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

S. Moein Moghimi^*,1, Islam Hamad^*, Thomas L. Andresen, Kent Jørgensen and Janos Szebeni

^* Molecular Targeting and Polymer Toxicology Group, School of Pharmacy, University of Brighton, Brighton, UK;

LiPlasome Pharma A/S, Technical University of Denmark, Lyngby, Denmark; and

Nephrology Research Group, Hungarian Academy of Sciences and Institute of Pathophysiology, Semmelweis University, Budapest, Hungary

¹Correspondence: Molecular Targeting and Polymer Toxicology Group, School of Pharmacy, University of Brighton, Cockcroft Bldg., Lewes Rd., Brighton BN2 4GJ, UK. E-mail: s.m.moghimi{at}brighton.ac.uk

SPECIFIC AIMS

Infusion of long circualting methoxypoly(ethylene glycol)-grafted liposomes (PEGylated liposomes) in to a substantial percentage of human subjects may induce cardiopulmonary distress, which is a manifestation of "complement activation-related pseudoallergy." The phospholipid-methoxypoly(ethylene glycol), mPEG, conjugate seems to be responsible for PEGylated liposome-mediated complement activation since vesicles of similar size distribution and bilayer composition as their PEGylated counterparts but without the phospholipid-mPEG conjugates rarely activate the human complement system. In view of numerous medical applications for PEGylated liposomes and the clinical importance of the observed complement-mediated hypersensitivity reactions that can lead to anaphylactoid shock and cardiac anaphylaxis, we sought to investigate and identify which structural features of the phospholipid-mPEG conjugate are responsible for PEGylated liposome-induced complement activation in human sera. Our efforts were particularly focused on design and synthesis of novel lipid-mPEG conjugates where the phospholipid moiety of the conjugates is made from prodrug ether lipids, as a first critical step toward development of safer long circulating vesicles for drug release at pathological sites with elevated secretory phospholipase-A2 activity.

PRINCIPAL FINDINGS

1. Conjugate design
A key structural feature of the mPEG-phospholipid conjugate is the presence of a net anionic charge localized on the phosphate oxygen moiety of the mPEG-phospholipid conjugate, which could be responsible for complement activation. Indeed, anionic vesicles are potent activators of the human complement system. We designed two lipid-mPEG conjugates. The phospholipid component of both conjugates (Conj-A and Conj-B) was dipalmitoylphosphatidylethanoloamine (DPPE) with a nonhydrolyzable ether bond in the 1-position (1-O-DPPE), a substrate for sPLA2. The phosphate moiety of Conj-A was anionic, and its phosphodiester and amide linkage groups were the same as classical phospholipid-mPEG conjugates used for construction of long circulating liposomes. In Conj-B, the phosphate oxygen was methylated to eliminate the net negative charge, thus yielding a nonionic species (Fig. 1 ). The mPEG segments of both Conj-A and -B were purposely short in length (7 ethylene glycol units, molecular mass=350 kDa) for better exposure of phosphodiester linkage and liposome surface to natural antibodies and complement proteins, thus testing the validity of the proposed complement activation hypothesis. 1-O-DPPE-mPEG conjugates were incorporated into DPPC liposomes, since pure DPPC vesicles do not activate the human complement system.

View larger version (36K): [in this window] [in a new window]   Figure 1. Schematic representation of complement activation by anionic lipid-mPEG conjugates. In A, antibodies bind to both phospholipid head-groups in bilayer as well as to the phosphate oxygen moiety of the lipid-mPEG conjugates. All 3 modules of C1q may subsequently interact with antibody (Ab)-coated liposomes leading to activation of the classical pathway of the complement. In addition, C1q (through its basic head) may electrostatically interact with the anionic charge localized on phosphate oxygen of lipid-mPEG conjugate (B). Ether oxygen groups of the mobile mPEG may also accommodate C1q via hydrogen bonding (B). Methylation of phosphate oxygen could sterically interfere with Ab and C1q binding (C).

