EXTH-38. ENGINEERED EXOSOMES FOR THERAPEUTIC GENE DELIVERY IN BRAIN TUMORS

Abstract The poor outcome of patients with glioblastoma (GBM) is at least partly due to the inability to deliver therapeutic agents to the tumor. We have shown that exosomes, naturally occurring nano-size extracellular vesicles, are capable of delivering antiglioma microRNAs (MiRs) to brain tumors (Lang, FM et al. Neuro Oncol, 2018;20(3):380–390). However, our studies suggested that there is significant opportunity to increase packaging efficiency and delivery specificity of exosomes. To this end, we engineered exosomes to express specific viral proteins (called eExos) in order to enhance packaging and delivery capabilities of antiglioma genes. These eExos are created by transfecting HEK 293 cells with plasmids containing viral proteins and a plasmid of the therapeutic gene. After 72 hrs, differential ultracentrifugation was used to isolate the exosomes. To test the efficacy of these novel eExos, we transfected them with a plasmid containing Cre recombinase (as the therapeutic gene), and treated U87 cells harboring a dsRed/eGFP Cre recombinase/LoxP site (U87dsR/GFP). In in vitro studies, treatment of U87dsR/GFP with a single dose of eExos resulted in 82% conversion rate of cells from red to green, compared to control exosomes (< 18% green cells). In in vivo studies, a single intratumoral injection of eExos into mice harboring 7-day old intracranial U87dsR/GFP gliomas, resulted in significant increases in green cells compared to control exosomes when tumors were harvested at day 10. Mechanistic studies employing florescent microscopy demonstrate that in contrast to natural exosomes, eExos deliver their cargo to the nucleus rather than to lysosomes, avoiding degradation of the delivered agent and facilitating expression of the plasmid. We conclude that eExos, engineered to contain specific viral proteins, are capable of packaging and delivering antiglioma genes more effectively than natural exosomes and may overcome the current inability to deliver biological therapeutic agents to brain tumors.

Download Full-text

Development of an LDL Receptor-Targeted Peptide Susceptible to Facilitate the Brain Access of Diagnostic or Therapeutic Agents

Biology ◽

10.3390/biology9070161 ◽

2020 ◽

Vol 9 (7) ◽

pp. 161

Author(s):

Séverine André ◽

Lionel Larbanoix ◽

Sébastien Verteneuil ◽

Dimitri Stanicki ◽

Denis Nonclercq ◽

...

Keyword(s):

Ldl Receptor ◽

Density Lipoprotein ◽

Therapeutic Agents ◽

In Vivo Studies ◽

Phage Display Technology ◽

Brain Penetration ◽

Wide Range ◽

The Brain

Blood-brain barrier (BBB) crossing and brain penetration are really challenging for the delivery of therapeutic agents and imaging probes. The development of new crossing strategies is needed, and a wide range of approaches (invasive or not) have been proposed so far. The receptor-mediated transcytosis is an attractive mechanism, allowing the non-invasive penetration of the BBB. Among available targets, the low-density lipoprotein (LDL) receptor (LDLR) shows favorable characteristics mainly because of the lysosome-bypassed pathway of LDL delivery to the brain, allowing an intact discharge of the carried ligand to the brain targets. The phage display technology was employed to identify a dodecapeptide targeted to the extracellular domain of LDLR (ED-LDLR). This peptide was able to bind the ED-LDLR in the presence of natural ligands and dissociated at acidic pH and in the absence of calcium, in a similar manner as the LDL. In vitro, our peptide was endocytosed by endothelial cells through the caveolae-dependent pathway, proper to the LDLR route in BBB, suggesting the prevention of its lysosomal degradation. The in vivo studies performed by magnetic resonance imaging and fluorescent lifetime imaging suggested the brain penetration of this ED-LDLR-targeted peptide.

Download Full-text

Inhibition of Human Lymphoma Cell Growth by the PKC-Beta Selective Inhibitor Enzastaurin (LY317615) in Combination with Multiple Therapeutic Agents.

