In this study, we identified the proton-coupled folate transporter (PCFT) as a route for targeted delivery of drugs to some gliomas. Using the techniques of confocal imaging, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and small interfering (siRNA) knockdown against the PCFT, we demonstrated that Gl261 and A172 glioma cells, but not U87 and primary cultured astrocytes, express the PCFT, which provides selective internalization of folic acid (FA)-conjugated cytochrome c-containing nanoparticles (FA-Cyt c NPs), followed by cell death. The FA-Cyt c NPs (100 µg/mL), had no cytotoxic effects in astrocytes but caused death in glioma cells, according to their level of expression of PCFT. Whole-cell patch clamp recording revealed FA-induced membrane currents in FA-Cyt c NPs-sensitive gliomas, that were reduced by siRNA PCFT knockdown in a similar manner as by application of FA-Cyt c NPs, indicating that the PCFT is a route for internalization of FA-conjugated NPs in these glioma cells. Analysis of human glioblastoma specimens revealed that at least 25% of glioblastomas express elevated level of either PCFT or folate receptor (FOLR1). We conclude that the PCFT provides a mechanism for targeted delivery of drugs to some gliomas as a starting point for the development of efficient methods for treating gliomas with high expression of PCFT and/or FOLR1.

The delivery of Cytochrome c (Cyt c) to the cytosol stimulates apoptosis in cells where its release from mitochondria and apoptotic induction is inhibited. We developed a drug delivery system consisting of Cyt c nanoparticles decorated with folate-poly(ethylene glycol)-poly(lactic-co-glycolic acid)-thiol (FA-PEG-PLGA-SH) to deliver Cyt c into cancer cells and tested their targeting in the Lewis Lung Carcinoma (LLC) mouse model. Cyt c-PLGA-PEG-FA nanoparticles (NPs) of 253 ± 55 and 354 ± 11 nm were obtained by Cyt c nanoprecipitation, followed by surface decoration with the co-polymer SH-PLGA-PEG-FA. The internalization of Cyt c-PLGA-PEG-FA nanoparticles (NPs) in LLC cells was confirmed by confocal microscopy. NP caspase activation was more efficient than the NP-free formulation. Caspase activity assays showed NPs retained 88–96% Cyt c activity. The NP formulations were more effective in decreasing LLC cell viability than NP-free formulation, with IC50 49.2 to 70.1 μg/mL versus 129.5 μg/mL, respectively. Our NP system proved to be thrice as selective towards cancerous than normal cells. In vivo studies using near infrared-tagged nanoparticles show accumulation in mouse LLC tumor 5 min post-injection. In conclusion, our NP delivery system for Cyt c shows superiority over the NP-free formulation and reaches a folic acid-overexpressing tumor in an immune-competent animal model.

rugosa lipase nanoparticles as robust catalyst for biodiesel production in organic solvents.Inexpensive but resourceful sources of lipids, for example, used cooking oil (UCO) and brown grease (BG), which often contain large amounts of free fatty acids (FFA), are difficult to convert into biodiesel economically and in good yield. Candida rugosa lipase nanoparticles (cNP) were formed first and subsequently cross-linked nanoparticles (CLNP) were obtained by crosslinking of them. Alternatively, cNP were conjugated to magnetic nanoparticles (mNP) to achieve a cNP-mNP conjugate. All three formulations were employed in three different organic solvents (n-heptane, 1,4-dioxane, and t-butanol) to produce biodiesel using BG and UCO in the transesterification reaction with ethanol and methanol. The radii of nanoparticles (NP) were 5.5, 75, 100, 85 nm for mNP, cNP, CLNP, and cNP-mNP, respectively, as measured by scanning/transmission electron microscopy and dynamic light scattering. The catalytic efficiency (Kcat/KM) of cNP, CLNP, and cNP-mNP was increased ca. -25, -68, -176 folds in n-heptane and -35, -131, -262 folds in 1,4-dioxane compared to the lyophilized lipase in the model transesterification reaction of p-nitrophenyl palmitate (PNPP) with ethanol. In biodiesel formation, the best performance with 100% conversion of BG was achieved under optimum conditions with cNP-mNP, ethanol at a 1:3 molar ratio of lipid-to-alcohol, NP at a 1:0.1 weight ratio of lipid-to-enzyme, and water at a 1:0.04 weight ratio of enzyme-to-water at 30 o C for 35 h. The operational stability of the CLNP and cNP-mNP was sustained even after five consequent biodiesel batch conversions while 50% and 82% residual activity (storage stability) were retained after 40 d.

