BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 16: Intracellular drug delivery Last time nano- and micro-particle drug carriers Delivery to tissues from systemic circulation Today. Intracellular drug delivery Reading A.S. Hoffman et aL., Design of "smart polymers that can direct intracellular drug delivery, Polym. Adv. Technol. 13, 992-999(2002) Ph gradients and drug delivery: cancer res. 56, 1194(1996): adv drug deliv rev 25, 3 (1997); see asokan minireview J Pharm. sci 2002 Intracellular delivery of molecules Pathways of import into the cell Uptake of extracellular material by cells Size limitations -500 nm or less Occurs in clathrin-coated pits Can be triggered by receptor binding Environment within endocytic vesicle PH lowered in pathw Extracellular fluid DNAses, proteases, peptidases Endosomes 5.5-6.5 Proteases lysosomes 3.0-5 Proteases(e. g cathepsins) cialized scavengers(macrophages, neutrophilsand antigen presenting cells limitations: up to the size of the cell Lecture 16-intracellular delivery 1 of 8
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Lecture 16: Intracellular drug delivery Last time: nano- and micro-particle drug carriers Delivery to tissues from systemic circulation Today: Intracellular drug delivery Reading: A.S. Hoffman et al., ‘Design of “smart” polymers that can direct intracellular drug delivery,’ Polym. Adv. Technol. 13, 992-999 (2002) Ph gradients and drug delivery: cancer res. 56, 1194 (1996); adv drug deliv rev 25, 3(1997); see asokan minireview J. Pharm. Sci 2002 Intracellular delivery of molecules Pathways of import into the cell Uptake of extracellular material by cells o Endocytosis Size limitations: ~500 nm or less Occurs in clathrin-coated pits Can be triggered by receptor binding Environment within endocytic vesicles: x PH lowered in pathway Compartment Approximate pH Contents relevant for therapeutic delivery Extracellular fluid 7.4 DNAses, proteases, peptidases Endosomes ~5.5-6.5 Proteases lysosomes ~3.0-5.5 Proteases (e.g. cathepsins) cytosol o macropinocytosis, phagocytosis Specialized scavengers (macrophages, neutrophils) and antigen presenting cells Size limitations: up to the size of the cell Lecture 16 – intracellular delivery 1 of 8
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Endocytosis CYTOSOL (nearly all cells Can be triggered by transp receptor binding cell and retuning to the plant Engulfs volumes -500 ethrane, whether it nm diam or smaller Phagocytosis dendritic cells) ENgulf volumes up to the size of the cel Access to the cytosol is tightly regulated o Typically, internalized material dOES NOT ever reach the cytosol- confined to vesicles For mouse fibroblasts, only 5%of tested protein and 20% of oligonucleotides internalized by a cell could reach the cytosol (Cancer Res 59, 1180(1999); Nucleic Acids Res 25, 3290(1997)) o Special case: dendritic cells and(maybe)macrophages Cross-priming: triggering of certain receptors by pathogens leads to delivery of antigens to the o Drug delivery has been attempted by using high doses to obtain a small 'leak' current into cytosol o Leishmania(Alving 1988 Adv Drug Deliv Rev 2, 107) o Pathway to attack intracellular bacteria Phagocytosis of carrier Fusion of endome with parasite-loaded lysosomes Binding of liposomal carrier to bacterial cell wall and disruption of cell wall o commercial product: Ambisome(Gilead, Boulder CO liposomal formulation of amphotericin b to treat leishmaniasis lipid-like drug inserts in liposomal wall as well as within liposomal internal aqueous compartment Lecture 16-intracellular delivery 2 of 8
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Endocytosis: (nearly all cells) receptor binding ¥Can be triggered by ¥Engulfs volumes ~500 nm diam. or smaller Phagocytosis: (macrophages, neutrophils, dendritic cells) ¥Engulf volumes up to the size of the cell Access to the cytosol is tightly regulated o Typically, internalized material DOES NOT ever reach the cytosol- confined to vesicles For mouse fibroblasts, only 5% of tested protein and 20% of oligonucleotides internalized by a cell could reach the cytosol (Cancer Res. 59, 1180 (1999); Nucleic Acids Res. 25, 3290 (1997)) o Special case: dendritic cells and (maybe) macrophages Cross-priming: triggering of certain