Development of Nanocapsule Vehicles for Targeted-Delivery
of Therapeutic Agents, 01-9304

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Principal Investigator
(Joe McDonough)
Neal K. Vail

Inclusive Dates: 04/01/02 - 09/30/03

Background - One of the more challenging public health issues today is maintaining skeletal health. Osteoporosis is a major public health problem associated with significant morbidity and mortality. Adult women can undergo accelerated bone loss beginning at menopause, resulting in the loss of 30 to 40 percent of maximum bone mass in the five years following menopause and a marked increase in the occurrence of debilitating fractures. Similarly, maintaining skeletal health during long-term space flight is a major concern. Astronauts typically experience a 1- to 2-percent decrease in bone volume per month at selected skeletal sites, suggesting a total loss of more than 50 percent during a typical interplanetary mission. Such skeletal degradation can dramatically affect the ability to perform both rudimentary tasks and critical extravehicular activities during long-term space missions. Furthermore, this bone loss is not fully recovered on return to Earth.

The pathogenesis of both osteoporosis and space-related bone loss is complex, multifactorial, and not well understood. However, the explanation must certainly lie in an absolute or relative diminution in the level of osteoblastic bone-forming activity when compared with osteoclastic bone-resorbing activity. In osteoporosis, bone resorption remains essentially constant, while bone formation decreases slightly, leading to an overall loss of bone. In space, bone formation stops completely.

Although there is no cure for bone loss, several approaches prevent or slow its progress. Adequate calcium, vitamin D, appropriate exercise, and certain medications may aid in maintaining bone health. Currently, estrogens, a few bisphosphonates, and raloxifene are FDA approved for the prevention and treatment of postmenopausal osteoporosis. Calcitonin is approved for treatment only. Each of these medications is classified as an anti-resorptive medication, since each affects only the resorptive component of the bone-remodeling cycle. This means there is considerable opportunity to approach bone mass maintenance from the perspective of maintaining normal bone homeostasis rather than stopping already occurring bone loss.

Approach - The goal of this research is to develop nanocapsules designed to target the systemic skeleton and demonstrate that these targeting nanocapsules selectively bind to specific components of the system skeleton. The concept is illustrated in Figures 1a-c. The specific aims of the research are to:

  1. Develop small unilamellar liposomes as model targeted-delivery nanocapsules;
  2. Demonstrate encapsulation of model compounds and determine their in vitro release kinetics;
  3. Demonstrate the in vitro targeting and adherence capabilities of targeted-delivery nanocapsule vehicles;
  4. Enhance provisional patent applications.

Skeletally targeted therapies have significant opportunity in the areas of osteoporosis prevention, cartilage repair, cancer treatment, fracture repair, and tissue engineering. Furthermore, targeted nanocapsule delivery vehicles have broad application in new drug therapies because they limit systemic exposure to active agents, increase agent efficacy at local sites, and introduce opportunities for triggered release of active agents in response to external signals, administered co-factors, or local metabolic signals.

Accomplishments - Liposomes were selected as model vehicles because of their available chemistries for modification and the established methods for their performance characterization both in vitro and in vivo. A lipid formulation was developed, and an extrusion method was used to prepare liposomes with typical particle diameters of approximately 125 nanometers. A model protein, bovine serum albumin, was encapsulated with the liposome formulation, resulting in an encapsulation efficiency of nearly 25 percent.

Two different compounds were selected as bone-targeting ligands based on their known affinity for hydroxyapatite, the major constituent of the calcified matrix of bone. The first ligand was methylene bisphosphonate, a nontherapeutic compound commonly used in bone imaging. The other ligand was an aspartic acid oligomer of length 4 - 6. The ligands were synthesized and characterized. Fluorescent-labeled ligands were shown to preferentially adsorb to various hydroxyapatite substrates in vitro and were shown to do so as a function of available surface area (Figure 2). Ligand-containing liposomes were prepared and were shown to preferentially adsorb to hydroxyapatite substrates in vitro (Figure 3). The ligands were also formulated into micelles, which were shown to encapsulate a lipophilic bone anabolic agent. Continued work is focused on demonstrating efficacy of the technology in vivo.

Fig 1a. Bone-targeting nanocarriers are comprised of targeting ligands (red cylinders protruding from carrier surface), a membrane, and a payload. The targeting ligands are chosen to selectively bind to specific sites within the systemic skeleton, such as the mineral phase. Fig 1b. The bone-targeting nanocarriers preferentially attach to the bone matrix via the surface-bound targeting ligands. Fig 1c. The nanocarrier payload is released following targeted site attachment. Release may occur by natural nanocarrier degradation, application of external stimuli, administration of a complementary factor in schedule, or in response to local biochemical signals.
Fig 2. Surface area normalized adsorption of FITC-labeled ligands onto various hydroxyapatite substrates. Fig 3. Absolute adsorption of methylene bisphosphonate-containing liposomes onto various hydroxyapatite substrates.

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