Carola Leuschner

Membrane disrupting lytic peptides for cancer treatments.

Membrane disrupting lytic peptides are abundant in nature and serve insects, invertebrates, vertebrates and humans as defense molecules. Initially, these peptides attracted attention as antimicrobial agents; later, the sensitivity of tumor cells to lytic peptides was discovered. In the last decade intensive research has been conducted to determine how lytic peptides lyse bacteria and tumor cells. A number of synthetic peptides have been designed to optimize their antibiotic and anti-tumor properties and improve their therapeutic capabilities. The sequences of alpha-helical cationic membrane disrupting peptides has been discussed, their proposed mechanisms of action reviewed, and their roles in cell selectivity and tumor cell destruction considered. Parameters important for the selection and design of lytic peptides for cancer treatments include increased activities against tumor cells, low cytolytic activities to normal mammalian cells and erythrocytes. The conjugation of lytic peptides with hormone ligands and the production of pro-peptides provide methods for targeting of cancer cells. The therapeutic possibilities in cancer treatment by targeted lytic peptides are broad and offer improvement to currently used chemotherapeutical drugs. Lytic peptides interact with the tumor cell membrane within minutes, and their activity is independent of multi-drug resistance. Lytic peptide-chorionic gonadotropin (CG) conjugates destroy primary tumors, prevent metastases and kill dormant and metastatic tumor cells. These conjugates do not destroy vital organs; they are not antigenic, and are more toxic to tumor cells than to non-malignant cells.

Molecular MRI for sensitive and specific detection of lung metastases

Early and specific detection of metastatic cancer cells in the lung (the most common organ targeted by metastases) could significantly improve cancer treatment outcomes. However, the most widespread lung imaging methods use ionizing radiation and have low sensitivity and/or low specificity for cancer cells. Here we address this problem with an imaging method to detect submillimeter-sized metastases with molecular specificity. Cancer cells are targeted by iron oxide nanoparticles functionalized with cancer-binding ligands, then imaged by high-resolution hyperpolarized 3He MRI. We demonstrate in vivo detection of pulmonary micrometastates in mice injected with breast adenocarcinoma cells. The method not only holds promise for cancer imaging but more generally suggests a fundamentally unique approach to molecular imaging in the lungs.

Membrane disrupting lytic peptide conjugates destroy hormone dependent and independent breast cancer cells in vitro and in vivo.

We have prepared conjugates of a membrane disrupting lytic peptide (hecate) and a 15-amino acid segment of the β-chain of CG and hecate and the decapeptide, luteinizing hormone releasing hormone (LHRH). We have tested the concept that these conjugates will target breast cancer cells expressing LH/CG or LHRH receptors. In previous studies, we were able to destroy prostate cancers in vitro and in vivo with lytic peptide conjugates [1]. Hecate, hecate–βCG and LHRH–hecate were added to cultures of the human breast cancer cell lines MCF-7 and MDA-MB-435S. Hecate and its conjugates showed concentration dependent toxicity to both cell lines. The lytic peptide alone showed similar EC50 values for both cell lines; however, there was a significant difference between the EC50 values when the conjugates were tested. The hormone dependent MCF-7 cell line was less sensitive to the βCG conjugate than to the LHRH conjugate; the reverse was found for the hormone independent MDA-MB-435S cells. Removal of steroids decreased the sensitivity of MCF-7 cells to both lytic peptide conjugates and this sensitivity could be restored by adding estradiol. Activation of protein kinase C further increased the sensitivity to the drug. MDA-MB-435S xenografts were established in intact female athymic nude mice, which were treated once a week for 3 weeks with hecate–βCG via the lateral tail vein. The ability of hecate–βCG to destroy xenografts of human breast cancer cells (MDA-MB-435S) in nude mice was demonstrated for the first time. We conclude that hecate–βCG and LHRH–hecate conjugates could serve as useful drugs for the treatment of breast cancer.

Destruction of breast cancers and their metastases by lytic peptide conjugates in vitro and in vivo

In a series of in vivo and in vitro experiments, the concept has been established that breast cancer cells that express LH/CG or LHRH receptors can be targeted and destroyed by constructs consisting of a lytic peptide moiety and a 15-amino acid segment of the beta-chain of CG or by an LHRH lytic peptide conjugate. Data obtained in vitro established the validity of this concept, showed the specificities of the Hecate-betaCG, and Phor14 and Phor21-betaCG conjugates in killing cells that express functional LH/CG receptors and proved that the LH/CG receptor capacity is directly related to the compounds specificity. In in vivo experiments, Hecate-betaCG, Phor14-betaCG, and Phor21-betaCG(ala) each caused highly significant reductions of tumor volume and tumor burden in nude mice bearing breast cancer xenografts; Hecate and Phor21 alone or conjugated with non-specific peptides were not effective. Most importantly, the lytic peptide conjugates were all highly effective in targeting and destroying disseminated breast cancer metastases in lymph nodes, bones, lungs and other organs.

