Low-density Lipoprotein Docosahexaenoic Acid Research Articles

Repeated trans-arterial treatments of LDL-DHA nanoparticles induce multiple pathways of tumor cell death in hepatocellular carcinoma bearing rats.

Repeated hepatic arterial delivery of therapeutic agents to the liver by percutaneously implanted port-catheter systems has been widely used to treat unresectable liver cancer. This approach is applied to assess the therapeutic efficacy of repeated low-density lipoprotein-docosahexaenoic acid (LDL-DHA) nanoparticle treatments in a rat model of hepatocellular carcinoma. N1S1 hepatoma bearing rats underwent placement of a percutaneously implanted hepatic artery port-catheter system and were allocated to untreated, control LDL-triolein (LDL-TO) or LDL-DHA nanoparticle infusions groups. Treatments were performed every three days over a nine day study period. MRI was performed at baseline and throughout the study. At the end of the study tissue samples were collected for analyses. Implantation of the port catheters was successful in all rats. MRI showed that repeated infusions of LDL-DHA nanoparticles significantly impaired the growth of the rat hepatomas eventually leading to tumor regression. The tumors in the LDL-TO treated group showed delayed growth, while the untreated tumors grew steadily throughout the study. Histopathology and MRI support these findings demonstrating extensive tumor necrosis in LDL-DHA treated groups while the control groups displayed minor necrosis. Molecular and biochemical analyses also revealed that LDL-DHA treated tumors had increased levels of nuclear factor-kappa B and lipid peroxidation and depletion of glutathione peroxidase 4 relative to the control groups. Evidence of both ferroptosis and apoptosis tumor cell death was observed following LDL-DHA treatments. In conclusion repeated transarterial infusions of LDL-DHA nanoparticles provides sustained repression of tumor growth in a rat hepatoma model.

Assessing the safety of transarterial locoregional delivery of low-density lipoprotein docosahexaenoic acid nanoparticles to the rat liver

Hepatic-arterial infusion (HAI) of low-density lipoprotein (LDL) nanoparticles reconstituted with docosahexaenoic acid (DHA) (LDL-DHA) has been shown in a rat hepatoma model to be a promising treatment for hepatocellular carcinoma. To date, little is known regarding the safety of HAI of LDL-DHA to the liver. Therefore, we aimed to investigate the deposition, metabolism and safety of HAI of LDL-DHA (2, 4 or 8 mg/kg) in the rat. Following HAI, fluorescent labeled LDL nanoparticles displayed a biexponential plasma concentration time curve as the particles were rapidly extracted by the liver. Overall, increasing doses of HAI of LDL-DHA was well tolerated in the rat. Body weight, plasma biochemistry and histology were all unremarkable and molecular markers of inflammation did not increase with treatment. Lipidomics analyses showed that LDL-DHA was preferentially oxidized to the anti-inflammatory mediator, protectin DX. We conclude that HAI of LDL-DHA nanoparticles is not only safe, but provides potential hepatoprotective benefits.

An implanted port-catheter system for repeated hepatic arterial infusion of low-density lipoprotein-docosahexaenoic acid nanoparticles in normal rats: A safety study

BackgroundIn recent years, small animal arterial port-catheter systems have been implemented in rodents with reasonable success. The aim of the current study is to employ the small animal port-catheter system to evaluate the safety of multiple hepatic-artery infusions (HAI) of low-density lipoprotein-docosahexaenoic acid (LDL-DHA) nanoparticles to the rat liver. MethodsWistar rats underwent surgical placement of indwelling HAI ports. Repeated administrations of PBS or LDL-DHA nanoparticles were performed through the port at baseline and days 3 and 6. Rats were sacrificed on day 9 at which point blood and various organs were collected for histopathology and biochemical analyses. ResultsThe port-catheter systems were implanted successfully and repeated infusions of PBS or LDL-DHA nanoparticles were tolerated well by all animals over the duration of the study. Measurements of serum liver/renal function tests, glucose and lipid levels did not differ between control and LDL-DHA treated rats. The liver histology was unremarkable in the LDL-DHA treated rats and the expression of hepatic inflammatory regulators (NF-κβ, IL-6 and CRP) were similar to control rats. Repeated infusions of LDL-DHA nanoparticles did not alter liver glutathione content or the lipid profile in the treated rats. The DHA extracted by the liver was preferentially metabolized to the anti-inflammatory DHA-derived mediator, protectin DX. ConclusionOur findings indicate that repeated HAI of LDL-DHA nanoparticles is not only well tolerated and safe in the rat, but may also be protective to the liver.

