Using in Vitro live-cell imaging to explore chemotherapeutics delivered by lipid-based nanoparticles

Conventional imaging techniques can provide detailed information about cellular processes. However, this information is based on static images in an otherwise dynamic system, and successive phases are easily overlooked or misinterpreted. Live-cell imaging and time-lapse microscopy, in which living cells can be followed for hours or even days in a more or less continuous fashion, are therefore very informative. The protocol described here allows for the investigation of the fate of chemotherapeutic nanoparticles after the delivery of doxorubicin (dox) in living cells. Dox is an intercalating agent that must be released from its nanocarrier to become biologically active. In spite of its clinical registration for more than two decades, its uptake, breakdown, and drug release are still not fully understood. This article explores the hypothesis that lipid-based nanoparticles are taken up by the tumor cells and are slowly degraded. Released dox is then translocated to the nucleus. To prevent fixation artifacts, live-cell imaging and time-lapse microscopy, described in this experimental procedure, can be applied

Cell membrane modulation as adjuvant in cancer therapy

Cancer is a complex disease involving numerous biological processes, which can exist in parallel, can be complementary, or are engaged when needed and as such can replace each other. This redundancy in possibilities cancer cells have, are fundamental to failure of therapy. However, intrinsic features of tumor cells and tumors as a whole provide also opportunities for therapy. Here we discuss the unique and specific makeup and arrangement of cell membranes of tumor cells and how these may help treatment. Interestingly, knowledge on cell membranes and associated structures is present already for decades, while application of membrane modification and manipulation as part of cancer therapy is lagging. Recent developments of scientific tools concerning lipids and lipid metabolism, opened new and previously unknown aspects of tumor cells and indicate possible differences in lipid composition and membrane function of tumor cells compared to healthy cells. This field, coined Lipidomics, demonstrates the importance of lipid components in cell membrane in several illnesses. Important alterations in cancer, and specially in resistant cancer cells compared to normal cells, opened the door to new therapeutic strategies. Moreover, the ability to modulate membrane components and/or properties has become a reality. Here, developments in cancer-related Lipidomics and strategies to interfere specifically with cancer cell membranes and how these affect cancer treatment are discussed. We hypothesize that combination of lipid or membrane targeted strategies with available care to improve chemotherapy, radiotherapy and immunotherapy will bring the much needed change in treatment in the years to come

Inhomogeneous crystal grain formation in DPPC-DSPC based thermosensitive liposomes determines content release kinetics

Thermosensitive liposomes (TSL) receive attention due to their rapid externally controlled drug release at transition temperature in combination with hyperthermia. This rapid release feature of TSL occurs when the liposome membrane is going through a phase change which results in numerous interfaces, at so-called crystal grain boundaries. Based on experience with TSLs, our group found that thermosensitive liposomes formulated by binary compositions of DPPC and DSPC at proper ratios are able to exhibit rapid release without incorporation of release-promoting components. The aim of this study was to understand the mechanism of rapid release from bi-component DPPC-DSPC based TSL. Based on the investigation of a series of TSLs formulated by different DPPC-DSPC ratios, and through the analysis of binary-phase diagrams of DPPC-DSPC TSLs, we conclude that inhomogeneous crystal grains are formed in bi-component TSL membranes rather than mono-component, thereby facilitating content release. The resulting inhomogeneous membrane pattern is affected by DPPC/DSPC ratio, i.e. this determines the number of interfaces between solid and liquid phases at transition temperature, which can be diminished by addition of cholesterol. At appropriate DPPC/DSPC ratio, substantive solid/liquid interfaces can be generated not only between membrane domains but also between crystal grains in each domain of the liposome membranes, therefore improving content release from the TSL at transition temperatures

A moderate thermal dose is sufficient for effective free and TSL based thermochemotherapy

Hyperthermia, i.e. heating the tumor to a temperature of 40–43 °C is considered by many a valuable treatment to sensitize tumor cells to radiotherapy and chemotherapy. In recent randomized trials the great potential of adding hyperthermia to chemotherapy was demonstrated for treatment of high risk soft tissue sarcoma: +11.4% 5 yrs. overall survival (OS) and for ovarian cancer with peritoneal involvement nearly +12 months OS gain. As a result interest in combining chemotherapy with hyperthermia, i.e. thermochemotherapy, is growing. Extensive biological research has revealed that hyperthermia causes multiple effects, from direct cell kill to improved oxygenation, whereby each effect has a specific temperature range. Thermal sensitization of the tumor cell for chemotherapy occurs for many drugs at temperatures ranging from 40 to 42 °C with little additional increase of sensitization at higher temperatures. Increasing perfusion/oxygenation and increased extravasation are two other important hyperthermia induced mechanisms. The combination of free drug and hyperthermia has not been found to increase tumor drug concentration. Hence, enhanced effectiveness of free drug will de

Catalytic imaging-guided cancer therapy using non-coordinated and coordinated nanozymes

