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.

Elucidating the structural organization of a novel low-density lipoprotein nanoparticle reconstituted with docosahexaenoic acid.

Low-density lipoprotein nanoparticles reconstituted with unesterified docosahexaenoic acid (LDL-DHA) is promising nanomedicine with enhanced physicochemical stability and selective anticancer cytotoxic activity. The unique functionality of LDL-DHA ultimately relates to the structure of this nanoparticle. To date, however, little is known about the structural organization of this nanoparticle. In this study chemical, spectroscopic and electron microscopy analyses were undertaken to elucidate the structural and molecular organization of LDL-DHA nanoparticles. Unesterified DHA preferentially incorporates into the outer surface layer of LDL, where in this orientation the anionic carboxyl end of DHA is exposed to the LDL surface and imparts an electronegative charge to the nanoparticles surface. This negative surface charge promotes the monodisperse and homogeneous distribution of LDL-DHA nanoparticles in solution. Further structural analyses with cryo-electron microscopy revealed that the LDL-DHA nanostructure consist of a phospholipid bilayer surrounding an aqueous core, which is distinctly different from the phospholipid monolayer/apolar core organization of plasma LDL. Lastly, apolipoprotein B-100 remains strongly associated with this complex and maintains a discrete size and shape of the LDL-DHA nanoparticles similar to plasma LDL. This preliminary structural assessment of LDL-DHA now affords the opportunity to understand the important structure-function relationships of this novel nanoparticle.

Investigation into the distinct subcellular effects of docosahexaenoic acid loaded low-density lipoprotein nanoparticles in normal and malignant murine liver cells.

BACKGROUND: Recent studies have shown that low density lipoproteins reconstituted with the natural omega 3 fatty acid docosahexaenoic acid (LDL-DHA) is selectively cytotoxic to liver cancer cells over normal hepatocytes. To date, little is known about the subcellular events which transpire following LDL-DHA treatment. METHODS: Herein, murine noncancer and cancer liver cells, TIB-73 and TIB-75 respectively, were investigated utilizing confocal microscopy, flow cytometry and viability assays to demonstrate differential actions of LDL-DHA nanoparticles in normal versus malignant cells. RESULTS: Our studies first showed that basal levels of oxidative stress are significantly higher in the malignant TIB-75 cells compared to the normal TIB-73 cells. As such, upon entry of LDL-DHA into the malignant TIB-75 cells, DHA is rapidly oxidized precipitating global and lysosomal lipid peroxidation along with increased lysosomal permeability. This leakage of lysosomal contents and lipid peroxidation products trigger subsequent mitochondrial dysfunction and nuclear injury. The cascade of LDL-DHA mediated lipid peroxidation and organelle damage was partially reversed by the administration of the antioxidant, N-acetylcysteine, or the iron-chelator, deferoxamine. LDL-DHA treatment in the normal TIB-73 cells was well tolerated and did not elicit any cell or organelle injury. CONCLUSION: These studies have shown that LDL-DHA is selectively cytotoxic to liver cancer cells and that increased levels of ROS and iron catalyzed reactions promote the peroxidation of DHA which lead to organelle dysfunction and ultimately the demise of the cancer cell. GENERAL SIGNIFICANCE: LDL-DHA selectively disrupts lysosomal, mitochondrial and nuclear function in cancer cells as a novel pathway for eliminating cancer cells.

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.

BACKGROUND & AIMS: 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 selective cytotoxicity toward HCC cells. We investigated the ability of LDL-DHA to reduce growth of orthotopic hepatomas in rats. METHODS: 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. RESULTS: 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 significantly down-regulating the antioxidant enzyme glutathione peroxidase-4. Remarkably, the redox balance in the surrounding liver was not disrupted. CONCLUSION: 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.

AIM: 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. MATERIALS & METHODS: 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. RESULTS & DISCUSSION: 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. CONCLUSION: In summary, these findings indicate that LDL-DHA nanoparticles show great promise as a selective anticancer agent against hepatocellular carcinoma.

ApoE3 mediated polymeric nanoparticles containing curcumin: apoptosis induced in vitro anticancer activity against neuroblastoma cells.

Curcumin, a natural phytoconstituent, is known to be therapeutically effective in the treatment of various cancers such as, breast cancer, lung cancer, pancreatic cancer, brain cancer, etc. However, low bioavailability and photodegradation of curcumin hampers its overall therapeutic efficacy. Anionic polymerization method was employed for the preparation of apolipoprotein-E3 mediated curcumin loaded poly(butyl)cyanoacrylate nanoparticles (ApoE3-C-PBCA) and characterized for size, zeta potential, entrapment efficiency, photostability, morphology, and in vitro release study. ApoE3-C-PBCA were found to be effective against SH-SY5Y neuroblastoma cells compared to curcumin solution (CSSS) and curcumin loaded PBCA nanoparticles (C-PBCA) from in vitro cell culture investigations. Flow cytometry techniques employed for the detection of anticancer activity revealed enhanced activity of curcumin against SH-SY5Y neuroblastoma cells with ApoE3-C-PBCA compared to CSSS and C-PBCA, and apoptosis being the underlying mechanism. Present study revealed that ApoE3-C-PBCA has tremendous potential to develop into an effective therapeutic treatment modality against brain cancer.

