Increased Drug Uptake Research Articles

Abstract 3627: Development of CLENs for future treatment & diagnostic detection using cell models of drug-resistant ovarian cancer

Abstract Current guidelines suggest the use of a platinum agent and a taxane as first line therapy for ovarian cancer where total surgical resection is limited. Liposomal preparations of these drugs have demonstrated therapeutic value by increasing drug uptake and reducing toxicity. The following research aims to improve target specificity by employing a multi-strategic approach to drug targeting and treatment in vitro. Cellular membrane lipid-extracted nanoliposomes (CLENs) were constructed in part from lipid extracts (LE) derived from the membrane of multidrug resistant ovarian cancer (SKOV-3-MDR) cell lines, and more conventional components of liposomes. Formulations of greatest interest were selected after comparing cellular uptake studies of conventional liposomes to various types of CLENs composed of mixed ratios of DOPC, cholesterol, and LE. Cellular uptake was measured by rhodamine fluorescence (RF) following 24 hours incubation in 48 well plates, where RF was used as an indicator of cellular uptake. To determine effectiveness of LE, formulations with and without synthetic cationic lipid DOTAP were compared. Flow cytometry was used to evaluate liposome binding. Additional studies include drug loading efficiency and SRB cytotoxicity studies involving cisplatin, and the effect of additional inclusion of MAG-C (magnetic material) in the SKOV-3 lipid extract-containing liposomes. CLENs containing 15% LE demonstrated greater uptake compared to CLENs prepared with 0% and 5% LE content. Minimal uptake was observed in non-target cancer cell lines (prostate cancer-CRL-1740 and gliobastoma-U87MG), as well as with normal pancreas, non-malignant (hTERT) cell line. The fluorescence intensity values reported for SKOV-3-MDR, CRL-1740, U87-MG and hTERT, were 437 ± 153, 295 ± 67, 57 ± 6, and 29 ± 6, respectively. The additional inclusion of DOTAP showed significant cellular uptake with and without LE. Flow cytometry showed greater initial binding of CLENs to target cells when LE was included in preparations. Incorporation efficiency of cisplatin into SKOV-3 CLENS was 67%. Chemotherapy drug-loaded CLENs is an appealing vehicle as it addresses many parameters. Early findings suggest that CLENS is a suitable vehicle for targeting and treatment using a drug-resistant ovarian cancer cell line. CLENS allows for faster initial nanoparticle binding, cell-type specific uptake, and enhances cytotoxicity. Our encouraging results corroborate this theorem, showing greater targeting with reduced uptake by non-target cells. Additional studies involving MAG-C are currently underway. Citation Format: Negar Amini, Joshua Emerson, Jenel Clement. Development of CLENs for future treatment & diagnostic detection using cell models of drug-resistant ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3627.

Neuropilin-1 drives tumor-specific uptake of chlorotoxin

BackgroundChlorotoxin (Cltx) isolated from scorpion venom is an established tumor targeting and antiangiogenic peptide. Radiolabeled Cltx therapeutic (131I-TM601) yielded promising results in human glioma clinical studies, and the imaging agent tozuleristide, is under investigation in CNS cancer studies. Several binding targets have previously been proposed for Cltx but none effectively explain its pleiotropic effects; its true target remains ambiguous and is the focus of this study.MethodsA peptide-drug conjugate (ER-472) composed of Cltx linked to cryptophycin as warhead was developed as a tool to probe the molecular target and mechanism of action of Cltx, using multiple xenograft models.ResultsNeuropilin-1 (NRP1), an endocytic receptor on tumor and endothelial cells, was identified as a novel Cltx target, and NRP1 binding by Cltx increased drug uptake into tumor. Metabolism of Cltx to peptide bearing free C-terminal arginine, a prerequisite for NRP1 binding, took place in the tumor microenvironment, while native scorpion Cltx with amidated C-terminal arginine did not bind NRP1, and instead acts as a cryptic peptide. Antitumor activity of ER-472 in xenografts correlated to tumor NRP1 expression. Potency was significantly reduced by treatment with NRP1 blocking antibodies or knockout in tumor cells, confirming a role for NRP1-binding in ER-472 activity. Higher cryptophycin metabolite levels were measured in NRP1-expressing tumors, evidence of NRP1-mediated enhanced drug uptake and presumably responsible for the superior antitumor efficacy.ConclusionsNRP1 was identified as a novel Cltx target which enhances tumor drug uptake. This finding should facilitate tumor selection for chlorotoxin-based therapeutics and diagnostics.

