Perspectives on complement and phagocytic cell responses to nanoparticles: From fundamentals to adverse reactions.

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.

The Interplay Between Blood Proteins, Complement, and Macrophages on Nanomedicine Performance and Responses.

In the blood, depending on their physicochemical characteristics, nanoparticles attract a wide range of plasma biomolecules. The majority of blood biomolecules bind nonspecifically to nanoparticles. On the other hand, biomolecules such as pattern-recognition complement-sensing proteins may recognize some structural determinants of the pristine surface, causing complement activation. Adsorption of nonspecific blood proteins could also recruit natural antibodies and initiate complement activation, and this seems to be a global process with many preclinical and clinical nanomedicines. We discuss these issues, since complement activation has ramifications in nanomedicine stability and pharmacokinetics, as well as in inflammation and disease progression. Some studies have also predicted a role for complement systems in infusion-related reactions, whereas others show a direct role for macrophages and other immune cells independent of complement activation. We comment on these discrepancies and suggest directions for exploring the underlying mechanisms.

Metabolism of steroidal lactones by the fungus Corynespora cassiicola CBS 161.60 results in a mechanistically unique intramolecular ring-D cyclization resulting in C-14 spiro-lactones.

The fungus Corynespora cassiicola metabolises exogenous steroids in a unique and highly specific manner. Central to this, is the ability of this organism to functionalise substrates (androgens, progestogens) at the highly stereochemically hindered 8ß-position of the steroid nucleus. A recent study has identified that 8ß-hydroxylation occurs through inverted binding in a 9α-hydroxylase. In order to discern the metabolic fate of more symmetrical molecules, we have investigated the metabolism of a range of steroidal analogues functionalised with ring-D lactones, but differing in their functional group stereochemistry at carbon-3. Remarkably, the 3α-functionalised steroidal lactones underwent a mechanistically unique two step intramolecular cyclisation resulting in the generation of a ring-D spiro-carbolactone. This rapid rearrangement initiated with hydroxylation at carbon 14 followed by transesterification, resulting in ring contraction with formation of a butyrolactone at carbon-14. Remarkably this rearrangement was found to be highly dependent on the stereochemistry at carbon-3, with the ß-analogues only undergoing 9α-hydroxylation. The implications of these findings and their mechanistic bases are discussed.

AFM visualization of sub-50nm polyplex disposition to the nuclear pore complex without compromising the integrity of the nuclear envelope.

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.

Metabolic fate of 3α,5-cycloandrostanes in the endogenous lactonization pathway of Aspergillus tamarii KITA.

A series of 3α,5-cycloandrostane analogues with a range of functionality (6α and 6ß alcohols and ketone) at carbon 6 were tested in the endogenous lactonization pathway in Aspergillus tamarii KITA. This metabolic route converts progesterone to testololactone in high yield through a four step enzymatic pathway. To date, no studies have looked at the effect of steroids devoid of polar functionality at carbon 3 and their subsequent metabolic fate by fungi which contain Baeyer-Villiger monooxygenases. Incubation of all of the cycloandrostane analogues resulted in lactonization of ring-D irrespective of C-6 stereochemistry or absence of C-3 functionality. Presence of 6ß-hydroxy group and the C-17 ketone was required in order for these analogues to undergo hydroxylation at C-15ß position. All metabolites were isolated by column chromatography and were identified by (1)H, (13)C NMR, DEPT analysis and other spectroscopic data.

Complement monitoring of Pluronic 127 gel and micelles: suppression of copolymer-mediated complement activation by elevated serum levels of HDL, LDL, and apolipoproteins AI and B-100.

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.

Single-walled carbon nanotube surface control of complement recognition and activation.

Carbon nanotubes (CNTs) are receiving considerable attention in site-specific drug and nucleic acid delivery, photodynamic therapy, and photoacoustic molecular imaging. Despite these advances, nanotubes may activate the complement system (an integral part of innate immunity), which can induce clinically significant anaphylaxis. We demonstrate that single-walled CNTs coated with human serum albumin activate the complement system through C1q-mediated classical and the alternative pathways. Surface coating with methoxypoly(ethylene glycol)-based amphiphiles, which confers solubility and prolongs circulation profiles of CNTs, activates the complement system differently, depending on the amphiphile structure. CNTs with linear poly(ethylene glycol) amphiphiles trigger the lectin pathway of the complement through both L-ficolin and mannan-binding lectin recognition. The lectin pathway activation, however, did not trigger the amplification loop of the alternative pathway. An amphiphile with branched poly(ethylene glycol) architecture also activated the lectin pathway but only through L-ficolin recognition. Importantly, this mode of activation neither generated anaphylatoxins nor induced triggering of the effector arm of the complement system. These observations provide a major step toward nanomaterial surface modification with polymers that have the properties to significantly improve innate immunocompatibility by limiting the formation of complement C3 and C5 convertases.

