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Systemic administration of nanomedicines results in the activation of the complement cascade, promoting phagocytic uptake and triggering proinflammatory responses. Identifying the biomarkers that can predict the "risk" of abnormally high complement responders can improve the safety and efficacy of nanomedicines. Polyethylene glycol (PEG) and dextran are two types of clinically approved polymer coatings that trigger complement activation. We performed a multifaceted analysis of the factors affecting the complement activation by PEGylated liposomal doxorubicin (PLD) and dextran-coated superparamagnetic iron oxide nanoworms (SPIO NWs) in plasma from patients with different inflammatory disease conditions and healthy donors. The complement activation (measured as deposition of the complement protein C3) varied greatly, with 29-fold and 26-fold differences for PLD and SPIO NWs, respectively. Chronic inflammation, acute infection, use of steroids, and sex had minor effects on the variable complement activation, whereas age inversely correlated with the complement activation. C-reactive protein level was not predictive of high (top 20th percentile) complement responses. Plasma concentrations of the main complement factors, as well as total IgG and IgM, showed no correlation with the activation by either nanoparticle. On the other hand, plasma concentrations of anti-PEG IgG and IgM showed a strong positive correlation with the activation by PLD. Particularly, titers of anti-PEG IgM showed the best predictive value for the "risk" of high complement activation by PLD. Titers of antidextran IgG and IgM showed a lower correlation with the activation by SPIO NWs and poor predictive value of the top 20% complement responses. Nanoparticle-bound immunoglobulins showed the best correlation with complement activation and a strong predictive value, supporting the critical role of immunoglobulins in inciting complement. The opsonization of PLD with C3 in plasma with high anti-PEG antibodies was predominantly via the alternative pathway. Characterizing the nature of nanoparticle-binding antibodies has important implications in mitigating and stratifying nanomedicine safety.

In recent years, steady progress has been made in synthesizing and characterizing engineered nanoparticles, resulting in several approved drugs and multiple promising candidates in clinical trials. Regulatory agencies such as the Food and Drug Administration and the European Medicines Agency released important guidance documents facilitating nanoparticle-based drug product development, particularly in the context of liposomes and lipid-based carriers. Even with the progress achieved, it is clear that many barriers must still be overcome to accelerate translation into the clinic. At the recent conference workshop "Mechanisms and Barriers in Nanomedicine" in May 2023 in Colorado, U.S.A., leading experts discussed the formulation, physiological, immunological, regulatory, clinical, and educational barriers. This position paper invites open, unrestricted, nonproprietary discussion among senior faculty, young investigators, and students to trigger ideas and concepts to move the field forward.

There is considerable interest in quantifying anti-PEG antibodies, given their potential involvement in accelerated clearance, complement activation, neutralization, and acute reactions associated with drug delivery systems. Published and commercially available anti-PEG enzyme-linked immunosorbent assays (ELISAs) differ significantly in terms of reagents and conditions, which could be confusing to users who want to perform in-house measurements. Here, we optimize the ELISA protocol for specific detection of anti-PEG IgG and IgM in sera from healthy donors and in plasma from cancer patients administered with PEGylated liposomal doxorubicin. The criterion of specificity is the ability of free PEG or PEGylated liposomes to inhibit the ELISA signals. We found that coating high-binding plates with monoamine methoxy-PEG 5000 , as opposed to bovine serum albumin-PEG 20000 , and blocking with 1% milk, as opposed to albumin or lysozyme, significantly improve the specificity, with over 95% of the signal being blocked by competition. Despite inherent between-assay variability, setting the cutoff value of the optical density at the 80th percentile consistently identified the same subjects. Using the optimized assay, we longitudinally measured levels of anti-PEG IgG/IgM in cancer patients before and after the PEGylated liposomal doxorubicin chemotherapy cycle (1 month apart, three cycles total). Antibody titers did not show any increase but rather a decrease between treatment cycles, and up to 90% of antibodies was bound to the infused drug. This report is a step toward harmonizing anti-PEG assays in human subjects, emphasizing the cost-effectiveness and optimized specificity.

