BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 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 ary according to the specific type of receptor (1) Most receptors return Transcytosis brane domain from which they came; (2) some recep- mes, where they are degraded; and (3)some recepto uutmain of the plasma membrane, thereby mediating Figure 13-32 Possible fates for transmembrane receptor proteins that 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)) upffer cells- macrophages in liver o Particulate removal aided by process of opsonization Process of tagging' foreign particles for efficient removal by macrophages Lecture 15-stealth' particles 1 of 8
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 spleen s tagged by different opsonins removed by cells in different organs e.g.liver vs Complement proteins, particularly C3 and C5 Immunoglobulins Other proteins known to facilitate particle uptake o Fibronectin o C-reactive protein Tuftsin Protein adsorption to particles is key Hydrophobic particles quickly removed from circulation in vivo(Int/ J. Pharm. 29, 53 Penetration through capillary walls into tissues o Passive delivery due to leakiness of vessels at sites of inflammation or tumor vasulature abnormalities 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 Stealth particles"," 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 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 ptors of macrophages unable to bind particle/molec 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 Lecture15-‘ stealth’ particles 2 of 8
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 abnormalities1 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 work1 Stealth particles2,3 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 nonimmunogenic and have greatly increased half-lives in vivo Concept4,5 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 g Pluronics PEO PPO 瀏餐灤 PEG block copoly Block copolymer rbed micelles/nanoparticles (Stolnik et al. 1995) PEGylated carriers PEG chain PEG binds 2-3 water molecules per repeat unit o Causes PEGylated compounds to function as though they are 5-10 times larger than their true molar Observed in SEC and gel electrophoresis experiments PEG/ater '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 Theory of Halperin, building on previous analyses of Alexander/de gennes and Szleifer Lecture 15-stealth' particles 3 of 8
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 unit5 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 experiments4 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 carriers6,7 o Theory of Halperin7 , building on previous analyses of Alexander/de Gennes and Szleifer
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 Primary =髻B second ad Figure 2. Large (c) he effertive potential /eXperiencer hy interaction potential between the bare surface and the protel Wigu re je smell ar tesr s all co can rat itra ter n ertion mechanism favors primary adsorption at the o Experimental measurements and comparison with theory o Measurements made using surface plasmon resonance o Experimental details o=on gr PEG MW= 2000 g/mole(N= 45) Layer thicknesses Lo=40-60 A of tested pre BPTI(bovine pancreatic trypsin inhibitor): MW=6000-g/mole;R-21x21x30 A HSA(human serum albumin ) MW=66, 200 g/mole; R=38X38x150 A FBN(Fibrinogen): MW=340,000 g/mole: R-55x55x460 A rod-link protein Comparison of theory stance between grafting sites, A with experiment FIGURE 9 lymer- Distance between grafting sites, A FIGURE 7: Characteristic time course for fibrinogen adsorption onto a supported DSPE monoLayer measured by SE FIaURE 10 on of HsA (D and FN ( o)as race pol ymer-lp (Efremova et al. 2000) Lecture15-‘ stealth’ particles 4 of 8
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 resonance6 o Experimental details: σ = on graphs! [area/chain] PEG MW = 2000 g/mole (N = 45) Layer thicknesses L0 = 40-60 Å Sizes of tested proteins: • BPTI (bovine pancreatic trypsin inhibitor) : MW = 6000- g/mole; R ~ 21x21x30 Å • HSA (human serum albumin): MW = 66,200 g/mole; R = 38x38x150 Å • FBN (Fibrinogen): MW = 340,000 g/mole; R ~ 55x55x460 Å rod-link protein Comparison of theory with experiment (Efremova et al. 2000)
