University of Nebraska Medical Center Omaha, Nebraska, United States
Tessa Liner (University of Nebraska Medical Center)| Chuhan Zhang (University of Nebraska Medical Center)| Bentia McVicker (University of Nebraska Medical Center, University of Nebraska Medical Center)| David Oupicky (University of Nebraska Medical Center)| Robert Bennett (University of Nebraska Medical Center, University of Nebraska Medical Center)
Hepatic fibrosis is a progressive disease which can result in end-stage cirrhosis, a leading cause of death in the US. It is caused by the activation of myofibroblasts (HSC), that produce excess collagen under perpetual injury. Relaxin, a member of the insulin/IGF/relaxin superfamily of peptide hormones, has been shown to play a role in deactivating HSC through its receptor, RXFP1. The deactivation of HSC reduces expression of collagen, therefore slowing the progression of fibrosis.
Significance of Problem
Relaxin has been shown to be antifibrotic in some rodent models of hepatic fibrosis, however it is unstable in circulation and requires long-term continuous treatment. By developing a stabilized transport system, it would improve the efficacy and provide an HSC-specific targeting system through RXFP1.
Relaxin tethered to nanoparticles (NP) will direct the particles to the HSC and activate RXFP1, thereby inducing an antifibrotic response.
NP were prepared from rhodamine-labled poly(lactide-co-glycolide) (PLGA 16kDa)-b-PEG (3.4kDa)-COOH copolymers to produce control NP (C-NP). Relaxin was attached to the carboxylic group by carbodiimide crosslinking (R-NP). The particles were subjected to hydrodynamic analysis to determine their size. Human HEK-293T cells stably expressing RXFP1 (HEK-RXFP1) were used for NP binding. RXFP1 activation in HEK-RXFP1 cells, control cells (HEK), and the hepatic stellate cell line, LX2, which endogenously expresses RXFP1, was determined by cAMP production using a TR-FRET assay. To determine the localization of NP in diseased liver, hepatic fibrosis was induced in mice by injection of carbon tetrachloride for 2 weeks. NP were injected via the tail vein, and 2 hours later the liver and other organs were removed, and fluorescence detected using an IVIS imager. Frozen sections were used to determine colocalization of NP with cell marker proteins by fluorescence immunohistochemistry.
The conjugation of relaxin caused a small increase in particle size. TR-FRET analysis showed R-NP increase cAMP production, indicative of RXFP1 activation. HEK cells showed no response to either NP, however HEK-RXFP1 cells produced a concentration-dependent response to R-NP. Similarly, R-NP induced a dose-dependent cAMP increase in LX2 cells. Contrary to the C-NP, there was increased binding of R-NP to HEK-RXFP1 cells. IVIS imaging showed that both NP homed primarily to the liver, however R-NP had increased homing to kidney and adipose tissue, other sites of RXFP1 expression. Immunohistochemistry revealed that C-NP primarily colocalized with macrophages in the liver, while R-NP had increased association with HSC.
This study showed that covalent attachment of relaxin to NP retains biological activity and allows for HSC targeting both in vitro and in vivo. This proof-of-concept shows that R-NP can be used as both a targeting moiety and antifibrotic agent. Future studies aim to optimize the configurations of the NP and load antifibrotic agents as cargo to be delivered to desired profibrotic cells.