Structural Properties and Surface Modification Decided Pharmacokinetic Behavior and Bio-Distribution of DNA Origami Frameworks in Mice.

This study establishes and validates a series of three dimentional (3D) DNA origami frameworks (DOFs) carrying imaging probes to evaluate their pharmacokinetics and real-time bio-distribution in mice. Three typical DOFs with distinguished structural properties are subjected to mice intravenous injection to systematically investigate their in vivo behaviors. Tracing the radioisotope zirconium-89 (89 Zr) trapped at the inner space of the frameworks, positron emission tomography (PET) imaging is employed to record the real-time bio-distribution of the structures and acquire their pharmacokinetic parameters in the major metabolic organs. The 3D DOFs show different behavior compared to previous structures, with lower kidney accumulation and higher liver retention. Modifications to the structures, such as exposed ssDNA or polyethylene glycol (PEG) moieties, impact their behavior, but are structure-dependent. The 43 nm icosahedra framework among the DOFs perform the best in liver targeting, with the ssDNA extensions enhancing this tendency. The modification of triantennary N-acetylgalactosamine (GalNAc), further improves its uptake in liver cells, especially in hepatocytes over other cell types, discovered by flow cytometry analysis.

Multiplexing Nanodrug Ameliorates Liver Fibrosis via ROS Elimination and Inflammation Suppression.

Liver fibrosis is the leading risk factor for hepatocellular carcinoma. Both oxidative stress and inflammation promote the progression of liver fibrosis, but existing therapeutic strategies tend to focus solely on one issue. Additionally, targeting of pathological microstructures is often neglected. Herein, an esterase-responsive carbon quantum dot-dexamethasone (CD-Dex) is developed for liver fibrosis therapy to simultaneously target pathological microstructures, scavenge reactive oxygen species (ROS), and suppress inflammation. Hepatocyte-targeting CD-Dex can efficiently eliminate the intrahepatic ROS, thereby inhibiting the activation of Kupffer cells, preventing further inflammation progression. Moreover, released dexamethasone (Dex) also suppresses inflammatory response by inhibiting the infiltration of inflammatory cells. Antifibrotic experiments demonstrate that CD-Dex significantly alleviates liver injury and collagen deposition, consequently preventing the progression of liver fibrosis. Taken together, these findings suggest that via ROS elimination and inflammation suppression, the newly developed multiplexing nanodrug exhibits great potential in liver fibrosis therapy.

Systemic biodistribution and hepatocyte-specific gene editing with CRISPR/Cas9 using hyaluronic acid-based nanoparticles.

The goal of this study was to evaluate hepatocyte-specific gene editing, via systemic administration of hyaluronic acid (HA)-based nanoparticles in naïve CD-1 mice. Using HA-poly(ethylene imine) (HA-PEI) and HA-PEI-mannose nanoparticles with differential mannose density (1X and 2X), we have evaluated systemic biodistribution and hepatocyte-specific delivery using IVIS imaging and flow cytometry. Additionally, we have investigated hepatocyte-specific delivery and transfection of CRISPR/Cas9 gene editing plasmid and eGFP gene payload to integrate at the Rosa26 locus. IVIS imaging showed uptake of HA-PEI nanoparticles primarily by the liver, and with addition of mannose at different concentrations, the nanoparticles showed increased uptake in both the liver and spleen. HA-PEI-mannose nanoparticles showed 55-65% uptake by hepatocytes, along with uptake by resident macrophage regardless of the mannose concentration. One of two gRNA targets showed 15% genome editing and obtained similar results for all three nanoparticle formulations. Cells positive for our gene payload were greatest with HA-PEI-mannose-1X nanoparticles where 16.2% of cells were GFP positive. The results were encouraging as proof of concept for the development of a non-viral biodegradable and biocompatible polymeric delivery system for gene editing specifically targeting hepatocytes upon systemic administration.

A near-infrared hepatocyte-targeting probe based on Tricyanofuran to detect cysteine in vivo: Design, synthesis and evaluation.

In this work, a near-infrared hepatocyte-targeting fluorescence probe TCF-Gal-Cys was developed. The TCF-Gal-Cys exhibited a low detection limit, good sensitivity and selectivity toward Cys. The responsive mechanism of TCF-Gal-Cys was proposed that the acrylate of TCF-Gal-Cys was subsequently attacked by the thiol group and the amino group of Cys, releasing a strong near-infrared fluorescent group. TCF-Gal-Cys displayed a good hepatocyte-targeting capacity and could specifically distinguish hepatocytes from A549, Hela, SGC-7901 cells because the galactose group of TCF-Gal-Cys can be recognized by HepG2 cells overexpressing ASGPR. The TCF-Gal-Cys has achieved excellently imaging performance to Cys in the zebrafish, so TCF-Gal-Cys has potential to be an effective tool to in real time monitor Cys-related diseases in vitro and in vivo.