130 - Local regional delivery of low-density lipoprotein docosahexaenoic acid nanoparticles selectively kills tumors by lipid peroxidation

Metabolic reprogramming within malignant tumors favors the expression of saturated and mono-unsaturated fatty acids in their lipid membranes over polyunsaturated fatty acids (PUFAs). The paucity of PUFAs within tumor membranes is suggested to protect tumor cells from endogenous and drug-induced oxidative stress. In recent years our lab has demonstrated that low-density lipoprotein (LDL) nanoparticle delivery of the natural PUFA docosahexaenoic acid (DHA) to hepatoma bearing rats introduces omega-3 PUFAs to tumors with lethal tumoricidal effects. Following local regional hepatic artery delivery of DHA loaded LDL nanoparticles (LDL-DHA) the hepatoma and surrounding normal liver readily extract and internalize the administered nanoparticles through LDL receptor mediated endocytosis. Within these tissues DHA was rapidly metabolized to primary oxidation products, monohydroxylated DHA isomers. Overall, the hepatoma generated significantly higher concentrations of these oxidized DHA derivatives and the pattern of the oxidized isomers, namely the excessive amounts of C20- and C4-monohydroxylated DHA, suggest autoxidation was primarily responsible for DHA metabolism within the tumor. Furthermore, copious amounts of aldehydes, terminal products of lipid peroxidation, were present in the tumor. In particular, 4-hydroxy hexenal (4-HHE), a direct end product of omega-3 peroxidation was detected at levels far exceeding (2-10 X) that in the surrounding liver. Subsequent observation of LDL-DHA treated rats reveal extensive damage and necrosis of the hepatoma, meanwhile, the surrounding liver remained healthy not showing any evidence of injury. Collectively, these findings show that LDL-DHA is a safe and promising nanomedicine that selectively mediates lethal lipid peroxidation in malignant tumors.

Effects of low-density lipoprotein docosahexaenoic acid nanoparticles on cancer stem cells isolated from human hepatoma cell lines.

Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid with anti-cancer properties. Recently, DHA packaged within low-density lipoprotein (LDL) nanoparticles (LDL-DHA) was demonstrated to be effective in a murine model of hepatocellular carcinoma (HCC). Cancer stem cells (CSCs) are a subpopulation of tumor cells that are resistant to most cancer therapies and thereby, contribute to tumor recurrences. To determine whether LDL-DHA is effective against CSCs derived from human HCC cell lines and tumor bearing rats. Epithelial cellular adhesion molecule positive and CD133 negative (EpCAM+CD133-) CSCs were isolated from HuH-7 and HepG2 human HCC lines and exposed to varying concentrations (1-60µM) of LDL-DHA nanoparticles for 0-72h. HCC tumor bearing rats were treated with 2mg/kg LDL-DHA nanoparticles for 3 days. Regardless of the cell line employed, LDL-DHA nanoparticles achieved 70-100% killing of EpCAM+CD133- CSCs at a concentration of 40µM after 48h of exposure while DHA and LDL alone had minimal or no cytotoxic effects. Similar results were obtained with LDL-DHA nanoparticle treatment of EpCAM-CD133- adult cancer cells (ACCs). In keeping with these findings were similar levels of low density lipoprotein receptor expression and LDL-DHA nanoparticle induced lipid peroxidation activity and reactive oxygen species in the CSC and ACC populations. However, differences in sensitivity to LDL-DHA treatment were observed in vivo experiments where LDL-DHA nanoparticle treated tumors had a higher percent of surviving CSCs relative to ACCs. Further research on LDL-DHA nanoparticle therapy for human HCC is warranted.