receptors by pathogens leads to delivery of antigens to the cytosol o Drug delivery has been attempted by using high doses to obtain a small ‘leak’ current into cytosol Delivery of proteins, DNA, small-molecule drugs to the cytosol Example motivation: treatment of leishmania bacterial infections o Leishmania (Alving 1988 Adv Drug Deliv Rev 2, 107) o Pathway to attack intracellular bacteria: Phagocytosis of carrier Fusion of endome with parasite-loaded lysosomes Binding of liposomal carrier to bacterial cell wall and disruption of cell wall o commercial product: Ambisome (Gilead, Boulder CO) 1 liposomal formulation of amphotericin B to treat leishmaniasis lipid-like drug inserts in liposomal wall as well as within liposomal internal aqueous compartment Lecture 16 – intracellular delivery 2 of 8
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Leishmania liposomes Disruption of bacterial cell wall lysosome (1) endosome Leishmania-infected macrophage Amphotericin B cutaneous leishmaniasis Mechanisms for intracellular delivery Cross the plasma membrane Direct entry to cytosol Viral peptide transporters o Typically use highly stable hydrophobic helix peptide Structural similarity to transmembrane protein tails o Difficult to mimic selective activation of membrane-penetration activity that viruses have- conjugates always on'-can't control which membranes are crossed Hydrophobic sequences used by viruses to enter cells of the signal peptide(represented by the helix- us or the carboxyl to alected sequences of intracellular proteins Outside Outside Carem opinon n Chemial Bolg (Hawiger, 1999) Generally believed to be a more dangerous strategy than endosomal escape: Lecture 16-intracellular delivery 3 of 8
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 liposomes (2) (3) endosome lysosome (4) (1) Drug-loaded Disruption of bacterial cell wall Amphotericin B cutaneous leishmaniasis Leishmania-infected macrophage Mechanisms for intracellular delivery Cross the plasma membrane 2 Direct entry to cytosol Viral peptide transporters o Typically use highly stable hydrophobic helix peptide Structural similarity to transmembrane protein tails o Difficult to mimic selective activation of membrane-penetration activity that viruses have- conjugates always ‘on’- can’t control which membranes are crossed Hydrophobic sequences used by viruses to enter cells: (Hawiger, 1999) Generally believed to be a more dangerous strategy than endosomal escape: Lecture 16 – intracellular delivery 3 of 8
BEH. 462/3.962J Molecular Principles of Biomaterials Spring 2003 Potential to destroy electrical potential gradient maintained by cell across plasma membrane causing cell death Escape from endosomes/lysosomes Enter endocytic pathway, cargo released from vesicles once taken inside the cel Dangers of the endocytic pathway(Asokan 2002) PH: surface 7. 4-> endosomes->6.5-5.5-> lysosomes-50 o Lysosomes reached in 30-60 min. typically Endosomes and lysosomes contain proteases(e.g cathepsins), lipases, glycolases phophatases Routes Viral peptides evolved for endosomal escape o HIV-tat peptide(J Biol Chem 276, 3254(2001)) GRKKRRQRRRPPQC Current mechanism hypothesis Positively-charged residues bind polyanionic proteoglycans, triggering rapid internalization Unclear how escape from endosome occurs o Influenza hemaglutinin peptide Undergoes conformational change at reduced pH nserts in membrane reduced ph causes a membrane- destabilizing change in conformation Source for'model of virus-induced biomembrane fusion graphic: http://www.erin.utoronto.ca/-w3bio315/biomembrane%20fusion 3. The change n poten shape binge Cel2 thermon uencesauter leaflet of pld laer. esT 4.The outer leaflets of lipd laer fus loody desolating te membrane pletng the fusion va a anmembcne anchor peps Model of virus-Induced Biomembrane Fusion Fusion with endosomal membranes o Liposomes that become unstable and fusion-competent at reduced pH o Yatvin Fig. 1 and Fig. 2 Disruption of endosomal compartments o pH-triggered membrane-destabilizing component o hemolysin from listeria monocytogenes bacterium.7 Lecture 16-intracellular delivery 4 of 8
