Overcoming Nanoparticle-Mediated Complement Activation by Surface PEG Pairing.

Many PEGylated nanoparticles activate the complement system, which is an integral component of innate immunity. This is of concern as uncontrolled complement activation is potentially detrimental and contributes to disease pathogenesis. Here, it is demonstrated that, in contrast to carboxyPEG2000-stabilized poly(lactic-co-glycolic acid) nanoparticles, surface camouflaging with appropriate combinations and proportions of carboxyPEG2000 and methoxyPEG550 can largely suppress nanoparticle-mediated complement activation through the lectin pathway. This is attributed to the ability of the short, rigid methoxyPEG550 chains to laterally compress carboxyPEG2000 molecules to become more stretched and assume an extended, random coil configuration. As supported by coarse-grained molecular dynamics simulations, these conformational attributes minimize statistical protein binding/intercalation, thereby affecting sequential dynamic processes in complement convertase assembly. Furthermore, PEG pairing has no additional effect on nanoparticle longevity in the blood and macrophage uptake. PEG pairing significantly overcomes nanoparticle-mediated complement activation without the need for surface functionalization with complement inhibitors.

Non-Lamellar Liquid Crystalline Nanocarriers for Thymoquinone Encapsulation.

Owing to their unique structural features, non-lamellar liquid crystalline nanoparticles comprising cubosomes and hexosomes are attracting increasing attention as versatile investigative drug carriers. BACKGROUND: Depending on their physiochemical characteristics, drug molecules on entrapment can modulate and reorganize structural features of cubosomes and hexosomes. Therefore, it is important to assess the effect of guest molecules on broader biophysical characteristics of non-lamellar liquid crystalline nanoparticles, since drug-induced architectural, morphological, and size modifications can affect the biological performance of cubosomes and hexosomes. METHODS: We report on alterations in morphological, structural, and size characteristics of nanodispersions composed from binary mixtures of glycerol monooleate and vitamin E on thymoquinone (a molecule with wide therapeutic potentials) loading. RESULTS: Thymoquinone loading was associated with a slight increase in the mean hydrodynamic nanoparticle size and led to structural transitions from an internal biphasic feature of coexisting inverse cubic Fd3m and hexagonal (H2) phases to an internal inverse cubic Fd3m phase (micellar cubosomes) or an internal inverse micellar (L2) phase (emulsified microemulsions, EMEs). We further report on the presence of "flower-like" vesicular populations in both native and drug-loaded nanodispersions. CONCLUSIONS: These nanodispersions have the potential to accommodate thymoquinone and may be considered as promising platforms for the development of thymoquinone nanomedicines.

Cisplatin Encapsulation Generates Morphologically Different Multicompartments in the Internal Nanostructures of Nonlamellar Liquid-Crystalline Self-Assemblies.

Cisplatin ( cis-diamminedichloroplatinum(II)) is among the most potent cytotoxic agents used in cancer chemotherapy. The encapsulation of cisplatin in lipid-based drug carriers has been challenging owing to its low solubility in both aqueous and lipid phases. Here, we investigated cisplatin encapsulation in nonlamellar liquid-crystalline (LC) nanodispersions formed from a ternary mixture of phytantriol (PHYT), vitamin E (Vit E), and an anionic phospholipid [either phosphatidylglycerol (DSPG) or phosphatidylserine (DPPS)]. We show an increase in cisplatin encapsulation efficiency (EE) in nanodispersions containing 1.5-4 wt % phospholipid. The EE was highest in DPPS-containing nanodispersions (53-98%) compared to DSPG-containing counterparts (25-40%) under similar experimental conditions. Through structural and morphological characterizations involving synchrotron small-angle X-ray scattering and cryogenic transmission electron microscopy, we further show that varying the phospholipid content of cisplatin-free nanodispersions triggers an internal phase transition from a neat hexagonal (H2) phase to a biphasic phase (internal H2 phase coexisting with the lamellar (Lα) phase). However, cisplatin encapsulation in both DPPS- and DSPG-containing nanodispersions generates the coexistence of morphologically different multicompartments in the internal nanostructures comprising vesicles as a core, enveloped by an inverted-type surface bicontinuous cubic Im3 m (primitive, QIIP) phase or H2 phase. We discuss the biophysical basis of these drug-induced morphological alterations and provide insights into the potential development of inverted-type LC nanodispersions for cisplatin delivery.

Polyplex Evolution: Understanding Biology, Optimizing Performance.

