Hepatic injury and fibrosis develops in response to a wide array of etiologies such as viral infection, toxin exposure, alcohol abuse and metabolic diseases.59
Persistent injury leads to inflammation, fibrosis and compensatory hepatocyte hyperplasia usually cumulating in cirrhosis. Any treatment that can prevent hepatocellular injury, inflammation or fibrosis is a potential therapeutic for preventing cirrhosis in the setting of chronic liver disease. Chronic liver disease is the third leading cause for hospital visits in the United States, resulting in 5.9 million visits in 2004 and nearly 30 000 annual deaths.60
Given this considerable disease burden, there is intense interest in developing therapies aimed at chronic liver disease.
Plant-derived polyphenols are increasingly being recognized for their medicinal potential.16
Curcumin is a yellow polyphenol extracted from the rhizome of turmeric (C. longa
), which has been used as a spice, coloring agent and as a therapeutic agent in traditional Indian medicine.3
Curcumin’s promising antineoplastic,4
activities have been explored in animals. In vitro
and in vivo
studies have explored curcumin’s antifibrotic potential in the liver.10–15
However, its promise is plagued by the lack of bioavailable curcumin in liver even following massive oral doses.18
Although curcumin formulated with phosphatidylcholine (Meriva®
) enhances oral absorption and results in detectable intrahepatic curcumin, it is only detectable up to 2 h after dosing.17
The lack of water solubility is the primary problem limiting curcumin’s therapeutic development.
To address the lack of solubility, we developed a biocompatible nanomaterial to provide an opportunity for harnessing the full potential of a potent, yet poorly soluble compounds like curcumin.61–63
We have developed polymeric nanoparticles comprised of NIPAAM, MMA and AA that are capable of solubilizing a broad range of poorly water-soluble drugs. Curcumin encapsulated in these polymeric nanoparticles, NanoCurc™, is fully soluble in aqueous media, and shows essentially no toxicity upon daily systemic administration through the intraperitoneal route in mice.
In this study, we found that NanoCurc™ delivers a substantial concentration of curcumin to the liver. As early as 6 h after multiple doses and as long as 12 h after a signal dose, a significant amount of curcumin is detected in the liver. The curcumin is detectable in hepatocytes isolated from the liver. This might explain why we see a significant reduction in CCl4
-induced hepatocellular injury and production of proinflammatory cytokines (). The exact mechanism by which curcumin induces a protective hepatocellular environment is not clear. The ability of GSH to prevent CCl4
liver injury is limited as the low oxygen tensions in the centrilobular zones limit formation of the CCl3
that GSH could reduce. Another possibility is the inhibition of the CCl3
free radical formation, which is required for CCl4
-mediated liver injury. The formation of the free radical requires cytochrome P
450, which is a known target of curcumin.64–66
Although we see the delivery of curcumin to hepatocytes as overcoming a significant limitation in the therapeutic development of curcumin, the need to deliver curcumin to hepatocytes is not clear. Others studies have shown large daily oral doses of FC can inhibit CCl4
-induced fibrosis in rats.11
However, this delivery route results in detectable curcumin in the gastrointestinal tract, but not in the liver.10
This suggests that curcumin might work through multiple mechanisms.
We also found that NanoCurc™ treatment results in a significant amount of curcumin in the NPC compartment. Although the cellular make-up of this compartment is complex, it does contain pro-fibrotic stellate cells and myofibroblasts. Lipid-laden cells, likely stellate cells, were evident in the NPC fraction (Supplementary Figure 1
, green arrows). This is important, as there is considerable in vitro
evidence that curcumin directly induces stellate cell apoptosis and blocks stellate cell activation.10,67
These previous studies bolster our conclusions as we know that the NanoCurc™ formulation does not alter its biological activity. NanoCurc™ is equally effective as the unencapsulated form at inducing stellate cell apoptosis (). However, this is not entirely surprising as NanoCurc™ bio-effectiveness is comparable to the free parent compound in a cancer system.26
We also provide in vivo
evidence of NanoCurc™ effectiveness in preventing stellate cell activation as it induces PPAR-γ transcription, an essential mediator of curcumin’s effects on eliminating stellate cells.15
As our data show that NanoCurc™ protects the liver from injury, it could be argued that NanoCurc™ is more hepatoprotective than antifibrotic. However, it is likely that NanoCurc™ is working at multiple steps in the development of fibrosis. First, as already mentioned, NanoCurc™ prevents hepatocellular injury. However, this is not a complete suppression of injury. There is slight but measurable hepatocellular injury in NanoCurc™-treated mice as evidence by the increase in serum ALT and intrahepatic TNF-α compared with untreated mice (). The same is true for the production of the potent pro-fibrotic cytokine TGF-β (). We also present evidence of effects of curcumins on pro-fibrotic stellate cells through the induction of PPAR-γ (). Collectively, our findings suggest that NanoCurc™ works at multiple steps in the injury and fibrosis pathway. This agent could be particularly useful in preventing the progression of chronic liver disease in patients with active on-going liver injury such as in viral or autoimmune hepatitis as it targets both liver injury and fibrosis mechanisms.
Hepatic inflammation is central to many liver diseases.32
Interestingly, our data show that NanoCurc™ has little effect on NKT cells and neutrophils in the liver. There is a comparable decrease in NKT and increase in Gr-1high
cells in void nanoparticle and NanoCurc™-treated mice (Supplementary Figure 2
). This is consistent with what has been reported in wild-type CCl4
This could indicate that a low level of injury is sufficient to affect the immune system or that another important component of the innate immune system, particularly Kupffer cells,68
might be targeted by NanoCurc™. More immediate 24 h studies and analysis of chemokines might be more revealing in dissecting NanoCurc™’s potential role in altering the livers inflammatory milieu.
The results of this study show NanoCurc™ feasibility in treating chronic liver diseases. Being fully water soluble, NanoCurc™ overcomes the most important road block that prevented curcumin’s clinical development, further showing that NanoCurc™ prevents liver injury and fibrosis is a significant developmental step in bringing this natural compound to clinical trials.