RESUMO
Subcutaneous (SC) injection of protein-based therapeutics is a convenient and clinically established drug delivery method. However, progress is needed to increase the bioavailability. Transport of low molecular weight (Mw) biotherapeutics such as insulin and small molecule contrast agents such as lipiodol has been studied using X-ray computed tomography (CT). This analysis, however, does not translate to the investigation of higher Mw therapeutics, such as monoclonal antibodies (mAbs), due to differences in molecular and formulation properties. In this study, an iodinated fluorescein analog rose bengal (RB) was used as a radiopaque and fluorescent label to track the distribution of bovine serum albumin (BSA) compared against unconjugated RB and sodium iodide (NaI) via CT and confocal microscopy following injection into ex vivo porcine SC tissue. Importantly, the high concentration BSA-RB exhibited viscosities more like that of viscous biologics than the small molecule contrast agents, suggesting that the labeled protein may serve as a more suitable formulation for the investigation of injection plumes. Three-dimensional (3D) renderings of the injection plumes showed that the BSA-RB distribution was markedly different from unconjugated RB and NaI, indicating the need for direct visualization of large protein therapeutics using conjugated tags rather than using small molecule tracers. Whereas this proof-of-concept study shows the novel use of RB as a label for tracking BSA distribution, our experimental approach may be applied to high Mw biologics, including mAbs. These studies could provide crucial information about diffusion in SC tissue and the influence of injection parameters on distribution, transport, and downstream bioavailability.
RESUMO
Therapeutic macromolecules possess properties such as size and electrostatic charge that will dictate their transport through subcutaneous (SC) tissue and ultimate bioavailability and efficacy. To improve therapeutic design, platforms that systematically measure the transport of macromolecules as a function of both drug and tissue properties are needed. We utilize a Transwell chamber with tunable collagen-hyaluronic acid (ColHA) hydrogels as an in vitro model to determine mass transport of macromolecules using non-invasive UV spectroscopy. Increasing hyaluronic acid (HA) concentration from 0 to 2 mg/mL within collagen gels decreases the mass transport of five macromolecules independent of size and charge and results in a maximum decrease in recovery of 23.3% in the case of bovine immunoglobulin G (IgG). However, in a pure 10 mg/mL HA solution, negatively-charged macromolecules bovine serum albumin (BSA), ß-lactoglobulin (BLg), dextran (Dex), and IgG had drastically increased recovery by 20-40% compared to their performance in ColHA matrices. This result was different from the positively-charged macromolecule Lysozyme (Lys), which, despite its small size, showed reduced recovery by 3% in pure HA. These results demonstrate two distinct regimes of mass transport within our tissue model. In the presence of both collagen and HA, increasing HA concentrations decrease mass transport; however, in the absence of collagen, the high negative charge of HA sequesters and increases residence time of positively-charged macromolecules and decreases residence time of negatively-charged macromolecules. Through our approach, ColHA hydrogels serve as a platform for the systematic evaluation of therapeutic macromolecule transport as a function of molecular characteristics.