Polyethylenimine (PEI) is a gold standard polycationic transfectant. However, the highly efficient transfecting activity of PEI and many of its derivatives is accompanied by serious cytotoxic complications and safety concerns at innate immune levels, which impedes the development of therapeutic polycationic nucleic acid carriers in general and their clinical applications. In recent years, the dilemma between transfection efficacy and adverse PEI activities has been addressed from in-depth investigations of cellular processes during transfection and elucidation of molecular mechanisms of PEI-mediated toxicity and translation of these integrated events to chemical engineering of novel PEI derivatives with an improved benefit-to-risk ratio. This review addresses these perspectives and discusses molecular events pertaining to dynamic and multifaceted PEI-mediated cytotoxicity, including membrane destabilization, mitochondrial dysfunction, and perturbations of glycolytic flux and redox homeostasis as well as chemical strategies for the generation of better tolerated polycations. We further examine the effect of PEI and its derivatives on complement activation and interaction with Toll-like receptors. These perspectives are intended to lay the foundation for an improved understanding of interlinked mechanisms controlling transfection and toxicity and their translation for improved engineering of polycation-based transfectants.

Polyethylenimine architecture-dependent metabolic imprints and perturbation of cellular redox homeostasis.

Polyethylenimines (PEIs) are among the most efficient polycationic non-viral transfectants. PEI architecture and size not only modulate transfection efficiency, but also cytotoxicity. However, the underlying mechanisms of PEI-induced multifaceted cell damage and death are largely unknown. Here, we demonstrate that the central mechanisms of PEI architecture- and size-dependent perturbations of integrated cellular metabolomics involve destabilization of plasma membrane and mitochondrial membranes with consequences on mitochondrial oxidative phosphorylation (OXPHOS), glycolytic flux and redox homeostasis that ultimately modulate cell death. In comparison to linear PEI, the branched architectures induced greater plasma membrane destabilization and were more detrimental to glycolytic activity and OXPHOS capacity as well as being a more potent inhibitor of the cytochrome c oxidase. Accordingly, the branched architectures caused a greater lactate dehydrogenase (LDH) and ATP depletion, activated AMP kinase (AMPK) and disturbed redox homeostasis through diminished availability of nicotinamide adenine dinucleotide phosphate (NADPH), reduced antioxidant capacity of glutathione (GSH) and increased burden of reactive oxygen species (ROS). The differences in metabolic and redox imprints were further reflected in the transfection performance of the polycations, but co-treatment with the GSH precursor N-acetyl-cysteine (NAC) counteracted redox dysregulation and increased the number of viable transfected cells. Integrated biomembrane integrity and metabolomic analysis provides a rapid approach for mechanistic understanding of multifactorial polycation-mediated cytotoxicity, and could form the basis for combinatorial throughput platforms for improved design and selection of safer polymeric vectors.

Platelet mimicry: The emperor's new clothes?

Here we critically examine whether coating of nanoparticles with platelet membranes can truly disguise them against recognition by elements of the innate immune system. We further assess whether the "cloaking technology" can sufficiently equip nanoparticles with platelet-mimicking functionalities to include in vivo targeting of damaged blood vessels and binding to platelet-adhering opportunistic pathogens. We present views for improved, and pharmaceutically viable nanoparticle design strategies.

Dendrimers in Medicine: Therapeutic Concepts and Pharmaceutical Challenges.

Dendrimers are three-dimensional macromolecular structures originating from a central core molecule and surrounded by successive addition of branching layers (generation). These structures exhibit a high degree of molecular uniformity, narrow molecular weight distribution, tunable size and shape characteristics, as well as multivalency. Collectively, these physicochemical characteristics together with advancements in design of biodegradable backbones have conferred many applications to dendrimers in formulation science and nanopharmaceutical developments. These have included the use of dendrimers as pro-drugs and vehicles for solubilization, encapsulation, complexation, delivery, and site-specific targeting of small-molecule drugs, biopharmaceuticals, and contrast agents. We briefly review these advances, paying particular attention to attributes that make dendrimers versatile for drug formulation as well as challenging issues surrounding the future development of dendrimer-based medicines.

Modulatory effect of human plasma on the internal nanostructure and size characteristics of liquid-crystalline nanocarriers.

