCA) assay and showed no significant effect by length and lipophilicity of HBPs. The relative amount of HBP-immobilized EGFP was found to be 10-15 fold that of adsorbed EGFP, whereas the relative amount of ß-lactamase immobilized through HBPs (2, 3, 4, 6, and 7) was not significantly different than adsorbed ß-lactamase. The enzymatic activity of HBP-immobilized ß-lactamase was measured with cefazolin as substrate, and it was found that the catalytic efficiency of HBP-immobilized ß-lactamase improved 2-5 fold over adsorbed ß-lactamase. The results obtained demonstrate the feasibility of our oriented immobilization approach and showed an increased activity of the oriented proteins in comparison with adsorbed proteins on the same hydroxyapatite surface matrix.

Enhanced affinity bifunctional bisphosphonates for targeted delivery of therapeutic agents to bone.

Skeletal diseases have a major impact on the worldwide population and economy. Although several therapeutic agents and treatments are available for addressing bone diseases, they are not being fully utilized because of their uptake in nontargeted sites and related side effects. Active targeting with controlled delivery is an ideal approach for treatment of such diseases. Because bisphosphonates are known to have high affinity to bone and are being widely used in treatment of osteoporosis, they are well-suited for drug targeting to bone. In this study, a targeted delivery of therapeutic agent to resorption sites and wound healing sites of bone was explored. Toward this goal, bifunctional hydrazine-bisphosphonates (HBPs), with spacers of various lengths, were synthesized and studied for their enhanced affinity to bone. Crystal growth inhibition studies showed that these HBPs have high affinity to hydroxyapatite, and HBPs with shorter spacers bind more strongly than alendronate to hydroxyapatite. The HBPs did not affect proliferation of MC3T3-E1 preosteoblasts, did not induce apoptosis, and were not cytotoxic at the concentration range tested (10(-6)-10(-4) M). Furthermore, drugs can be linked to the HBPs through a hydrazone linkage that is cleavable at the low pH of bone resorption and wound healing sites, leading to release of the drug. This was demonstrated using hydroxyapatite as a model material of bone and 4-nitrobenzaldehyde as a model drug. This study suggests that these HBPs could be used for targeted delivery of therapeutic agents to bone.