DQAsomes as the Prototype of Mitochondria-Targeted Pharmaceutical Nanocarriers : An Update.

DQAsomes (dequalinium-based liposome-like vesicles) are the prototype for all mitochondria-targeted vesicular pharmaceutical nanocarrier systems. First described in 1998 in a paper which has been cited as of May 2020 over 150 times, DQAsomes have been successfully explored for the delivery of DNA and low-molecular weight molecules to mitochondria within living mammalian cells. Moreover, they also appear to have triggered the design and development of a large variety of similar mitochondria-targeted nanocarriers . Potential areas of application of DQAsomes and of related mitochondria-targeted pharmaceutical nanocarriers involve mitochondrial gene therapy , antioxidant and updated therapy as well as apoptosis-based anticancer chemotherapy. Here, detailed protocols for the preparation, characterization, and application of DQAsomes are given and most recent developments involving the design and use of DQAsome-related particles are highlighted and discussed.

Synthesis of Triphenylphosphonium Phospholipid Conjugates for the Preparation of Mitochondriotropic Liposomes.

Surface modification of liposomes with a ligand is facilitated by the conjugation of the ligand to a hydrophobic molecule that serves to anchor the ligand to the liposomal bilayer. We describe here a simple protocol to conjugate a triphenylphosphonium group to several commercially available functionalized phospholipids. The resulting triphenylphosphonium-conjugated lipids can be used to prepare liposomes that preferentially associate with mitochondria when exposed to live mammalian cells in culture.

Nanomechanical Analysis of Extracellular Matrix and Cells in Multicellular Spheroids.

INTRODUCTION: Over the last decade, atomic force microscopy (AFM) has played an important role in understanding nanomechanical properties of various cancer cell lines. This study is focused on Lewis lung carcinoma cell tumours as 3D multicellular spheroid (MS). Not much is know about the mechanical properties of the cells and the surrounding extracellular matrix (ECM) in rapidly growing tumours. METHODS: Depth-dependent indentation measurements were conducted with the AFM. Force-vs.-indentation curves were used to create stiffness profiles as a function of depth. Here studies were focused on the outer most layer, i.e., proliferation zone of the spheroid. RESULTS: Both surface and sub-surface stiffness profiles of MS were created. This study revealed three nanomechanical topographies, Type A-high modulus due to collagen fibers, Type B-high stiffness at cell membrane and ECM interface and Type C-increased modulus due to cell lying deep inside matrix at a depth of 1.35 µm. Both Type and Type-B topographies result from collagen-based structures in ECM. CONCLUSION: This study has first time revealed mechanical constitution of an MS. Depth-dependent indentation studies have the revealed role of various molecular and cellular components responsible for providing mechanical stability to MS. Nanomechanical heterogeneities revealed in this investigation can shed new light in developing correct dosage regime for collagenase treatment of tumours and designing better controlled artificial extracellular matrix systems for replicating tissue growth in-vitro.

In Vitro Testing of Anticancer Nanotherapeutics Using Tumor Spheroids.

Tumor cells grown as spheroids more closely resemble in vivo solid tumors and present physical and physiological barriers to drug action that conventional monolayer cell cultures do not. The physiological relevance of the model can be further increased by incorporating fluid flow and multiple cell types. The protocols described in this chapter use simple and easily available equipment to reproducibly generate spheroid models that can be used for the in vitro testing of a variety of preparations.

Dynamic and Depth Dependent Nanomechanical Properties of Dorsal Ruffles in Live Cells and Biopolymeric Hydrogels.

The nanomechanical properties of various biological and cellular surfaces are increasingly investigated with Scanning Probe Microscopy. Surface stiffness measurements are currently being used to define metastatic properties of various cancerous cell lines and other related biological tissues. Here we present a unique methodology to understand depth dependent nanomechanical variations in stiffness in biopolymers and live cells. In this study we have used A2780 and NIH3T3 cell lines and 0.5% and 1% Agarose to investigate depth dependent stiffness and porosity on nanomechanical properties in different biological systems. This analytical methodology can circumvent the issue associated with the contribution of substrates on cell stiffness. Here we demonstrate that by calculating 'continuous-step-wise-modulus' on force versus distance curves one can observe minute variation as function of depth. Due to the presence of different kinds of cytoskeletal filament, dissipation of contact force might vary from one portion of a cell to another. On NIH3T3 cell lines, stiffness profile of Circular Dorsal Ruffles could be observed in form of large parabolic feature with changes in stiffness at different depth. In biopolymers like agarose, depending upon the extent of polymerization in there can be increase or decrease in stiffness due variations in pore size and extent to which crosslinking is taking place at different depths. 0.5% agarose showed gradual decrease in stiffness whereas with 1% agarose there was slight increase in stiffness as one indents deeper into its surface.

