BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 15 – ‘stealth’ particles 1 of 8 Lecture 15: ‘Stealth’ particles Last time: Nano- and micro-particle carriers Today: Delivery of drugs to tissue from circulation ‘stealth’ particles theory and function Reading: S. Stolnik et al. ‘Long circulating microparticulate drug carriers,’ Adv. Drug. Deliv. Rev. 16, 195 (1995) Delivery of drugs to tissues via systemic circulation Avenues of systemic molecule delivery 1. Intravenous injection 2. oral delivery to lumen of gut Transcytosis: objectives for systemic delivery to tissues/organs ? avoid premature elimination by kidneys o kidneys filter out molecules smaller than X nm ? Avoiding reticuloendothelial system (RES) o Particles larger than 200 nm screened by monocytes and macrophages in liver and spleen (Biochem. Biophys. Res. Commun. 177, 861 (1991)) ? Kupffer cells – macrophages in liver o Particulate removal aided by process of opsonization ? Process of ‘tagging’ foreign particles for efficient removal by macrophages BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 15 – ‘stealth’ particles 2 of 8 ? Particles tagged by different opsonins removed by cells in different organs e.g. liver vs. spleen ? Components: ? Complement proteins, particularly C3 and C5 ? Immunoglobulins ? Other proteins known to facilitate particle uptake: o Fibronectin o C-reactive protein o Tuftsin ? Protein adsorption to particles is key ? Hydrophobic particles quickly removed from circulation in vivo (Intl. J. Pharm. 29, 53 (1986)) ? Penetration through capillary walls into tissues o Passive delivery due to ‘leakiness’ of vessels at sites of inflammation or tumor vasulature abnormalities 1 ? Avoid induction of antibodies against molecules/particles o FUTURE OBJECTIVE: o Pre-targeting drugs that trigger permeability of vasculature at desired sites to allow tissue access – a needed advance to make tissue-specific targeting truly work 1 Stealth particles 2,3 ? How to avoid uptake by scavenger cells? o Van Oss showed in 1978 (Ann. Rev. Microbiol. 32, 19 (1978)) that many bacteria have a highly hydrophilic hydrated surface layer of protein, polysaccharide, and glycoprotein that reduces interactions with blood components and inhibits phagocytosis o Davis at about the same time (J. Biol. Chem. 252, 3578 (1977)) showed that PEGylated proteins are non- immunogenic and have greatly increased half-lives in vivo Concept 4,5 ? Similar to design of protein adsorption-resistant surfaces, provide entropic barrier o Eliminate protein binding to particle/molecule o Enhanced solubility (proteins)/stability (particles) in water ? Functionalization of particles with PEG increases in vivo circulation time o Reduced protein adsorption to particle/molecule surface o Receptors of macrophages unable to bind particle/molecule ? Same polymers employed as in the design of protein-resistant surfaces o Most studied: poly(ethylene glycol) o Others: dextran ? PEGylation applied to all forms of molecular, nano-, and micro-particulate carriers BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 15 – ‘stealth’ particles 3 of 8 (Stolnik et al. 1995) Adsorbed PEG block copolymers PEG block copolymer micelles/nanoparticles Covalently grafted PEG Block copolymer adsorbed Block copolymer entangled Carrier surface e.g. Pluronics: PEO PPO PEGylated carriers: protein PEG chain (Shi et al. 2002) liposomes Potential for membrane fusion ? PEG binds 2-3 water molecules per repeat unit 5 o Causes PEGylated compounds to function as though they are 5-10 times larger than their true molar mass ? Observed in SEC and gel electrophoresis experiments 4 ? PEG/water ‘shield’ can reduce activity of protein, but generally the increased circulation time makes up for this Theory of stealth particle repulsion of protein binding to carriers 6,7 o Theory of Halperin 7 , building on previous analyses of Alexander/de Gennes and Szleifer BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 15 – ‘stealth’ particles 4 of 8 (Halperin 1999) Primary adsorption secondary adsorption o Experimental measurements and comparison with theory o Measurements made using surface plasmon resonance 6 o Experimental