Ian R. Corbin

Biomimetic Nanocarrier for Direct Cytosolic Drug Delivery

Dedicated to Professor Britton Chance on the occasion of his 96th birthdayThe ability to transport a large quantity of drug moleculesinto cytosolic compartments of cancer cells has powerfulimplications in modern molecular therapeutics because thesites of action of the drugs are often cytosolic organelles.

HDL‐Mimicking Peptide–Lipid Nanoparticles with Improved Tumor Targeting

Targeted delivery of intracellularly active diagnostics and therapeutics in vivo is a major challenge in cancer nanomedicine. A nanocarrier should possess long circulation time yet be small and stable enough to freely navigate through interstitial space to deliver its cargo to targeted cells. Herein, it is shown that by adding targeting ligands to nanoparticles that mimic high-density lipoprotein (HDL), tumor-targeted sub-30-nm peptide-lipid nanocarriers are created with controllable size, cargo loading, and shielding properties. The size of the nanocarrier is tunable between 10 and 30 nm, which correlates with a payload of 15-100 molecules of fluorescent dye. Ligand-directed nanocarriers targeting epidermal growth factor receptor (EGFR) are confirmed both in vitro and in vivo. The nanocarriers show favorable circulation time, tumor accumulation, and biodistribution with or without the targeting ligand. The EGFR targeting ligand is proved to be essential for the EGFR-mediated tumor cell uptake of the nanocarriers, a prerequisite of intracellular delivery. The results demonstrate that targeted HDL-mimetic nanocarriers are useful delivery vehicles that could open new avenues for the development of clinically viable targeted nanomedicine.

Mitochondrial metabolism mediates oxidative stress and inflammation in fatty liver

Mitochondria are critical for respiration in all tissues; however, in liver, these organelles also accommodate high-capacity anaplerotic/cataplerotic pathways that are essential to gluconeogenesis and other biosynthetic activities. During nonalcoholic fatty liver disease (NAFLD), mitochondria also produce ROS that damage hepatocytes, trigger inflammation, and contribute to insulin resistance. Here, we provide several lines of evidence indicating that induction of biosynthesis through hepatic anaplerotic/cataplerotic pathways is energetically backed by elevated oxidative metabolism and hence contributes to oxidative stress and inflammation during NAFLD. First, in murine livers, elevation of fatty acid delivery not only induced oxidative metabolism, but also amplified anaplerosis/cataplerosis and caused a proportional rise in oxidative stress and inflammation. Second, loss of anaplerosis/cataplerosis via genetic knockdown of phosphoenolpyruvate carboxykinase 1 (Pck1) prevented fatty acid-induced rise in oxidative flux, oxidative stress, and inflammation. Flux appeared to be regulated by redox state, energy charge, and metabolite concentration, which may also amplify antioxidant pathways. Third, preventing elevated oxidative metabolism with metformin also normalized hepatic anaplerosis/cataplerosis and reduced markers of inflammation. Finally, independent histological grades in human NAFLD biopsies were proportional to oxidative flux. Thus, hepatic oxidative stress and inflammation are associated with elevated oxidative metabolism during an obesogenic diet, and this link may be provoked by increased work through anabolic pathways.

Lipoprotein nanoplatform for targeted delivery of diagnostic and therapeutic agents.

Low-density lipoprotein (LDL) provides a highly versatile natural nanoplatform for delivery of visible or near-infrared fluorescent optical and magnetic resonance imaging (MRI) contrast agents and photodynamic therapy and chemotherapeutic agents to normal and neoplastic cells that overexpress low-density lipoprotein receptors (LDLRs). Extension to other lipoproteins ranging in diameter from about 10 nm (high-density lipoprotein [HDL]) to over a micron (chylomicrons) is feasible. Loading of contrast or therapeutic agents onto or into these particles has been achieved by protein loading (covalent attachment to protein side chains), surface loading (intercalation into the phospholipid monolayer), and core loading (extraction and reconstitution of the triglyceride/cholesterol ester core). Core and surface loading of LDL have been used for delivery of optical imaging agents to tumor cells in vivo and in culture. Surface loading was used for delivery of gadolinium-bis-stearylamide contrast agents for in vivo MRI detection in tumor-bearing mice. Chlorin and phthalocyanine near-infrared photodynamic therapy agents (≤ 400/LDL) have been attached by core loading. Protein loading was used to reroute the LDL from its natural receptor (LDLR) to folate receptors and could be used to target other receptors. A semisynthetic nanoparticle has been constructed by coating magnetite iron oxide nanoparticles with carboxylated cholesterol and overlaying a monolayer of phospholipid to which apolipoprotein A1 or E was adsorbed for targeting HDL or adsorbing synthetic amphipathic helical peptides ltargeting LDL or folate receptors. These particles can be used for in situ loading of magnetite into cells for MRI-monitored cell tracking or gene expression.