Development of an in vitro tumor spheroid culture model amenable to high-throughput testing of potential anticancer nanotherapeutics.

CONTEXT: Three-dimensional tumor spheroid cultures are a better representative of in vivo solid tumors than monolayer cultures and should be used for testing potential nanotherapeutics in vitro. OBJECTIVE: To develop techniques to test the disposition and efficacy of nanocarrier formulations in spheroids in a cost-effective manner amenable to high-throughput testing. METHODS: Spheroids were obtained using a modified liquid overlay technique in a 96-well plate. Several nanocarrier formulations were prepared and tested in the spheroid model. The disposition of the formulations in the spheroids was determined by confocal microscopy while the effect of the drug-loaded formulations was assessed in terms of the cell viability, loss of membrane integrity, induction of caspases and inhibition of growth of the spheroids. RESULTS: The surface charge of the formulations influenced the accumulation of the nanocarrier and drug in the spheroid, with the cationic formulation accumulating to the greatest extent. Also, the smallest particle size formulation, micelles, penetrated to the greatest extent in the spheroid. The iRGD tumor-penetrating peptide co-administered with unmodified liposomes exhibited both high accumulation and penetration. The effect studies revealed that the formulations that penetrated or accumulated to the highest extent in the spheroid exhibited better antitumor activity compared to the other formulations. CONCLUSION: The 96-well plate format spheroid model developed in the study can be used toward the rational selection of nanocarrier therapeutics prior to their testing in in vivo models.

Hydrophobized triphenyl phosphonium derivatives for the preparation of mitochondriotropic liposomes: choice of hydrophobic anchor influences cytotoxicity but not mitochondriotropic effect.

CONTEXT: Nanocarrier-based strategies to achieve delivery of bioactives specifically to the mitochondria are being increasingly explored due to the importance of mitochondria in critical cellular processes. OBJECTIVE: To test the ability of liposomes modified with newly synthesized triphenylphosphonium (TPP)-phospholipid conjugates and to test their use in overcoming the cytotoxicity of stearyl triphenylphosphonium (STPP)-modified liposomes when used for delivery of therapeutic molecules to the mitochondria. METHODS: TPP-phospholipid conjugates with the dioleoyl, dimyristoyl or dipalmitoyl lipid moieties were synthesized and liposomes were prepared with these conjugates in a 1 mol% ratio. The subcellular distribution of the liposomes was tested by confocal microscopy. Furthermore, the liposomes were tested for their effect on cell viability using a MTS assay, on cell membrane integrity using a lactate dehydrogenase assay and on mitochondrial membrane integrity using a modified JC-1 assay. RESULTS: The liposomes modified with the new TPP-phospholipid conjugates exhibited similar mitochondriotropism as STPP-liposomes but they were more biocompatible as compared to the STPP liposomes. While the STPP-liposomes had a destabilizing effect on cell and mitochondrial membranes, the liposomes modified with the TPP-phospholipid conjugates did not demonstrate any such effect on biomembranes. CONCLUSIONS: Using phospholipid anchors in the synthesis of TPP-lipid conjugates can provide liposomes that exhibit the same mitochondrial targeting ability as STPP but with much higher biocompatibility.

Synthesis of triphenylphosphonium phospholipid conjugates for the preparation of mitochondriotropic liposomes.

Surface modification of liposomes with a ligand is facilitated by the conjugation of the ligand to a hydrophobic molecule that serves to anchor the ligand to the liposomal bilayer. We describe here a simple protocol to conjugate a triphenylphosphonium group to several commercially available functionalized phospholipids. The resulting triphenylphosphonium conjugated lipids can be used to prepare liposomes that preferentially associate with mitochondria when exposed to live mammalian cells in culture.

In Vitro assessment of the utility of stearyl triphenyl phosphonium modified liposomes in overcoming the resistance of ovarian carcinoma Ovcar-3 cells to paclitaxel.

