S. Moein Moghimi

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.—Moghimi, S. M. Hunter, A. C., Murray, J. C. Nanomedicine: current status and future prospects. FASEB J. 19, 311‐330 (2005)

Poloxamers and poloxamines in nanoparticle engineering and experimental medicine

Poloxamers and poloxamine nonionic surfactants have diverse applications in various biomedical fields ranging from drug delivery and medical imaging to management of vascular diseases and disorders. Although this is a progressive, rapidly advancing field in biotechnology, the future will depend on the recognition and rectification of a range of toxicity issues, which have to be addressed but have frequently been ignored until now.

Methylation of the phosphate oxygen moiety of phospholipid-methoxy(polyethylene glycol) conjugate prevents PEGylated liposome-mediated complement activation and anaphylatoxin production

Methoxy(polyethylene glycol), mPEG,‐grafted liposomes are known to exhibit prolonged circulation time in the blood, but their infusion into a substantial percentage of human subjects triggers immediate non‐IgE‐mediated hypersensitivity reactions. These reactions are strongly believed to arise from anaphylatoxin production through complement activation. Despite the general view that vesicle surface camouflaging with mPEG should dramatically suppress complement activation, here we show that bilayer enrichment of noncomplement activating liposomes [dipalmitoylphosphatidylcholine (DPPC) vesicles] with phospholipid‐mPEG conjugate induces complement activation resulting in vesicle recognition by macrophage complement receptors. The extent of vesicle uptake, however, is dependent on surface mPEG density. We have delineated the likely structural features of phospholipid‐mPEG conjugate responsible for PEGylated liposome‐induced complement activation in normal as well as C1q‐deficient human sera, using DPPC vesicles bearing the classical as well as newly synthesized lipid‐mPEG conjugates. With PEGylated DPPC vesicles, the net anionic charge on the phosphate moiety of phospholipid‐mPEG conjugate played a key role in activation of both classical and alternative pathways of complement and anaphylatoxin production (reflected in significant rises in SC5b‐9, C4d, and C3a‐desarg levels in normal human sera as well as SC5b‐9 in EGTA‐chelated/Mg2+ supplemented serum), since methylation of the phosphate oxygen of phospholipid‐mPEG conjugate, and hence the removal of the negative charge, totally prevented complement activation. To further corroborate on the role of the negative charge in complement activation, vesicles bearing anionic phospholipid‐mPEG conjugates, but not the methylated phospholipid‐mPEG, were shown to significantly decrease serum hemolytic activity and increase plasma thromboxane B2 levels in rats. In contrast to liposomes, phospholipid‐mPEG micelles had no effect on complement activation, thus suggesting a possible role for vesicular zwitterionic phospholipid head‐groups as an additional factor contributing to PEGylated liposome‐mediated complement activation. Our findings provide a rational conceptual basis for development of safer vesicles for site‐specific drug delivery and controlled release at pathological sites.—Moghimi, S. M., Hamad, I., Andresen, T. L., Jørgensen, K., Szebeni, J. Methylation of the phosphate oxygen moiety of phospholipid‐methoxy(polyethylene glycol) conjugate prevents PEGylated liposome‐mediated complement activation and anaphylatoxin production FASEB J. 20, E2057–E2067 (2006)

Hyaluronan-coated nanoparticles: The influence of the molecular weight on CD44-hyaluronan interactions and on the immune response

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.

Tissue specific opsonins for phagocytic cells and their different affinity for cholesterol-rich liposomes

In accordance with the finding of our in vivo experiments reported earlier [(1983) Biochim. Biophys. Acta 761, 142–157 (1986) Biochim. Biophys. Acta 888, 184–190], the results of in vitro experiments show that Kupffer cells avidly take up cholesterol‐poor but not cholesterol‐rich liposomes, whereas splenic phagocytic cells take up preferentially cholesterol‐rich rather than cholesterol‐poor liposomes in the presence of serum. Evidence presented here suggests that serum contains opsonins specific for hepatic and splenic phagocytic cells and these opsonins have different affinities for cholesterol‐rich and cholesterol‐poor liposomes.

