Engineering HepG2 spheroids with injectable fiber fragments as predictable models for drug metabolism and tumor infiltration.

In vitro cell and tissue models are playing essential roles in the identification of active pharmaceutical ingredients. Though HepG2 cells have attractive profiles over primary hepatocytes in the availability and viability retention, the expression of metabolizing enzymes is quite low. In the current study, three-dimensional (3D) HepG2 spheroids with smaller sizes of 150 µm (3Ds) and bigger sizes of 300 µm (3Db) are engineered using injectable fiber fragments as the substrate. In contrast to two-dimensional (2D) culture, the enzyme activities for drug metabolisms are restored in 3Ds and the pathophysiological profiles of tumor tissues are rebuilt in 3Db spheroids. Compared with spheroid culture without fiber fragments, 3Ds spheroids show higher activities of metabolizing enzymes (CYP3A4, CYP2A9, and phase II) and higher sensitivities to enzyme inducers (rifampicin and glutathione) and inhibitors (ketoconazole and probenecid). The drug clearance and toxicity to 3Ds spheroids predict better the clinical observations and drug-drug interactions. In addition, compared to scaffold-free spheroid culture, stronger expressions of E-cadherin and hypoxia-inducible factor-1α (HIF-1α) and higher fibronectin secretions are determined in 3Db spheroids, displaying apparent hypoxic and apoptotic regions similar to those found in solid tumors. In contrast to the overestimated drug toxicity in other systems, the infiltrations of free drug and drug-loaded micelles are apparently restricted in 3Db spheroids, exhibiting drug resistance just like in tumor tissues. Thus, this study demonstrates HepG2 spheroids with different sizes as predictable and physiologically relevant models for high-throughput screening of drug metabolism and tumor infiltration.

Glucose and lipid metabolism screening models of hepatocyte spheroids after culture with injectable fiber fragments.

With the rise of obesity, diabetes, and other metabolic diseases, in vitro hepatic cell and tissue models play an essential role in the identification of active pharmaceutical ingredients. Up to now, three-dimensional (3D) culture models have rarely focused on hepatic glucose and lipid metabolism. In addition, primary human liver cells suffer from limited availability and interdonor difference for establishing reproducible models. Thus, in the current study, the most available human liver cancer cell line (HepG2) and primary hepatocytes from rats (rPH) were proposed to construct 3D spheroids using injectable fiber fragments with galactose grafts (gSF) as the substrate. rPH and HepG2 spheroids show strong cell-cell and cell-fiber fragment interactions to promote the cell viability, albumin, and urea syntheses. Compared with HepG2 spheroids, rPH spheroids indicate stronger glucose metabolism abilities in terms of glucose consumption, intracellular glycogen content, gluconeogenesis rate, and sensitivity to glucose modulator hormones like insulin and glucagon. On the other hand, HepG2 spheroids display strong lipid metabolism abilities in producing significantly higher levels of total cholesterol and triglyceride. Compared with those without fiber fragments, the gSF-supported 3D culture establishes effective models for in vitro glucose (rPH spheroids) and lipid metabolisms (HepG2 spheroids). The screening models are confirmed from the respective enzyme activities and gene expressions and show significantly higher sensitivity and clinically related responses to hypoglycemic and lipid-lowering drugs. Thus, the culture configuration demonstrates a predictable in vitro platform for defining glucose and lipid metabolism profiles and screening therapeutic agents for metabolism disorders like diabetes and obesity.

Tumor pH-Responsive Release of Drug-Conjugated Micelles from Fiber Fragments for Intratumoral Chemotherapy.

The tumor accumulation of micelles is essential to enhance the cellular uptake and extend the release of chemotherapeutic agents. In the previous study camptothecin (CPT)-conjugated micelles (MCPT) were constructed with disulfide linkages and folate moieties for reduction-sensitive release and cell-selective uptake. This study proposes a strategy to integrate the promicelle polymers (PMCPT) into fiber fragments for intratumoral injection, realizing acid-liable release of PMCPT in response to acidic tumor microenvironment and spontaneous self-assembly into MCPT. Acid-liable 2-propionic-3-methylmaleic anhydride (CDM)-linked poly(ethylene glycol) initiates the ring-opening polymerization of dl-lactide as the fiber matrix. There is no apparent burst release of MCPT from fiber fragments and around 80% of accumulated releases after incubation in pH 6.5 buffers for 40 days. Compared to MCPT freshly prepared via solvent evaporation, the micelles released from fiber fragments reveal similar profiles, such as folate-mediated cellular uptake and glutathione-sensitive drug release. Taking advantage of the aggregation-induced emission (AIE) effect of tetraphenylethylene (TPE) derivatives, TPE-conjugated micelles (MTPE) have been successfully been used to track the self-assembly into micelles after release from fibers and subsequent cell internalization into cytosol. The self-assembly induced fluorescence light-up was also detected after intratumoral injection of fiber fragments. Compared with CPT-loaded fiber fragments and intratumoral or intravenous injection of free MCPT, the sustained release from fiber fragments and high accumulation of micelles in tumors result in significantly higher inhibition of tumor growths, prolongation of animal survival, and induction of tumor cell apoptosis. Thus, the integration of double targeting and double stimuli responsiveness into fragmented fibers provides a feasible strategy to realize the sustained micelle release from fibers and promote the therapeutic efficacy.

Tunable release of chemotherapeutic and vascular disrupting agents from injectable fiber fragments potentiates combination chemotherapy.

Cancer progression and metastasis relies much on vasculature networks in tumor microenvironment, and the combination treatment with chemotherapeutic drugs and vascular disrupting agents represents apparent clinical benefits. In the current study, fiber fragments with loadings of hydroxycamptothecin (HCPT) or combretastatin A-4 (CA4) were proposed for tumor inhibition and blood vessel disruption after local administration in tumors. To address challenges in balancing the disruption of tumor vessels and intratumoral uptake of chemotherapeutic agents, this study is focus on release tuning of HCPT and CA4 from the fiber fragment mixtures. Hydroxypropyl-ß-cyclodextrin (HPCD) was blended at ratios from 0 to 10% into CA4-loaded fiber fragments (Fc) to modulate CA4 release durations from 0.5 to 24days, and HCPT-loaded fiber fragments (Fh) indicated a sustained release for over 35days. In vitro cytotoxicity tests indicated a sequential inhibition on the endothelial and tumor cell growth, and the growth inhibition of tumor cells was more significant after treatment with mixtures of Fh and Fc containing 2% HPCD (Fc2) than that of other mixtures. In an orthotopic breast tumor model, compared with those of free CA4, or Fc with a fast or slow release of CA4, Fh/Fc mixtures with CA4 release durations from 2 to 12days indicated a lower tumor growth rate, a prolonged animal survival, a lower vessel density in tumors, and a less significant tumor metastasis. In addition, the tumor cell proliferation rate, hypoxia-inducible factor-1α expression within tumors, and the number of surface metastatic nodules in lungs were significantly lower after treatment with Fh/Fc2 mixtures with a CA4 release duration of 5days than those of other mixtures. It demonstrates the advantages of fiber fragment mixtures in independently modulating the release of multiple drugs and the essential role of release tuning of chemotherapeutic drugs and vascular disrupting agents in improving the therapeutic efficacy.