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.

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.

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.

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.

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.

Transformation of 5-ene steroids by the fungus Aspergillus tamarii KITA: mixed molecular fate in lactonization and hydroxylation pathways with identification of a putative 3beta-hydroxy-steroid dehydrogenase/Delta5-Delta4 isomerase pathway.

The fungus Aspergillus tamarii metabolizes progesterone to testololactone in high yield through a sequential four step enzymatic pathway which, has demonstrated flexibility in handling a range of steroidal probes. These substrates have revealed that subtle changes in the molecular structure of the steroid lead to significant changes in route of metabolism. It was therefore of interest to determine the metabolism of a range of 5-ene containing steroidal substrates. Remarkably the primary route of 5-ene steroid metabolism involved a 3beta-hydroxy-steroid dehydrogenase/Delta(5)-Delta(4) isomerase (3beta-HSD/isomerase) enzyme(s), generating 3-one-4-ene functionality and identified for the first time in a fungus with the ability to handle both dehydroepiansdrosterone (DHEA) as well as C-17 side-chain containing compounds such as pregnenolone and 3beta-hydroxy-16alpha,17alpha-epoxypregn-5-en-20-one. Uniquely in all the steroids tested, 3beta-HSD/isomerase activity only occurred following lactonization of the steroidal ring-D. Presence of C-7 allylic hydroxylation, in either epimeric form, inhibited 3beta-HSD/isomerase activity and of the substrates tested, was only observed with DHEA and its 13alpha-methyl analogue. In contrast to previous studies of fungi with 3beta-HSD/isomerase activity DHEA could also enter a minor hydroxylation pathway. Pregnenolone and 3beta-hydroxy-16alpha,17alpha-epoxypregn-5-en-20-one were metabolized solely through the putative 3beta-HSD/isomerase pathway, indicating that a 17beta-methyl ketone functionality inhibits allylic oxidation at C-7. The presence of the 3beta-HSD/isomerase in A. tamarii and the transformation results obtained in this study highlight an important potential role that fungi may have in the generation of environmental androgens.

Distinct metabolic handling of 3beta-hydroxy-17a-oxa-D-homo-5alpha-androstan-17-one by the filamentous fungus Aspergillus tamarii KITA: Evidence in support of steroid/hydroxylase binding hypothesis.

Aspergillus tamarii KITA transforms progesterone in to testololactone in high yield through a sequential four-step enzymatic pathway which also has the flexibility to transform a range of steroidal substrates. This study has investigated the further metabolism of testololactone and a range of fully saturated steroidal lactone analogues. In contrast to testololactone, which even after 120 h incubation did not undergo further metabolism, the lactone analogues entered the minor hydroxylation pathway. Uniquely, after forming 3beta-hydroxy-17a-oxa-D-homo-5alpha-androstan-17-one (48 h) 4 distinct positions on the steroid skeleton were monohydroxylated (11beta, 6beta, 7beta, 11alpha) which geometrically relate to the four binding positions (normal, reverse, inverted normal and inverted reverse) possible within the steroidal hydroxylase(s). This is the first evidence demonstrating the four possible steroid/hydroxylase(s) binding interactions with a single molecule that has previously been hypothesized with a single organism. In addition a rare 1beta-monohydroxylation was observed, this may be indicative of dehydration generating 1-ene functionality in A. tamarii rather than dehydrogenation as reported in man and microorganisms. The importance of these findings in relation to steroid/hydroxylase binding interactions is discussed.

Predominant allylic hydroxylation at carbons 6 and 7 of 4 and 5-ene functionalized steroids by the thermophilic fungus Rhizomucor tauricus IMI23312.

This paper demonstrates for the first time transformation of a series of steroids (progesterone, androst-4-en-3,17-dione, testosterone, pregnenolone and dehydroepiandrosterone) by the thermophilic fungus Rhizomucor tauricus. All transformations were found to be oxidative (monohydroxylation and dihydroxylation) with allylic hydroxylation the predominant route of attack functionalizing the steroidal skeleta. Timed experiments demonstrated that dihydroxylation of progesterone, androst-4-en-3,17-dione and pregnenolone all initiated with hydroxylation on ring-B followed by attack on ring-C. Similar patterns of steroidal transformation to those observed with R. tauricus have been observed with some species of thermophilic Bacilli and mesophilic fungi. All metabolites were isolated by column chromatography and were identified by (1)H, (13)C NMR, DEPT analysis and other spectroscopic data. The application of thermophilic fungi to steroid transformation may represent a potentially rich source for the generation of new steroidal compounds as well as for uncovering inter and intraspecies similarities and differences in steroid metabolism.

Novel quartz crystal microbalance based biosensor for detection of oral epithelial cell-microparticle interaction in real-time.