Paclitaxel loaded in liposomes modified with stearyl triphenyl phosphonium (STPP) showed improved mitochondrial colocalization and cytotoxicity in a paclitaxel resistant cell line. The improvement in cytotoxicity was not solely due to the increased accumulation of paclitaxel in mitochondria but also due to the specific toxicity of STPP towards the resistant cell line. Mechanistic studies revealed that the cytotoxicity of STPP was associated with a decrease in mitochondrial membrane potential and other hallmarks related to caspase-independent cell death (CICD). This specific toxicity of STPP towards the paclitaxel resistant cell line was also maintained in three-dimensional in vitro spheroid cultures.

Palmitoyl ascorbate liposomes and free ascorbic acid: comparison of anticancer therapeutic effects upon parenteral administration.

PURPOSE: To evaluate and compare anticancer therapeutic effect of palmitoyl ascorbate liposomes (PAL) and free ascorbic acid (AA). METHODS: Liposomes incorporating palmitoyl ascorbate (PA) were prepared and evaluated for PA content by HPLC. To elucidate mechanism of action of cell death in vitro, effect of various H(2)O(2) scavengers and metal chelators on PA-mediated cytotoxicity was studied. Effect of various combinations of PAL and free AA on in vitro cytotoxicity was evaluated on 4T1 cells. In vivo, PAL formulation was modified with polyethylene glycol; effect of PEGylation on in vitro cytotoxicity was evaluated. Biodistribution of PEG-PAL formulation was investigated in female Balb/c mice bearing murine mammary carcinoma (4T1 cells). In vivo anticancer activity of PEG-PAL (PEG-PAL equivalent to 20 mg/kg of PA injected intravenously on alternate days) was compared with free AA therapy in same model. RESULTS: PEG-PAL treatment was significantly more effective than free AA treatment in slowing tumor growth. CONCLUSIONS: Nanoparticle formulations incorporating PA can kill cancer cells in vitro. The mechanism of PA cytotoxicity is based on production of extracellular reactive oxygen species and involves intracellular transition metals.

The utility of an isolated mitochondrial fraction in the preparation of liposomes for the specific delivery of bioactives to mitochondria in live mammalian cells.

PURPOSE: To develop and evaluate liposomal formulations prepared with an isolated mitochondrial fraction as a mitochondria-specific delivery vehicle. METHODS: Liposomes were prepared with either a crude mitochondrial fraction extracted from cells or lipids extracted from the crude mitochondrial fraction and were then characterized by determining their size and zeta potential. The cell uptake of the liposomes and loaded bioactive was studied using flow cytometry and confocal microscopy. The cytotoxicity of the formulations was tested by MTS cytotoxicity assay. RESULTS: Liposomes prepared with the mitochondrial extracts were non-toxic and colocalized with mitochondria in F98 cells. Addition of DOTAP to the liposomes facilitated DNA complexation and the DNA delivered intracellularly co-localized with mitochondria. CONCLUSION: The results from this study establish the potential of using a mitochondrial fraction isolated from cells to prepare liposomes capable of delivering biologically active molecules to mitochondria of live mammalian cells.

On the mechanism of targeting of phage fusion protein-modified nanocarriers: only the binding peptide sequence matters.

The integration of pharmaceutical nanocarriers with phage display techniques is emerging as a new paradigm for targeted cancer nanomedicines. We explored the direct use of landscape phage fusion proteins for the self-assembly of phage-derived binding peptides to liposomes for cancer cell targeting. The primary purpose of this study was to elucidate the targeting mechanism with a particular emphasis on the relative contributions of the two motifs that make up the landscape phage fusion protein (a binding peptide and the phage pVIII coat protein) to the targeting efficiency. Using transmission electron microscopy and dynamic light scattering, we confirmed the formation of phage-liposomes. Using FACS analysis, fluorescence microscopy, and fluorescence photospectrometry, we found that liposomes modified with MCF-7-specific phage fusion proteins (MCF-7 binding peptide, DMPGTVLP, fused to the phage PVIII coat protein) provided a strong and specific association with target MCF-7 cancer cells but not with cocultured, nontarget cells including C166-GFP and NIH3T3. The substitution for the binding peptide fused to phage pVIII coat protein abolished the targeting specificity. The addition of free binding peptide, DMPGTVLP, competitively inhibited the interaction of MCF-7-specific phage-liposomes with target MCF-7 cells but showed no reduction of MCF-7-associated plain liposomes. The proteolysis of the binding peptide reduced MCF-7 cell-associated phage-liposomes in a proteinase K (PK) concentration-dependent manner with no effect on the binding of plain liposomes to MCF-7 cells. Overall, only the binding peptide motif was involved in the targeting specificity of phage-liposomes. The presence of phage pVIII coat protein did not interfere with the targeting efficiency.