113 - Low-Density Lipoprotein Docosahexaenoic Acid Nanoparticles Kill Liver Cancer Cells by Ferroptosis

Low-density lipoprotein nanoparticles reconstituted with the natural omega-3 fatty acid, docosahexaenoic acid (LDL-DHA), is a novel experimental therapeutic that has been reported to selectively kill malignant hepatoma cells over normal hepatocytes and effectively treat orthotopic liver tumors in the rat. To date, little is known about the cell death pathways by which LDL-DHA nanoparticles kill tumor cells. Here we show that the LDL-DHA nanoparticles are cytotoxic to both rat and human hepatocellular carcinoma (HCC) cell lines. Following LDL-DHA treatment both rat and human HCC cells experience pronounced lipid peroxidation, depletion of glutathione and inactivation of the lipid antioxidant glutathione peroxidase-4 (GPX4) . Inhibitor studies revealed that treated HCC cells die independent of apoptotic, necroptotic or autophagic pathways, but require the presence of cellular iron. These hallmark features are consistent and were later confirmed to reflect ferroptosis, a novel form of nonapoptotic iron-dependent cell death. In keeping with the mechanisms of ferroptosis cell death, GPX4 was also found to be a central regulator of LDL-DHA induced cytotoxicity. We also investigated the effects of LDL-DHA treatments in mice bearing human HCC tumor xenografts. In vivo treatments of LDL-DHA were extremely cytotoxic to the HCC tumors and severely inhibited their growth long term. Consistent with our in vitro findings, the LDL-DHA treated HCC tumors experienced ferroptotic cell death characterized by increased levels of lipid hydroperoxides and suppression of GPX4 expression. Supplementary studies with specific ferrrpotosis inhibitors also confirmed this mechanism of tumor killing by LDL-DHA. Conclusion LDL-DHA induces cell death in HCC cells through the ferroptosis pathway, this represents a novel molecular mechanism of anticancer activity for LDL-DHA nanoparticles.

Low-density lipoprotein docosahexaenoic acid nanoparticles induce ferroptotic cell death in hepatocellular carcinoma

Low-density lipoprotein nanoparticles reconstituted with the natural omega-3 fatty acid, docosahexaenoic acid (LDL-DHA), have been reported to selectively kill hepatoma cells and reduce the growth of orthotopic liver tumors in the rat. To date, little is known about the cell death pathways by which LDL-DHA nanoparticles kill tumor cells. Here we show that the LDL-DHA nanoparticles are cytotoxic to both rat hepatoma and human hepatocellular carcinoma (HCC) cell lines. Following LDL-DHA treatment both rat and human HCC cells experience pronounced lipid peroxidation, depletion of glutathione and inactivation of the lipid antioxidant glutathione peroxidase-4 (GPX4) prior to cell death. Inhibitor studies revealed that the treated HCC cells die independent of apoptotic, necroptotic or autophagic pathways, but require the presence of cellular iron. These hallmark features are consistent and were later confirmed to reflect ferroptosis, a novel form of nonapoptotic iron-dependent cell death. In keeping with the mechanisms of ferroptosis cell death, GPX4 was also found to be a central regulator of LDL-DHA induced tumor cell killing. We also investigated the effects of LDL-DHA treatments in mice bearing human HCC tumor xenografts. Intratumoral injections of LDL-DHA severely inhibited the growth of HCC xenografts long term. Consistent with our in vitro findings, the LDL-DHA treated HCC tumors experienced ferroptotic cell death characterized by increased levels of tissue lipid hydroperoxides and suppression of GPX4 expression. Conclusion: LDL-DHA induces cell death in HCC cells through the ferroptosis pathway, this represents a novel molecular mechanism of anticancer activity for LDL-DHA nanoparticles.

Localized delivery of low-density lipoprotein docosahexaenoic acid nanoparticles to the rat brain using focused ultrasound