The delivery of Cytochrome c (Cyt c) to the cytosol stimulates apoptosis in cells were its release from mitochondria and apoptosis induction is inhibited. We developed a drug delivery system consisting of Cyt c nanoparticles decorated with folate-poly(ethylene glycol)-poly(lactic-co-glycolic acid)-thiol (FA-PEG-PLGA-SH) to deliver Cyt c into cancer cells and test their targeting in the Lewis Lung Carcinoma (LLC) mouse model. Cyt c-PLGA-PEG-FA nanoparticles (NPs) of 253 ± 55 and 354 ± 11 nm were obtained by Cyt c nanoprecipitation, followed by surface decoration with the co-polymer SH-PLGA-PEG-FA, and compared to a nanoparticle-free formulation. Overexpression of FA in LLC cells and internalization of Cyt c-PLGA-PEG-FA nanoparticles (NPs) was confirmed by confocal microscopy. Caspase activation assays show NPs retain 88-96% Cyt c activity. The NP formulations were more efficient in decreasing LLC cell viability than the NP-free formulation, with IC50: 49.2 to 70.1 μg/ml versus 129.5 μg/ml, respectively. Our NP system is thrice as selective towards cancerous than normal cells. In-vivo studies using tagged nanoparticles show accumulation in mouse LLC tumor 5 min post-injection. In conclusion, our NP delivery system for Cyt c shows superiority over the NP-free formulation and reaches a folic acid-overexpressing tumor in an immune-competent animal model.

Botryococcus braunii (B. braunii) is a green microalga primarily found in freshwater, reservoirs, and ponds. Photosynthetic pigments from algae have shown many bioactive molecules with therapeutic potential. Herein, we report the purification, characterization, and anticancer properties of photosystem I light-harvesting complex I (PSI-LHCI) from the green microalga B. braunii UTEX2441. The pigment–protein complex was purified by sucrose density gradient and characterized by its distinctive peaks using absorption, low-temperature (77 K) fluorescence, and circular dichroism (CD) spectroscopic analyses. Protein complexes were resolved by blue native-PAGE and two-dimensional SDS-PAGE. Triple-negative breast cancer MDA-MB-231 cells were incubated with PSI-LHCI for all of our experiments. Cell viability was assessed, revealing a significant reduction in a time- and concentration-dependent manner. We confirmed the internalization of PSI-LHCI within the cytoplasm and nucleus after 12 h of incubation. Cell death mechanism by oxidative stress was confirmed by the production of reactive oxygen species (ROS) and specifically superoxide. Furthermore, we monitored autophagic flux, apoptotic and necrotic features after treatment with PSI-LHCI. Treated MDA-MB-231 cells showed positive autophagy signals in the cytoplasm and nucleus, and necrotic morphology by the permeabilization of the cell membrane. Our findings demonstrated for the first time the cytotoxic properties of B. braunii PSI-LHCI by the induction of ROS and autophagy in breast cancer cells.