2. Macrophage uptake studies
Human serum stimulated recognition of radiolabeled DPPC liposomes containing 5 mol% anionic phospholipid-mPEG conjugates by rat peritoneal macrophagesprincipally via CD11b (Mac-1) receptor, but liposome uptake decreased dramatically with increasing the bilayer content of phospholipid-mPEG conjugate to 15 mol%. Serum, however, exerted no stimulatory effect on macrophage uptake of liposomes bearing the methylated conjugate. All liposomes remained intact in serum.

3. Liposome- and micelle-mediated complement activation
DPPC vesicles (70–110 nm) with bilayer content of 5 mol% anionic phospholipid-mPEG conjugate activated complement system in human serum as reflected in significant rises in SC5b-9 and C3a-desarg. Complement activation did proceed via both classical and alternative pathways as demonstrated in normal, C1q-deficient, and factor B depleted C1q-deficient sera. Elevation of serum SC5b-9 levels was more dramatic when bilayer content of Conj-A was increased to 10–15 mol%. Also, larger liposomes (260 nm) were more potent in activating complement. No complement activation, however, was observed with liposomes bearing the methylated conjugate (Conj-B); this was regardless of liposome size and bilayer concentration of Conj-B. To further corroborate the role of the negative charge in complement activation, vesicles bearing anionic phospholipid-mPEG conjugates, but not the methylated Conj-B, were shown to significantly decrease serum hemolytic activity and increase plasma thromboxane B2 levels in rats (Fig. 2 ).

View larger version (14K): [in this window] [in a new window]   Figure 2. Plasma TXB2 levels in rats following bolus intravenous (iv) injection of liposomes (80 mg/kg in 0.5 ml). Liposomes were composed of DPPC and 5 mol% of designated conjugates (size range 80–130 nm). Similar results were obtained with liposomes bearing 10–15 mol% DPPE-mPEG350 conjugate or Conj-B (not shown). The 50% serum hemolytic complement activity (CH50) represents values at 8 min post-liposome injection and is expressed as % of respective baseline level. With the exception of Conj-B, CH50 values were significantly different from baseline (2-sided t test, P<0.05).

In contrast to liposomes, anionic phospholipid-mPEG350 and -mPEG2000 conjugates in monomeric state and in micellar form as well as free PEG molecules were incapable of rising serum levels of complement activating products.

CONCLUSIONS AND SIGNIFICANCE

To our knowledge, this is the first study that elaborates on the involvement of the anionic charge localized on the phosphate oxygen moiety of phospholipid-mPEG conjugates in PEGylated liposome-mediated complement activation and anaphylatoxin production. Subsequently, we designed liposomes bearing a nonionic 1-O-phospholipid-mPEG conjugate that do not activate complement in human and rat sera in vitro as well as in vivo (rat model). This is a critical step toward development of safer zwitterionic vesicles, which are temperature sensitive as well as susceptible to degradation by sPLA2. After observation that phospholipid-mPEG micelles have no effect on complement activation, we suggest a possible role for vesicular zwitterionic phospholipid head groups as an additional or prerequisite factor contributing to PEGylated liposome-mediated complement activation. Methylation could either prevent the binding of natural antibodies to the phosphate oxygen or interfere with spatial organization of surface-bound antibodies for subsequent recognition by all three modules of globular C1q domain (Fig. 1) . Since the top of C1q is predominantly basic, our results may also indicate lack of C1q binding to Conj-B bearing liposomes.

Our approach not only provides a rational conceptual basis for design of safer PEGylated liposomes for site-specific drug delivery and targeting but also highlights the importance of linkage chemistry in complement activation. The latter is of importance for surface engineering of implants and nanodevices with mPEG conjugates and related polymers for in vivo applications.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.06-6186fje

This Article Abstract Full Text Full Text (PDF) All Versions of this Article: fj.06-6186fjev1 20/14/2591    most recent Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Moghimi, S. M. Articles by Szebeni, J. Search for Related Content PubMed PubMed Citation Articles by Moghimi, S. M. Articles by Szebeni, J.