The inverted-type liquid-crystalline dispersions comprising cubosomes and hexosomes hold much potential for drug solubilization and site-specific targeting on intravenous administration. Limited information, however, is available on the influence of plasma components on nanostructural and morphological features of cubosome and hexosome dispersions, which may modulate their stability in the blood and their overall biological performance. Through an integrated approach involving SAXS, cryo-TEM, and nanoparticle tracking analysis (NTA) we have studied the time-dependent effect of human plasma (and the plasma complement system) on the integrity of the internal nanostructure, morphology, and fluctuation in size distribution of phytantriol (PHYT)-based nonlamellar crystalline dispersions. The results indicate that in the presence of plasma the internal nanostructure undergoes a transition from the biphasic phase (a bicontinuous cubic phase with symmetry Pn3m coexisting with an inverted-type hexagonal (H2) phase) to a neat hexagonal (H2) phase, which decreases the median particle size. These observations were independent of a direct effect by serum albumin and dispersion-mediated complement activation. The implication of these observations in relation to soft nanocarrier design for intravenous drug delivery is discussed.

Differential Modulation of Cellular Bioenergetics by Poly(L-lysine)s of Different Molecular Weights.

Poly(L-lysine)s (PLLs), and related derivatives, have received considerable attention as nonviral vectors. High molecular weight PLLs (H-PLLs) are superior transfectants compared with low Mw PLLs (L-PLLs), but suggested to be more cytotoxic. Through a pan-integrated metabolomic approach using Seahorse XF technology, we studied the impact of PLL size on cellular bioenergetic processes in two human cell lines. In contrast to L-PLLs (1-5 kDa), H-PLLs (15-30 kDa) were more detrimental to both mitochondrial oxidative phosphorylation (OXPHOS) and glycolytic activity resulting in considerable intracellular ATP depletion, thereby initiating necrotic-type cell death. The cellular differences to polycation sensitivity were further related to the mitochondrial state, where the impact was substantial on cells with hyperpolarized mitochondria. These medium-throughput approaches offer better opportunities for understanding inter-related intracellular and cell type-dependent processes instigating a bioenergetics crisis, thus, aiding selection (from available libraries) and improved design of safer biodegradable polycations for nucleic acid compaction and cell type-specific delivery.

Mechanisms of complement activation by dextran-coated superparamagnetic iron oxide (SPIO) nanoworms in mouse versus human serum.

BACKGROUND: The complement system is a key component of innate immunity implicated in the neutralization and clearance of invading pathogens. Dextran coated superparamagnetic iron oxide (SPIO) nanoparticle is a promising magnetic resonance imaging (MRI) contrast agent. However, dextran SPIO has been associated with significant number of complement-related side effects in patients and some agents have been discontinued from clinical use (e.g., Feridex™). In order to improve the safety of these materials, the mechanisms of complement activation by dextran-coated SPIO and the differences between mice and humans need to be fully understood. METHODS: 20 kDa dextran coated SPIO nanoworms (SPIO NW) were synthesized using Molday precipitation procedure. In vitro measurements of C3 deposition on SPIO NW using sera genetically deficient for various components of the classical pathway (CP), lectin pathway (LP) or alternative pathway (AP) components were used to study mechanisms of mouse complement activation. In vitro measurements of fluid phase markers of complement activation C4d and Bb and the terminal pathway marker SC5b-C9 in normal and genetically deficient sera were used to study the mechanisms of human complement activation. Mouse data were analyzed by non-paired t-test, human data were analyzed by ANOVA followed by multiple comparisons with Student-Newman-Keuls test. RESULTS: In mouse sera, SPIO NW triggered the complement activation via the LP, whereas the AP contributes via the amplification loop. No involvement of the CP was observed. In human sera the LP together with the direct enhancement of the AP turnover was responsible for the complement activation. In two samples out of six healthy donors there was also a binding of anti-dextran antibodies and C1q, suggesting activation via the CP, but that did not affect the total level of C3 deposition on the particles. CONCLUSIONS: There were important differences and similarities in the complement activation by SPIO NW in mouse versus human sera. Understanding the mechanisms of immune recognition of nanoparticles in mouse and human systems has important preclinical and clinical implications and could help design more efficient and safe nano-formulations.

Just so stories: the random acts of anti-cancer nanomedicine performance.

Contrary to high expectations, the majority of clinically approved anti-cancer nanomedicine, and those under clinical trials, have shown limited therapeutic efficacy in humans. So, why these nanomedicine are not delivering their promise? Here, we discuss likely factors, and call for a paradigm shift in approach and design of future cancer nanotherapeutics based on realistic cancer models representing human disease, and better understanding of integrated pathophysiological processes, including systems immunology, that modulate human tumor functionality and growth. FROM THE CLINICAL EDITOR: This critical review of the current state of translational oncology research utilizing nanomedicine-based approaches provides a comprehensive discussion of the multiple factors that are responsible for poor outcomes when translating these approaches models to the actual human disease.

Complement activation by PEG-functionalized multi-walled carbon nanotubes is independent of PEG molecular mass and surface density.