Focused ultrasound exposures in the presence of microbubbles can achieve transient, non-invasive, and localized blood–brain barrier (BBB) opening, offering a method for targeted delivery of therapeutic agents into the brain. Low-density lipoprotein (LDL) nanoparticles reconstituted with docosahexaenoic acid (DHA) could have significant therapeutic value in the brain, since DHA is known to be neuroprotective. BBB opening was achieved using pulsed ultrasound exposures in a localized brain region in normal rats, after which LDL nanoparticles containing the fluorescent probe DiR (1,1′-Dioctadecyl-3,3,3′,3′-Tetramethylindotricarbocyanine Iodide) or DHA were administered intravenously. Fluorescent imaging of brain tissue from rats administered LDL-DiR demonstrated strong localization of fluorescence signal in the exposed hemisphere. LDL-DHA administration produced 2 × more DHA in the exposed region of the brain, with a corresponding increase in Resolvin D1 levels, indicating DHA was incorporated into cells and metabolized. Histological evaluation did not indicate any evidence of increased tissue damage in exposed brain regions compared to normal brain. This work demonstrates that localized delivery of DHA to the brain is possible using systemically-administered LDL nanoparticles combined with pulsed focused ultrasound exposures in the brain. This technology could be used in regions of acute brain injury or as a means to target infiltrating tumor cells in the brain.

Hepatic Arterial Infusion of Low-Density Lipoprotein Docosahexaenoic Acid Nanoparticles Selectively Disrupts Redox Balance in Hepatoma Cells and Reduces Growth of Orthotopic Liver Tumors in Rats

Dietary intake of the natural omega-3 fatty acid docosahexaenoic acid (DHA) has been implicated in protecting patients with viral hepatitis B or C from developing hepatocellular carcinoma (HCC). Little is known about the effects of DHA on established solid tumors. Here we describe a low-density lipoprotein-based nanoparticle that acts as a transporter for unesterified DHA (LDL-DHA) and demonstrates selectivecytotoxicity toward HCC cells. We investigated the ability of LDL-DHA to reduce growth of orthotopic hepatomas in rats. AxC-Irish (ACI) rats were given intrahepatic injections of rat hepatoma cells (H4IIE); 24 tumor-bearing rats (mean tumor diameter, ∼1 cm) were subject to a single hepatic artery injection of LDL nanoparticles (2 mg/kg) loaded with DHA (LDL-DHA), triolein (LDL-TO), or sham surgery controls. Tumor growth was measured by magnetic resonance imaging and other methods; tumor, liver, and serum samples were collected and assessed by histochemical, immunofluorescence, biochemical, and immunoblot analyses. Three days after administration of LDL-TO or sham surgery, the control rats had large, highly vascularized tumors that contained proliferating cells. However, rats given LDL-DHA had smaller, pale tumors that were devoid of vascular supply and >80% of the tumor tissue was necrotic. Four to 6 days after injection of LDL-DHA, the tumors were 3-fold smaller than those of control rats. The liver tissue that surrounded the tumors showed no histologic or biochemical evidence of injury. Injection of LDL-DHA into the hepatic artery of rats selectively deregulated redox reactions in tumor tissues by increasing levels of reactive oxygen species and lipid peroxidation, depleting and oxidizing glutathione and nicotinamide adenine dinucleotide phosphate, and significantlydown-regulating the antioxidant enzyme glutathione peroxidase-4. Remarkably, the redox balance in the surrounding liver was not disrupted. LDL-DHA nanoparticle selectively kills hepatoma cells and reduces growth of orthotopic liver tumors in rats. It induces tumor-specific necrosis by selectively disrupting redox balance within the cancer cell.

Low-Density Lipoprotein-Mediated Delivery of Docosahexaenoic Acid Selectively Kills Murine Liver Cancer Cells

The natural omega-3 polyunsaturated fatty acid, docosahexaenoic acid (DHA), has recently been credited for possessing anticancer properties. Herein, we investigate the cytotoxic actions of DHA-loaded low-density lipoprotein (LDL) nanoparticles in normal and liver cancer cells. LDL-DHA nanoparticles were prepared and subjected to extensive biophysical characterization. The therapeutic utility of LDL-DHA nanoparticles was evaluated in normal and malignant murine hepatocyte cell lines, TIB-73 and TIB-75, respectively. The engineered LDL-DHA nanoparticles possessed enhanced physical and oxidative stabilities over native LDL and free DHA. Dose-response studies showed that therapeutic doses of LDL-DHA nanoparticles that completely killed TIB-75 were innocuous to TIB-73. The selective induction of lipid peroxidation and reactive oxygen species in the cancer cells was shown to play a central role in LDL-DHA nanoparticle-mediated cytotoxicity. In summary, these findings indicate that LDL-DHA nanoparticles show great promise as a selective anticancer agent against hepatocellular carcinoma.