Recent applications of quartz crystal resonant sensor technology to monitor cell adhesion and specific ligand interaction processes has triggered the development of a new category of quartz crystal microbalance (QCM) based biosensors. In this study human oral epithelial cells (H376) were cultured on quartz sensors and their response to microspheres investigated in situ using the QCM technique. The results demonstrated that this novel biosensor was able to follow cell-microsphere interactions in real-time and under conditions of flow as would occur in the oral cavity. Unique frequency profiles generated in response to the microspheres were postulated to be due to phases of mass addition and altered cellular rigidity. Supporting microscopic evidence demonstrated that the unique frequency responses obtained to these interactions were in part due to binding between the cell surface and the microspheres. Furthermore, a cellular uptake process, in response to microsphere loading was identified and this, by influencing the rigidity of the cellular cytoskeleton, was also detectable through the frequency responses obtained.

Ring-B functionalized androst-4-en-3-ones and ring-C substituted pregn-4-en-3-ones undergo differential transformation in Aspergillus tamarii KITA: ring-A transformation with all C-6 substituted steroids and ring-D transformation with C-11 substituents.

The fungus Aspergillus tamarii transforms progesterone 1 into testololactone 5 in high yield through a four-step enzymatic pathway which is flexible to a range of steroidal substrates. To date, no studies have investigated the fate of C-6 (ring-B) and C-11 (ring-C) functionalized steroidal substrates on metabolism. Remarkably all of the C-6 functionalized substrates underwent reductive metabolism on ring-A in contrast to C-11 functionalized steroids where only ring-D oxidative or reductive transformation occurred. In order to discern the precise role of the functional groups in directing metabolism 6-ketoprogesterone 10 with functionality at C-6 and the ring-D methyl ketone underwent reductive and oxidative transformation on both terminal A and D rings showing that this functionality was directing metabolism. Androst-4-en-3,6-dione 12 devoid of ring-D functionality underwent reductive metabolism on ring-A proving that the C-6 functionality was directing metabolism to this ring with the ring-D methyl ketone responsible for generating transformation at this position. Functionality at C-11 exclusively controlled entry into and degree of metabolism on the lactonization pathway. These novel findings may have important bearing in the future understanding of structure activity relationships in revealing new metabolic pathways and further affords a unique opportunity for generation of novel bioactive steroidal compounds.

Molecular hurdles in polyfectin design and mechanistic background to polycation induced cytotoxicity.

Synthetic polymer based Polyfectins (cationic polymer-DNA complex) have received intensive scientific research as they can potentially circumvent problems associated with viral vectors for gene therapy. These cationic macromolecules can readily condense DNA or RNA into stable nanostructures for use in gene delivery. Recently two commonly used polycations, poly(ethylenimine) (PEI) and poly(L-lysine) have demonstrated their ability to induce apoptosis in a range of human cell lines. This may be the explanation for short-term gene transfection observed with polyfectins. It is the aim of this review to discuss these and other factors behind observed toxicities including the inherent polydisperse nature of polymeric macromolecules and their behaviour in vivo. Strategies for reduction of toxicity are included such as new polymeric synthetic technologies and vector pegylation. There is a clear and immediate need for understanding of the mechanisms which cause polyfectin toxicity which will ultimately facilitate improved vector design and safer gene delivery.

Fate of novel Quasi reverse steroidal substrates by Aspergillus tamarii KITA: bypass of lactonisation and an exclusive role for the minor hydroxylation pathway.

The fungus Aspergillus tamarii transforms progesterone to testololactone in high yield through a flexible four-step enzymatic pathway. To date no studies have investigated the effect of transposition of steroidal functionality between ring-A and ring-D in order to determine the effect on steroidal metabolism. A series of novel quasi reverse steroids (7-9) were synthesised through Linz and Schafer oxidation where 14-en-16-one functionality is generated on ring-D of the steroid. To retain parity with the normal series ring-D functionality was substituted onto ring-A of the analogues. All of the analogues (7-9) were handled through a minor 11beta-hydroxylation pathway with no lactones being formed. In previous studies testololactone is produced within the first 12 h of metabolism. A time course experiment demonstrated that the transformation of these steroids initiated with the formation of a 3beta-hydroxy group after which (48-96 h) hydroxylation through a minor pathway occurred, indicating that this hydroxylase was only then being induced. This is in contrast to the normal fungal metabolism of xenobiotic steroidal substrates where they are primarily hydroxylated. Furthermore, ring-D hydrogenation is reported for the first time as is reverse metabolism on this pathway. All metabolites were isolated by column chromatography and were identified by 1H and 13C NMR spectroscopy, DEPT analysis and other spectroscopic and crystallographic data.

Nanomedicine: current status and future prospects.

