Islam Hamad

Poly(ethylene glycol)s generate complement activation products in human serum through increased alternative pathway turnover and a MASP-2-dependent process

Poly(ethylene glycol) (PEG) is receiving increasing attention as an intravenous therapeutic agent per se in a variety of experimental therapeutics and veterinary settings, such as spinal cord injury and traumatic axonal brain injury. PEG is often perceived to be immunologically safe, but here we demonstrate that near-monodisperse endotoxin-free PEGs, at concentrations relevant to above-mentioned settings, can generate complement activation products in human serum on a time scale of minutes (reflected in significant rises in serum levels of C4d, Bb, C3a-desArg and SC5b-9). With the aid of sera depleted from either C2 or C1q, and devoid of anti-PEG antibodies, we further demonstrate that, depending on PEG concentration and M(wt), generation of complement activation products occur either exclusively through the lectin pathway activation or through both the lectin pathway and increased fluid phase turnover of the alternative pathway. Inhibition of PEG-mediated C4d elevation in C1q-depleted serum by the broad serine protease inhibitor Futhan and anti-MASP-2 antibodies as well as competitive studies with d-mannose and N-acetylglucosamine indicated a likely role for ficolins/MASP-2 in PEG-mediated triggering of the lectin pathway and independent of calcium. PEG-mediated amplification of the alternative pathway is a complex process related to protein partitioning and exclusion effect, but factor H depletion/exclusion seems to play a minor role. Our results are relevant to the proposed potential therapeutic applications of intravenous PEG and warn about possible acute PEG infusion-related reactions in sensitive individuals and animals. PEG-mediated generation of complement activation products further provides a plausible explanation to the previously reported unexplained anaphylaxis or the referred cardiovascular collapse in sensitive animals that have received medicines containing high levels of PEG as solubilizer/carrier.

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)

Complement activation cascade triggered by PEG-PL engineered nanomedicines and carbon nanotubes: The challenges ahead

Since their introduction, poly(ethylene glycol)-phospholipid (PEG-PL) conjugates have found many applications in design and engineering of nanosized delivery systems for controlled delivery of pharmaceuticals especially to non-macrophage targets. However, there are reports of idiosyncratic reactions to certain PEG-PL engineered nanomedicines in both experimental animals and man. These reactions are classified as pseudoallergy and may be associated with cardiopulmonary disturbance and other related symptoms of anaphylaxis. Recent studies suggest that complement activation may be a contributing, but not a rate limiting factor, in eliciting hypersensitivity reactions to such nanomedicines in sensitive individuals. This is rather surprising since PEGylated structures are generally assumed to suppress protein adsorption and blood opsonization events including complement. Here, we examine the molecular basis of complement activation by PEG-PL engineered nanomedicines and carbon nanotubes and discuss the challenges ahead.

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.

Activation of the Human Complement System by Cholesterol-Rich and PEGylated Liposomes—Modulation of Cholesterol-Rich Liposome-Mediated Complement Activation by Elevated Serum LDL and HDL Levels

Intravenously infused liposomes may induce cardiopulmonary distress in some human subjects, which is a manifestation of “complement activation-related pseudoallergy.” We have now examined liposome-mediated complement activation in human sera with elevated lipoprotein (LDL and HDL) levels, since abnormal or racial differences in serum lipid profiles seem to modulate the extent of complement activation and associated adverse responses. In accordance with our earlier observations, cholesterol-rich (45 mol% cholesterol) liposomes activated human complement, as reflected by a significant rise in serum level of S-protein-bound form of the terminal complex (SC5b-9). However, liposome-induced rise of SC5b-9 was significantly suppressed when serum HDL cholesterol levels increased by 30%. Increase of serum LDL to levels similar to that observed in heterozygous familial hypercholesterolemia also suppressed liposome-mediated SC5b-9 generation considerably. While intravenous injection of cholesterol-rich liposomes into pigs was associated with an immediate circulatory collapse, the drop in systemic arterial pressure following injection of liposomes preincubated with human lipoproteins was slow and extended. Therefore, surface-associated lipoprotein particles (or apolipoproteins) seem to lessen liposome-induced adverse haemodynamic changes, possibly as a consequence of suppressed complement activation in vivo. PEGylated liposomes were also capable of activating the human complement system, and the presence of surface projected methoxypoly(ethylene glycol) chains did not interfere with generation of C3 opsonic fragments. We also show that poly(ethylene glycol) is not responsible for PEGylated liposome-mediated complement activation. The net anionic charge on the phosphate moiety of the phospholipid-mPEG conjugate seemed to play a critical role in activation of both the classical and alternative pathways of the complement system.