Novel inhibitors of ABC transporters increase accumulation of transport substrates in multidrug resistant cancer cells and blood brain barrier cells

Multidrug resistance (MDR) is a major cause of chemotherapy failure. Overexpression of ATP‐binding cassette (ABC) transporters, P‐glycoprotein (P‐gp) and breast cancer resistance protein (BCRP) are two well‐studied drug transporters which are associated with MDR. These two transporters also act as a major functional unit of the blood brain barrier to protect the brain from xenobiotics and toxins. Lack of clinically approved P‐gp and BCRP inhibitors renders chemotherapy treatments of many MDR cancers ineffective and obstructs drug uptake into the brain. Using computational methods, we previously identified a novel class of P‐gp inhibitors that were not transport substrates of the pump. Here we describe the effects of chemical variants of the previously identified P‐gp inhibitor, SMU‐29. The variants were generated using computational approaches or by structure‐based design to improve protein binding interactions. All variants showed improved efficacy in reversing paclitaxel resistance in the P‐gp over‐expressing DU145 TXR prostate cancer cell line and their ability of increasing P‐gp substrate calcein in these cells. Five of these variants were not transported by P‐gp and three of them were substrates of the pump. Four variants only affected P‐gp, but not BCRP function, making them more selective than the parental compound, SMU 29. The three variants that affected BCRP reversed MDR in a BCRP over‐expressing breast cancer cell line, MCF‐7 M100, and increased accumulation of BCRP substrates such as Hoechst 33342, mitoxantrone and daunorubicin in these cells, indicating the potential to be used as BCRP inhibitors. The variants that affected both P‐gp and BCRP also showed higher accumulation of P‐gp and BCRP substrates in a blood brain barrier model cell line, hCMEC/D3, showing the potential to open the blood brain barrier for increased drug uptake into the brain.Support or Funding InformationThis work was supported by NIH NIGMS [R15GM094771‐02] to PVD and JGW, SMU University Research Council, the SMU Center for Drug Discovery, Design and Delivery, the Communities Foundation of Texas, and a private gift from Ms. Suzy Ruff of Dallas, Texas.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Dual drug-loaded PLA nanoparticles bypassing drug resistance for improved leukemia therapy

Drug resistance is still a bottle-neck hindering successful chemotherapy in leukemia treatment. Nanocarriers have emerged as promising candidates to circumvent drug resistance and discover potent drug combinations. Here, we showed that co-encapsulation of daunorubicin (DNR) and glycyrrhizic acid (GA) in polylactic acid (PLA) nanoparticles effectively bypassed drug resistance and remarkably inhibited the growth of drug-resistant leukemia cells. The quantification of intracellular drug found that the encapsulation effectively increased drug uptake in the resistant K562/A02 cells. Modification of P-glycoprotein antibody on nanoparticles further enhanced drug accumulation in the leukemia cells, which was also confirmed by fluorescent microscopy imaging. More importantly, loaded in PLA nanoparticles, DNR and GA exerted an excellent synergistic effect, leading to significantly improved cell inhibition compared to the treatments without nanocarriers and those with a single drug. Correlative mechanism study revealed that the drug delivery system drastically enhanced cell apoptosis by regulating apoptotic genes but did not influence MDR1 expression. The present study proposes a versatile strategy to improve the therapeutic efficacy against drug-resistant leukemia and inspires the discovery of more potent drug combinations for enhanced leukemia therapy.