Apoptosis-induced anticancer effect of transferrin-conjugated solid lipid nanoparticles of curcumin.

Broad spectrum therapeutic potential of curcumin is usually hampered by its photodegradation and low bioavailability. Present investigation was designed with an objective to develop transferrin-mediated solid lipid nanoparticles (Tf-C-SLN) resistant to the photostability and capable of enhancing the bioavailability by targeted drug delivery to elicit anticancer activity against SH-SY5Y neuroblastoma cells in vitro. Hot homogenization method was used for the formulation of Tf-C-SLN and evaluated physicochemically using parameters such as, size, zeta potential, entrapment efficiency and photostability, transmission electron microscopy (TEM), nuclear magnetic resonance (NMR), differential scanning colorimetry (DSC), and in vitro release study. In vitro cytotoxicity and apoptosis investigations were performed using microplate analysis and flow cytometry techniques. The physicochemical characterization confirmed the suitability of formulation method and various parameters therein. TEM investigation revealed the spherical morphology while NMR and DSC study confirmed the entrapment of curcumin inside the nanoparticles. The cytotoxicity, reactive oxygen species, and cell uptake were found to be increased considerably with Tf-C-SLN compared with curcumin-solubilized surfactant solution, and curcumin-loaded SLN (C-SLN) suggesting the targeting effect. AnnexinV-FITC/PI double staining, DNA analysis, caspase detection, and reduced mitochondrial potential confirmed the induction of apoptosis with nanoparticle treatment. Enhanced anticancer activity with Tf-C-SLN compared with curcumin-solubilized surfactant solution and C-SLN was observed from flow cytometry investigations with apoptosis being the major underlying mechanism. The in vitro observations of our investigation are very compelling and concrete to advocate the potential of Tf-C-SLN in enhancing the anticancer effect of curcumin against neuroblastoma in vivo and possible clinical applications.

Transferrin mediated solid lipid nanoparticles containing curcumin: enhanced in vitro anticancer activity by induction of apoptosis.

Photodegradation and low bioavailability are major hurdles for the therapeutic use of curcumin. Aim of the present study was to formulate transferrin-mediated solid lipid nanoparticles (Tf-C-SLN) to increase photostability, and enhance its anticancer activity against MCF-7 breast cancer cells. Tf-C-SLN were prepared by homogenization method and characterized by size, zeta potential, entrapment efficiency and stability, transmission electron microscopy (TEM), X-ray diffraction (XRD) and in vitro release study. Microplate analysis and flow cytometry techniques were used for cytotoxicity and apoptosis study. The physical characterization showed the suitability of method of preparation. TEM and XRD study revealed the spherical nature and entrapment of curcumin in amorphous form, respectively. The cytotoxicity, ROS and cell uptake was found to be increased considerably with Tf-C-SLN compared to curcumin solubilized surfactant solution (CSSS) and curcumin-loaded SLN (C-SLN) suggesting the targeting effect. AnnexinV-FITC/PI double staining, DNA analysis and reduced mitochondrial potential confirmed the apoptosis. The flow cytometric studies revealed that the anticancer activity of curcumin is enhanced with Tf-C-SLN compared to CSSS and C-SLN, and apoptosis is the mechanism underlying the cytotoxicity. The present study indicated the potential of Tf-C-SLN in enhancing the anticancer effect of curcumin in breast cancer cells in vitro.

ApoE3 mediated poly(butyl) cyanoacrylate nanoparticles containing curcumin: study of enhanced activity of curcumin against beta amyloid induced cytotoxicity using in vitro cell culture model.

Beta amyloid plays a main role in the pathophysiology of Alzheimer's disease by inducing oxidative stress in the brain. Curcumin, a natural antioxidant, is known to inhibit beta amyloid and beta amyloid induced oxidative stress. However, low bioavailability and photodegradation are the major concerns for the use of curcumin. In the present study, we have formulated apolipoprotein E3 mediated poly(butyl) cyanoacrylate nanoparticles containing curcumin (ApoE3-C-PBCA) to provide photostability and enhanced cell uptake of curcumin by targeting. Prepared nanoparticles were characterized for particle size, zeta potential, entrapment efficiency and in vitro drug release. The entrapment of curcumin inside the nanoparticles was confirmed by X-ray diffraction analysis. Physicochemical characterization confirmed the suitability of the method of preparation. The photostability of curcumin was increased significantly in nanoparticles compared to plain curcumin. In vitro cell culture study showed enhanced therapeutic efficacy of ApoE3-C-PBCA against beta amyloid induced cytotoxicity in SH-SY5Y neuroblastoma cells compared to plain curcumin solution. Beta amyloid is known to induce apoptosis in neuronal cells, therefore antiapoptotic activity of curcumin was studied using flow cytometry assays. From all the experiments, it was found that the activity of curcumin was enhanced with ApoE3-C-PBCA compared to plain curcumin solution suggesting enhanced cell uptake and a sustained drug release effect. The synergistic effect of ApoE3 and curcumin was also studied, since ApoE3 also possesses both antioxidant and antiamyloidogenic activity. It was found that ApoE3 did indeed have activity against beta amyloid induced cytotoxicity along with curcumin. Hence, ApoE3-C-PBCA offers great advantage in the treatment of beta amyloid induced cytotoxicity in Alzheimer's disease.