Lipid nanoparticle-mediated hepatocyte delivery of siRNA and silibinin in metabolic dysfunction-associated steatotic liver disease.

Lipid nanoparticle-mediated co-delivery of siRNA and small molecule holds a great potential to treat metabolic dysfunction-associated steatotic liver disease (MASLD). However, targeted delivery of therapeutics to hepatocytes remains challenging. Taking the advantage of rising low density lipoprotein receptor/very-low density lipoprotein receptor (LDLR/VLDR) levels in MASLD, the biological fate of dinonylamine-ethylene glycol chlorophosphate-1-nonanol (DNNA-COP-NA) based lipid nanoparticles (LNPs) was oriented to liver tissues via apolipoprotein E (ApoE)-LDLR/VLDLR pathway. We then adopted a three-round screening strategy to optimize the formulation with both high potency and selectivity to deliver siRNA-HIF-1α (siHIF1α) and silibinin (SLB) payloads to hepatocytes. The optimized SLB/siHIF1α-LNPs mediates great siRNA delivery and transfection of hepatocytes. In high fat diet (HFD)- and carbon tetrachloride (CCl4)-induced mouse models of MASLD, SLB/siHIF1α-LNPs enabled the silencing of hypoxia inducible factor-1α (HIF-1α), a therapeutic target primarily expressed by hepatocytes, leading to significantly reduced inflammation and liver fibrosis synergized with SLB. Moreover, it is demonstrated the hepatocyte-targeting delivery of SLB/siHIF1α-LNPs has the potential to restore the immune homeostasis by modulating the population of Tregs and cytotoxic T cells in spleen. This proof-of-concept study enable siRNA and small molecule co-delivery to hepatocytes through intrinsic variation of targeting receptors for MASLD therapy.

Ligand-free high loading capacity ursolic acid self-carried nanovesicles enable hepatocyte targeting via absorbing apolipoproteins.

Ursolic acid (UA), a natural pentacyclic terpenoid carboxylic acid that can exert a potent hepatoprotective activity, has been developed into various types of nanoparticles to improve its pharmacological effects, however, the phagocytosis of nanoparticles by Kupffer cells greatly limits its efficacy. Herein, UA/Tween 80 nanovesicles (V-UA) were constructed and despite its simple composition, it fulfills multiple functions simultaneously: UA served as not only an active ingredient in the nanovesicle drug delivery system, but also acts as part of the carrier to stabilize UA/Tween 80 nanostructure; with a molar ratio of UA to Tween 80 up to 2:1, the formulation possesses a significant advantage of higher drug loading capacity; relative to liposomal UA (Lipo-UA), a conditional cellular uptake and higher accumulation of V-UA in hepatocytes provide insights into the hepatocytes targeting mechanisms of this nanovesicles. Favorable hepatocyte targeting ability also facilitates the treatment of liver diseases, which was well validated in three liver disease models.

Design, synthesis and biological evaluation of novel dual-targeting fluorescent probes for detection of Fe3+ in the lysosomes of hepatocytes mediated by galactose-morpholine moieties.

In this work, novel dual-targeting probes composed of galactose and morpholine were designed and synthesized for monitoring Fe3+ levels in the lysosome of hepatocyte. MP-Gal-1, MP-Gal-2 and MP-Gal-3 showed good selectivity and sensitivities toward Fe3+ with the detection limits of 9.40 × 10-8 M, 7.68 × 10-8 M and 7.10 × 10-8 M, respectively. 1:2 stoichiometry is the most likely recognition mode between probe and Fe3+. Low toxic MP-Gal-1, MP-Gal-2 and MP-Gal-3 exhibited favorable hepatic targeting effect in both cell and tissue levels, which was because the galactose group of probe could be recognized by ASGPR overexpressed on the hepatocytes. The hepatocyte-targeting capacity followed MP-Gal-1 < MP-Gal-2 < MP-Gal-3 trend, which was attributed to the galactose cluster effect. MP-Gal-1, MP-Gal-2 and MP-Gal-3 also displayed good lysosomes-targeting capacities, because the basic morpholine moiety of probes could be easily attracted by the acidic lysosome. Therefore, MP-Gal-1, MP-Gal-2 and MP-Gal-3 have good dual targeting capacities (liver and lysosome) and could be used to detect lysosomal Fe3+ in the liver, which is great significant for precise diagnosis and treatment of liver lysosomal iron-related diseases.

