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: (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 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 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 membrane- destabilizing 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 bacterium 6,7 Lecture 16 – intracellular delivery 4 of 8 BEH.462/3.962J Molecular Principles of Biomaterials Spring 2003 (Bhakdi 1996) x Targeting to antigen presenting cells that cross-prime 8,9 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 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 t 1/2 drops by 60-fold (JACS 77, 5590 (1955)) o t 1/2 = 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 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 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 pore- forming 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