Polymeric particulate technologies for oral drug delivery and targeting: a pathophysiological perspective.

The oral route for delivery of pharmaceuticals is the most widely used and accepted. Nanoparticles and microparticles are increasingly being applied within this arena to optimize drug targeting and bioavailability. Frequently the carrier systems used are either constructed from or contain polymeric materials. Examples of these nanocarriers include polymeric nanoparticles, solid lipid nanocarriers, self-nanoemulsifying drug delivery systems and nanocrystals. It is the purpose of this review to describe these cutting edge technologies and specifically focus on the interaction and fate of these polymers within the gastrointestinal system.

Polymeric particulate technologies for oral drug delivery and targeting: a pathophysiological perspective.

The oral route for delivery of pharmaceuticals is the most widely used and accepted. Nanoparticles and microparticles are increasingly being applied within this arena to optimize drug targeting and bioavailability. Frequently the carrier systems used are either constructed from or contain polymeric materials. Examples of these nanocarriers include polymeric nanoparticles, solid lipid nanocarriers, self-nanoemulsifying drug delivery systems and nanocrystals. It is the purpose of this review to describe these cutting edge technologies and specifically focus on the interaction and fate of these polymers within the gastrointestinal system.

Genomic perspectives in inter-individual adverse responses following nanomedicine administration: The way forward.

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.

Complement system and the brain: selected pathologies and avenues toward engineering of neurological nanomedicines.

Several nanoparticle systems and supramolecular assemblies are under investigation as potential therapeutic entities for Alzheimer's disease and other neurological disorders through both brain-specific targeting and peripheral effects. However, activation of the complement system, a complex innate immune network of over 30 circulating and membrane-bound proteins, remains a serious concern related to the use of these prospective neurological nanomedicines. The role of complement in processes of neurodegeneration in the injured or aged and diseased central nervous system is well known. Nanoparticle-mediated complement activation cannot only induce adverse cardiopulmonary distress in sensitive subjects, but may further aggravate the already-compromised condition of neurological disorders and diseases. This minireview briefly examines the role of complement in neurological diseases and outlines the current status of the development of key neurological nanomedicines with respect to complement activation. Understanding of these topics is crucial for rational design and development of safe neurological nanomedicines.

Polyethylenimine-mediated impairment of mitochondrial membrane potential, respiration and membrane integrity: implications for nucleic acid delivery and gene therapy.

The 25 kDa branched polyethylenimine (PEI) is a highly efficient synthetic polycation used in transfection protocols, but also triggers mitochondrial-mediated apoptotic cell death processes where the mechanistic issues are poorly understood. We now demonstrate that PEI in a concentration- and time-dependent manner can affect functions (membrane potential, swelling and respiration) and ultrastructural integrity of freshly isolated rat liver mitochondria. The threshold concentration for detection of PEI-mediated impairment of rat liver mitochondrial functions is 3 µg/mL, however, lower PEI levels still exert some effects on mitochondrial morphology and respiration, and these may be related to the inherent membrane perturbing properties of this polycation. The PEI-mediated mitochondrial swelling phase is biphasic, with a fast decaying initial period (most prominent from 4 µg/mL PEI) followed by a slower, linear swelling response. The slow phase is presumably the result of a time-dependent transition permeability opening in mitochondria initially resistant to swelling/depolarization, but may further be related to PEI-induced nanoscale structural defects and/or formation of pores in the outer membrane. Respiration assessments further suggested that PEI in the presence of exogenous ADP behaves in a similar fashion to a slow-acting inhibitory compound. PEI further shows an uncoupling property that is detectable at low respiration rates. The relevance of these findings to PEI-mediated initiation of intrinsic apoptotic pathway is discussed.

Transformation of structurally diverse steroidal analogues by the fungus Corynespora cassiicola CBS 161.60 results in generation of 8ß-monohydroxylated metabolites with evidence in favour of 8ß-hydroxylation through inverted binding in the 9α-hydroxylase.