The mechanism of Hepatocyte-Targeting and safety profile of Phospholipid-Free small unilamellar vesicles.

Phospholipid-free small unilamellar vesicles (PFSUVs) composed of cholesterol and TWEEN80 (5:1 mol ratio), with an average diameter of 60 nm, displayed targeted delivery to the hepatocytes after intravenous (i.v.) injection. Here, we conducted a series of experiments to elucidate the hepatocyte targeting mechanism. The uptake of PFSUVs by HepG2 cells was increased by 3-fold in the presence of serum. The plasma protein corona adsorbed to PFSUVs was analyzed and subtypes of apolipoproteins were found enriched, specifically apolipoprotein AII (ApoA2). The cellular uptake was increased by 1.5-fold when the culture medium was supplemented with ApoA2, but not ApoC1 and ApoE. Furthermore, the cellular uptake of PFSUVs increased with increasing concentrations of ApoA2 in the medium and was almost completely blocked in the presence of BLT-1, an inhibitor for the scavenger receptor B-1 (SR-B1), which is a receptor for ApoA2. The data suggest that upon i.v. delivery, PFSUVs adsorbed plasma ApoA2 to the surface, which was recognized by SR-B1 expressed by the hepatocytes and then internalized. After internalization, mainly through the clathrin-mediated endocytosis, PFSUVs were found in the endosomes after 1-2 h post treatment and then lysosomes in 4 h. We also examined the cytotoxicity, hemolytic toxicity and complement activation of PFSUVs by incubating the formulation with HepG2 cells, red blood cells and human plasma, respectively, demonstrating no toxicity at concentrations higher than the therapeutic doses.

Multivalent rubber-like RNA nanoparticles for targeted co-delivery of paclitaxel and MiRNA to silence the drug efflux transporter and liver cancer drug resistance.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide. Analogous to the border customs, liver mainly functions as a filter to detoxify chemicals and metabolite administered orally or intravenously. Besides, the liver cancer cells overexpress the drug exporters which cause high drug effluxion from liver cancer cells, leading to chemoresistance and a diminished chemotherapeutic effect on liver cancer. Recently, we found that RNA nanoparticles display rubber-like property that can rapidly deliver therapeutics to tumor site efficiently and the rest of the RNA nanoparticle were cleared by renal excretion within half hour after systemic injection. Therefore, we designed a new multivalent RNA nanoparticle harboring three copies of hepatocyte targeting-ligands, one copy of miR122, and 24 copies of Paclitaxel to overcome the drug effluxion and chemoresistance thus, synergistically treating HCC. The hepatocyte targeting ligands introduce tumor specificity to the RNA nanoparticles as they selectively bind and internalize into liver cancer cells. The rubber-like RNA nanoparticles allow for enhanced targeting ability to the HCC tumors. The RNA nanoparticles carrying miR122 and PTX were delivered to the liver cancer cells efficiently due to their rubber-like property to enhance their EPR as well as the receptor-mediated endocytosis by hepatocyte targeting-ligands. The miR122 efficiently silenced the drug exporters and the oncogenic proteins. The synergistic effect between miR122 and PTX was confirmed by HSA (Highest Single Agent) synergy model. IC50 was determined to be 460 nM. In vivo studies on mice xenografts revealed that the RNA nanoparticle predominantly accumulated in HCC tumor sites and efficiently inhibited the tumor growth after multiple IV injection. This demonstrates the potential of the rubber-like multivalent RNA nanoparticles to conquest the liver cancer, a currently incurable lethal disease.

Design, synthesis and evaluation of a novel fluorescent probe to accurately detect H2S in hepatocytes and natural waters.