Moxifloxacin imprinted silicon based hydrogels for sustained ocular release

Introduction: Traditional eye drop formulations are poorly efficient for ocular drug delivery, mainly due to the low residence time of the drug on the eye which results in a low bioavailability (1–5%) [1]. Soft contact lenses (SCLs) could overcome the rapid clearance of the drug in the eye. However, their insufficient capacity as drug reservoir may prevent the release of therapeutic concentrations [2]. Drug imprinted hydrogels have shown an increase in drug uptake and prolonged drug release [3]. In this work imprinted and non-imprinted silicon based hydrogels were designed for the loading of the antibiotic drug moxifloxacin hydrochloride (MXF) and evaluated regarding their potential use as therapeutic SCLs. Materials and methods: A silicon based (TRIS) hydrogel was prepared as previously reported [4]. An alternative composition containing acrylic acid, a functional monomer that may establish potential interactions with MXF was also prepared (TRIS/AA). A molecular imprinting strategy for MXF was followed and materials containing the drug were also synthesized (TRIS + D and TRIS/AA + D). After extensive washing, all hydrogels were loaded with the drug by soaking in MFX solution (2.5 mg/mL) at room temperature for 72 h. Release kinetics was investigated under sink conditions in NaCl solution. Transmittance, swelling and oxygen permeability were recorded. A mathematical model was applied to predict the in vivo efficacy of the drug release profile. Results: All hydrogels showed adequate physical properties for daily contact lenses wearing. No significant variations were observed in the release profile between TRIS + D and TRIS/AA hydrogels, while TRIS hydrogels showed faster release. The TRIS/AA + D hydrogel provided the most sustained release. The MXF concentration profile in the tear fluid estimated from the mathematical model indicated that the release of MXF from TRIS/AA + D hydrogel may be above the minimal inhibitory concentration (MIC) of different microorganisms typically colonizers of the eye for 48 h, while TRIS + D hydrogel was capable of maintaining the necessary concentration of MXF for ≈24 h and the non-imprinted hydrogels for ≈ 10 h. Discussion and conclusions: Imprinted silicone hydrogels bearing AA functional mers were demonstrated to be able to host large amount of MXF and to provide controlled release. These imprinted materials could be useful for the design of daily therapeutic SCLs.

Dysregulation of Macropinocytosis Processes in Glioblastomas May Be Exploited to Increase Intracellular Anti-Cancer Drug Levels: The Example of Temozolomide.

Macropinocytosis is a clathrin-independent endocytosis of extracellular fluid that may contribute to cancer aggressiveness through nutrient supply, recycling of plasma membrane and receptors, and exosome internalization. Macropinocytosis may be notably triggered by epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR), two well-known markers for glioblastoma aggressiveness. Therefore, we studied whether the expression of key actors of macropinocytosis is modified in human glioma datasets. Strong deregulation has been evidenced at the mRNA level according to the grade of the tumor, and 38 macropinocytosis-related gene signatures allowed discrimination of the glioblastoma (GBM) samples. Honokiol-induced vacuolization was then compared to vacquinol-1 and MOMIPP, two known macropinocytosis inducers. Despite high phase-contrast morphological similarities, honokiol-induced vacuoles appeared to originate from both endocytosis and ER. Also, acridine orange staining suggested differences in the macropinosomes’ fate: their fusion with lysosomes appeared very limited in 3-(5-methoxy -2-methyl-1H-indol-3-yl)-1-(4-pyridinyl)-2-propen-1-one (MOMIPP)-treated cells. Nevertheless, each of the compounds markedly increased temozolomide uptake by glioma cells, as evidenced by LC-MS. In conclusion, the observed deregulation of macropinocytosis in GBM makes them prone to respond to various compounds affecting their formation and/or intracellular fate. Considering that sustained macropinocytosis may also trigger cell death of both sensitive and resistant GBM cells, we propose to envisage macropinocytosis inducers in combination approaches to obtain dual benefits: increased drug uptake and additive/synergistic effects.

Liposomes targeted to MHC-restricted antigen improve drug delivery and antimelanoma response.

PurposeMelanoma is the most aggressive form of skin cancer. Chemotherapy at a late stage fails due to low accumulation in tumors, indicating the need for targeted therapy.Materials and methodsTo increase drug uptake by tumor cells, we have targeted doxorubicin-containing liposomes using a T-cell receptor (TCR)-like antibody (scFv G8 and Hyb3) directed against melanoma antigen A1 (MAGE-A1) presented by human leukocyte antigen A1 (M1/A1). With the use of flow cytometry and confocal microscopy, we have tested our formulation in vitro. In vivo pharmacokinetics was done in tumor-free nu/nu mice, while biodistribution and efficacy study was done in nu/nu mice xenograft.ResultsWe demonstrated two to five times higher binding and internalization of these immunoliposomes by M1+/A1+ melanoma cells in vitro in comparison with nontargeted liposomes. Cytotoxicity assay showed significant tumor cell kill at 10 µM doxorubicin (DXR) for targeted vs nontargeted liposomes. In vivo pharmacokinetics of nontargeted and targeted liposomes were similar, while accumulation of targeted liposomes was 2- to 2.5-fold and 6.6-fold enhanced when compared with nontargeted liposomes and free drug, respectively. Notably, we showed a superior antitumor activity of MAGE-A1-targeted DXR liposomes toward M1+/A1+ expressing tumors in mice compared with the treatment of M1−/A1+ tumors. Our results indicate that targeted liposomes showed better cytotoxicity in vitro and pharmacokinetics in vivo.ConclusionLiposomes decorated with TCR-mimicking scFv antibodies effectively and selectively target antigen-positive melanoma. We showed that DXR-loaded liposomes coupled to anti-M1/-A1 scFv inflict a significant antitumor response. Targeting tumor cells specifically promotes internalization of drug-containing nanoparticles and may improve drug delivery and ultimately antitumor efficacy. Our data argue that targeting MAGE in A1 context, by nanosized carriers decorated with TCR-like antibodies mimicking scFv, can be used as a theragnostic platform for drug delivery, immunotherapy, and potentially imaging, and diagnosis of melanoma.