Corynespora cassiicola has a unique but unexplored ability amongst fungi, in that it can hydroxylate 17α-hydroxyprogesterone at the highly hindered C-8 position of the steroid nucleus. In order to gain greater understanding of the mechanistic basis and capability of the 8ß-hydroxylase we have transformed a range of structurally diverse androgens and progestogens with this organism. This has revealed that both steroid types can be hydroxylated at the 8ß-position. The collective data has demonstrated the first time that 8ß-hydroxylation occurs through inverted binding within a 9α-hydroxylase of the fungus. In the case of the progestogens, for this to occur, the presence of 17α-oxygen functionality (alcohol or epoxide) was essential. Remarkably monohydroxylation of 17α-hydroxyprogesterone at carbons 8ß and 15ß has strongly indicated that the responsible hydroxylase has 2 different binding sites for the ring-A ketone. Unusually, with one exception, all hydroxylation occurred at axial protons and in the case of the progestogens, all above the plane of the ring system. In general all maximally oxidised metabolites contained four oxygen atoms. The importance of these findings in relation to 8ß-hydroxylation of these steroids is discussed.

Material properties in complement activation.

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.

Distinct polymer architecture mediates switching of complement activation pathways at the nanosphere-serum interface: implications for stealth nanoparticle engineering.

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.

Transformation of a series of saturated isomeric steroidal diols by Aspergillus tamarii KITA reveals a precise stereochemical requirement for entrance into the lactonization pathway.

Four isomers of 5α-androstan-3,17-diol have been transformed by the filamentous fungus Aspergillus tamarii, an organism which has the ability to convert progesterone to testololactone in high yield through an endogenous four step enzymatic pathway. The only diol handled within the lactonization pathway was 5α-androstan-3α,17ß-diol which, uniquely underwent oxidation of the 17ß-alcohol to the 17-ketone prior to its Baeyer-Villiger oxidation and the subsequent production of 3α-hydroxy-17a-oxa-D-homo-5α-androstan-17-one. This demonstrated highly specific stereochemical requirements of the 17ß-hydroxysteroid dehydrogenase for oxidation of this specific steroidal diol to occur. In contrast, the other three diols were transformed within the hydroxylation pathway resulting in functionalization at C-11ß. Only 5α-androstan-3ß,17α-diol could bind to the hydroxylase in multiple binding modes undergoing monohydroxylation in 6ß and 7ß positions. Evidence from this study has indicated that hydroxylation of saturated steroidal lactones may occur following binding of ring-D in its open form in which an α-alcohol is generated with close spatial parity to the C-17α hydroxyl position. All metabolites were isolated by column chromatography and were identified by (1)H, (13)C NMR and DEPT analysis and further characterized using infra-red, elemental analysis and accurate mass measurement.

Cationic carriers of genetic material and cell death: a mitochondrial tale.

Central to gene therapy technology has been the use of cationic polymers as vectors for DNA and RNA (polyfectins). These have been presumed to be safer than viral systems which, for example, have been found to switch on oncogenes. Two key polycations that have been intensively researched for use as synthetic vectors are poly(ethylenimine) and poly(L-lysine). A frequent stumbling block with these polyfectins is that long-term gene expression in cell lines has not been achieved. Recently it has transpired that both of these polycations can induce mitochondrially mediated apoptosis. It is the aim of this review to discuss the mechanisms behind the observed polycation toxicity including roles for little studied cellular organelles in the process such as the lysosome and endoplasmic reticulum.

An unusual ring--a opening and other reactions in steroid transformation by the thermophilic fungus Myceliophthora thermophila.

A series of steroids (progesterone, testosterone acetate, 17beta-acetoxy-5 alpha-androstan-3-one, testosterone and androst-4-en-3,17-dione) have been incubated with the thermophilic ascomycete Myceliophthora thermophila CBS 117.65. A wide range of biocatalytic activity was observed with modification at all four rings of the steroid nucleus and the C-17beta side-chain. This is the first thermophilic fungus to demonstrate the side-chain cleavage of progesterone. A unique fungal transformation was observed following incubation of the saturated steroid 17beta-acetoxy-5 alpha-androstan-3-one resulting in 4-hydroxy-3,4-seco-pregn-20-one-3-oic acid which was the product generated following the opening of an A-homo steroid, presumably by lactonohydrolase activity. Hydroxylation predominated at axial protons of the steroids containing 3-one-4-ene ring-functionality. This organism also demonstrated reversible acetylation and oxidation of the 17beta-alcohol of testosterone. All steroidal metabolites were isolated by column chromatography and were identified by (1)H, (13)C NMR, DEPT analysis and other spectroscopic data. The range of steroidal modification achieved with this fungus indicates that these organisms may be a rich source of novel steroid biocatalysis which deserve greater investigation in the future.