Osteoarthritis is a degenerative disease of progressive joint damage in which the shock-absorbing cartilage that creates a friction-reducing barrier between joints becomes damaged and wears away. A disease for which there is no cure, nonsurgical osteoarthritis treatment often entails attempting to help ease patients’ discomfort and other symptoms. Still, treatment rarely provides complete relief. The toxicity profile of oral medications and poor penetration to joint tissue limit their benefits. Localized pharmaceutical treatments injected directly into the joint capsule are often limited by efficacy and rapid clearance of the drug from the active site.

Now researchers have found that, by manipulating/shielding the cationic charge of a drug delivery system containing an insulin-like growth factor-1 (IGF-1) to enhance IGF-1 and IGF-1 receptor interaction, both drug delivery and the ability to stimulate cartilage tissue to produce new, healthy cartilage cells improve. This study is reported in a recent issue of Science Translational Medicine.

Tissue regeneration is particularly challenging with damaged cartilage because it is avascular, lacking blood vessels that can supply the tissue with fresh nutrients to support repair and new cell turnover.

“Once you've passed maturity, the cartilage no longer has blood vessels connecting it directly to circulation, so all the nutrients have to come through the joint fluid,” says Brett Geiger, a graduate student in biological engineering at the Massachusetts Institute of Technology (MIT) and the study’s lead author. “Cartilage in adults is quiescent, intended only to maintain homeostasis, so its ability to heal itself is quite limited. You have to put something in the cartilage to heal it.”

The research group chose to use IGF-1 as the therapeutic agent to drive tissue regeneration. IGF-1 is a growth factor that exhibits anabolic, or tissue-building, behavior by encouraging the growth and survival of cartilage cells while encouraging the biosynthesis of the cartilage matrix. However, articular cartilage is densely comprised of proteoglycan—a hybrid sugar-protein molecule that bears a negative charge. To overcome the potential for poor interaction, researchers needed to find a vehicle small enough to penetrate the cartilage matrix while carrying a charge that would essentially allow the drug moiety to “stick” to the anionic proteoglycan. The researchers selected the densely cationic polyamidoamine (PAMAM) dendrimer, generation (Gen) 4 to Gen 6 due to its ideal size, measuring from 45 Å to 67 Å, so that the entire molecule averages 4-6 nm in diameter.

While one might expect the cationic nature of the PAMAM dendrimer to increase the interaction between the drug moiety and the anionic proteoglycan residues within the cartilage, a molecule with overwhelmingly positive charges could potentially become toxic by stabilizing defects in the anionic cellular membrane, leading to membrane perforation and cell death. Investigators hypothesized that partially shielding the positive charge of the molecule while still controlling its size might allow the molecule to navigate through the cartilage matrix to interact with the target site (IGF-1 receptors) without compromising the molecule’s bioactivity and allowing the molecule to unbind from the target site eventually.

To test this theory, the researchers attached a polyethylene glycol (PEG)-based polymer, that is, PEGylated 20-60% of the PAMAM’s end groups to shield the dendrimer’s surface charges both through covalent modification and steric hindrance. Nonreactive primary amine end groups retained and contributed to the molecules’ positive charge. The end result was the formation of a dendrimer-bioconjugated IGF-1 that did not denature the IGF-1 protein. The conjugate exhibited the induction of sulfated proteoglycan synthesis in bovine, or cow-derived, cartilage explants.

After examining the tissue of rats injected intra-articularly (or within the joint) with fluorescent IGF-1 and dendrimer IGF-1 formulations 2 to 6 days post-injection, the investigators made the following observations: Femoral cartilage—which is at the femoral joint head in the knee—still contained IGF-1 2 days after injection but not at 6 days. At 6 days, dendrimer-IGF-1 still remained in the tissue. Additionally, the less charged of the two dendrimer-IGF-1 conjugates, Gen 4 35% PEG-IGF-1, had diffused throughout the tissue with greater uniformity, but the more heavily charged Gen 6 45% PEG-IGF-1 compound displayed a concentrated gradient spanning the thickness of the cartilage. Gen 4 35% PEG-IGF-1 exhibited an intra-articular half-life of about 1 day and Gen 6 45% PEG-IGF-1 of about 4 days, 2.5 and 10 times that of free IGF-1, respectively. 

The research team also conducted experiments to evaluate the potential for tissue damage by injecting two formulations of PEGylated dendrimers into bovine cartilage tissue disks ex vivo and rats in vivo. Upon examining dendrimer-treated tissues, including those in rats euthanized 2 months post-injection, the researchers found that the joints exhibited normal histology and they found no evidence of histotoxic activity.

According to the researchers, their findings lay the groundwork in improving the development of new joint-nourishing biologics that may one day give people who have osteoarthritis better and safer treatment options.

“After PEGylation, our optimal system has 120+ positive charges on a single molecule, so we’re able to modulate a charge you can’t reproduce using a number of other systems,” says Paula T. Hammond, department head of chemical engineering at MIT and the study’s corresponding author. “Because we can use PEG to help modulate the charge, then we have a perfect way of balancing [therapeutic activity] and toxicity.”

Read the abstract in Science Translational Medicine.