Serum opsonins and phagocytosis of saturated and unsaturated phospholipid liposomes

Recently we reported that serum contains opsonins specific for hepatic and splenic phagocytic cells and that these opsonins have different properties and affinities for cholesterol-rich and cholesterol-free egg phosphatidylcholine liposomes (Moghimi, S.M. and Patel, H.M. (1988) FEBS Lett. 233, 143-147). In the present report we investigate the affinity of these opsonins for the liposomes prepared from sphingomyelin and saturated phospholipids, as measured by their effect on the uptake of these liposomes by hepatic and splenic phagocytic cells. Results presented here suggest that neither liver- nor spleen-specific opsonins have affinity for sphingomyelin or saturated phospholipid liposomes since serum fails to enhance their uptake in liver or splenic cells. On the contrary, these liposomes attract serum dysopsonins which inhibit their uptake by liver cells. Inclusion of cholesterol in these liposome preparations enhances their uptake in splenic cells but not in liver cells. It is suggested that fluidity and hydrophobicity of liposomal membranes play an important role in attracting the right opsonins which determine their phagocytic fate.

Complement activation by PEGylated single-walled carbon nanotubes is independent of C1q and alternative pathway turnover

We have investigated the interaction between long circulating poly(ethylene glycol)-stabilized single-walled carbon nanotubes (SWNTs) and the complement system. Aminopoly(ethylene glycol)(5000)-distearoylphosphatidylethanolamine (aminoPEG(5000)-DSPE) and methoxyPEG(5000)-DSPE coated as-grown HIPco SWNTs activated complement in undiluted normal human serum as reflected in significant rises in C4d and SC5b-9 levels, but not the alternative pathway split-product Bb, thus indicating activation exclusively through C4 cleavage. Studies in C2-depleted serum confirmed that PEGylated nanotube-mediated elevation of SC5b-9 was C4b2a convertase-dependent. With the aid of monoclonal antibodies against C1s and human serum depleted from C1q, nanotube-mediated complement activation in C1q-depleted serum was also shown to be independent of classical pathway. Nanotube-mediated C4d elevation in C1q-depleted serum, however, was inhibited by N-acetylglucosamine, Futhan (a broad-spectrum serine protease inhibitor capable of preventing complement activation through all three pathways) and anti-MASP-2 antibodies; this strongly suggests a role for activation of MASP-2 in subsequent C4 cleavage and assembly of C4b2a covertases. Intravenous injection of PEGylated nanotubes in some rats was associated with a significant rise in plasma thromboxane B2 levels, indicative of in vivo nanotube-mediated complement activation. The clinical implications of these observations are discussed.

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.

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.

Differential properties of organ-specific serum opsonins for liver and spleen macrophages

Earlier we reported that serum contains organ-specific opsonins which selectively enhance recognition of liposomes by macrophages in the specific organs of the reticuloendothelial system (Moghimi, S.M. and Patel, H.M. (1988) FEBS Lett. 233, 143-147). The results presented here describe the properties of these organ-specific opsonins which differentiate between liver-specific and spleen-specific opsonins responsible for the enhancement of phagocytosis of liposomes by Kupffer cells and spleen macrophage, respectively. Liver-specific opsonin is a heat-stable macromolecule which on heating or on freezing and thawing exhibits enhanced opsonic activity. Serum also contains a dialysable factor which inhibits its opsonic activity. On the other hand, the spleen-specific opsonin is a heat-labile macromolecule which is sensitive to freezing and thawing and requires a dialysable serum co-factor for its optimum opsonic activity on spleen macrophages. Removal of this factor from serum brings about an irreversible conformational change in the opsonin. Evidence suggests that the spleen-specific opsonin may be composed of more than one different opsonin molecule. It is suggested that the serum factor(s) that inhibits liver-specific opsonic activity and enhances the spleen-specific activity may not be the same molecule, but in both the cases the factor(s) may mediate its function by modifying the process of the opsonisation of liposomes or by influencing the interaction of the opsonised liposomes with the respective cells. We propose that purification of the organ-specific opsonins may provide an opportunity to target drug carriers selectively to a specific organ of the reticuloendothelial system, and help us to evaluate their role in the altered opsonin states known to exist in certain diseases.