BEH.462/3. 962J Molecular Principles of Biomaterials Spring 2003 Function of stealth particles o PEGylated molecules o PEGylated IFN-o2a 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 potency increased 500-fold Table 1 Influence of pegylation on pharmacokinetics and phar Pharmacokinetic effect Pharmacodynamic effect Interferon-a2a Sustained absorption In wio antiviral activity increased 12-to 135-times Increased half-life(from 3-8 h to 65 h) Antitumour activity increased 18-fold Decreased volume of distribution (from 31-73I to 8-12) mproved sustained response to chronic hepatitis C urEase systemic ch aI e(run 6.6-29.2 L008-0.10 Mi Interleukin-6 Increased half-life(rom 2. 1-206 min) Thrombopoietic potency ncreased 500-times Tumour necrosis factor Increased hali-life(tom 3 to 45-136min) Antitumour potency increased 4-to 100-times b三之出导 s injected every other day and its short ifetme in circulation leads to pulsed blood concentratons levelswhich cycle bebw level. The branched PEG40KDa IFN-a2a has a lng circulating ifetme due to the presence of the PEg, and the once weekly injection leads constant bod concentrations above the herapeutic level over the one-week penod aris,2003) PEGylated microspheres Li et al. showed significant increases in circulation time by preparing controlled release microparticles using PEG-PLGaA block copolymers 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? Lecture15-‘ stealth’ particles 5 of 8
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-α2a4 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 copolymers2 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 PEG 5KDa PLGA= 40 KDa CH,O-tCH: CH,-0-xtC-CH-0-*t-C-CH-0-tI Poly(vinyl alcohol) Adsorbs to surface of organic droplets to provide initial stability to forming spheres Block copolymer localizes at aqueous/polymer interfaces CHyO-tCH-CH-0-t-C-CH-0-tC-CH-0-H PEG 5KDa. PLGA=40 KDa Double emulsion PEG chains line inner ag. compartments- minimize protein denaturation Surface steric o Results Unmodified PLGa particles t1n=13.6 min PEG block copolymer particles t1/2= 270 min (4.5 hr) No PLGA particles remaining in blood after 12 hrs een/liver uptake Altered biodistribution: high blood availability and reduced Lecture15-‘ stealth’ particles 6 of 8
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. compartmentsminimize protein denaturation o Results: Unmodified PLGA particles t1/2 = 13.6 min. PEG block copolymer particles t1/2 = 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 TEM of nanoparticles Release properties of diblock particles 000000 Tine(days) 6. Release profles of BSA from PLGA(O Increased t in blood: Altered biodistribution 当125 日 PLGA Nanoparticles a PEG-PLGA Nanoparticles 0.75 型0.5 06888 5025 0 in。 20 Time after intravenous injection (hr) Fig. 7. Blood clearance curves of [I]BSA in PLGA (O)and PEG-PLGA(O) nanoparticle (Li et aL., 2001) 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 Use4, 5 Pegademase(Dagen o Pegylated adenosine deaminase(enzyme) 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-a2a(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 Lecture15-‘ stealth’ particles 7 of 8
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 t1/2 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 Use4,5 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 References Moghimi, S M, Hunter, A C.& Murray, J C Long-circulating and target-specific nanoparticles: theory to practice. Pharmacol Rev 53, 283-318(2001) Li, Y. et al. PEGylated PLGA nanoparticles as protein carriers: synthesis, preparation and biodistribution in rats. J Control Release 71, 203-11 (2001) Stolnik, S, Illum, L& Davis, S. S Long Circulating Microparticulate Drug Carriers. Advanced Drug Delivery 3456 Reviews16,195-214(1995) Kozlowski, A& Harris, J M. Improvements in protein PEGylation: pegylated interferons for treatment of hepatitis C. Control Release 72, 217-24(2001 Harris, J M.& Chess, R B Effect of pegylation on pharmaceuticals. Nat Rev Drug Discov 2, 214-21(2003) 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) Halperin, A Polymer brushes that resist adsorption of model proteins: Design parameters. Langmuir 15, 2525- 2533(1999) Lecture15-‘ stealth’ particles 8 of 8
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)