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Potential to destroy electrical potential gradient maintained by cell across plasma membrane causing cell death Escape from endosomes/lysosomes Enter endoycytic pathway, cargo released from vesicles once taken inside the cell Dangers of the endocytic pathway (Asokan 2002 3 ) x PH: surface 7.4 -> endosomes -> 6.5-5.5 -> lysosomes ~5.0 o Lysosomes reached in 30-60 min. typically x Endosomes and lysosomes contain proteases (e.g. cathepsins), lipases, glycolases, phophatases Routes x Viral peptides evolved for endosomal escape o HIV-tat peptide (J. Biol Chem 276, 3254 (2001)) 4 Polybasic Tat sequence : x GRKKRRQRRRPPQC Current mechanism hypothesis: x Positively-charged residues bind polyanionic proteoglycans, triggering rapid internalization x Unclear how escape from endosome occurs o Influenza hemagluttinin peptide Undergoes conformational change at reduced pH x Inserts in membrane, reduced pH causes a membranedestabilizing change in conformation x Source for ‘model of virus-induced biomembrane fusion’ graphic: http://www.erin.utoronto.ca/~w3bio315/biomembrane%20fusion. htm x Fusion with endosomal membranes 5 o Liposomes that become unstable and fusion-competent at reduced pH o Yatvin Fig.1 and Fig. 2 x Disruption of endosomal compartments o pH-triggered membrane-destabilizing component o hemolysin from listeria monocytogenes bacterium6,7 Lecture 16 – intracellular delivery 4 of 8
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 QQ-00 Fig. I A Assembly model for alpha-toxin in lipi bilayers. water. sion in the membrane plane to form pre-pore complexes (c) C Lecithin liposomes carrying reincorporate (Bhakdi 1996) Targeting to antigen presenting cells that cross-prime o Mechanism not yet known Figure 2. Pn Example Approach: ' release from endosomes Pat Stayton and Allan Hoffman U. Washington Murthy et al. encrypted polymers o concept: mask membrane-disruptive moieties on a drug-carrying polymer until endosomes are reached Lecture 16-intracellular delivery 5 of 8
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 (Bhakdi 1996) x Targeting to antigen presenting cells that cross-prime8,9 o Mechanism not yet known Example Approach: ‘smart’ release from endosomes 10 Pat Stayton and Allan Hoffman U. Washington- Murthy et al. ‘encrypted’ polymers o concept: mask membrane-disruptive moieties on a drug-carrying polymer until endosomes are reached Lecture 16 – intracellular delivery 5 of 8
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Multi-function molecular backbone("masked") UNmasked" backbone carriers disrupts endosomal Acid-dogradable acetal Enkerg R n endosome Peptide DRUG Free DRUG delivered PEG grafts mask" backbone丰 ODN DRUG Released in high ionic strength of cytosol murthy et al., 2003) endosomes 3 functionalities of polymer carrier 1. targeting ligand for receptor-mediated endocytosis 2. pH-responsive element for endosomal membrane disruption, exposed only when endosomes are reached 3. therapeutic drug attached, released in endosomes pH-responsive element: acetal linkages o degradation rate of acetal linkages sensitive to identity of para group on attached benzene ring N->o tu drops by 60-fold(JACS 77, 5590(1955)) o t1/2=15 min at pH 5. 4 for the given structure hydrolysis rate 100X at pH 5.4 compared to pH 7.4 Lecture 16-intracellular delivery 6 of 8
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 Multi-function molecular carriers: Reduced to -SH in cytosol Released in high ionic strength of cytosol (Murthy et al., 2003) (1) (2) (3) (4) endosomes Receptor targeting 3 functionalities of polymer carrier: 1. targeting ligand for receptor-mediated endocytosis 2. pH-responsive element for endosomal membrane disruption, exposed only when endosomes are reached 3. therapeutic drug attached, released in endosomes pH-responsive element: acetal linkages o degradation rate of acetal linkages sensitive to identity of para group on attached benzene ring N -> O t1/2 drops by 60-fold (JACS 77, 5590 (1955)) o t1/2 = 15 min at pH 5.4 for the given structure hydrolysis rate 100X at pH 5.4 compared to pH 7.4 o Lecture 16 – intracellular delivery 6 of 8