Blood ◽

10.1182/blood.v112.11.1595.1595 ◽

2008 ◽

Vol 112 (11) ◽

pp. 1595-1595

Author(s):

Randall M Rossi ◽

Marlene Balys ◽

Dean Franklin ◽

Valerie Grose ◽

Richard I Fisher ◽

...

Keyword(s):

Single Agent ◽

Large Cell ◽

Therapeutic Agents ◽

In Vivo Studies ◽

Therapeutic Drugs ◽

Potential Synergy ◽

Size Growth ◽

Reduce Tumor Growth

Abstract Previous studies in our lab have shown that the PKC-beta inhibitor, enzastaurin (LY317615), when used to treat a panel of human diffuse large cell lymphoma (DLCL) lines, was able to induce cell death in vitro and substantially reduce tumor growth in xenograft assays. These findings support the hypothesis that activation of PKC contributes to tumor cell survival and proliferation, which has been implicated in the pathogenesis of human B cell lymphomas. Specifically, PKC-beta activation is increased in tumor cells from patients with poor prognosis DLCL, suggesting that PKC-beta may be a target for therapeutic intervention. In the present study, we have explored the interaction of enzastaurin with a panel of well characterized therapeutic agents to evaluate whether its anti-tumor activity can potentially be enhanced. Drugs were chosen for analysis based either on known single agent activity in lymphoma, or by preclinical evaluation indicating potential synergy with enzastaurin. For in vitro culture assays (48–72 hr treatment), the addition of gemcitabine, rapamycin, or bortezomib, increased the cytotoxicity of enzastaurin from 2 to 7 fold. This effect was evident with multiple human DLCL cell lines, (OCI-Ly3, 7, 10, 19, and SUDHL-4, and 6), as well as two independent primary DLCL cultures. For in vivo studies, subcutaneous transplantation of the DLCL cell line OCI-Ly19, (previously engineered to express luciferase which allows for real time in vivo imaging), or a primary DLCL isolate, into immune deficient NOD/SCID mice formed reproducible tumors. Recipient animals were separated into uniform cohorts when the tumors were of <=500 cubic mm in size. The animals were then simultaneously or sequentially treated with enzastaurin, (150 mg/kg b.i.d. via oral gavage) and a secondary drug, either gemcitabine, (2.5 or 5.0 mg/kg 1x/3 days IP), bortezomib, (0.4 mg/kg twice weekly IP), rapamycin, (1.0 mg/kg, daily IP), or rituxan, (5 mg/kg, weekly IP). Imaging and analysis of tumor volumes showed that addition of either rituxan or rapamycin provided no additional benefit in comparison to enzastaurin alone during the course of treatment. In contrast, the combination of either gemcitabine or bortezomib with enzastaurin demonstrated significantly reduced tumor volumes in comparison to enzastaurin alone (17% to 38% greater decrease with enzastaurin + gemcitabine, and 50% greater decrease in tumor volume with enzastaurin + bortezomib). These data suggest that the use of enzastaurin in combination with existing therapeutic drugs (gemcitabine or bortezomib) has the potential to limit tumor size/growth while lowering dose levels and thereby reducing potential side effects associated with traditional treatments.

Download Full-text

Gold Nanoparticles as Targeted Delivery Systems and Theranostic Agents in Cancer Therapy

Current Medicinal Chemistry ◽

10.2174/0929867326666190506123721 ◽

2019 ◽

Vol 26 (35) ◽

pp. 6493-6513 ◽

Cited By ~ 6

Author(s):

Alexandra Mioc ◽

Marius Mioc ◽

Roxana Ghiulai ◽

Mirela Voicu ◽

Roxana Racoviceanu ◽

...