Complement plays a critical role in the immune response toward nanomaterials. The complement attack on a foreign surface results in the deposition of C3, assembly of C3 convertases, the release of anaphylatoxins C3a and C5a, and finally, the formation of membrane attack complex C5b-9. Various technologies can measure complement activation markers in the fluid phase, but measurements of surface C3 deposition are less common. Previously, we developed an ultracentrifugation-based dot blot immunoassay (DBI) to measure the deposition of C3 and other protein corona components on nanoparticles. Here, we validate the repeatability of the DBI and its correlation with pathway-specific and common fluid phase markers. Moreover, we discuss the advantages of DBI, such as cost-effectiveness and versatility, while addressing potential limitations. This study provides insights into complement activation at the nanosurface level, offering a valuable tool for nanomedicine researchers in the field., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Biodegradable poly(alkyl cyanoacrylate) (PACA) nanoparticles (NPs) are receiving increasing attention in anti-cancer nanomedicine development not only for targeted cancer chemotherapy, but also for modulation of the tumor microenvironment. We previously reported promising results with cabazitaxel (CBZ) loaded poly(2-ethylbutyl cyanoacrylate) NPs (PEBCA-CBZ NPs) in a patient derived xenograft (PDX) model of triple-negative breast cancer, and this was associated with a decrease in M2 macrophages. The present study aims at comparing two endotoxin-free PACA NP variants (PEBCA and poly(2-ethylhexyl cyanoacrylate); PEHCA), loaded with CBZ and test whether conjugation with folate would improve their effect., Methods: Cytotoxicity assays and cellular uptake of NPs by flow cytometry were performed in different breast cancer cells. Biodistribution and efficacy studies were performed in PDX models of breast cancer. Tumor associated immune cells were analyzed by multiparametric flow cytometry., Results: In vitro studies showed similar NP-induced cytotoxicity patterns despite difference in early NP internalization. On intravenous injection, the liver cleared the majority of NPs. Efficacy studies in the HBCx39 PDX model demonstrated an enhanced effect of drug-loaded PEBCA variants compared with free drug and PEHCA NPs. Furthermore, the folate conjugated PEBCA variant did not show any enhanced effects compared with the unconjugated counterpart which might be due to unfavorable orientation of folate on the NPs. Finally, analyses of the immune cell populations in tumors revealed that treatment with drug loaded PEBCA variants affected the myeloid cells, especially macrophages, contributing to an inflammatory, immune activated tumor microenvironment., Conclusion: We report for the first time, comparative efficacy of PEBCA and PEHCA NP variants in triple negative breast cancer models and show that CBZ-loaded PEBCA NPs exhibit a combined effect on tumor cells and on the tumor associated myeloid compartment, which may boost the anti-tumor response., Competing Interests: Dr Ýrr Mørch reports NaDeNo Nanoscience is a SINTEF-spin off established in 2022 commercializing PACA-encapsulated cabazitaxel nanoparticles (currently in pre-clinical phase). In addition, Dr Ýrr Mørch has a patent WO 2014/191502 issued to NaDeNo Nanoscience AS, a patent WO 2019/185685 pending to NaDeNo Nanoscience AS. The authors declare that they have no other competing interests., (© 2024 Valsalakumari et al.)

Effective inhibition of the complement system is needed to prevent the accelerated clearance of nanomaterials by complement cascade and inflammatory responses. Here we show that a fusion construct consisting of human complement receptor 2 (CR2) (which recognizes nanosurface-deposited complement 3 (C3)) and complement receptor 1 (CR1) (which blocks C3 convertases) inhibits complement activation with picomolar to low nanomolar efficacy on many types of nanomaterial. We demonstrate that only a small percentage of nanoparticles are randomly opsonized with C3 both in vitro and in vivo, and CR2-CR1 immediately homes in on this subpopulation. Despite rapid in vivo clearance, the co-injection of CR2-CR1 in rats, or its mouse orthologue CR2-Crry in mice, with superparamagnetic iron oxide nanoparticles nearly completely blocks complement opsonization and unwanted granulocyte/monocyte uptake. Furthermore, the inhibitor completely prevents lethargy caused by bolus-injected nanoparticles, without inducing long-lasting complement suppression. These findings suggest the potential of the targeted complement regulators for clinical evaluation., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

The complement system is a multicomponent and multifunctional arm of the innate immune system. Complement contributes to non-specific host defence and maintains homeostasis through multifaceted processes and pathways, including crosstalk with the adaptive immune system, the contact (coagulation) and the kinin systems, and alarmin high-mobility group box 1. Complement is also present intracellularly, orchestrating a wide range of housekeeping and physiological processes in both immune and nonimmune cells, thus showing its more sophisticated roles beyond innate immunity, but its roles are still controversial. Particulate drug carriers and nanopharmaceuticals typically present architectures and surface patterns that trigger complement system in different ways, resulting in both beneficial and adverse responses depending on the extent of complement activation and regulation as well as pathophysiological circumstances. Here we consider the role of complement system and complement regulations in host defence and evaluate the mechanisms by which nanoparticles trigger and modulate complement responses. Effective strategies for the prevention of nanoparticle-mediated complement activation are introduced and discussed., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [P.N.T. declares financial interests in CosmoPHOS Ltd (Greece). Z.S.F. & S.M.M. declare financial interests in S. M. Discovery Inc. and S. M. Discovery Ltd. H.B.H., E.D. & D.S. declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.]., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Nonlamellar lyotropic liquid crystalline (LLC) nanoparticles are a family of versatile nano-self-assemblies, which are finding increasing applications in drug solubilization and targeted drug delivery. LLC nanodispersions are heterogeneous with discrete nanoparticle subpopulations of distinct internal architecture and morphology, frequently coexisting with micelles and/or vesicles. Diversity in the internal architectural repertoire of LLC nanodispersions grants versatility in drug solubilization, encapsulation, and release rate. However, drug incorporation contributes to the heterogeneity of LLC nanodispersions, and on exposure to biological media, LLC nanodispersions often undergo nanostructural and morphological transformations. From a pharmaceutical perspective, coexistence of multiple types of nanoparticles with diverse structural attributes, together with media-driven transformations in colloidal characteristics, brings challenges in dissecting biological and therapeutic performance of LLC nanodispersions in a spatiotemporal manner. Here, we outline innate and acquired heterogeneity of LLC nanodispersions and discuss technological developments and alternative approaches needed to improve homogeneity of LLC formulations for drug delivery applications.