Liposome-mediated triggering of complement cascade

The complement system is one of the most ancient defense strategies, which serves as an important effector arm of both innate and acquired immunity. Three consequences ensue when liposomes trigger the complement system in the blood. These are priming of the vesicular surface by opsonic complement fragments for recognition and clearance by phagocytic cells, generation of anaphylatoxins, and chemoattractants, which may subsequently initiate pseudoallergy-related anaphylaxis in sensitive individuals, and insertion of the lytic complex C5b-9 into the liposomal bilayer, which may result in substantial leakage of vesicular encapsulated aqueous cargo. Here, we briefly review and discuss the molecular basis of complement activation by phospholipid vesicles and reflect on sensitive and rapid Enzyme-Linked Immunosorbent Assays (ELISA) for assessing and monitoring liposome-mediated triggering of a complement cascade in human serum.

Critical issues in site-specific targeting of solid tumours: the carrier, the tumour barriers and the bioavailable drug

Background: The concept of passive and active targeting of solid tumours with intravenously administered particulate and macromolecular carriers is an attractive one that has received considerable attention and promising results have emerged from such attempts at the clinical level. Particulate and polymeric drug carriers have the capability to deliver from 2- to 10-times more drug to solid tumours compared with the administered drug in its free form, and it is through the altered pharmacokinetics and pharmacodynamics of the encapsulated/conjugated drugs relative to free drugs that anticancer drug-induced toxicity is dramatically reduced. Objectives: It is the intention of this article to examine the role of selected particulate and macromolecular entities as carriers of anticancer drugs and their ability to target different components of solid tumours following the intravenous route of injection, and release their cargo in a bioavailable form at levels that exceed the minimum cytotoxic concentration. Methods: The authors of this paper have focused on carrier behaviour (pharmacokinetics of single and multiple injections, and new toxicity issues that may arise from different dosing schedules and dose intensities, as well as from the carrier itself), pathophysiological factors regulating particulate and macromolecular transport into tumours (structural arrangements of tumour vasculature, tumour vascular permeability, interstitial hypertension and interstitial transport), and biochemical and physicochemical factors controlling drug release from extravasated carriers (the bioavailable drug). Conclusion: Nanoscale drug carriers can passively target solid tumours, but achieving therapeutic responses involves pathophysiological processes that control carrier transport into tumours and biochemical factors regulating drug release from extravasated carriers and maintaining free drug levels above the minimum cytotoxic concentration. It is conceivable that future sophistication in tumour targeting and the outcome of end results will depend on an improved understanding of tumour biology and biological barriers, as well as advances in carrier design and nanoengineering.

Complement monitoring of Pluronic gel and micelle copolymer-mediated complement activation by elevated HDL, LDL, apolipoproteins A1 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.

Hypersensitivity Reactions to Nanomedicines: Causative Factors and Optimization of Design Parameters

Administration of clinically approved nanomedicines often induces non-IgE-mediated hypersdensitivity reactions in some patients. Current research strongly suggests that complement activation may be a contributing, but not a rate limiting factor in eliciting hypersensitivity reactions to nanomedicines in sensitive individuals. The molecular basis of complement activation by nanomedicines is complex and depends on nanomedicine structure and dose. These issues are discussed in relation to liposomal, micellar and polymeric nanomedicines as well as current structural design strategies to circumvent nanomedicine- mediated complement activation.