Targeting Breast and Prostate Cancers Through Their Hormone Receptors

Abstract A targeted treatment that effectively destroys human breast, prostate, ovarian, and testicular cancer cells that express luteinizing hormone/chorionic gonadotropin (LH/CG) receptors has been developed. The treatment consists of a conjugate of a membrane-disrupting lytic peptide (Hecate, Phor14, or Phor21) and a 15-amino acid segment of the beta chain of CG. Because these conjugates act primarily by destroying cell membranes, their effects are independent of cell proliferation. The conjugates are relatively small molecules, are rapidly metabolized, and are not antigenic. In a series of independent experiments conducted in three different laboratories, the validity of the concept has been established, and it has been shown that the LH/CG receptor capacity of the cancer cells is directly related to the sensitivity of the lytic peptide conjugates. Sensitivity to the drugs can be increased by pretreating prostate or breast cancer cells with FSH or estradiol to up-regulate LH/CG receptors. A series of 23 in vivo experiments involving a total of 1630 nude mice bearing xenografts of human prostate or breast cancer cells showed convincingly that all three lytic peptide-betaCG compounds were highly effective in destroying tumors and reducing tumor burden. Hecate-betaCG was less effective in mice bearing ovarian epithelial cancer cell xenografts, but was highly effective in treating granulosa cell tumors in transgenic mice. In addition, Hecate-betaCG and Phor14-betaCG were highly effective in targeting and destroying prostate and breast cancer cell metastases in the presence or absence of the primary tumors. Although effective in vitro, neither Hecate nor Phor14 alone were effective in reducing primary tumor volume or burden in nude mice bearing prostate or breast cancer xenografts.

Targeted destruction of androgen-sensitive and -insensitive prostate cancer cells and xenografts through luteinizing hormone receptors

We have prepared a conjugate of a lytic peptide (hecate) and a 15‐amino acid segment of the β‐chain of LH to test the concept that this conjugate will target cancer cells expressing LH receptors.

Conjugates of lytic peptides and LHRH or βCG target and cause necrosis of prostate cancers and metastases

In a series of in vivo and in vitro experiments, it was shown that membrane disrupting lytic peptides (Hecate, Phor14, or Phor21) conjugated to a 15 amino acid segment of the beta chain of CG or to LHRH were able to target and destroy hormone dependent and independent human prostate cancer xenografts in nude mice. In vitro sensitivity of the cells to the drugs was directly related to LH/CG receptor expression, and pretreatment in vitro or in vivo with estrogens or FSH to enhance LH/CG receptor expression capacity and increased sensitivity to the drugs. Administration of unconjugated Hecate and LHRH was ineffective. Most importantly, all of the lytic peptide-betaCG conjugates tested were highly effective in destroying prostate cancer metastatic cells in lymph nodes, bones and lungs.

Nanofabrication towards biomedical applications : techniques, tools, applications, and impact