The Membrane-Active Phytopeptide Cycloviolacin O2 Simultaneously Targets HIV-1-infected Cells and Infectious Viral Particles to Potentiate the Efficacy of Antiretroviral Drugs.

Background: Novel strategies to increase the efficacy of antiretroviral (ARV) drugs will be of crucial importance. We hypothesize that membranes of HIV-1-infected cells and enveloped HIV-1 particles may be preferentially targeted by the phytopeptide, cycloviolacin O2 (CyO2) to significantly enhance ARV efficacy. Methods: Physiologically safe concentrations of CyO2 were determined via red blood cell (RBC) hemolysis. SYTOX-green dye-uptake and radiolabeled saquinavir (3H-SQV) uptake assays were used to measure pore-formation and drug uptake, respectively. ELISA, reporter assays and ultracentrifugation were conducted to analyze the antiviral efficacy of HIV-1 protease and fusion inhibitors alone and co-exposed to CyO2. Results: CyO2 concentrations below 0.5 μM did not show substantial hemolytic activity, yet these concentrations enabled rapid pore-formation in HIV-infected T-cells and monocytes and increased drug uptake. ELISA for HIV-1 p24 indicated that CyO2 enhances the antiviral efficacy of both SQV and nelfinavir. CyO2 (< 0.5 μM) alone decreases HIV-1 p24 production, but it did not affect the transcription regulatory function of the HIV-1 long terminal repeat (LTR). Ultracentrifugation studies clearly showed that CyO2 exposure disrupted viral integrity and decreased the p24 content of viral particles. Furthermore, direct HIV-1 inactivation by CyO2 enhanced the efficacy of enfuvirtide. Conclusions: The membrane-active properties of CyO2 may help suppress viral load and augment antiretroviral drug efficacy.

Antimony resistance in Leishmania (Viannia) braziliensis clinical isolates from atypical lesions associates with increased ARM56/ARM58 transcripts and reduced drug uptake.

BACKGROUND In addition to the limited therapeutic arsenal and the side effects of antileishmanial agents, drug resistance hinders disease control. In Brazil, Leishmania braziliensis causes atypical (AT) tegumentary leishmaniasis lesions, frequently refractory to treatment.OBJECTIVES The main goal of this study was to characterise antimony (Sb)-resistant (SbR) L. braziliensis strains obtained from patients living in Xakriabá indigenous community, Minas Gerais, Brazil.METHODS The aquaglyceroporin 1-encoding gene (AQP1) from L. braziliensis clinical isolates was sequenced, and its function was evaluated by hypo-osmotic shock. mRNA levels of genes associated with Sb resistance were measured by quantitative reverse transcription polymerase chain reaction (qRT-PCR). Atomic absorption was used to measure Sb uptake.FINDINGS Although clinical isolates presented delayed recovery time in hypo-osmotic shock, AQP1 function was maintained. Isolate 340 accumulated less Sb than all other isolates, supporting the 65-fold downregulation of AQP1 mRNA levels. Both 330 and 340 isolates upregulated antimony resistance marker (ARM) 56/ARM58 and multidrug resistant protein A (MRPA); however, only ARM58 upregulation was an exclusive feature of SbR field isolates. CA7AE seemed to increase drug uptake in L. braziliensis and represented a tool to study the role of glycoconjugates in Sb transport.MAIN CONCLUSIONS There is a clear correlation between ARM56/58 upregulation and Sb resistance in AT-harbouring patients, suggesting the use of these markers as potential indicators to help the treatment choice and outcome, preventing therapeutic failure.

Titanium Dioxide Nanoparticles Enhance Leakiness and Drug Permeability in Primary Human Hepatic Sinusoidal Endothelial Cells.