Nanozymes with and without coordinated structures, in addition to performing enzyme-mimic activities through converting O2[rad]– to H2O2 or H2O2 into O2 and [rad]OH, are of interest in the development of diagnostic imaging and image-guided therapy platforms. Controlled catalytic activity, high selectivity/sensitivity, low cost, easy generation, stability and biodegradability, and combination therapy, make the use of nanozymes promising for imaging-guided cancer therapy. Therefore, in this review, we attempt to describe the improved imaging resolution relied on the catalytic activity of nanozymes in the TME mediated by the accumulation of contrast agents (such as metal ions, contrast agents, and organic compounds) or molecules that provide the high-resolution imaging of tumor cells. The following section delves into important approaches, including tumor inhibition, nanocarrier clearance, biocompatibility, as well as the efficacy of nanozymes with and without coordinated structures in catalytic cancer therapy. Finally, with an overview of the challenges, we highlighted the main obstacles in the development of nanoscale catalysts in the advancement of multimodal imaging for improved diagnosis and cancer therapy. We hope that this review may clarify the important aspects regarding the utilization of nanozymes, both with and without coordinated structures in multi-modal imaging and image-guided cancer therapy.</p

Liposomes as delivery systems in the prevention and treatment of infectious diseases

Research on the potential application of liposomes in the prevention and treatment of infectious diseases has focussed on improvement of the therapeutic index of antimicrobial drugs and immunomodulators and on stimulation of the immune response to otherwise weak antigens in vaccines composed of purified micro-organism subunits. In this review current approaches in this field are outlined. The improved therapeutic index of antimicrobial drugs after encapsulation in liposomes is a result of enhanced drug delivery to infected tissue or infected cells and/or a reduction of drug toxicity of potentially toxic antibiotics. Liposomal encapsulation of immunomodulators that activate macrophages aims at reducing the toxicity of these agents and targeting them to the cells of the mononuclear phagocyte system in order to increase the nonspecific resistance of the host against infections. Studies on the immunogenicity of liposomal antigens have demonstrated that liposomes can potentiate the humoral and cell mediated immunity to a variety of antigens

Precision Localization of Lipid-Based Nanoparticles by Dual-Fluorescent Labeling for Accurate and High-Resolution Imaging in Living Cells

In nanomedicine, lipid-based nanoparticles (NPs) such as liposomes (LPs) have established an important position. Precise delineation of NP interaction with cells and detailed characterization of activity are becoming essential, which mainly rely on labeling with lipophilic fluorescent molecules and assuming stable association with NPs. However, because of label separation from NPs in (biological) media, or when processed by cells, fluorescence-based detection of an NP incorporating a single label may not necessarily indicate the actual presence of an NP but may be from the dissociated label, rendering results unreliable. Herein, flow cytometry and confocal microscopy are employed to demonstrate that to verify the localization of LPs in a cell with perfect accuracy, dual-labeling, and contemporaneous detection of both fluorescent signals in one pixel are required. This is combined with size exclusion chromatography (SEC) and mass spectrometry measurements to indicate factors involved in label dissociation, which helps to understand the possible conditions of dissociated label and NP. It is shown that determining label colocalization with, and label dissociation from, dual-labeled NPs are needed to provide accurate spatiotemporal insight into targeting destination (colocalized signals) and disintegration (separated signals) of NPs during intracellular processing and in studying payload delivery with precision in nanomedicine.</p

Systemic toxicity and cytokine/acute phase protein levels in patients after isolated limb perfusion with tumor necrosis factor-alpha complicated by high leakage

BACKGROUND: Since the introduction of high-dose tumor necrosis factor-alpha (TNFalpha) in the setting of isolated limb perfusion (ILP) in the clinic, prevention of leakage to the body of the patient is monitored with great precision for fear of TNF-mediated toxicity. That we observed remarkably little toxicity in patients with and without leakage prompted us to determine patterns of cytokines and acute phase proteins in patients with high leakage and in patients without any leakage. METHODS: TNFalpha, interleukin (IL)-6, IL-8, C-reactive protein, and secretory (s)-phospholipase A2 were measured at several time points during and after (until 7 days) ILP in 10 patients with a leakage to the systemic circulation varying in percentage from 12% to 65%. As a control, the same measurements, both in peripheral blood and in perfusate, were performed in nine patients without systemic leakage. RESULTS: In patients with systemic leakage, levels of TNFalpha increased during ILP, reaching values to 277 ng/ml. IL-6 and IL-8 peaked 3 hours after ILP with values significantly higher compared with patients without systemic leakage. C-reactive protein and s-phospholipase A2 peaked at day 1 in both patient groups, s-phospholipase A2 with significant higher levels and C-reactive protein, in contrast, with lower levels in the leakage patients. CONCLUSIONS: High leakage of TNFalpha to the systemic circulation, caused by a complicated ILP, led to 10-fold to more than 100-fold increased levels of TNFalpha, IL-6, and IL-8 in comparison with patients without leakage. The increase of the acute phase proteins was limited. Even when high leakage occurs, this procedure should not