The complement system, professional phagocytes and other cells such as Natural killer cells and mast cells are among the important components of the innate arm of the immune system. These constituents provide an orchestrated array of defences and responses against tissue injury and foreign particles, including nanopharmaceuticals. While interception of nanopharmaceuticals by the immune system is beneficial for immunomodulation and treatment of phagocytic cell disorders, it is imperative to understand the multifaceted mechanisms by which nanopharmaceuticals interacts with the immune system and evaluate the subsequent balance of beneficial versus adverse reactions. An example of the latter is adverse infusion reactions to regulatory-approved nanopharmaceuticals seen in human subjects. Here, we discuss collective opinions and findings from our laboratories in mapping nanoparticle-mediated complement and leucocyte/macrophage responses., Competing Interests: Declaration of Competing Interest S.M.M. & P.N.T. are named inventors on PCT, EPO and US patent filings on dendrimers. P.N.T. declares financial interests in CosmoPHOS Ltd. (Greece). S.M.M. is named inventor on EPO, divisional and US granted patents on NanoLigand Carriers. Z.S.F. & S.M.M declares financial interests in S. M. Discovery Inc. and S. M. Discovery Ltd. H.B.H., A.Y., A.C.H., E.P. & D.S. declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

The contribution of the complement system to non-specific host defence and maintenance of homeostasis is well appreciated. Many particulate systems trigger complement activation but the underlying mechanisms are still poorly understood. Activation of the complement cascade could lead to particle opsonisation by the cleavage products of the third complement protein and might promote inflammatory reactions. Antibody binding in a controlled manner and/or sensing of particles by the complement pattern-recognition molecules such as C1q and mannose-binding lectin can trigger complement activation. Particle curvature and spacing arrangement/periodicity of surface functional groups/ligands are two important parameters that modulate complement responses through multivalent engagement with and conformational regulation of surface-bound antibodies and complement pattern-recognition molecules. Thus, a better fundamental understanding of nanometer- and angstrom-scale parameters that modulate particle interaction with antibodies and complement proteins could portend new possibilities for engineering of particulate drug carriers and biomedical platforms with tuneable complement responses and is discussed here., Competing Interests: Declaration of Competing Interest S.M.M. & P.N.T. are named inventors on PCT, EPO and US patent filings on dendrimers. P.N.T. declares financial interests in CosmoPHOS Ltd. (Greece). H.B.H., A.Y. & D.S. declare that they have no competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Many aspects of innate immune responses to SARS viruses remain unclear. Of particular interest is the role of emerging neutralizing antibodies against the receptor-binding domain (RBD) of SARS-CoV-2 in complement activation and opsonization. To overcome challenges with purified virions, here we introduce "pseudovirus-like" nanoparticles with ∼70 copies of functional recombinant RBD to map complement responses. Nanoparticles fix complement in an RBD-dependent manner in sera of all vaccinated, convalescent, and naı̈ve donors, but vaccinated and convalescent donors with the highest levels of anti-RBD antibodies show significantly higher IgG binding and higher deposition of the third complement protein (C3). The opsonization via anti-RBD antibodies is not an efficient process: on average, each bound antibody promotes binding of less than one C3 molecule. C3 deposition is exclusively through the alternative pathway. C3 molecules bind to protein deposits, but not IgG, on the nanoparticle surface. Lastly, "pseudovirus-like" nanoparticles promote complement-dependent uptake by granulocytes and monocytes in the blood of vaccinated donors with high anti-RBD titers. Using nanoparticles displaying SARS-CoV-2 proteins, we demonstrate subject-dependent differences in complement opsonization and immune recognition.

The complement system plays a key role in opsonization and immune clearance of engineered nanoparticles. Understanding the efficiency, inter-subject, and inter-strain differences of complement opsonization in preclinical species can help with translational nanomedicine development and improve our ability to model complement response in humans. Dextran-coated superparamagnetic iron oxide (SPIO) nanoparticles and a wide range of non-magnetic iron oxide nanoparticle formulations are widely used in magnetic resonance imaging and as clinically approved iron supplements. Previously we found that opsonization of SPIO nanoworms (NW) with the third complement protein (C3) proceeds mostly via the alternative pathway in humans, and via the lectin pathway in mice. Here, we studied the pathway and efficiency of opsonization of 106 nm SPIO NW with C3 in different preclinical species and commonly used laboratory strains. In sera of healthy human donors (n = 6), C3 opsonization proceeded exclusively through the alternative pathway. On the other hand, the C3 opsonization in dogs (6 breeds), rats (4 strains) and mice (5 strains) sera was either partially or completely dependent on the complement Ca 2+ -sensitive pathways (lectin and/or classical). Specifically, C3 opsonization in sera of Long Evans rat strain, and mouse strains widely used in nanomedicine research (BALB/c, C57BL/6 J, and A/J) was only through the Ca 2+ -dependent pathways. Dogs and humans had the highest between-subject variability in C3 opsonization levels, while rat and mouse sera showed the lowest between-strain variability. Furthermore, using a panel of SPIO nanoparticles of different sizes and dextran coatings, we found that the level of C3 opsonization (C3 molecules per milligram Fe) in human sera was lower than in animal sera. At the same time, there was a strong predictive value of complement opsonization in dog and rat sera; nanoparticles with higher C3 deposition in animals showed higher deposition in humans, and vice versa. Notably, the opsonization decreased with decreasing size in all sera. The studies highlight the importance of the consideration of species and strains for predicting human complement responses (opsonization) towards nanomedicines., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Recent infectious disease outbreaks, such as COVID-19 and Ebola, have highlighted the need for rapid and accurate diagnosis to initiate treatment and curb transmission. Successful diagnostic strategies critically depend on the efficiency of biological sampling and timely analysis. However, current diagnostic techniques are invasive/intrusive and present a severe bottleneck by requiring specialist equipment and trained personnel. Moreover, centralised test facilities are poorly accessible and the requirement to travel may increase disease transmission. Self-administrable, point-of-care (PoC) microneedle diagnostic devices could provide a viable solution to these problems. These miniature needle arrays can detect biomarkers in/from the skin in a minimally invasive manner to provide (near-) real-time diagnosis. Few microneedle devices have been developed specifically for infectious disease diagnosis, though similar technologies are well established in other fields and generally adaptable for infectious disease diagnosis. These include microneedles for biofluid extraction, microneedle sensors and analyte-capturing microneedles, or combinations thereof. Analyte sampling/detection from both blood and dermal interstitial fluid is possible. These technologies are in their early stages of development for infectious disease diagnostics, and there is a vast scope for further development. In this review, we discuss the utility and future outlook of these microneedle technologies in infectious disease diagnosis., Competing Interests: The authors have no conflicts of interest to declare., (© 2021 Chinese Pharmaceutical Association and Institute of Materia Medica, Chinese Academy of Medical Sciences. Production and hosting by Elsevier B.V.)