Synthesis and evaluation of a stable bacteriochlorophyll-analog and its incorporation into high-density lipoprotein nanoparticles for tumor imaging.

The syntheses of novel near-infrared (NIR) dyes with excellent optical properties in biological tissues have driven the continued improvement of fluorescence imaging of deeply seated tumors. Bacteriochlorophyll a (Bchl), a dye synthesized by the phototrophic bacteria, R. sphaeroids, is particularly suited for deep tissue imaging due to its high absorbance coefficient and good fluorescence quantum yield in the NIR spectrum. However, obstacles that impede the development of this fluorophore are its poor stability and lack of tumor specificity. These issues ultimately limit its utility for tumor detection. Herein we describe a robust synthesis of a novel Bchl analog, bacteriochlorin e(6) bisoleate (BchlBOA), which is chemically stable, has excellent photophysical properties (ex, 752 nm; em, 762 nm) and is tailored for the incorporation into a tumor targetable high-density lipoprotein (HDL)-like nanoparticle (NP). Incorporating BchlBOA into HDL (HDL-BchlBOA) yielded 12 nm sized particles, corresponding well with the diameter of native HDL. Functional cell uptake studies showed that HDL-BchlBOA was taken up by cells expressing the HDL receptor, scavenger receptor B type I (SR-BI), and was inhibited by 25-fold excess native HDL. Furthermore, the NP was successfully detected in KB cancer cells both in vitro and in tumor xenografts. Taken together, these results demonstrate that we successfully synthesized and formulated a stable analog of Bchl that is capable of being incorporated within HDL-like NPs for tumor-targeted imaging.

In vivo assessment of hepatic triglycerides in murine non-alcoholic fatty liver disease using magnetic resonance spectroscopy

In vivo(1)H magnetic resonance spectroscopy (MRS) was used to examine the progression of fatty liver in two murine models of progressive hepatic steatosis: leptin-deficient obese (ob/ob) mice and mice maintained on a diet deficient in methionine and choline (MCDD). Ob/ob mice displayed high levels of intracellular hepatic triglycerides as early as 9 weeks after birth, as observed with MRS and histopathology. Single voxel spectra of ob/ob liver displayed strong resonances arising from saturated (1.3 ppm) and unsaturated (2.8 and 5.3 ppm) fatty acyl chains that could be resolved in the absence of water suppression. Hepatic inflammation, induced by lipopolysaccharide administration, led to a significant increase in unsaturated and polyunsaturated fatty acyl chain resonances (P<0.05), indicating a change in the composition of hepatic triglycerides in lipid droplets. Mice maintained on the MCDD displayed histological evidence of hepatic steatosis as early as two weeks, progressing to macrovesicular steatohepatitis at 10 weeks. The histological changes were accompanied by significant increases in saturated and unsaturated fatty acyl chain resonances and a significant decrease in the lipid/(water+lipid) ratio (P<0.05). These results indicate that in vivo(1)H MRS may be a suitable method to monitor the progression of steatohepatitis.

LIPOPROTEIN NANOPLATFORM FOR TARGETED DELIVERY OF DIAGNOSTIC AND THERAPEUTIC AGENTS

Low-density lipoprotein (LDL) provides a highly versatile natural nanoplatform for delivery of optical and MRI contrast agents, photodynamic therapy agents and chemotherapeutic agents to normal and neoplastic cells that over express LDL receptors (LDLR). Extension to other lipoproteins ranging in diameter from approximately 5-10 nm (high density lipoprotein, HDL) to over a micron (chilomicrons) is feasible. Loading of contrast or therapeutic agents has been achieved by covalent attachment to protein side chains, intercalation into the phospholipid monolayer and extraction and reconstitution of the triglyceride/cholesterol ester core. Covalent attachment of folate to the lysine side chain amino groups was used to reroute the LDL from its natural receptor (LDLR) to folate receptors and could be utilized to target other receptors. A semi-synthetic nanoparticle has been constructed by coating magnetite iron oxide nanoparticles (MIONs) with carboxylated cholesterol and overlaying a monolayer ofphospholipid to which Apo A1, Apo E or synthetic amphoteric alpha-helical polypeptides were adsorbed for targeting HDL, LDL or folate receptors, respectively. These particles can be utilized for in situ loading of magnetite into cells for MRI monitored cell tracking or gene therapy.