Design and synthesis of fluorescent probe with fast response, excellent water solubility and good hepatocyte-targeting capacity to detect hydrogen sulfide (H2S) in hepatocytes and water samples is of great significance. Here, a novel fluorescent probe QL-Gal-N3 for detection of H2S was designed and synthesized based on H2S-mediated azide reduction strategy. This sensor demonstrated low toxicity, fast response (within 1 min), high selectivity and low detection limit (as low as 126 nM in water) for the detection of H2S. HeLa, A549 and HepG-2 cells were chosen to investigate the hepatocyte-targeting ability of QL-Gal-N3 respectively. The results indicated that the specific recognition of ASGPR over-expressed in hepatocytes by galactose group was an important reason for the good targeting ability of probe QL-Gal-N3. Furthermore, due to the introduction of glycosyl moiety, the water solubility of fluorescent probe was enhanced obviously. It was successfully applied for the detection of H2S in environmental water samples including river water, tap water, lake water and waste water.

Development of Novel Galactosylated PLGA Nanoparticles for Hepatocyte Targeting Using Molecular Modelling.

Doxorubicin-loaded PLGA nanoparticles conjugated with a new galactose-based ligand for the specific recognition by human hepatoma cellular carcinoma cells (Hep G2) were successfully produced. The new targeting compound was selected using molecular docking combined with quantum chemical calculations for modelling and comparing molecular interactions among the H1 subunit of the asialoglycoprotein receptor containing the carbohydrate recognition domain and the ligand. The ligand, bis(1-O-ethyl-ß-D-galactopyranosyl)amine, was synthetized, characterized, and subsequently linked to PLGA. Unloaded (PLGA-di-GAL NP) and doxorubicin-loaded (DOX-PLGA-di-GAL NP) nanoparticles were prepared using an emulsion method and characterized. The produced DOX-PLGA-di-GAL NP are spherical in shape with a size of 258 ± 47 nm, a zeta potential of -62.3 mV, and a drug encapsulation efficiency of 83%. The in vitro drug release results obtained show a three-phase release profile. In vitro cell studies confirmed the interaction between Hep G2 cells and PLGA-di-GAL NP. Cell cytotoxicity tests showed that unloaded NP are nontoxic and that DOX-PLGA-di-GAL NP caused a decrease of around 80% in cellular viability. The strategy used in this work to design new targeting compounds represents a promising tool to develop effective hepatocyte targeting drug delivery systems and can be applied to other tissues/organs.

Optimization-by-design of hepatotropic lipid nanoparticles targeting the sodium-taurocholate cotransporting polypeptide.

Active targeting and specific drug delivery to parenchymal liver cells is a promising strategy to treat various liver disorders. Here, we modified synthetic lipid-based nanoparticles with targeting peptides derived from the hepatitis B virus large envelope protein (HBVpreS) to specifically target the sodium-taurocholate cotransporting polypeptide (NTCP; SLC10A1) on the sinusoidal membrane of hepatocytes. Physicochemical properties of targeted nanoparticles were optimized and NTCP-specific, ligand-dependent binding and internalization was confirmed in vitro. The pharmacokinetics and targeting capacity of selected lead formulations was investigated in vivo using the emerging zebrafish screening model. Liposomal nanoparticles modified with 0.25 mol% of a short myristoylated HBV derived peptide, that is Myr-HBVpreS2-31, showed an optimal balance between systemic circulation, avoidance of blood clearance, and targeting capacity. Pronounced liver enrichment, active NTCP-mediated targeting of hepatocytes and efficient cellular internalization were confirmed in mice by 111In gamma scintigraphy and fluorescence microscopy demonstrating the potential use of our hepatotropic, ligand-modified nanoparticles.

Bolstering cholesteryl ester hydrolysis in liver: A hepatocyte-targeting gene delivery strategy for potential alleviation of atherosclerosis.

Current atherosclerosis treatment strategies primarily focus on limiting further cholesteryl esters (CE) accumulation by reducing endogenous synthesis of cholesterol in the liver. No therapy is currently available to enhance the removal of CE, a crucial step to reduce the burden of the existing disease. Given the central role of hepatic cholesteryl ester hydrolase (CEH) in the intrahepatic hydrolysis of CE and subsequent removal of the resulting free cholesterol (FC), in this work, we applied galactose-functionalized polyamidoamine (PAMAM) dendrimer generation 5 (Gal-G5) for hepatocyte-specific delivery of CEH expression vector. The data presented herein show the increased specific uptake of Gal-G5/CEH expression vector complexes (simply Gal-G5/CEH) by hepatocytes in vitro and in vivo. Furthermore, the upregulated CEH expression in the hepatocytes significantly enhanced the intracellular hydrolysis of high density lipoprotein-associated CE (HDL-CE) and subsequent conversion/secretion of hydrolyzed FC as bile acids (BA). The increased CEH expression in the liver significantly increased the flux of HDL-CE to biliary as well as fecal FC and BA. Meanwhile, Gal-G5 did not induce hepatic or renal toxicity. It was also not immunotoxic. Because of these encouraging pre-clinical testing results, using this safe and highly efficient hepatocyte-specific gene delivery platform to enhance the hepatic processes involved in cholesterol elimination is a promising strategy for the alleviation of atherosclerosis.