Preface.List of Contributors.I Fabrication of Nanomaterials.1 Synthetic Approaches to Metallic Nanomaterials (Ryan Richards and Helmut Bonnemann).1.1 Introduction.1.2 Wet Chemical Preparations.1.3 Reducing Agents.1.4 Electrochemical Synthesis.1.5 Decomposition of Low-Valency Transition Metal Complexes.1.6 Particle Size Separations.1.7 Potential Applications in Materials Science.2 Synthetic Approaches for Carbon Nanotubes (Bingqing Wei, Robert Vajtai, and Pulickel M. Ajayan).2.1 Introduction.2.2 Family of Carbon Nanomaterials.2.3 Synthesis of Carbon Nanotubes.2.4 Controllable Synthesis of Carbon Nanotube Architectures.2.5 Perspective on Biomedical Applications.2.6 Conclusion.3 Nanostructured Systems from Low-Dimensional Building Blocks (Donghai Wang, Maria P. Gil, Guang Lu, and Yunfeng Lu).3.1 Introduction.3.2 Nanostructured System by Self-Assembly.3.3 Biomimetic and Biomolecular Recognition Assembly.3.4 Template-Assisted Integration and Assembly.3.5 External-Field-Induced Assembly.3.6 Direct Synthesis of 2D/3D Nanostructure.3.7 Applications.3.8 Concluding Remarks.4 Nanostructured Collagen Mimics in Tissue Engineering (Sergey E. Paramonov and Jeffrey D. Hartgerink).4.1 Introduction.4.2 Collagen Structural Hierarchy.4.3 Amino Acid Sequence and Secondary Structure.4.4 Experimental Observation of the Collagen Triple Helix.4.5 Folding Kinetics.4.6 Stabilization Through Sequence Selection.4.7 Stabilization via Hydroxyproline: The Pyrrolidine Ring Pucker.4.8 Triple Helix Stabilization Through Forced Aggregation.4.9 Extracellular Matrix and Collagen Mimics in Tissue Engineering.4.10 Sticky Ends and Supramolecular Polymerization.4.11 Conclusion.5 Molecular Biomimetics: Building Materials Natures Way, One Molecule at a Time (Candan Tamerler and Mehmet Sarikaya).5.1 Introduction.5.2 Inorganic Binding Peptides via Combinatorial Biology.5.3 Physical Specificity and Molecular Modeling.5.4 Applications of Engineered Polypeptides as Molecular Erectors.5.5 Future Prospects and Potential Applications in Nanotechnology.II Characterization Tools for Nanomaterials and Nanosystems.6 Electron Microscopy Techniques for Characterization of Nanomaterials (Jian-Min (Jim) Zuo).6.1 Introduction.6.2 Electron Diffraction and Geometry.6.3 Theory of Electron Diffraction.6.4 High-Resolution Electron Microscopy.6.5 Experimental Analysis.6.6 Applications.6.7 Conclusions and Future Perspectives.7 X-Ray Methods for the Characterization of Nanoparticles (Hartwig Modrow).7.1 Introduction.7.2 X-Ray Diffraction: Getting to Know the Arrangement of Atoms.7.3 Small-Angle X-Ray Scattering: Learning About Particle Shape and Morphology.7.4 X-Ray Absorption: Exploring Chemical Composition and Local Structure.7.5 Applications.7.6 Summary and Conclusions.A.1 General Approach.A.2 X-Ray Diffraction.A.3 Small-Angle Scattering.A.4 X-Ray Absorption.8 Single-Molecule Detection and Manipulation in Nanotechnology and Biology (Christopher L. Kuyper, Gavin D. M. Jeffries, Robert M. Lorenz, and Daniel T. Chiu).8.1 Introduction.8.2 Optical Detection of Single Molecules.8.3 Single-Molecule Manipulations Using Optical Traps.8.4 Applications in Single-Molecule Spectroscopy.8.5 Single-Molecule Detection with Bright Fluorescent Species.8.6 Nanoscale Chemistry with Vesicles and Microdroplets.8.7 Perspectives.9 Nanotechnologies for Cellular and Molecular Imaging by MRI (Patrick M. Winter, Shelton D. Caruthers, Samuel A. Wickline, and Gregory M. Lanza).9.1 Introduction.9.2 Cardiovascular Disease.9.3 Cellular and Molecular Imaging.9.4 Cellular Imaging with Iron Oxides.9.5 Molecular Imaging with Paramagnetic Nanoparticles.9.6 Conclusions.III Application of Nanotechnology in Biomedical Research.10 Nanotechnology in Nonviral Gene Delivery (Latha M. Santhakumaran, Alex Chen, C. K. S. Pillai, Thresia Thomas, Huixin He, and T. J. Thomas).10.1 Introduction.10.2 Agents That Provoke DNA Nanoparticle Formation.10.3 Characterization of DNA Nanoparticles.10.4 Mechanistic Considerations in DNA Nanoparticle Formation.10.5 Systemic Gene Therapy Applications.10.6 Future Directions.11 Nanoparticles for Cancer Drug Delivery (Carola Leuschner and Challa Kumar).11.1 Introduction.11.2 Cancer: A Fatal Disease and Current Approaches to Its Cure.11.3 Characteristics of Tumor Tissues.11.4 Drug Delivery to Tumors.11.5 Physicochemical Properties of Nanoparticles in Cancer Therapy.11.6 Site-Specific Delivery of Chemotherapeutic Agents Using Nanoparticles.11.7 Nonviral Gene Therapy with Nanoparticles.11.8 Hyperthermia.11.9 Controlled Delivery of Chemotherapeutic Drugs Using Nanoparticles.11.10 Nanoparticles to Circumvent MDR.11.11 Potential Problems in Using Nanoparticles for Cancer Treatment.11.12 Future Outlook.12 Diagnostic and Therapeutic Applications of Metal Nanoshells (Christopher Loo, Alex Lin, Leon Hirsch, Min-Ho Lee, Jennifer Barton, Naomi Halas, Jennifer West, and Rebekah Drezek).12.1 Introduction.12.2 Methodology.12.3 Results and Discussion.12.4 Conclusions.13 Decorporation of Biohazards Utilizing Nanoscale Magnetic Carrier Systems (Axel J. Rosengart and Michael D. Kaminski).13.1 introduction.13.2 Technological Need.13.3 Technical Basis.13.4 Technology Specifications.14 Nanotechnology in Biological Agent Decontamination (Peter K. Stoimenov and Kenneth J. Klabunde).14.1 Introduction.14.2 Standard Methods for Chemical Decontamination of Biological Agents.14.3 Nanomaterials for Decontamination.14.4 Magnesium Oxide.14.5 Mechanism of Action.14.6 Titanium Dioxide.14.7 Summary.IV Impact of Biomedical Nanotechnology on Industry, Society, and Education.15 Too Small to See: Educating the Next Generation in Nanoscale Science and Engineering (Anna M. Waldron, Keith Sheppard, Douglas Spencer, and Carl A. Batt).15.1 Introduction.15.2 Nanotechnology as a Motivator for Engaging Students.15.3 The Nanometer Scale.15.4 Understanding Things Too Small to See.15.5 Creating Hands-On Science Learning Activities to Engage the Mind.15.6 Things That Scare Us.15.7 The Road Ahead.16 Nanobiomedical Technology: Financial, Legal, Clinical, Political, Ethical, and Societal Challenges to Implementation (Steven A. Edwards).16.1 Introduction.16.2 Drexler and the Dreaded Universal Assembler.16.3 Financial.16.4 Legal and Regulatory.16.5 Operational.16.6 Clinical.16.7 Political, Ethical And Social Challenges.16.8 Summary.Abbreviations.Index.