The filamentous bacteriophage fd bind a cell target with exquisite specificity through its few copies of display peptides, whereas nanoparticles functionalized with hundreds to thousands of synthetically generated phage display peptides exhibit variable and often-weak target binding. We hypothesise that some phage peptides in a hierarchical structure rather than in monomeric form recognise and bind their target. Here we show hierarchial forms of a brain-specific phage-derived peptide (herein as NanoLigand Carriers, NLCs) target cerebral endothelial cells through transferrin receptor and the receptor for advanced glycation-end products, cross the blood-brain-barrier and reach neurons and microglial cells. Through intravenous delivery of NLC-β-secretase 1 (BACE1) siRNA complexes we show effective BACE1 down-regulation in the brain without toxicity and inflammation. Therefore, NLCs act as safe multifunctional nanocarriers, overcome efficacy and specificity limitations in active targeting with nanoparticles bearing phage display peptides or cell-penetrating peptides and expand the receptor repertoire of the display peptide.

Complement activation plays an important role in pharmacokinetic and performance of intravenously administered nanomedicines. Significant efforts have been directed toward engineering of nanosurfaces with low complement activation, but due to promiscuity of complement factors and redundancy of pathways, it is still a major challenge. Cell membrane-anchored Decay Accelerating Factor (DAF, a.k.a. CD55) is an efficient membrane bound complement regulator that inhibits both classical and alternative C3 convertases by accelerating their spontaneous decay. Here we tested the effect of various short consensus repeats (SCRs, "sushi" domains) of human CD55 on nanoparticle-mediated complement activation in human sera and plasma. Structural modeling suggested that SCR-2, SCR-3 and SCR-4 are critical for binding to the alternative pathway C3bBb convertase, whereas SCR-1 is dispensable. Various domains were expressed in E.coli and purified by an affinity column. SCRs were added to lepirudin plasma or sera from different healthy subjects, to monitor nanoparticle-mediated complement activation as well as C3 opsonization. Using superparamagnetic iron oxide nanoworms (SPIO NWs), we found that SCR-2-3-4 was the most effective inhibitor (IC 50 ~0.24 μM for C3 opsonization in sera), followed by SCR-1-2-3-4 (IC 50 ~0.6 μM), whereas shorter domains (SCR-3, SCR-2-3, SCR-3-4) were ineffective. SCR-2-3-4 also inhibited C5a generation (IC 50 ~0.16 μM in sera). In addition to SPIO NWs, SCR-2-3-4 effectively inhibited C3 opsonisation and C5a production by clinically approved nanoparticles (Feraheme, LipoDox and Onivyde). SCR-2-3-4 inhibited both lectin and alternative pathway activation by nanoparticles. When added to lepirudin-anticoagulated blood from healthy donors, it significantly reduced the uptake of SPIO NWs by neutrophils and monocytes. These results suggest that soluble domains of membrane-bound complement inhibitors are potential candidates for preventing nanomedicine-mediated complement activation in human subjects., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Cationic polyplexes and lipoplexes are widely used as artificial systems for nucleic acid delivery into the cells, but they can also induce cell death. Mechanistic understanding of cell toxicity and biological side effects of these cationic entities is essential for optimization strategies and design of safe and efficient nucleic acid delivery systems. Numerous methods are presently available to detect and delineate cytotoxicity and cell death-mediated signals in cell cultures. Activation of caspases is part of the classical apoptosis program and increased caspase activity is therefore a well-established hallmark of programmed cell death. Additional methods to monitor cell-death related signals must, however, also be carried out to fully define the type of cell toxicity in play. These may include methods that detect plasma membrane damage, loss of mitochondrial membrane potential, phosphatidylserine exposure, and cell morphological changes (e.g., membrane blebbing, nuclear changes, cytoplasmic swelling, cell rounding). Here we describe a 96-well format protocol for detection of caspase-3/7 activity in cell lysates, based on a fluorescent caspase-3 assay, combined with a method to simultaneously determine relative protein contents in the individual wells.