Recent progress in the therapeutic applications of nanotechnology.

PURPOSE OF REVIEW: The field of pharmaceutical and medical nanotechnology has grown rapidly in recent decades and offers much promise for therapeutic advances. This review is intended to serve as a quick summary of the major areas in the therapeutic application of nanotechnology. RECENT FINDINGS: Nanotechnology for therapeutic application falls into two broad categories of particulate systems and nanoengineered devices. Recent studies appear to focus on the development of multifunctional particles for drug delivery and imaging and the development of nanotechnology-based biosensors for diagnostic applications. Cancer treatment and diagnosis appears to be the principal focus of many of these applications, but nanotechnology is also finding application in tissue engineering and surface engineering of medical implants. SUMMARY: Particulate drug delivery systems in general appear to be poised for increased use in the clinic, whereas nanoengineered implants and diagnostic sensors might well be the next major wave in the medical use of nanotechnology.

In vitro optimization of liposomal nanocarriers prepared from breast tumor cell specific phage fusion protein.

Fusion proteins created by phage display peptides with tumor cell specificity and the pVIII major coat protein of filamentous phages have been explored recently as a simple and cost-effective means for preparing tumor-targeted liposomes that improve the cytotoxicity of anticancer drugs in vitro. The next step in the development of this approach is the optimization of the liposome composition for the maximum targeting activity and subsequent testing in vivo. This study aimed to investigate the impact of preparation protocols, lipid composition and phage protein content on the targeting efficiency of phage protein-modified liposomes. Analysis of size, zeta potential and morphology was used to investigate the effect of preparation protocols on the stability and homogeneity of the phage liposomes. A previously developed coculture targeting assay and a factorial design approach were used to determine the role of lipid composition of the liposomal membrane on the target cell specificity of the phage liposomes. Western blot combined with proteinase K treatment detected the orientation of targeted phage protein in liposomal membrane. Phage protein, DPPG and PEG(2k)-PE showed positive effects on target specificity of phage liposomes. The results served to identify optimal formulation that offer an improved liposomal affinity for target tumor cells over the non-optimized formulation.

Palmitoyl ascorbate-loaded polymeric micelles: cancer cell targeting and cytotoxicity.

PURPOSE: To evaluate the potential of palmitoyl ascorbate (PA)-loaded micelles for ascorbate-mediated cancer cell targeting and cytotoxicity. METHODS: PA was incorporated in polyethylene glycol-phosphatidyl ethanolamine micelles at varying concentrations. The formulations were evaluated for PA content by RP-HPLC. A stable formulation was selected based on size and zeta potential measurements. A co-culture of cancer cells and GFP-expressing non-cancer cells was used to determine the specificity of PA micelle binding. In vitro cytotoxicity of the micellar formulations towards various cancer cell lines was investigated using a cell viability assay. To elucidate the mechanism of action of cell death in vitro, the effect of various H(2)O(2) scavengers and metal chelators on PA-mediated cytotoxicity was studied. The in vivo anti-cancer activity of PA micelles was studied in female Balb/c mice bearing a murine mammary carcinoma (4T1 cells). RESULTS: PA micelles associated preferentially with various cancer cells compared to non-cancer cells in co-culture. PA micelles exhibited anti-cancer activity in cancer cell lines both in vitro and in vivo. The mechanism of cell death was due primarily to generation of reactive oxygen species (ROS). CONCLUSIONS: The anti-cancer activity of PA micelles associated with its enhanced cancer cell binding and subsequent generation of ROS.

Approaches for targeting mitochondria in cancer therapy.

The recognition of the role that mitochondria play in human health and disease is evidenced by the emergence in recent decades of a whole new field of "Mitochondrial Medicine". Molecules located on or inside mitochondria are considered prime pharmacological targets and a wide range of efforts are underway to exploit these targets to develop targeted therapies for various diseases including cancer. However the concept of targeting, while seemingly simple in theory, has multiple subtly different practical approaches. The focus of this article is to highlight these differences in the context of a discussion on the current status of various mitochondria-targeted approaches to cancer therapy.

