(357f) Development of Cationic PAMAM Dendrimers As an Avascular Tissue Drug Delivery Platform

Avascular tissues, such as articular cartilage, pose an interesting problem for drug delivery: therapeutics introduced into system circulation do not reach the target tissue at efficacious levels. Furthermore, due to the lack of vascularity, the natural healing of such tissues is hindered by the poor supply of nutrients, making sufficient drug delivery of the utmost importance for tissue damage and tissue diseases. For example, osteoarthritis is a debilitating joint disease that degrades a patient’s articular cartilage until a large lesion forms in the otherwise smooth surface, causing pain, stiffness, and immobility. Currently, there are no disease modifying osteoarthritis drugs (DMOADS) that are capable of slowing the progression of the disease. Potential DMOADs exist, but local injection of these therapies result in quick elimination from the joint space by synovial fluid turnover without efficacious interaction with the dense, negatively charged matrix. As a result, a number of drug delivery formulations have been explored for sustained delivery of osteoarthritic therapies.

Previously, positively charged, multivalent carriers less than 15 nm in size have been shown to utilize electrostatic interactions to bind to and penetrate through articular cartilage faster than they can be cleared from the joint space. Among those studied are poly(amido amine) dendrimers (PAMAM) which consist of a hierarchically branched polymer with a high surface density of cationic primary amines, ideal for electrostatic-based drug delivery and carrier covalent modification. The cytotoxicity of the polymer’s high charge density can be mitigated by covalently conjugating poly(ethylene glycol) (PEG) to its surface. It has been hypothesized and tested computationally that PEG chains bound to the surface of PAMAM are capable of forming hydrogens bonds with the peripheral primary amines of the dendrimer, reducing the effective charge of the dendrimers beyond a single charge per covalently bound PEG chain, but this has not been experimentally demonstrated. To explore this, a novel salt-based method was developed and used to quantify the number of PAMAM primary amines accessible to the physiological environment for a library of PEG-PAMAM conjugates consisting of two dendrimer generations, five PEG chain lengths, and a wide range of PEG grafting densities. From these data, conclusions were drawn about the extent to which PEG non-covalently interacts with the PAMAM surface. It was found that longer PEG chains form a greater number of hydrogen bonds with PAMAM primary amines, whereas shorter chains form very few. Longer PEG chains contain a greater number of oxygen atoms, which act as proton acceptors and lead to more hydrogen bonds. Additionally, the greatest number of hydrogen bonds between individual PEG chains and PAMAM primary amines was found at low grafting densities, which then decreased as grafting density increased. This finding is attributed to the steric repulsion between PEG chains as grafting density increases, causing PEG chains to elongate and protrude away from the surface.

Though literature shows that dendrimer-bound PEG oligomers enhance tissue penetration while reducing overall binding and cytotoxicity, the role that the PEG corona plays in these interactions has not been explored. With a greater understanding of the PEG corona of PEG-PAMAM conjugates, a thorough investigation into the effects the corona has on drug delivery properties was warranted. When tested in an ex vivo bovine cartilage model, the dendrimer-cartilage interactions were found to be solely dependent on the PEG chain length and conjugate’s number of accessible charged amines. Cartilage uptake, which takes into account all binding events between dendrimer and cartilage including absorption and adsorption, and chondrocyte cytotoxicity both increase with increasing accessible charged amines, independent of PEG chain length. The kinetics of tissue uptake is enhanced by higher accessible charged amines and shorter PEG chain lengths, whereas diffusion through cartilage is hindered. From these data, it is determined that cartilage uptake and diffusion is dictated by the strength and reversibility of dendrimer-cartilage interactions. PEG-PAMAM conjugates with a large number of accessible charged amines have high cartilage binding strengths, enhancing cartilage uptake but hindering diffusion. Longer PEG chains disrupt the electrostatic interactions between dendrimer and cartilage, introducing reversibility to the dendrimer-cartilage binding, and allowing for greater diffusion of conjugates through cartilage but reduced uptake. When formulations with substantial cartilage diffusion were tested in a rat model, it was found that conjugates with a greater number of accessible charged amines and shorter PEG chains exhibited greater pharmacokinetics, increasing rat joint residence times from three days for free drug, to well over 30 days. These data suggest that, in order to overcome barriers associated with local delivery of osteoarthritis therapies, sufficient cartilage binding strength is necessary for rapid uptake into cartilage. However, reversibility of electrostatic binding is necessary for diffusion through cartilage.

Finally, to test whether therapeutically relevant biologics can be covalently loaded onto the dendrimers, a bioconjugation protocol utilizing dibenzocyclooctyne (DBCO)-azide click chemistry was developed and used to load both anti-catabolic interleukin-1 receptor antagonist (IL-1RA) and anabolic insulin-like growth factor 1 onto PEG-PAMAM nanocarriers with a protein-to-dendrimer ratio ranging from 1.2 to 2.5. Both of these biologics have been identified as potential DMOADs. Conservation of protein bioactivity was confirmed using both in vitro and ex vivo methods. When intra-articularly injected into the joints of healthy rats, free drugs were retained within the joint space for only three days, whereas the dendrimer-bound proteins were retained for weeks. This finding confirmed that the cationic PEG-PAMAM conjugates increase retention time within the joint space for a variety of biologics.

With a greater understanding of the PEG corona, including its influence on PEG-PAMAM’s electrostatic-based drug delivery to cartilage, researchers can tune the drug delivery properties to fit various delivery profiles. For biologic delivery, a more prolonged joint residence time may be optimal for sustained delivery of the dendrimer-bound protein, whereas specific diffusion profiles or uptake kinetics might be optimal for small molecule therapies that must be cleaved from the dendrimer. Furthermore, these findings may be translatable to drug delivery to other avascular tissues, such as intervertebral discs or cornea.

Research Interests: drug delivery, assay development, nanoparticles, materials development, polymer synthesis, polymer engineering, self assembly