Liver sinusoidal endothelial cells (LSECs) represent the permeable interface that segregates the blood compartment from the hepatic cells, regulating hepatic vascular tone and portal pressure amidst changes in the blood flow. In the presence of pathological conditions, phenotypic changes in LSECs contribute to the progression of chronic liver diseases, including the loss of endothelial permeability. Therefore, modulating LSECs offers a possible way to restore sinusoidal permeability and thereby improve hepatic recovery. Herein, we showed that titanium dioxide nanoparticles (TiO2 NPs) could induce transient leakiness in primary human hepatic sinusoidal endothelial cells (HHSECs). Interestingly, HHSECs exposed to these NPs exhibited reduced protein kinase B (Akt) phosphorylation, an important protein kinase which regulates cell attachment. Using a 3D co-culture system, we demonstrated that TiO2 NPs diminished the attachment of HHSECs onto normal human hepatic cell LO2. To further illustrate the significance of leakiness in liver sinusoids, we showed that NP-induced leakiness promoted Sunitinib transport across the HHSEC layer, resulting in increased drug uptake and efficacy. Hence, TiO2 NPs have the potential to modulate endothelial permeability within the specialized sinusoidal endothelium, especially during events of fibrosis and occlusion. This study highlighted the possible use of inorganic NPs as a novel strategy to promote drug delivery targeting the diseased liver.

Surfactant Free Delivery of Docetaxel by Poly[(R)-3-hydroxybutyrate-(R)-3-hydroxyhexanoate]-Based Polymeric Micelles for Effective Melanoma Treatments.

Docetaxel (DTX) is a new semisynthetic chemical in the taxoid family and serves a wide spectrum of chemotherapeutics. Current commercial formulation of DTX is based on the addition of the nonionic surfactants (i.e., ethanol and Tween 80), which are reported to cause severe hemolysis, hypersensitivity reactions, or neurotoxic toxicity and greatly hinders patient tolerance or compliance. In this report, a novel low-toxic, biodegradable, and amphiphilic poly[(R)-3-hydroxybutyrate-(R)-3-hydroxyhexanoate] (PHBHx)-based polyurethane (a copolymer made of hydrophobic PHBHx with biocompatible D-3-hydroxybutyric acid as degradation product, thermosensitive polypropylene glycol (PPG), and hydrophilic polyethylene glycol (PEG) segments) with nanosized micelle formation ability to encapsulate DTX, as a surfactant free formulation, is reported. Interestingly, this DTX-loaded poly(PHBHx/PEG/PPG urethane) micelle formulation with >90% drug loading efficiency shows significantly improved DTX solubility in aqueous medium, reduced hemolysis for better blood compatibility, and increased drug uptake in A375 melanoma cells, which provides the possibility of systematic delivery of DTX. As a proof-of-concept, an A375 melanoma xenograft mouse model is established to verify the therapeutic effect of this DTX-loaded poly(PHBHx/PEG/PPG urethane) micelle formulation, indicating the promising application of PHBHx-based polymeric nanosized micelle as a surfactant free formulation of chemotherapeutics which might greatly be beneficial for controllable delivery of pharmaceutics and cancer therapy.

Novel Combinations of Agents Targeting Translation That Synergistically Inhibit Fungal Pathogens.

A range of fungicides or antifungals are currently deployed to control fungi in agriculture or medicine, but resistance to current agents is growing so new approaches and molecular targets are urgently needed. Recently, different aminoglycoside antibiotics combined with particular transport inhibitors were found to produce strong, synergistic growth-inhibition of fungi, by synergistically increasing the error rate of mRNA translation. Here, focusing on translation fidelity as a novel target for combinatorial antifungal treatment, we tested the hypothesis that alternative combinations of agents known to affect the availability of functional amino acids would synergistically inhibit growth of major fungal pathogens. We screened 172 novel combinations against three phytopathogens (Rhizoctonia solani, Zymoseptoria tritici, and Botrytis cinerea) and three human pathogens (Cryptococcus neoformans, Candida albicans, and Aspergillus fumigatus), showing that 48 combinations inhibited strongly the growth of the pathogens; the growth inhibition effect was significantly greater with the agents combined than by a simple product of their individual effects at the same doses. Of these, 23 combinations were effective against more than one pathogen, including combinations comprising food-and-drug approved compounds, e.g., quinine with bicarbonate, and quinine with hygromycin. These combinations [fractional inhibitory combination (FIC) index ≤0.5] gave up to 100% reduction of fungal growth yield at concentrations of agents which, individually, had negligible effect. No synergy was evident against bacterial, plant or mammalian cells, indicating specificity for fungi. Mode-of-action analyses for quinine + hygromycin indicated that synergistic mistranslation was the antifungal mechanism. That mechanism was not universal as bicarbonate exacerbated quinine action by increasing drug uptake. The study unveils chemical combinations and a target process with potential for control of diverse fungal pathogens, and suggests repurposing possibilities for several current therapeutics.