Enhanced binding and killing of target tumor cells by drug-loaded liposomes modified with tumor-specific phage fusion coat protein.

AIM: To explore cancer cell-specific phage fusion pVIII coat protein, identified using phage display, for targeted delivery of drug-loaded liposomes to MCF-7 breast cancer cells. MATERIAL & METHODS: An 8-mer landscape library f8/8 and a biopanning protocol against MCF-7 cells were used to select a landscape phage protein bearing MCF-7-specific peptide. Size and morphology of doxorubicin-loaded liposomes modified with the tumor-specific phage fusion coat protein (phage-Doxil) were determined by dynamic light scattering and freeze-fraction electron microscopy. Topology of the phage protein in liposomes was examined by western blot. Association of phage-Doxil with MCF-7 cells was evaluated by fluorescence microscopy and fluorescence spectrometry. Selective targeting to MCF-7 was shown by FACS using a coculture model with target and nontarget cells. Phage-Doxil-induced tumor cell killing and apoptosis were confirmed by CellTiter-Blue Assay and caspase-3/CPP32 fluorometric assay. RESULTS: A chimeric phage fusion coat protein specific towards MCF-7 cells, identified from a phage landscape library, was directly incorporated into the liposomal bilayer of doxorubicin-loaded PEGylated liposomes (Doxil) without additional conjugation with lipophilic moieties. Western blotting confirmed the presence of both targeting peptide and pVIII coat protein in the phage-Doxil, which maintained the liposomal morphology and retained a substantial part of the incorporated drug after phage protein incorporation. The binding activity of the phage fusion pVIII coat protein was retained after incorporation into liposomes, and phage-Doxil strongly and specifically targeted MCF-7 cells, demonstrating significantly increased cytotoxicity towards target cells in vitro. CONCLUSION: We present a novel and straightforward method for making tumor-targeted nanomedicines by anchoring specific phage proteins (substitute antibodies) on their surface.

Liposomes for drug delivery to mitochondria.

Efficacy of therapeutically active drugs known to act on intracellular targets can be enhanced by specific delivery to the site of action. Triphenylphosphonium cations can be used to create subcellular targeted liposomes that efficiently deliver drugs to mitochondria, thus enhancing their therapeutic action.

Mitochondria-targeted liposomes improve the apoptotic and cytotoxic action of sclareol.

Current efforts toward improving the effectiveness of drug therapy are increasingly relying on drug-targeting strategies to effectively deliver bioactive molecules to their molecular targets. Pharmaceutical nanocarriers represent a major tool toward this aim, and our efforts have been directed toward achieving nanocarrier-mediated subcellular delivery of drug molecules with mitochondria as the primary subcellular target. Meeting the need for specific subcellular delivery is essential to realizing the full potential of many poorly soluble anticancer drugs. In this article, we report that mitochondria-targeted liposomes significantly improve the apoptotic and cytotoxic action of sclareol, a poorly soluble potential anticancer drug. The results support the broad applicability of our nanocarrier-mediated subcellular targeting approach as a means to improve the effectiveness of certain anticancer therapeutics.

Subcellular targeting: a new frontier for drug-loaded pharmaceutical nanocarriers and the concept of the magic bullet.

The ability of a pharmacologically active molecule selectively to find its target is closely linked with its potential as a successful therapeutic drug. It has become increasingly evident that there are several pharmacologically active molecules that exert their action on molecular targets inside cell organelles. In the case of a drug molecule with no defined specificity for a particular organelle, the molecule would either need to have sufficiently long metabolic stability to allow for random interaction with the organelle to occur, or a targeting strategy for the intended subcellular compartment would need to be devised in order to potentiate therapeutic effect. In the case of molecules with a stronger affinity for a non-target subcellular compartment, there exists even greater need for the ability to control subcellular disposition. Subcellular or organelle-specific targeting has thus emerged as a new frontier in drug delivery. In this review selected examples of recent work are discussed that the authors believe might eventually lead to the application of pharmaceutical nanocarriers to create the next generation of 'magic bullets' that are capable of delivering a drug payload to a molecular target at a subcellular location.