Formation of protein corona in vivo affects drug release from temperature-sensitive liposomes.

Thermally triggered drug release from temperature-sensitive liposomes (TSL) holds great promise for cancer therapy. Different types of TSL have been designed recently for heat triggered drug release inside tumor blood vessels or after accumulation into the tumor interstitium. However, justification of drug release profiles is for far mainly based on in vitro release data. While these methods could be good enough to give early indication about the thermal sensitivity of TSL, they are still far from being optimum. This is because these methods do not take into consideration the actual adsorption of proteins onto the surface of TSL after their in vivo administration, also known as "protein corona" and the influence this could have on drug release. Therefore, in this study we compared thermal triggered drug release profile of two different types of doxorubicin encapsulated TSL; namely the lysolipid-containing TSL (LTSL) and traditional TSL (TTSL) after their in vivo recovery from the blood circulation of CD-1 mice. Ex vivo release profile at 42 °C was then tested either in the presence of full plasma or after removal of unbound plasma proteins (i.e. protein corona coated TSL). Our data showed that the influence of the environment on drug release profile was very much dependent on the type of TSL. LTSL release profile was consistently characterized by ultrafast drug release independent on the conditions tested. On the contrary, TTSL release profile changed significantly. Doxorubicin release from in vivo recovered TTSL was slow and incomplete in the presence of unbound plasma proteins, whereas very rapid drug release was detected from in vivo recovered and purified protein corona-coated TTSL in the absence of unbound proteins. Using mass spectrometry and quantification of protein adsorption, we confirmed that this discrepancy is due to the changes in protein adsorption onto TTSL when heated in the presence of unbound proteins leading to reduction in drug release. In summary this study showed that the formation of the in vivo corona on TSL will have a dramatic impact on their release profile and is dependent on both their lipid composition and the protein content of the environment in which drug release is triggered.

Activation of hepatic stellate cell in Pten null liver injury model.

BACKGROUND: Hepatic fibrosis is a prominent pathological feature associated with chronic liver disease including non-alcoholic hepatosteatosis (NASH), and a precursor for liver cancer development. We previously reported that PTEN loss in the liver, which leads to hyperactivated liver insulin signaling results in NASH development. Here we used the same mouse model to study the progression from steatosis to fibrosis. RESULTS: The Pten null livers develop progressive liver fibrosis as indicated by Sirius Red staining and increased expression of collagen I, Timp 1, SMAα, and p75NTR. Consistently, hepatic stellate cells (HSCs) isolated from Pten null livers are readily activated when compared with that from mice with intact PTEN. Deletion of AKT2, the downstream target of PTEN signal, blocked NASH development, and alleviated fibrosis. HSCs from the Pten/Akt2 double null mice are quiescent like those isolated from the control livers. Our analysis shows that the activation of HSCs does not depend on the intrinsic signals regulated by PI3K/AKT, the target of PTEN, but does depend on steatosis and injury to the liver. During the progression of liver fibrosis in the Pten null model, Wnt ligands and signaling receptor are induced, concurrent with the reduction of sFRP5, a Wnt antagonist. We showed that treatment of HSCs with Wnt receptor antagonist blocks the observed morphological changes when HSCs undergo activation in culture. This signal appears to be mediated by ß-catenin, as manipulating ß-catenin signaling alters marker gene expressions of HSC activation. CONCLUSIONS: Wnt/ß-catenin activation serves as an important mediator for fibrosis development resulting from NASH using a mouse model where NASH is mimicked by PTEN loss.

Temporal and spatial evolution of therapy-induced tumor apoptosis detected by caspase-3-selective molecular imaging.

PURPOSE: Induction of apoptosis in tumors is considered a desired goal of anticancer therapy. We investigated whether the dynamic temporal and spatial evolution of apoptosis in response to cytotoxic and mechanism-based therapeutics could be detected noninvasively by the caspase-3 radiotracer [(18)F]ICMT-11 and positron emission tomography (PET). EXPERIMENTAL DESIGN: The effects of a single dose of the alkylating agent cyclophosphamide (CPA or 4-hydroperoxycyclophosphamide), or the mechanism-based small molecule SMAC mimetic birinapant on caspase-3 activation was assessed in vitro and by [(18)F]ICMT-11-PET in mice bearing 38C13 B-cell lymphoma, HCT116 colon carcinoma, or MDA-MB-231 breast adenocarcinoma tumors. Ex vivo analysis of caspase-3 was compared to the in vivo PET imaging data. RESULTS: Drug treatment increased the mean [(18)F]ICMT-11 tumor uptake with a peak at 24 hours for CPA (40 mg/kg; AUC40-60: 8.04 ± 1.33 and 16.05 ± 3.35 %ID/mL × min at baseline and 24 hours, respectively) and 6 hours for birinapant (15 mg/kg; AUC40-60: 20.29 ± 0.82 and 31.07 ± 5.66 %ID/mL × min, at baseline and 6 hours, respectively). Voxel-based spatiotemporal analysis of tumor-intrinsic heterogeneity suggested that discrete pockets of caspase-3 activation could be detected by [(18)F]ICMT-11. Increased tumor [(18)F]ICMT-11 uptake was associated with caspase-3 activation measured ex vivo, and early radiotracer uptake predicted apoptosis, distinct from the glucose metabolism with [(18)F]fluorodeoxyglucose-PET, which depicted continuous loss of cell viability. CONCLUSION: The proapoptotic effects of CPA and birinapant resulted in a time-dependent increase in [(18)F]ICMT-11 uptake detected by PET. [(18)F]ICMT-11-PET holds promise as a noninvasive pharmacodynamic biomarker of caspase-3-associated apoptosis in tumors.