Cellular toxicity and/or cell death entail complex mechanisms that require multifaceted characterization. A detailed mechanistic assessment of cytotoxicity is essential for design and construction of more effective polycations for nucleic acid delivery. A single toxicity assay cannot stand alone in determining the type and extent of damage or cell death mechanism. In this chapter we describe a lactate dehydrogenase (LDH) assay for high-throughput screening that can be used as a starting point for further detailed cytotoxicity determination. LDH release is considered an early event in necrosis but a late event in apoptosis. An accurate temporal assessment of the toxic responses is crucial as late apoptosis may convert into necrosis as well as in situations where cell death is initiated without any visible cell morphological changes or responses in assays measuring late events, resulting in early ongoing toxicity being overlooked.

A better understanding of the molecular basis of polycation-mediated impairment of mitochondrial bioenergetics might improve the design and synthesis of more efficient and safer polymeric transfectants. Here we utilize the phosphorylation control protocol for studying the effect of polycations on mitochondrial respiration in intact mammalian cells using Oxygraph-2k (OROBOROS). The protocol offers an opportunity to comprehensively monitor mitochondrial respiration through consecutive additions of various cell membrane permeable compounds that alter mitochondrial respiration, thus providing useful information on different states of mitochondrial respiration. Furthermore, we demonstrate how to analyze the data obtained with the phosphorylation control protocol and how to calculate the respiratory flux ratios, which can be used as indicators of respiratory functionality and mitochondrial health.

It has been questioned as to whether polyplexes in the cytoplasm can reach the nuclear compartment and if so in what form. By applying atomic force microscopy (AFM) to the nuclear envelope and the nuclear pore complexes, we demonstrate that disposition of polyethylenimine (PEI)/DNA polyplexes that were microinjected into the oocytes of Xenopus laevis, as an example of a non-dividing cell, is exclusive to the nuclear pore complex (NPC). AFM images show NPCs clogged only with sub-50nm polyplexes. This mode of disposition neither altered the morphology/integrity of the nuclear membrane nor the NPC. AFM images further show polyplexes on the nucleoplasmic side of the envelope, presumably indicating species in transit. Transmission electron microscopy studies of ruptured nuclei from transfected human cell lines demonstrate the presence of sub-50nm particles resembling polyplexes in morphology compared with control preparations., (Copyright © 2016. Published by Elsevier B.V.)

Graphene oxide (GO) is believed to become applicable in biomedical products and medicine, thereby necessitating appropriate safety evaluation dependent on their applications and the route of administration. We have examined the effect of GO form (in solution versus immobilized) and oxidation state on two related elements of innate immunity: the complement system and interleukin-6 (IL-6) release in human blood. In solution, there was a decrease in GO-mediated complement activation with decreasing surface oxygen content (and altered oxygen functionality), whereas with immobilized GO complement response were reversed and increased with decreasing oxygen content. GO solutions, at concentrations below complement activating threshold, did not induce IL-6 release from human blood leukocytes, and further dampened lipopolysaccharide-induced IL-6 release in the whole blood. The latter effect became more profound with GO's having higher oxygen content. This protective role of GO solutions, however, disappeared at higher concentrations above complement-activating threshold. We discuss these results in relation to GO surface structure and properties, and implications for local administration and development of GO-based implantable devices., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