Carboxylated (4%) multi-walled carbon nanotubes were covalently functionalized with poly(ethylene glycol)1000 (PEG1000), PEG1500 and PEG4000 with a PEG loading of approximately 11% in all cases. PEG loading generated non-uniform and heterogeneous higher surface structures and increased nanotube width considerably, but all PEGylated nanotube species activated the complement system in human serum equally. Increased PEG loading, through adsorption of methoxyPEG2000(or 5000)-phospholipid conjugates, generated fewer complement activation products; however, complement activation was never completely eliminated. Our observations address the difficulty in making carbon nanotubes more compatible with innate immunity through covalent PEG functionalization as well as double PEGylation strategies. FROM THE CLINICAL EDITOR: Complement-mediated toxicity is a major limiting factor in certain nanomedicine applications. This study clarifies that PEGylation of carbon nanotubes is unlikely to address this complication.

High-resolution bioenergetics correlates the length of continuous protonatable diaminoethane motif of four-armed oligo(ethanamino)amide transfectants to cytotoxicity.

Recent clinical success with Onpattro and cationic ionizable lipid nanoparticle-based mRNA vaccines has rejuvenated research in the design and engineering of broader synthetic cationic vectors for nucleic acid compaction and transfection. However, perturbation of metabolic processes and cytotoxicity are still of concern with synthetic cationic vectors. Here, through an integrated bioenergetic and biomembrane integrity probing in three different human cell lines we reveal the dynamic effect of a library of sequence-defined four-arm oligo(ethanamino)amide transfectant on cell homeostasis, and identify metabolically safe building units over wide concentration ranges. The results show differential effects of the oligo(ethanamino)amide structure of comparable molecular weight on cell energetics. The severity of polycation effect on bioenergetic crisis follows with the length of continuous protonatable diaminoethane motif in the ascending order of glutaryl-triethylene tetramine, succinyl-tetraethylene pentamine and succinyl-pentaethylene hexamine. We further identify oligomeric structures that do not induce bioenergetic crisis even at high concentrations. Finally, transfection studies with a library of polyplexes carrying a reporter gene show no correlation between transfection efficiency and cytotoxicity. These observations demonstrate the usefulness of integrated high-resolution respirometry and plasma membrane integrity probing as a highly sensitive medium-throughput screening strategy for identification and selection of safe building units for transfectant engineering.

Cell medium-dependent dynamic modulation of size and structural transformations of binary phospholipid/ω-3 fatty acid liquid crystalline nano-self-assemblies: Implications in interpretation of cell uptake studies.

Lyotropic non-lamellar liquid crystalline (LLC) nanoparticles, with their tunable structural features and capability of loading a wide range of drugs and reporter probes, are emerging as versatile injectable nanopharmaceuticals. Secondary emulsifiers, such as Pluronic block copolymers, are commonly used for colloidal stabilization of LLC nanoparticles, but their inclusion often compromises the biological safety (e.g., poor hemocompatibility and enhanced cytotoxicity) of the formulation. Here, we introduce a library of colloidally stable, structurally tunable, and pH-responsive lamellar and non-lamellar liquid crystalline nanoparticles from binary mixtures of a phospholipid (phosphatidylglycerol) and three types of omega-3 fatty acids (ω-3 PUFAs), prepared in the absence of a secondary emulsifier and organic solvents. We study formulation size distribution, morphological heterogeneity, and the arrangement of their internal self-assembled architectures by nanoparticle tracking analysis, synchrotron small-angle X-ray scattering, and cryo-transmission electron microscopy. The results show the influence of type and concentration of ω-3 PUFAs in nanoparticle structural transitions spanning from a lamellar (Lα) phase to inverse discontinuous (micellar) cubic Fd3m and hexagonal phase (H2) phases, respectively. We further report on cell-culture medium-dependent dynamic fluctuations in nanoparticle size, number and morphology, and simultaneously monitor uptake kinetics in two human cell lines. We discuss the role of these multiparametric biophysical transformations on nanoparticle-cell interaction kinetics and internalization mechanisms. Collectively, our findings contribute to the understanding of fundamental steps that are imperative for improved engineering of LLC nanoparticles with necessary attributes for pharmaceutical development.

PEGylation of Phosphatidylglycerol/Docosahexaenoic Acid Hexosomes with d-α-Tocopheryl Succinate Poly(ethylene glycol)2000 Induces Morphological Transformation into Vesicles with Prolonged Circulation Times.