Applications of nanotechnology for treatment, diagnosis, monitoring, and control of biological systems has recently been referred to as "nanomedicine" by the National Institutes of Health. Research into the rational delivery and targeting of pharmaceutical, therapeutic, and diagnostic agents is at the forefront of projects in nanomedicine. These involve the identification of precise targets (cells and receptors) related to specific clinical conditions and choice of the appropriate nanocarriers to achieve the required responses while minimizing the side effects. Mononuclear phagocytes, dendritic cells, endothelial cells, and cancers (tumor cells, as well as tumor neovasculature) are key targets. Today, nanotechnology and nanoscience approaches to particle design and formulation are beginning to expand the market for many drugs and are forming the basis for a highly profitable niche within the industry, but some predicted benefits are hyped. This article will highlight rational approaches in design and surface engineering of nanoscale vehicles and entities for site-specific drug delivery and medical imaging after parenteral administration. Potential pitfalls or side effects associated with nanoparticles are also discussed.

An efficient one-pot synthesis generating 4-ene-3,6-dione functionalised steroids from steroidal 5-en-3beta-ols using a modified Jones oxidation methodology.

Steroids with 4-ene-3,6-dione functionality have application in natural product chemistry, as synthetic intermediates and as aromatase inhibitors. Here, we report a two-phase oxidation of a range of steroidal 5-en-3beta-ols into corresponding 4-ene-3,6-diones using a modified Jones oxidation. The new reaction affords high yields (77-89%) of product in relatively short reaction times (1-2h). The simplicity of this reaction gives significant advantages over previously reported methodologies.

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.

Causative factors behind poloxamer 188 (Pluronic F68, Flocor)-induced complement activation in human sera. A protective role against poloxamer-mediated complement activation by elevated serum lipoprotein levels.

Poloxamer 188 is a complex polydisperse mixture of non-ionic macromolecules. Adverse non-IgE-mediated hypersensitivity reactions occur in some individuals following intravenous injection of poloxamer 188-based pharmaceuticals, presumably via complement activation. Here we have delineated potential causal chemical and biological interactive factors behind poloxamer 188-induced complement activation in human serum specimens. We identified the molecular constituents inherent in poloxamer 188 preparations and studied their effect on generation of the two complement split products, SC5b-9 and Bb. Poloxamer 188 activated complement at sub-micellar concentrations and the results indicated the potential involvement of all three known complement activation pathways. The poloxamer-induced rise of SC5b-9 in human sera was abolished in the presence of a recombinant truncated soluble form of complement receptor type 1, thus confirming the role of C3/C5 convertases in the activation process. Poloxamer 188-mediated complement activation is an intrinsic property of these macromolecules and was independent of the degree of sample polydispersity, as opposed to other non-polymeric constituents. Poloxamer 188 preparations also contained unsaturated chains of diblock copolymers capable of generating SC5b-9 in human sera; this effect was terminated following the removal of double bonds by catalytic hydrogenation. By quasi-elastic light scattering, we established interaction between poloxamer and lipoproteins; interestingly, poloxamer-induced rise in SC5b-9 was significantly suppressed when serum HDL and LDL cholesterol levels were increased above normal to mimic two relevant clinical situations. This observation was consistent with previously reported data from patients with abnormal or elevated lipid profiles where no or poor complement activation by poloxamer 188 occurred. Our findings could provide the basis of novel approaches to the prevention of poloxamer-mediated complement activation.

Low and high molecular weight poly(L-lysine)s/poly(L-lysine)-DNA complexes initiate mitochondrial-mediated apoptosis differently.

Poly(L-lysine)s, PLLs, are commonly used for DNA compaction and cell transfection. We report that, although PLLs of low (2.9 kDa), L-PLL, and high (27.4 kDa), H-PLL, Mw in free form and DNA-complexed cannot only cause rapid plasma membrane damage in human cell lines, phosphatidylserine "scrambling" and loss of membrane integrity, but later (24 h) initiate stress-induced cell death via mitochondrial permeabilization without the involvement of processed caspase-2. Mitochondrially mediated apoptosis was confirmed by detection of cytochrome c (Cyt c) release, activation of caspases-9 and -3, and subsequent changes in mitochondrial membrane potential. Plasma membrane damage and apoptosis were most prominent with H-PLL. Cytoplasmic level of Cyt c was more elevated following H-PLL treatment, but unlike L-PLL case, inhibition of Bax channel-forming activity reduced the extent of Cyt c release from mitochondria by half. Inhibition of Bax channel-forming activity had no modulatory effect on L-PLL-mediated Cyt c release. Further, functional studies of isolated mitochondria indicate that H-PLL, but not L-PLL, can directly induce Cyt c release, membrane depolarization, and a progressive decline in the rate of uncoupled respiration. Combined, our data suggest that H-PLL and L-PLL are capable of initiating mitochondrially mediated apoptosis differently. The observed PLL-mediated late-phase apoptosis may provide an explanation for previously reported transient gene expression associated with PLL-based transfection vectors. The importance of our data in relation to design of novel and safer cationic non-viral vectors for human gene therapy is discussed.

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.