Digesting a Path Forward: The Utility of Collagenase Tumor Treatment for Improved Drug Delivery.

Collagen and hyaluronan are the most abundant components of the extracellular matrix (ECM) and their overexpression in tumors is linked to increased tumor growth and metastasis. These ECM components contribute to a protective tumor microenvironment by supporting a high interstitial fluid pressure and creating a tortuous setting for the convection and diffusion of chemotherapeutic small molecules, antibodies, and nanoparticles in the tumor interstitial space. This review focuses on the research efforts to deplete extracellular collagen with collagenases to normalize the tumor microenvironment. Although collagen synthesis inhibitors are in clinical development, the use of collagenases is contentious and clinically untested in cancer patients. Pretreatment of murine tumors with collagenases increased drug uptake and diffusion 2-10-fold. This modest improvement resulted in decreased tumor growth, but the benefits of collagenase treatment are confounded by risks of toxicity from collagen breakdown in healthy tissues. In this review, we evaluate the published in vitro and in vivo benefits and limitations of collagenase treatment to improve drug delivery.

Advances and Perspectives in Urinary Bladder Cancer Nanotherapy

Bladder cancer treatment remains a challenge in the pharmaceutical field due to the recurrence and progression of the disease, as well as the pronounced side effects associated with the available therapeutic modalities. Although important strategies have been investigated in different clinical trial phases, efficient and well-tolerated treatment approaches need to be developed to improve therapeutic efficacy and the quality of life for bladder cancer patients. This review discusses conventional protocols used in the clinical setting, detailing the use of Bacillus Calmette–Guérin, new immunomodulators, and drug delivery systems. New therapeutic approaches have been investigated with the aim of better therapeutic efficacy with low rates of recurrence and progression of non-muscle invasive bladder cancer and muscle invasive bladder cancer. Therefore, this review highlights the progression of therapy with the use of conventional treatments and the recent progress achieved from the use of innovative strategies, such as nanoparticles for sustained, controlled drug delivery and increased drug uptake by tumour cells.

Antisecretory Factor-Mediated Inhibition of Cell Volume Dynamics Produces Antitumor Activity in Glioblastoma.

Interstitial fluid pressure (IFP) presents a barrier to drug uptake in solid tumors, including the aggressive primary brain tumor glioblastoma (GBM). It remains unclear how fluid dynamics impacts tumor progression and can be targeted therapeutically. To address this issue, a novel telemetry-based approach was developed to measure changes in IFP during progression of GBM xenografts. Antisecretory factor (AF) is an endogenous protein that displays antisecretory effects in animals and patients. Here, endogenous induction of AF protein or exogenous administration of AF peptide reduced IFP and increased drug uptake in GBM xenografts. AF inhibited cell volume regulation of GBM cells, an effect that was phenocopied in vitro by the sodium-potassium-chloride cotransporter 1 (SLC12A2/NKCC1) inhibitor bumetanide. As a result, AF induced apoptosis and increased survival in GBM models. In vitro, the ability of AF to reduce GBM cell proliferation was phenocopied by bumetanide and NKCC1 knockdown. Next, AF's ability to sensitize GBM cells to the alkylating agent temozolomide, standard of care in GBM patients, was evaluated. Importantly, combination of AF induction and temozolomide treatment blocked regrowth in GBM xenografts. Thus, AF-mediated inhibition of cell volume regulation represents a novel strategy to increase drug uptake and improve outcome in GBM. Mol Cancer Res; 16(5); 777-90. ©2018 AACR.