Nano-sized cytochrome P450 3A4 inhibitors to block hepatic metabolism of docetaxel.

Most drugs are metabolized by hepatic cytochrome P450 3A4 (CYP3A4), resulting in their reduced bioavailability. In this study, we present the design and evaluation of bio-compatible nanocarriers trapping a natural CYP3A4-inhibiting compound. Our aim in using nanocarriers was to target the natural CYP3A4-inhibiting agent to hepatic CYP3A4 and leave drug-metabolizing enzymes in other organs undisturbed. In the design of such nanocarriers, we took advantage of the nonspecific accumulation of small nanoparticles in the liver. Specific targeting functionalization was added to direct nanocarriers toward hepatocytes. Nanocarriers were evaluated in vitro for their CYP3A4 inhibition capacity and in vivo for their biodistribution, and finally injected 24 hours prior to the drug docetaxel, for their ability to improve the efficiency of the drug docetaxel. Nanoparticles of poly(lactic-co-glycolic) acid (PLGA) with a hydrodynamic diameter of 63 nm, functionalized with galactosamine, showed efficient in vitro CYP3A4 inhibition and the highest accumulation in hepatocytes. When compared to docetaxel alone, in nude mice bearing the human breast cancer, MDA-MB-231 model, they significantly improved the delay in tumor growth (treated group versus docetaxel alone, percent treated versus control ratio [%T/C] of 32%) and demonstrated a major improvement in overall survival (survival rate of 67% versus 0% at day 55).

[Optimization of expression conditions and activity identification of hepatocyte-targeting peptide-human endostatin].

To obtain sufficient purified and active fusion protein-hepatocyte-targeting peptide-human endostatin (HTP-rES), we studied the growth curve and the optimal induction timing of BL21/pET21b-HTP-rES. Different conditions of pH value, induction time, induction concentration and induction temperature were optimized by univariate analysis. After washing, refolding and purifying, the activity of fusion protein was identified by flow cytometry and 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT). Results show that the logarithmic growth phase of BL21/pET21b-HTP-rES was from 1.5 h to 3.5 h, the optimum expression conditions were pH 8.0, 0.06 mmol/L IPTG, at 42 ℃ for 5 h. The purity of inclusion bodies was up to 60% after washing. The purity of target protein was more than 95% after refolding and purification. Our findings provide the foundation for further biological activity and drug development.

Hepatocyte-targeting gene transfer mediated by galactosylated poly(ethylene glycol)-graft-polyethylenimine derivative.

Biscarbamate cross-linked polyethylenimine derivative (PEI-Et) has been reported as a novel nonviral vector for efficient and safe gene transfer in our previous work. However, it had no cell-specificity. To achieve specific delivery of genes to hepatocytes, galactosylated poly(ethylene glycol)-graft-polyethylenimine derivative (GPE) was prepared through modification of PEI-Et with poly(ethylene glycol) and lactobionic acid, bearing a galactose group as a hepatocyte-targeting moiety. The composition of GPE was characterized by proton nuclear magnetic resonance. The weight-average molecular weight of GPE measured with a gel permeation chromatography instrument was 9489 Da, with a polydispersity of 1.44. GPE could effectively condense plasmid DNA (pDNA) into nanoparticles. Gel retardation assay showed that GPE/pDNA complexes were completely formed at weigh ratios (w/w) over 3. The particle size of GPE/pDNA complexes was 79-100 nm and zeta potential was 6-15 mV, values which were appropriate for cellular uptake. The morphology of GPE/pDNA complexes under atomic force microscopy appeared spherical and uniform in size, with diameters of 53-65 nm. GPE displayed much higher transfection efficiency than commercially available PEI 25 kDa in BRL-3A cell lines. Importantly, GPE showed good hepatocyte specificity. Also, the polymer exhibited significantly lower cytotoxicity compared to PEI 25 kDa at the same concentration or weight ratio in BRL-3A cell lines. To sum up, our results indicated that GPE might carry great potential in safe and efficient hepatocyte-targeting gene delivery.