In order to improve patient's benefit and safety, comprehensive regulatory guidelines on specificities of Non-Biological Complex Drugs (NBCDs), such as doxorubicin-encapsulated liposomes, and their follow-on versions are needed. Here, we compare Doxil® and its European analog Caelyx® with the two follow-on products DOXOrubicin (approved by the US Food and Drug Administration) and SinaDoxosome (produced in Iran) by cryogenic transmission electron microscopy, dynamic light scattering and Nanoparticle Tracking Analysis, and assess their potential in activating the complement system in human sera. We found subtle physicochemical differences between the tested liposomal products and even between the tested batches of Doxil® and Caelyx®. Notably, these included differences in vesicular population aspect ratios and particle number. Among the tested products, only SinaDoxosome, in addition to the presence of unilamellar vesicles with entrapped doxorubicin crystals, contained empty circular disks. Differences were also found in complement responses, which may be related to some morphological differences. This study has demonstrated an integrated biophysical and immunological toolbox for improved analysis and detection of physical differences among vesicular populations that may modulate their clinical performance. Combined, these approaches may help better product selection for infusion to the patients as well as for improved design and characterization of future vesicular NBCDs with enhanced clinical performance and safety., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The accumulation of extracellular amyloid-beta (Aβ) peptide and intracellular neurofibrillary tangles in the brain are two major neuropathological hallmarks of Alzheimer's disease (AD). It is thought that an equilibrium exists between Aβ in the brain and in the peripheral blood and thus, it was hypothesized that shifting this equilibrium towards the blood by enhancing peripheral clearance might reduce Aβ levels in the brain: the 'sink effect'. We tested this hypothesis by intraperitoneally injecting APP/PS1 transgenic mice with small unilamellar vesicles containing either phosphatidic acid or cardiolipin over 3weeks. This treatment reduced significantly the amount of Aβ in the plasma and the brain levels of Aβ were lighter affected. Nevertheless, this dosing regimen did modulate tau phosphorylation and glycogen synthase kinase 3 activities in the brain, suggesting that the targeting of circulating Aβ may be therapeutically relevant in AD., From the Clinical Editor: Intraperitoneal injection of small unilamellar vesicles containing phosphatidic acid or cardiolipin significantly reduced the amount of amyloid-beta (Aß) peptide in the plasma in a rodent model. Brain levels of Aß were also affected - although to a lesser extent - suggesting that targeting of circulating Aß may be therapeutically relevant of Alzheimer's disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In spite of significant insolubility and toxicity, carbon nanotubes (CNTs) erupt into the biomedical research, and create an increasing interest in the field of nanomedicine. Single-walled CNTs (SWCNTs) are highly hydrophobic and have been shown to be toxic while systemically administrated. Thus, SWCNTs have to be functionalized to render water solubility and biocompatibility. Herein, we introduce a method for functionalizing SWCNT using phospholipids (PL) conjugated to hyaluronan (HA), a hydrophilic glycosaminoglycan, with known receptors on many types of cancer and immune cells. This functionalization allowed for CNT solubilization, endowed the particles with stealth properties evading the immune system, and reduced immune and mitochondrial toxicity both in vitro and in vivo. The CNT-PL-HA internalized into macrophages and showed low cytotoxicity. In addition, CNT-PL-HA did not induce an inflammatory response in macrophages as evidenced by the cytokine profiling and the use of image-based high-content analysis approach in contrast to non-modified CNTs. In addition, systemic administration of CNT-PL-HA into healthy C57BL/6 mice did not alter the total number of leukocytes nor increased liver enzyme release as opposed to CNTs. Taken together, these results suggest an immune protective mechanism by the PL-HA coating that could provide future therapeutic benefit., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Poloxamer 407 is a non-ionic polyethylene oxide (PEO)/polypropylene oxide (PPO) block copolymer, which exhibits reversible thermogelation properties. Poloxamer gel has attracted many applications for controlled release of therapeutic agents as well as in surgical interventions such as controlled vascular occlusion. We show that poloxamer gel can trigger the complement system, which is an integral part of innate immunity and its inadvertent activation can induce clinically significant anaphylaxis. Complement activation by the poloxamer gel is through the alternative pathway, but material transformations from gel to the solution state further incite complement through calcium-sensitive pathways, where a role for C1q and antibodies has been eliminated. Poloxamer addition to plasma/serum (at levels above its critical micelle concentration, cmc) induced formation of large and diffused structures, which may have been responsible for triggering complement. Since poloxamer 407 administration has been reported to cause significant changes in plasma cholesterol and triglyceride levels we further examined the role of lipoproteins in poloxamer-mediated complement activation. Our results show a protective role for elevated serum HDL, LDL and their predominant apolipoproteins (apoAI and apoB-100, respectively) on poloxamer-mediated complement activation. Electron microscopy investigations indicated formation of two distinct populations of new structures on mixing of poloxamer (at concentrations above its cmc) with human LDL, which could have played a significant role in regulating complement activation. These observations are in line with the suggested modulatory role of lipoproteins in host defence and inflammatory processes. A better understanding of block copolymer interaction with lipoproteins/apolipoproteins could improve the immune safety of surgical and therapeutic interventions requiring PEO/PPO block copolymers and may provide new insights for combinatorial design of multifunctional copolymers., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Despite the preclinical success of adoptive therapy with T cells bearing chimeric nanoconstructed antigen receptors (CARs), certain limitations of this therapeutic approach such as the immunogenicity of the antigen binding domain, the emergence of tumor cell escape variants and the blocking capacity of soluble antigen still remain. Here, we address these issues using a novel CAR binding moiety based on the oligoclonal camelid single domain antibodies. A unique set of 13 single domain antibodies were selected from an immunized camel phage library based on their target specificity and binding affinity. A combination of these single domain antibodies was used to generate four tumor associated glycoprotein (TAG-72)-specific CARs harboring an identical antigen binding site, but with different signaling and spacer domains. Although all four CARs were functionally active against the TAG-72 expressing tumor cells, the combination of CD3ζ, OX40, CD28 as well as the CH3-CH2-hinge-hinge domains most efficiently triggered T cell activation. Importantly, CAR mediated functions were not blocked by the soluble TAG-72 antigen at a supraphysiological concentration. Our approach may have the potential to reverse multiple tumor immune evasion mechanisms, avoid CAR immunogenicity, and overcome problems in cancer gene therapy with engineered nanoconstructs., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Cationic polyplexes and lipoplexes are widely used as artificial systems for nucleic acid delivery into the cells, but they can also induce cell death. Mechanistic understanding of cell toxicity and biological side effects of these cationic entities is essential for optimization strategies and design of safe and efficient nucleic acid delivery systems. Numerous methods are presently available to detect and delineate cytotoxicity and cell death-mediated signals in cell cultures. Activation of caspases is part of the classical apoptosis program and increased caspase activity is therefore a well-established hallmark of programmed cell death. Additional methods to monitor cell death-related signals must, however, also be carried out to fully define the type of cell toxicity in play. These may include methods that detect plasma membrane damage, loss of mitochondrial membrane potential, phosphatidylserine exposure, and cell morphological changes (e.g., membrane blebbing, nuclear changes, cytoplasmic swelling, cell rounding). Here we describe a 96-well format protocol for detection of capsase-3/7 activity in cell lysates, based on a fluorescent caspase-3 assay, combined with a method to simultaneously determine relative protein contents in the individual wells.