ESTROGEN ANCHORED NANOMICELLES FOR COMBINATIONAL DRUG DELIVERY WITH ENHANCED THERAPEUTIC EFFICACY: A PROTEOMICS GUIDED INVESTIGATION FOR MULTIFUNCTIONAL NANOTHERAPEUTICS

The employment of the stable isotope labeling with amino acids in cell culture (SILAC) based proteomic analysis as a guidance tool for investigation of cellular response of a multifunctional nanodelivery system has been described for cancer therapy. The multifunctional nanodelivery system in this study was based on an estrogen anchored multimodal nanomicelle (NPG). The key components of the multifunctional nanomicelle consist: I²-cyclodextrin conjugated estrone (CDE1), an escort molecule; polymeric BH3 mimetic, a proapoptotic BcL-2 inhibitor; and the mitotic catastrophe agent paclitaxel. They were assembled into the nanomicelle by multiple weak interactions including hydrophobic/hydrophilic interaction and host-guest recognition. The resulting nanomicelle exhibited unimodal morphology with average size of 170 nm with sustained release. It was found that the nanoparticle exhibits excellent anti-tumor activities for the treatment of breast adenocarcinoma with exceptional targeting efficacy both in MCF-7 tumor bearing mice. NPG as a new drug delivery system demonstrated several merits such as the increased drug uptake in breast tumor tissue, low toxicity, potent tumor growth retardation and metastasis inhibition, as well as potential clinical practicality without compromising liver, kidney and immune function and ameliorating the conventional chemotherapeutics induced phlebitis in breast tumor bearing nude mice model. The systematic SILAC based proteomics study and the subsequent validation revealed that the synergistic induction of mitotic catastrophe through enhanced G2/M phase arrest and PI3K/Akt/mTOR mediated autophagy, account for the exceedingly potent anti-tumor activity of this convergent nanomicelle. Additionally, the verification of the top upregulated gene from the proteomics profiling revealed that the overexpression of zinc finger protein 350 (ZNF350/ZBRK1) is associated with the enhanced antitumor effect induced by NPG.KEYWORDS:Â Drug targeting, Estrogen Receptor, Proteomics, Paclitaxel, Gossypol, SynergismÂ

Copper Nanoparticles as Antibacterial Agents

Although antibiotics can treat most bacterial infections, development of microbial resistance restricts the advantages of the antibacterial agents in controlling infectious diseases. This is a major challenge that poses a serious threat prompting the search for alternative strategies to treat bacterial infections. Nanotechnology as an emerging field has been extensively used to overcome microbial resistance due to specific properties of nanoparticles such as increased drug uptake and high surface area to volume ratio. The metallic particles in nanoscale have demonstrated antibacterial activity against various bacterial species, including Gram-positive and Gramnegative bacteria, and fungi. Recently, copper nanoparticles have been widely investigated for use in fighting microbial infections. This article tries to briefly summarize the current studies related to the antibacterial properties of the copper nanoparticles. The reviewed papers reveal that the copper nanoparticles possess potent antimicrobial activities and can be used for controlling and treating different infectious diseases in the future.

Radiosensitizing Effect of Electrochemotherapy: A Systematic Review of Protocols and Efficiency.

Electrochemotherapy (ECT) is a combination of electroporation (EP) and chemotherapy and has been reported as a potential radiosensitizing agent for radiation therapy. The main objective of this study was to systematically review of the literature to evaluate the effectiveness of ECT in sensitization of tumors to ionization radiation. In addition, the clinical considerations and mechanisms of action of radiosensitizing effect are discussed. Nine studies were included in this review. Bleomycin and cisplatin showed radiosensitizing effects in combined protocols with EP. EP enhances the cytotoxicity of bleomycin and cisplatin by factor of 1000 and 100, respectively. The mechanism of action of these drugs is induction of single and double strand breaks in DNA molecule. Moreover, the two main mechanisms of EP are increasing drug uptake in the tumor cells and generating reactive oxygen species. A single session ECT before radiotherapy can significantly enhance the tumor response. ECT is effective for different cell lines and tumors with different levels of radiosensitivity. Our findings show that ECT can be further translated into the clinic and can be matched by singledose irradiation as well as in the fractionated regime.

Abstract 1087: CPX-351 works synergistically in combination with FLT3 inhibitors against AML with FLT3-ITD