iDQC anisotropy map imaging for tumor tissue characterization in vivo

Intermolecular double quantum coherences (iDQCs), signals that result from simultaneous transitions of two or more separated spins, are known to produce images that are highly sensitive to subvoxel structure, particularly local anisotropy. Here we demonstrate how iDQCs signal can be used to efficiently detect the anisotropy created in breast tumor tissues and prostate tumor tissues by targeted (LHRH‐conjugated) superparamagnetic nanoparticles (SPIONs), thereby distinguishing the necrotic area from the surrounding tumor tissue. Magn Reson Med, 2009.

Pharmacokinetics and pharmacodynamics of Phor21-βCG(ala), a lytic peptide conjugate

Phor21‐βCG(ala), a 36‐amino acid peptide comprised of a lytic peptide (Phor21) conjugated to a modified 15‐amino acid segment of the β‐chain of chorionic gonadotropin (βCG(ala)), selectively kills cancer cells that over‐express luteinizing hormone/chorionic gonadotropin (LH/CG) receptors by disrupting cellular membrane structure. These studies were designed to further characterize its in‐vitro inhibition and in‐vivo destruction of prostate cancer cells, biostability and pharmacokinetics to determine its pharmacokinetic and pharmacodynamic profile. Inhibitory effects of Phor21‐βCG(ala) were tested in PC‐3 and Caco‐2 cells as well as in nude mice bearing PC‐3 cells transfected with the luciferase gene (PC‐3.luc). Plasma stability, protease hydrolysis and pharmacokinetics of Phor21‐βCG(ala) were measured by using liquid chromatography mass spectrometry (LC/MS/MS). Phor21‐βCG(ala) selectively inhibited proliferation in‐vitro and in‐vivo metastases of PC‐3 cells. Phor21‐βCG(ala) was relatively stable in mouse, rat, dog and human plasma. Its degradation was partially due to protease hydrolysis and thermodynamic catalysis. Intravenous administration of Phor21‐βCG(ala) showed its blood Cmax and AUC0→∞ around the in‐vitro effective levels. In the tested rodents, Phor21‐βCG(ala) displayed a moderate volume of distribution at steady state (VdSS) and slow clearance (Cl) in the rodents. In conclusion, Phor21‐βCG(ala) displayed promising in‐vitro and in‐vivo anti‐cancer activity with favourable pharmacokinetics, and may offer a novel approach to metastatic cancer chemotherapy.