In Vitro Assessment of Poly-iodinated Triglyceride Reconstituted Low-Density Lipoprotein: Initial Steps Toward CT Molecular Imaging

RATIONALE AND OBJECTIVES Targeted molecular probes offer the potential for greater specificity in cancer imaging with contrast-enhanced computed tomography (CT). We investigate a low-density lipoprotein (LDL) nanoparticle loaded with poly-iodinated triglyceride (ITG) in a proof of concept study of targeted x-ray imaging. LDLs are targeted to the LDL cell surface receptor (LDLR), which is overexpressed in several tumor types. The LDL-LDLR pathway presents a high-capacity and self-renewing transport system for molecular imaging in CT. MATERIALS AND METHODS ITG was synthesized and loaded into the core of LDL particles to form a reconstituted nanoparticle, hereafter referred to as (rITG)LDL. Particle size was measured by dynamic light scattering. The x-ray attenuation of the (rITG)LDL solution was measured with CT imaging and signal enhancement was calibrated for equivalent iodine concentration. Cultured human hepatoblastoma G2 (HepG2) cells, which overexpress LDLR, were incubated with (rITG)LDL with or without native LDL. The cells were imaged with CT to characterize particle sequestration. RESULTS Reconstitution of LDL with ITG was successful and did not compromise the targeting function of the particle. Measurement of the x-ray attenuation properties of the (rITG)LDL solution revealed an effective iodine concentration of 0.78 mg/mL. In vitro studies of HepG2 cells demonstrated a significant increase in CT image intensity over control cells when incubated with (rITG)LDL. CONCLUSION The in vitro results of this study suggest that (rITG)LDL can provide adequate image enhancement for CT molecular imaging. Potential applications include breast imaging and small animal imaging at low x-ray energies. In vivo experiments will be required to verify that tumor uptake of (rITG)LDL is sufficient for enhanced detection. Use at higher x-ray energies, as used in conventional CT, will require a further increase in iodine loading.

Evaluation of bacteriochlorophyll-reconstituted low-density lipoprotein nanoparticles for photodynamic therapy efficacy in vivo

AIM To evaluate the novel nanoparticle reconstituted bacteriochlorin e6 bisoleate low-density lipoprotein (r-Bchl-BOA-LDL) for its efficacy as a photodynamic therapy agent delivery system in xenografts of human hepatoblastoma G2 (HepG2) tumors. MATERIALS & METHODS Bchl-BOA was encapsulated in the nanoparticle low-density lipoprotein (LDL), a native particle whose receptors overexpression is a cancer signature for a number of neoplasms. Evaluation of r-Bchl-BOA-LDL as a potential photosensitizer was performed using a tumor response and foot response assay. RESULTS & DISCUSSION When compared with controls, tumor regrowth was significantly delayed at injected murine doses of 2 µmole/kg r-Bchl-BOA-LDL after illumination at fluences of 125, 150 or 175 J/cm(2). Foot response assays showed that although normal tissue toxicity accompanied the higher fluences it was significantly reduced at the lowest fluence tested. CONCLUSION This research demonstrates that r-Bchl-BOA-LDL is an effective photosensitizer and a promising candidate for further investigation.

Original investigationIn Vitro Assessment of Poly-iodinated Triglyceride Reconstituted Low-Density Lipoprotein: Initial Steps Toward CT Molecular Imaging

RATIONALE AND OBJECTIVES Targeted molecular probes offer the potential for greater specificity in cancer imaging with contrast-enhanced computed tomography (CT). We investigate a low-density lipoprotein (LDL) nanoparticle loaded with poly-iodinated triglyceride (ITG) in a proof of concept study of targeted x-ray imaging. LDLs are targeted to the LDL cell surface receptor (LDLR), which is overexpressed in several tumor types. The LDL-LDLR pathway presents a high-capacity and self-renewing transport system for molecular imaging in CT. MATERIALS AND METHODS ITG was synthesized and loaded into the core of LDL particles to form a reconstituted nanoparticle, hereafter referred to as (rITG)LDL. Particle size was measured by dynamic light scattering. The x-ray attenuation of the (rITG)LDL solution was measured with CT imaging and signal enhancement was calibrated for equivalent iodine concentration. Cultured human hepatoblastoma G2 (HepG2) cells, which overexpress LDLR, were incubated with (rITG)LDL with or without native LDL. The cells were imaged with CT to characterize particle sequestration. RESULTS Reconstitution of LDL with ITG was successful and did not compromise the targeting function of the particle. Measurement of the x-ray attenuation properties of the (rITG)LDL solution revealed an effective iodine concentration of 0.78 mg/mL. In vitro studies of HepG2 cells demonstrated a significant increase in CT image intensity over control cells when incubated with (rITG)LDL. CONCLUSION The in vitro results of this study suggest that (rITG)LDL can provide adequate image enhancement for CT molecular imaging. Potential applications include breast imaging and small animal imaging at low x-ray energies. In vivo experiments will be required to verify that tumor uptake of (rITG)LDL is sufficient for enhanced detection. Use at higher x-ray energies, as used in conventional CT, will require a further increase in iodine loading.