Cellular toxicity and/or cell death entail complex mechanisms that require detailed evaluation for proper characterization. A detailed mechanistic assessment of cytotoxicity is essential for design and construction of more effective polycations for nucleic acid delivery. A single toxicity assay cannot stand alone in determining the type and extent of damage or cell death mechanism. In this chapter we describe a lactate dehydrogenase (LDH) assay for high-throughput screening that can be used as a starting point for further detailed cytotoxicity determination. LDH release is considered an early event in necrosis but a late event in apoptosis. An accurate temporal assessment of the toxic responses is crucial as late apoptosis may convert into necrosis as well as in situations where cell death is initiated without any visible cell morphological changes or responses in assays measuring late events, resulting in early ongoing toxicity being overlooked.

Carbon nanotubes are entities of different morphology and aspect ratios with anisotropic character. Due to their unique electronic, photonic, mechanical and chemical properties, carbon nanotubes are receiving increasing attention in nanomedicine research where examples include site-specific drug and nucleic acid delivery, photodynamic therapy and photoacoustic molecular imaging. The interaction of carbon nanotubes with the immune system, which plays a key role in the recognition and elimination of foreign materials, and consequential responses, is of central importance for the proposed successful biomedical applications of nanotubes. Research in this avenue, however, is scant and the limited available data are rather contradictory. In this progress article we have collected some of the most important experimental results obtained thus far on carbon nanotube-mediated immune toxicity with an emphasis on cardiovascular exposure, including activation of the complement system, macrophage recognition and clearance, and overall effects on the functionality of different immune cells. Mapping these immune-related risks as well as understanding their molecular mechanisms is a crucial step in the development of any carbon nanotube-containing nanopharmaceuticals., (Copyright © 2012 Elsevier B.V. All rights reserved.)

The underlying mechanism of intravenous infusion-related adverse reactions inherent to regulatory-approved nanomedicines still remains elusive. There are substantial inter-individual differences in observed adverse reactions, which may include cardiovascular, broncho-pulmonary, muco-cutaneous, neuro-psychosomatic and autonomic manifestations. Although nanomedicine-mediated triggering of complement activation has been suggested to be a significant contributing factor to these adverse events, complement activation may still proceed in non-responders. Whether these reactions share similar immunological mechanisms and underpinning genetic factors with drug hypersensitivity syndrome remains to be investigated. Genetic association studies could be a powerful tool to dissect causative factors and reveal the multiple molecular pathways that induce infusion related adverse reactions. It is envisaged that such research may lead to the design of reliable in vitro profiling tests for risk assessment and treatment decisions, thereby revolutionizing the practice of medicine with nanopharmaceuticals. Such procedures may further improve regulatory approval processes for nanomedicines currently in the pipeline and decrease the overall cost of health care. Here we discuss some key innate immunity genes and their polymorphisms in relation to nanomedicine infusion-mediated symptomatic responses., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Nanoparticulate systems are widely used for site-specific drug and gene delivery as well as for medical imaging. The mode of nanoparticle-cell interaction may have a significant effect on the pathway of nanoparticle internalization and subsequent intracellular trafficking. Total internal reflection fluorescence (TIRF) microscopy allows for real-time monitoring of nanoparticle-membrane interaction events, which can provide vital information in relation to design and surface engineering of therapeutic nanoparticles for cell-specific targeting. In contrast to other microscopy techniques, the bleaching effect by lasers in TIRF microscopy is considerably less when using fluorescent nanoparticles and it reduces photo-induced cytotoxicity during visualization of live-cell events since it only illuminates the specific area near or at the plasma membrane.

Hyaluronan (HA), a naturally occurring glycosaminoglycan, exerts different biological functions depending on its molecular weight ranging from 4000-10M Da. While high Mw HA (HMw-HA) is considered as anti-inflammatory, low Mw HA (LMw-HA) has been reported to activate an innate immune response. In addition, opposing effects on cell proliferation mediated by the HA receptor CD44, have also been reported for high and low Mw HA. We have previously demonstrated that HMw-HA can be covalently attached to the surface of lipid nanoparticles (NPs), endowing the carriers with long circulation and active targeting towards HA-receptors (CD44 and CD168) highly expressed on tumors. Here we present a small library of HA-coated NPs distinguished only by the Mw of their surface anchored HA ranging from 6.4 kDa to 1500 kDa. All types of NPs exerted no effect on macrophages, T cells and ovarian cancer cells proliferation. In addition, no induction of cytokines or complement activation was observed. The affinity towards the CD44 receptor was found to be solely controlled by the Mw of the NPs surface-bound HA, from extremely low binding for LMw-HA to binding with high affinity for HMw-HA. These findings have major implications for the use of HA in nanomedicine as LMw-HA surface modified-NPs could be a viable option for the replacement of polyethylene glycol (PEG) when passive delivery is required, lacking adverse effects such as complement activation and cytokine induction, while HMw-HA-coated NPs could be used for active targeting to CD44 overexpressing tumors and aberrantly activated leukocytes in inflammation., (Copyright © 2011 Elsevier B.V. All rights reserved.)