Amongst primary brain tumors, gliomas are considered the most lethal malignant tumors due to their resistance to radiation and chemotherapy and the difficulty to accurately localize them within the tissue. Chlorotoxin (Ctx), a small neurotoxin of 36 amino acids isolated from the venom of the Israeli scorpion Leiurus quinquestriatus, binds specifically and with high affinity a 72 kDa chloride channel membrane receptor in human gliomas and was proposed to cross the Blood Brain Barrier. These channels are reported to be absent or in low abundance in healthy tissues or in tumors of non‐glial origin. As a component of the scorpion venom, Ctx induces paralysis in small insects or other invertebrates however, no apparent signs of toxicity have been observed when injected in vertebrates. Cytochrome c (CytC), a small membrane impermeable mitochondrial protein (MW= 12 KDa) that facilitates electron transport in cellular respiration, is also known to mediate apoptosis, a normal process of controlled cell death, through the activation of a caspase cascade. In this work, a chemically synthesized‐Ctx is covalently conjugated to CytC through reversible disulfide bridges to accomplish targeted delivery to a glioma cell line 9L/lacZ from Rattus norvegicus and NIH 3T3 from Mus Musculus. Size exclusion chromatography and mass spectrometry characterization of the Ctx‐CytC conjugate show that the Ctx was successfully attached to CytC. Preliminary results from cell viability essay reveal that the Ctx‐Cytc was 40% more toxic than Ctx alone and more toxic to 9L/lacZ than the normal cell line NIH/3T3. These results suggest that the Ctx‐CytC conjugate can be further developed in order to be considered as an alternative potential targeted drug for treatment of gliomas.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Reactive oxygen species (ROS) are generated during normal metabolism; however, at high levels, they can promote cell damage and death. In green microalgae, such as Botryococcus braunii (B. braunii), the thylakoid membrane (TM) and particularly photosystem I (PSI) are the main contributors to the overall production of ROS. B. braunii PSI‐LHCI was purified by differential centrifugation in a 15–35% sucrose gradient for 16.5 hours, 4 ºC at 37,500 rpm and characterized by visible absorption spectroscopy, low temperature (77K) fluorescence emission spectroscopy and LC‐MS/MS. PSI can act as a light‐sensitive molecule called photosensitizer; that upon photoactivation by exposure to LED light (λmax=660 ± 10 nm) at a distance of 10 cm and a fluence rate of 50 mW/cm2 for 40 minutes triggered the formation of ROS species. ROS generation in TM and PSI, specifically singlet oxygen was monitored spectrophotometrically using the p‐nitrosodimethylaniline (RNO) assay at 440 nm, which produced RNO dye bleaching upon singlet oxygen generation. To test PSI‐LHCI use as a prospective anticancer drug, two cancer cell lines, HeLa and MDA‐MB‐231, and one normal cell line, NIH‐3T3 were utilized for in vitro cell viability studies. Each cell line was incubated for 24 h with various concentrations of PSI‐LHCI (3.125, 6.25, 12.5, 25, and 50 μg/ml) which demonstrated a concentration‐dependent reduction in cell viability. At a 12.5 μg/ml PSI‐LHCI concentration, cell viability experienced a steep drop to less than 5% in HeLa cells, and a 47% and 53% decreased cell viability in NIH‐3T3 and MDA‐MB‐231 cell lines. Confocal microscopy demonstrated PSI‐LHCI internalization, and apoptosis induction studies confirmed cellular damage due to morphological change hallmarks such as condensed nuclear chromatin, decreased and fragmented nucleus after DAPI staining and PI counterstaining positively identified dead or necrotic cells represented by the intense, condensed nucleus staining.Support or Funding InformationRISE Program: 5R25GM061151‐16This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Ribonuclease A is a very accessible protein that has been tested as a therapeutic for cancer in vitro and in vivo due to its ability to hinder protein production and induce cell death. However, the results in vivo were only positive when high amounts of enzyme were injected directly in the tumors. To overcome this limitation we are testing the use of a drug delivery system consisting of nanoparticles made out of the protein itself coated with poly(lactic‐co‐glycolic acid)‐poly(ethylene glycol)‐folate conjugates (PLGA‐PEG‐FA), in which the folate moiety confers selectivity towards cells overexpressing folic acid receptors on their membrane. Using a solvent displacement nanoprecipitation method we obtained nanoparticles constituted of ribonuclease A. Nanoparticles obtained under different conditions were analyzed by scanning electron microscope and dynamic light scattering, confirming spherical shape and an average size of 600 nm. The FA‐PEG‐PLGA copolymer to coat the nanoparticles was synthesized by conjugating the free alcohol group in PLGA with the carboxyl group in the folate‐conjugated‐PEG. The structure of the product was confirmed by 1H‐NMR. Experiments for coating optimization are in process and the efficacy of the system will be tested in HeLa cells as well as normal cells to confirm the selectivity. This drug delivery system does not have the limitation of a maximum loading capacity, and has been proven by us to efficiently deliver other proteins to cancer cells. Therefore, it is expected to selectively deliver higher amounts of ribonuclease A to cancer cells than previously reported systems.Support or Funding InformationSupported by the MSRC (Molecular Science Research Center)