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 pH-sensitive cleavage of PEG side chains: Time(min olymer Et s2 xs lesion Ma pH-sensitive membrane disruption he pfl- dependent hydrolysis and I s( or 74. In earht i to quantitate hemolysis is described i(73. Peptide- polymer conjugate Peptide only Figure 7. Cytoplasmic delivery of peptides with polymer E3. (a) Fluorescence microscopy (40X magnification) of RAW cells treated at40× magnification Lecture 16-intracellular delivery 7 of 8
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 pH-sensitive membrane disruption: pH-sensitive cleavage of PEG side chains: Lecture 16 – intracellular delivery 7 of 8
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) 2. Hawiger, J Noninvasive intracellular delivery of functional peptides and proteins. Curr Opin Chem Biol 3, 89-94 Asokan, A& Cho, M.J. Exploitation of intracellular pH gradients in the cellular delivery of macromolecules. J Pharm sc91,903-13(2002) andaren eng, F& Belting, M. Nuclear targeting of macromolecular polyanions by an HIV-Tat derived peptide. Role for cell-surface proteoglycans. J Biol Chem 277, 38877-83(2002) 5. Yatvin, M. B, Kreutz, W, Horwitz, B A.& Shinitzky, M. Ph-Sensitive Liposomes-Possible Clinical Implications Science210,1253-1254(1980) 6. Lee, K D, Oh, Y.K., Portnoy, D. A& Swanson, J. A Delivery of macromolecules into cytosol using liposomes containing hemolysin from Listeria monocytogenes. J Biol Chem 271, 7249-52 (1996) Bhakdi, set al. Staphylococcal alpha-toxin, streptolysin-O, and Escherichia coli hemolysin prototypes of pore- forming bacterial cytolysins. Arch Microbio/ 165, 73-9(1996) 8. Raychaudhuri, S& Rock, K. L Fully mobilizing host defense: building better vaccines. Nat Biotechnol 16, 1025 Falo, L.D., Jr, Kovacsovics-Bankowski, M, Thompson, K.& Rock, K L. Targeting antigen into the phagocyt 10. pathway in vivo induces protective tumour immunity. Nat Med 1, 649-53(1995) Murthy, N, Campbell, J, Fausto, N, Hoffman, A. S& Stayton, P S Bioinspired pH-Responsive Polymers for the Intracellular Delivery of Biomolecular Drugs. Bioconjug Chem 14, 412-9(2003) 11. Eniola, A.O.& Hammer, D A Artificial polymeric cells for targeted drug delivery. J Control Release 87, 15-22 (2003) 12. Shi, G, Guo, W, Stephenson, S M.& Lee, R J Efficient intracellular drug and gene delivery using folate receptor-targeted pH-sensitive liposomes composed of cationicanionic lipid combinations. J Control Release 80 309-19(2002) Lecture 16-intracellular delivery 8 of 8
BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 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. Hawiger, J. Noninvasive intracellular delivery of functional peptides and proteins. Curr Opin Chem Biol 3, 89-94 (1999). 3. Asokan, A. & Cho, M. J. Exploitation of intracellular pH gradients in the cellular delivery of macromolecules. J Pharm Sci 91, 903-13 (2002). 4. Sandgren, S., Cheng, F. & Belting, M. Nuclear targeting of macromolecular polyanions by an HIV-Tat derived peptide. Role for cell-surface proteoglycans. J Biol Chem 277, 38877-83 (2002). 5. Yatvin, M. B., Kreutz, W., Horwitz, B. A. & Shinitzky, M. Ph-Sensitive Liposomes - Possible Clinical Implications. Science 210, 1253-1254 (1980). 6. Lee, K. D., Oh, Y. K., Portnoy, D. A. & Swanson, J. A. Delivery of macromolecules into cytosol using liposomes containing hemolysin from Listeria monocytogenes. J Biol Chem 271, 7249-52 (1996). 7. Bhakdi, S. et al. Staphylococcal alpha-toxin, streptolysin-O, and Escherichia coli hemolysin: prototypes of poreforming bacterial cytolysins. Arch Microbiol 165, 73-9 (1996). 8. Raychaudhuri, S. & Rock, K. L. Fully mobilizing host defense: building better vaccines. Nat Biotechnol 16, 1025- 31 (1998). 9. Falo, L. D., Jr., Kovacsovics-Bankowski, M., Thompson, K. & Rock, K. L. Targeting antigen into the phagocytic pathway in vivo induces protective tumour immunity. Nat Med 1, 649-53 (1995). 10. Murthy, N., Campbell, J., Fausto, N., Hoffman, A. S. & Stayton, P. S. Bioinspired pH-Responsive Polymers for the Intracellular Delivery of Biomolecular Drugs. Bioconjug Chem 14, 412-9 (2003). 11. Eniola, A. O. & Hammer, D. A. Artificial polymeric cells for targeted drug delivery. J Control Release 87, 15-22 (2003). 12. Shi, G., Guo, W., Stephenson, S. M. & Lee, R. J. Efficient intracellular drug and gene delivery using folate receptor-targeted pH-sensitive liposomes composed of cationic/anionic lipid combinations. J Control Release 80, 309-19 (2002). Lecture 16 – intracellular delivery 8 of 8