details: ? σ = on graphs! [area/chain] ? PEG MW = 2000 g/mole (N = 45) ? Layer thicknesses L 0 = 40-60 ? ? Sizes of tested proteins: ? BPTI (bovine pancreatic trypsin inhibitor) : MW = 6000- g/mole; R ~ 21x21x30 ? ? HSA (human serum albumin): MW = 66,200 g/mole; R = 38x38x150 ? ? FBN (Fibrinogen): MW = 340,000 g/mole; R ~ 55x55x460 ? rod-link protein Comparison of theory with experiment (Efremova et al. 2000) BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 15 – ‘stealth’ particles 5 of 8 Function of stealth particles o PEGylated molecules o PEGylated IFN-α2a 4 ? Treatment of Hepatitis C- has antiviral activity (induces macrophages to kill virus) o PEGylated interleukin-6 ? 100-fold increase in blood half life with pegylation ? thrombopoietic potencty increased 500-fold (Harris, 2003) ? PEGylated microspheres: ? Li et al. showed significant increases in circulation time by preparing controlled release microparticles using PEG-PLGA block copolymers 2 o Formation of microspheres by double emulsion process ? Block copolymer self-emulsifies and PEG coats both internal and external aqueous interfaces ? Benefit of improved protein stability within microspheres? BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 15 – ‘stealth’ particles 6 of 8 PEG = 5KDa, PLGA = 40 KDa (1) (2) (3) (4) Poly(vinyl alcohol): Adsorbs to surface of organic droplets to provide initial stability to forming spheres Block copolymer localizes at organic/aq. solution interface Double emulsion synthesis PEG = 5KDa, PLGA = 40 KDa Surface steric barrier Block copolymer localizes at aqueous/polymer interfaces PEG chains line inner aq. compartments- minimize protein denaturation o Results: ? Unmodified PLGA particles t 1/2 = 13.6 min. ? PEG block copolymer particles t 1/2 = 270 min (4.5 hr) ? Altered biodistribution: high blood availability and reduced spleen/liver uptake ? No PLGA particles remaining in blood after 12 hrs BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 15 – ‘stealth’ particles 7 of 8 (Li et al., 2001) TEM of nanoparticles Release properties of diblock particles Increased t 1/2 in blood: Altered biodistribution: uncloaking PEGylated carriers ? Use of cleavable PEG-carrier linkages to ‘unmask’ carrier at selected site/time ? Allow full drug activity at site of action ? Example: ‘Stealth’ Carriers in Clinical Use 4,5 ? Pegademase (Adagen) o Pegylated adenosine deaminase (enzyme) o Treatment of severe combined immunodeficiency (SCID)- hereditary lack of adenosine deaminase ? Pegaspargase (Oncaspar) o Pegylated asparaginase (enzyme) o Treatment of leukemia ? Leukaemic cells cannot synthesize asparagines; asparaginase kills cells by depleting extracellular sources of this amino acid ? Pegylated IFN-α2a (Pegasys) o Treamtent of hepatitis C ? Doxil (Alza) o Pegylated liposomes carrying anti-cancer drug doxorubicin o Improves treatment from daily 30min injections for 5 days every 3 weeks to once-a-month single injections o Approved for treatment of Karposi’s sarcoma BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 15 – ‘stealth’ particles 8 of 8 References 1. Moghimi, S. M., Hunter, A. C. & Murray, J. C. Long-circulating and target-specific nanoparticles: theory to practice. Pharmacol Rev 53, 283-318 (2001). 2. Li, Y. et al. PEGylated PLGA nanoparticles as protein carriers: synthesis, preparation and biodistribution in rats. J Control Release 71, 203-11 (2001). 3. Stolnik, S., Illum, L. & Davis, S. S. Long Circulating Microparticulate Drug Carriers. Advanced Drug Delivery Reviews 16, 195-214 (1995). 4. Kozlowski, A. & Harris, J. M. Improvements in protein PEGylation: pegylated interferons for treatment of hepatitis C. J Control Release 72, 217-24 (2001). 5. Harris, J. M. & Chess, R. B. Effect of pegylation on pharmaceuticals. Nat Rev Drug Discov 2, 214-21 (2003). 6. Efremova, N. V., Bondurant, B., O'Brien, D. F. & Leckband, D. E. Measurements of interbilayer forces and protein adsorption on uncharged lipid bilayers displaying poly(ethylene glycol) chains. Biochemistry 39, 3441-51 (2000). 7. Halperin, A. Polymer brushes that resist adsorption of model proteins: Design parameters. Langmuir 15, 2525- 2533 (1999).