Abstract Introduction: CPX-351 (Vyxeos) is a liposomal combination of cytarabine and daunorubicin at a synergistic 5:1 molar ratio. Recently, CPX-351 has been shown to be significantly more effective than the 7+3 standard of care chemotherapy in treating high-risk AML patients, including patients with the FLT3-ITD mutation. We previously treated primary patient samples with CPX-351 ex vivo and found that FLT3-ITD+ samples were significantly more sensitive to CPX-351 and showed enhanced drug uptake. We hypothesized that dysregulated FLT3 signaling results in an activation of liposome uptake pathways, leading to increased sensitivity to CPX-351 and ultimately cell death. Furthermore, we examined the effect of combining CPX-351 with existing FLT3 inhibitors (e.g. quizartinib and midostaurin). Methods: To examine drug uptake dynamics, we exposed AML cell lines (including MOLM-13 and MOLM14 that contain FLT3-ITD, and ME1 that contains an activating FLT3 mutation) to varying concentrations of CPX-351, with or without pre-treatment of quizartinib and midostaurin. We evaluated cell viability using a colorimetric assay and measured intracellular daunorubicin fluorescence, an indicator of drug uptake, by flow cytometry. Additionally, we analyzed the synergy of exposing these cell lines to CPX-351 and FLT3 inhibitors in combination and at different dose schedules, followed by measuring cell viability and daunorubicin fluorescence. Results: We observed that cell lines containing FLT3-ITD or FLT3-activating mutation were more sensitive to CPX-351 and exhibited increased drug uptake compared to cell lines with other genetic abnormalities. Interestingly, we observed that pre-treatment with quizartinib for 16 hrs produced a population of cells (approximately 50% of the total population) that exhibited decreased daunorubicin fluorescence, suggesting that prolonged FLT3 inhibition may decrease CPX-351 uptake. Consistent with this, we observed robust synergy when combining CPX-351 with FLT3 inhibitors simultaneously or with CPX-351 exposure scheduled 24 hours prior to FLT3 inhibitor exposure. However, exposure to FLT3 inhibitors 24 hours prior to CPX-351 administration was less synergistic and even antagonistic at certain doses. Conclusions: These data provide additional supportive evidence that FLT3 activation results in increased uptake of CPX-351. This is consistent with results from the CPX-351 Phase III trial in which FLT3-ITD+ patients survived significantly longer when treated with CPX-351 compared to 7+3 chemotherapy. We also show that combining CPX-351 with existing FLT3 inhibitors can elicit a synergistic response when administered in dosing regimens where FLT3 inhibition does not precede CPX-351 treatment. Cumulatively, our data support further testing of CPX-351 in combination with FLT3 inhibitors for treating AML patients with genetic dysregulation of FLT3 signaling. Citation Format: David K. Edwards, Nathalie Javidi-Sharifi, Angela Rofelty, Max Gordon, Riley Roth-Carter, Paul Tardi, Lawrence Mayer, Jeffrey W. Tyner. CPX-351 works synergistically in combination with FLT3 inhibitors against AML with FLT3-ITD [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1087. doi:10.1158/1538-7445.AM2017-1087

Role of generation on folic acid-modified poly(amidoamine) dendrimers for targeted delivery of baicalin to cancer cells

Baicalin (BAI) has been reported to exert antitumor effects. However, BAI has limited water solubility, non-specific tumor targeting, and low bioavailability, which severely limited its clinical application. The aim of this study was to develop folic acid (FA) covalently conjugated-polyamidoamine (PAMAM) dendrimers (PAMAM-FA) as carrier systems for improvement of water solubility and tumor-specificity of BAI, and study the role of generation on the physiochemical properties and biological effects of PAMAM-FA/BAI complexes. In this work, four generations of PAMAM-FA were synthesized to entrap BAI. The average sizes of G3-FA/BAI, G4-FA/BAI, G5-FA/BAI, and G6-FA/BAI complexes were 174.4nm, 184.5nm, 258.8nm, and 247.5nm, respectively, and the zeta potentials of four PAMAM-FA/BAI complexes were −2.9mV, −6.6mV, −9.3mV, −9.0mV, respectively. The entrapment efficiencies of four PAMAM-FA/BAI complexes were 91.1%, 53.5%, 80.3%, and 91.9%, respectively, and the drug loading of PAMAM-FA/BAI complexes were about 22%. The formed PAMAM-FA/BAI complexes allowed sustained release of BAI in acidic PBS (pH5.4). In cellular uptake assay, PAMAM-FA/BAI complexes demonstrated increased drug uptake level in folate receptor (FR)-positive Hela cancer cells than FR-negative A549 cells, and the cellular uptake efficiency of PAMAM-FA is closely related with the generation of PAMAM. The MTT assay results showed that PAMAM-FA/BAI complexes demonstrated enhanced toxicity against Hela cells than non-FA-modified PAMAM/BAI complexes, and the G6-FA/BAI demonstrated the best inhibition efficiency. The cell cycle and cell apoptosis analysis further demonstrated the tumor-specific therapeutic efficacy of PAMAM-FA/BAI. These results suggested that the PAMAM-FA have the potential for targeted delivery of BAI into cancer cells to enhance its anti-tumor efficacy.