Considering the broad therapeutic potential of omega-3 polyunsaturated fatty acids such as docosahexaenoic acid (DHA), here we study the effect of PEGylation of DHA-incorporated hexosomes on their physicochemical characteristics and biodistribution following intravenous injection into mice. Hexosomes were formed from phosphatidylglycerol and DHA with a weight ratio of 3:2. PEGylation was achieved through the incorporation of either d-α-tocopheryl succinate poly(ethylene glycol)2000 (TPGS-mPEG2000) or 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-methoxy-poly(ethylene glycol)2000 (DSPE-mPEG2000) at a concentration of 1.5 wt %. Nanoparticle tracking analysis, synchrotron small-angle scattering, and cryo-transmission electron microscopy were employed to characterize the nanodispersions. The results show that PEGylated lipids induce a structural transition from an inverse hexagonal (H2) phase inside the nanoparticles (hexosomes) to a lamellar (Lα) phase (vesicles). We also followed the effect of mouse plasma on the nanodispersion size distribution, number, and morphology because changes brought by plasma constituents could regulate the in vivo performance of intravenously injected nanodispersions. For comparative biodistribution studies, fluorescently labeled nanodispersions of equivalent quantum yields were injected intravenously into healthy mice. TPGS-mPEG2000-induced vesicles were most effective in avoiding hepatosplenic clearance at early time points. In an orthotopic xenograft murine model of glioblastoma, TPGS-mPEG2000-induced vesicles also showed improved localization to the brain compared with native hexosomes. We discuss these observations and their implications for the future design of injectable lyotropic nonlamellar liquid crystalline drug delivery nanosystems for therapeutic interventions of brain and liver diseases.

Liposome triggering of innate immune responses: a perspective on benefits and adverse reactions.

This commentary focuses on therapeutic advantages as well as adverse reactions that entail liposome triggering of the innate immune system, notably the complement cascade and macrophage clearance mechanisms. Our discussion is centered on macrophage drug delivery, evasion of macrophage surveillance, immunogenicity, and hypersensitivity.

Pharmacokinetic analysis reveals limitations and opportunities for nanomedicine targeting of endothelial and extravascular compartments of tumours.

Targeting of nanoparticles to tumours can potentially improve the specificity of imaging and treatments. We have developed a multicompartmental pharmacokinetic model in order to analyse some of the factors that control efficiency of targeting to intravascular (endothelium) and extravascular (tumour cells and stroma) compartments. We make the assumption that transport across tumour endothelium is an important step for subsequent nanoparticle accumulation in the tumour (area-under-the-curve, AUC) regardless of entry route (interendothelial and transendothelial routes) and study this through a multicompartmental simulation. Our model reveals that increasing endothelial targeting efficiency has a much stronger effect on the AUC than increasing extravascular targeting efficiency. Furthermore, our analysis reveals that both extravasation and intratumoral diffusion rates need to be increased in order to significantly increase the AUC of extravascular-targeted nanoparticles. Increasing the nanoparticle circulation half-life increases the AUC independently of extravasation and intratumoral diffusion. Targeting the extravascular compartment leads to a buildup in the first layer surrounding blood vessels at the expense of deeper layers (binding site barrier). This model explains some of the limitations of tumour targeting and provides important guidelines for the design of targeted nanomedicines.

Perturbation of mitochondrial bioenergetics by polycations counteracts resistance to BRAFE600 inhibition in melanoma cells.

Acquired resistance to the oncogenic BRAFE600 inhibitor vemurafenib is a major clinical challenge in the treatment of melanoma. Vemurafenib resistance is poorly understood; however, available evidence indicates that reprogrammed mitochondrial metabolism could contribute to the resistance mechanism. Here we show that synthetic polycations, such as polyethylenimines and poly(l-lysine)s, prevent vemurafenib resistance in melanoma cells through induction of mitochondrial bioenergetic crisis. Polycations accumulate to a higher degree in hyperpolarized mitochondria (i.e. mitochondria with greater negative charge) which partly explains greater cellular uptake and mitochondrial accumulation of polycations in melanoma cells compared with epidermal melanocytes. Combined treatment of polycations and vemurafenib diminishes the metabolic flexibility of melanoma cells, making them unable to shift between glycolysis and mitochondrial oxidative phosphorylation according to energy demands. Thus, polycations exert considerable detrimental effects on melanoma cells at concentrations better tolerated by epidermal melanocytes and act synergistically with vemurafenib in effectuating bioenergetic crisis, DNA damage and cell death selectively in melanoma cells. Mechanistic understanding of this synergy could lead to the development of macromolecular and polymer therapeutics with structural attributes that encompass even greater cancer-specific cytotoxicity, and provide strategies for tailor-made combination therapies.