Keyword(s):

Gold Nanoparticles ◽

Anticancer Agents ◽

Targeted Delivery ◽

Therapeutic Agents ◽

Delivery Systems ◽

In Vivo Studies ◽

Theranostic Agents ◽

Targeted Delivery Systems

Cancer is still a leading cause of death worldwide, while most chemotherapies induce nonselective toxicity and severe systemic side effects. To address these problems, targeted nanoscience is an emerging field that promises to benefit cancer patients. Gold nanoparticles are nowadays in the spotlight due to their many well-established advantages. Gold nanoparticles are easily synthesizable in various shapes and sizes by a continuously developing set of means, including chemical, physical or eco-friendly biological methods. This review presents gold nanoparticles as versatile therapeutic agents playing many roles, such as targeted delivery systems (anticancer agents, nucleic acids, biological proteins, vaccines), theranostics and agents in photothermal therapy. They have also been outlined to bring great contributions in the bioimaging field such as radiotherapy, magnetic resonance angiography and photoacoustic imaging. Nevertheless, gold nanoparticles are therapeutic agents demonstrating its in vitro anti-angiogenic, anti-proliferative and pro-apoptotic effects on various cell lines, such as human cervix, human breast, human lung, human prostate and murine melanoma cancer cells. In vivo studies have pointed out data regarding the bioaccumulation and cytotoxicity of gold nanoparticles, but it has been emphasized that size, dose, surface charge, sex and especially administration routes are very important variables.

Download Full-text

Antisense Oligodeoxynucleotide Technology: Potential Use for the Treatment of Malignant Brain Tumors

Cancer Control ◽

10.1177/107327489800500207 ◽

1998 ◽

Vol 5 (2) ◽

pp. 163-170 ◽

Cited By ~ 19

Author(s):

Herbert H. Engelhard

Keyword(s):

Gene Expression ◽

Brain Tumors ◽

Relevant Information ◽

In Vivo Studies ◽

Antisense Oligodeoxynucleotide ◽

Malignant Brain Tumors ◽

Antisense Odns ◽

The Brain

Background: Antisense oligodeoxynucleotides (ODNs) have been proposed as a new therapy for patients with cancer, including malignant brain tumors. Antisense ODNs are taken up by tumor cells and selectively block gene expression. Use of ODNs for brain tumors is attractive due to their theoretical specificity, relative ease of production and, to date, paucity of reported adverse effects. This article presents current information regarding antisense ODNs and their possible future use for the treatment of brain tumors. Methods: The available published experimental and clinical information regarding antisense ODN treatment of glioblastoma cells and administration into the central nervous system (CNS) was reviewed. Other clinically relevant information pertaining to the molecular biology of antisense ODNs was also collected and summarized. Results: Targets for antisense ODN therapy in malignant glioma cells have included c-myc, c-myb, c-sis, c-erb B, CD44, p34cdc2, bFGF, PDGF, TGF-beta, IGF-1, PKC-alpha tumor necrosis factor, urokinase, and S100beta protein. Few in vivo studies of ODN treatment of brain tumors have yet been reported. Systemically administered ODNs enter the brain only in extremely small quantities; therefore, microinfusion into the brain has been recommended. Conclusions: Antisense ODNs have been used successfully to block glioblastoma gene expression in vitro and expression of multiple genes within the CNS of experimental animals. Upcoming clinical trials will address the safety of antisense ODN use against malignant brain tumors.

Download Full-text

In vitro 3D Spheroid Culture Developed on the Parafilm Surface Using HEK-293 Cells

Academic Perspective Procedia ◽

10.33793/acperpro.03.01.48 ◽

2020 ◽

Vol 3 (1) ◽

pp. 220-227

Author(s):

Erdal Eroğlu

Keyword(s):