We provide evidence for combining a single domain antibody (nanobody)-based targeting approach with transcriptional targeting as a safe way to deliver lethal transgenes to MUC1 over-expressing cancer cells. From a nanobody immune library, we have isolated an anti-DF3/Mucin1 (MUC1) nanobody with high specificity for the MUC1 antigen, which is an aberrantly glycosylated glycoprotein over-expressed in tumours of epithelial origin. The anti-MUC1 nanobody was covalently linked to the distal end of poly(ethylene glycol)(3500) (PEG(3500)) in PEG(3500)-25kDa polyethylenimine (PEI) conjugates and the resultant macromolecular entity successfully condensed plasmids coding a transcriptionally targeted truncated-Bid (tBid) killer gene under the control of the cancer-specific MUC1 promoter. The engineered polyplexes exhibited favourable physicochemical characteristics for transfection and dramatically elevated the level of Bid/tBid expression in both MUC1 over-expressing caspase 3-deficient (MCF7 cells) and caspase 3-positive (T47D and SKBR3) tumour cell lines and, concomitantly, induced considerable cell death. Neither transgene expression nor cell death occurred when the MUC1 promoter was replaced with the CNS-specific synapsin I promoter. Since PEGylated PEI was only responsible for DNA compaction and played no significant role in direct transfection and cell killing, our attempts overcome previously reported PEI-mediated apoptotic and necrotic cell death, which is advantageous for future in vivo transcriptional targeting as this will minimize (or eliminate) non-targeted cell damage., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Alzheimer's disease (AD) represents the most common form of dementia worldwide, affecting more than 35 million people. Advances in nanotechnology are beginning to exert a significant impact in neurology. These approaches, which are often based on the design and engineering of a plethora of nanoparticulate entities with high specificity for brain capillary endothelial cells, are currently being applied to early AD diagnosis and treatment. In addition, nanoparticles (NPs) with high affinity for the circulating amyloid-β (Aβ) forms may induce "sink effect" and improve the AD condition. There are also developments in relation to in vitro diagnostics for AD, including ultrasensitive NP-based bio-barcodes, immunosensors, as well as scanning tunneling microscopy procedures capable of detecting Aβ(1-40) and Aβ(1-42). However, there are concerns regarding the initiation of possible NP-mediated adverse events in AD, thus demanding the use of precisely assembled nanoconstructs from biocompatible materials. Key advances and safety issues are reviewed and discussed., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Uncontrolled complement activation can induce many inflammatory and life threatening conditions. Accordingly, the role of complement in initiation of adverse reactions to polymers and nanoparticulate drug carriers is receiving increasing attention and has prompted extensive 'structure-immune performance' relationship studies in nanomedicine research at many fronts. The interaction between nanomaterials and the complement system is complex and regulated by inter-related factors that include nanoscale size, morphology and surface characteristics. Each of these parameters may affect complement activation differently and through different sensing molecules and initiation pathways. The importance of material properties in triggering complement is considered and mechanistic aspects discussed. Mechanistic understanding of complement events could provide rational approaches for improved material design and nanoengineering strategies for clinical medicine., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Our ability to engineer nanomaterials for biological and medical applications is continuously increasing, and nanomaterial designs are becoming more and more complex. One very good example of this is the drug delivery field where nanoparticle systems can be used to deliver drugs specifically to diseased tissue. In the early days, the design of the nanoparticles was relatively simple, but today we can surface functionalize and manipulate material properties to target diseased tissue and build highly complex drug release mechanisms into our designs. One of the most promising strategies in drug delivery is to use ligands that target overexpressed or selectively expressed receptors on the surface of diseased cells. To utilize this approach, it is necessary to control the chemistry involved in surface functionalization of nanoparticles and construct highly specific functionalities that can be used as attachment points for a diverse range of targeting ligands such as antibodies, peptides, carbohydrates and vitamins. In this review we provide an overview and a critical evaluation of the many strategies that have been developed for surface functionalization of nanoparticles and furthermore provide an overview of how these methods have been used in drug delivery systems.

Nanoparticles with surface projected polyethyleneoxide (PEO) chains in "mushroom-brush" and "brush" configurations display stealth properties in systemic circulation and have numerous applications in site-specific targeting for controlled drug delivery and release as well as diagnostic imaging. We report on the "structure-activity" relationship pertaining to surface-immobilized PEO of various configurations on model nanoparticles, and the initiation of complement cascade, which is the most ancient component of innate human immunity, and its activation may induce clinically significant adverse reactions in some individuals. Conformational states of surface-projected PEO chains, arising from the block copolymer poloxamine 908 adsorption, on polystyrene nanoparticles trigger complement activation differently. Alteration of copolymer architecture on nanospheres from mushroom to brush configuration not only switches complement activation from C1q-dependent classical to lectin pathway but also reduces the level of generated complement activation products C4d, Bb, C5a, and SC5b-9. Also, changes in adsorbed polymer configuration trigger alternative pathway activation differently and through different initiators. Notably, the role for properdin-mediated activation of alternative pathway was only restricted to particles displaying PEO chains in a transition mushroom-brush configuration. Since nanoparticle-mediated complement activation is of clinical concern, our findings provide a rational basis for improved surface engineering and design of immunologically safer stealth and targetable nanosystems with polymers for use in clinical medicine.

Promising results have come from attempts to direct drugs, nucleic acids and diagnostic agents to tumours by using polymeric nanoparticles. Such carriers are versatile; their encapsulation capacity, drug release profile, and biological performance vary with their chemical makeup, morphology, and size. Polymeric nanoparticles may therefore be engineered for therapeutic and diagnostic purposes in accordance with the type, developmental stage and location of the cancer as well as the required route of administration. This article examines recent developments in design and engineering of polymeric nanoparticles and related platforms to include supramolecular systems such as nanocapsules and nanoparticle-based hydrogels, and assesses their potential diagnostic and therapeutic applications in experimental and clinical oncology.