Three Dimensional ◽

Culture Method ◽

In Vivo Studies ◽

Preclinical Research ◽

Hek 293 ◽

Hek 293 Cells ◽

293 Cells ◽

Hanging Drop Culture

Preclinical research to predict the effects of drugs and chemicals on humans is commonly carried out either by cell culture studies in vitro condition or on animals in vivo condition. While drug studies tested on cells cultured as a monolayer in plastic flasks are incompatible with realistic results, falsifying findings can also be achieved from in vivo studies performed on different species. In recent years, research on drug tests using spheroid cultures formed by growing cells in three-dimensional (3D) in vitro has attracted great interest. 3D spheroid structures are formed by growing the cells in a drop suspended on superhydrophobic surfaces. In this study, HEK-293 cells were investigated on parafilm surfaces displaying superhydrophobic properties by growing in 2 &micro;l volume using hanging drop culture method in terms of spheroid formation. Light microscopy images from spheroid structures were taken on different incubation days and the area of spheroids was measured using the ImageJ program. Our study holds important findings for a chip platform that can be developed for use in vitro drug tests.

Download Full-text

The Efficacy of Cholesterol-Based Carriers in Drug Delivery

Molecules ◽

10.3390/molecules25184330 ◽

2020 ◽

Vol 25 (18) ◽

pp. 4330

Author(s):

Ngonidzashe Ruwizhi ◽

Blessing Atim Aderibigbe

Keyword(s):

Drug Delivery ◽

Biological Activity ◽

Wide Distribution ◽

Therapeutic Agents ◽

The Body ◽

In Vivo Studies ◽

Routes Of Administration ◽

Bioactive Agents

Several researchers have reported the use of cholesterol-based carriers in drug delivery. The presence of cholesterol in cell membranes and its wide distribution in the body has led to it being used in preparing carriers for the delivery of a variety of therapeutic agents such as anticancer, antimalarials and antivirals. These cholesterol-based carriers were designed as micelles, nanoparticles, copolymers, liposomes, etc. and their routes of administration include oral, intravenous and transdermal. The biocompatibility, good bioavailability and biological activity of cholesterol-based carriers make them potent prodrugs. Several in vitro and in vivo studies revealed cholesterol-based carriers potentials in delivering bioactive agents. In this manuscript, a critical review of the efficacy of cholesterol-based carriers is reported.

Download Full-text

In Vitro Anti-cancer, Anti-hypertensive and Antihyperglycaemic effect of Hypoxis colchicifolia

Journal of Natural Remedies ◽

10.18311/jnr/2021/25315 ◽

2021 ◽

Vol 21 (3) ◽

pp. 209

Author(s):

Suggessan Moodley ◽

Depika Dwarka ◽

Himansu Baijnath ◽

John Jason Mellem

Keyword(s):

Methanolic Extract ◽

Therapeutic Potential ◽

Herbal Medicines ◽

A549 Cells ◽

Positive Control ◽

In Vivo Studies ◽

Hek 293 ◽

Anti Cancer

Economic challenges associated with non-communicable diseases and the sociocultural outlook of many patients especially in Africa has increased the dependence on traditional herbal medicines for these diseases. Hypoxis colchicifolia is a traditional medicinal plant used in Southern Africa against an array of ailments. This study evaluated the in vitro antidiabetic (α-amyalse and α-glucosidase), antihypertensive (angiotensin-converting enzyme) and anticancer potential of H. colchicifolia corm as well as leaf (acetone, methanol and aqueous) extracts. Results showed that extracts have a moderate anti-diabetic and anti-hypertensive potential, with great anti-cancer potential. The acetone extract of both fresh and dried corms produced significant α-amylase and α-glucosidase inhibition with ACE inhibited predominantly by the dried corms methanolic extract (IC50 368.2 μg/mL). Methanolic extract of dried leaves showed the least cytotoxicity against the noncancerous cell line HEK-293 while exhibiting the highest inhibition of MCF-7 cells (IC50 3.24 μg/mL). All extracts exhibited a greater inhibitory potential in A549 cells than the positive control camptothecin (IC50 304.2μg/mL). This study reveals that H. colchicifolia has therapeutic potential as an anti-diabetic and anticancer agent; however, further in vivo studies need to be conducted.

Download Full-text