The Innate Immune System and Drug Delivery Systems PDF

THE INNATE IMMUNE SYSTEM AND DRUG DELIVERY SYSTEMS Patrick Glassman, PhD September 19, 2024 LECTURE OBJECTIVES  Drug delivery system (DDS) applications  Role of complement  Role of phagocytes  Marginated leukocytes  Applications in stroke  WHAT DOES THE INNATE IMMUNE SYSTEM...

THE INNATE IMMUNE SYSTEM AND DRUG DELIVERY SYSTEMS Patrick Glassman, PhD September 19, 2024 LECTURE OBJECTIVES  Drug delivery system (DDS) applications  Role of complement  Role of phagocytes  Marginated leukocytes  Applications in stroke  WHAT DOES THE INNATE IMMUNE SYSTEM RECOGNIZE? PATTERNS Complement System Toll-Like Receptors (TLR) JE McInturff, RL Modlin et al. J Invest Dermat. 125(1):1-8 (2005). G Girardi, JJ Lingo et al. Front Immunol. 11:1681 (2020).  WHAT ARE DRUG DELIVERY SYSTEMS?  Drug delivery systems (DDS) are a formulation strategy that are used to modulate the disposition of drugs by:  Prolonging pharmacokinetics  Minimizing uptake into off-target tissues  Selectively targeting to sites of disease  Most particle-based DDS are lipid or protein-based complexes ~80-200 nm in diameter  This is similar in size to a virus! Image from https://ainslielab.web.unc.edu CLINICALLY-USED DRUG DELIVERY SYSTEMS LIPOSOMES Protein Nanoparticle LIPID NANOPARTICLES  Nanoparticles are used clinically to deliver small molecule drugs, mRNA, and siRNA  Approved therapeutic areas include oncology, infectious diseases, COVID-19, and hereditary transthyretin-mediated amyloidosis  Most approved carriers are lipid-based  MOST DRUG DELIVERY SYSTEMS ARE LIPID- Doxil BASED  Liposomes and lipid nanoparticles that are clinically used are generally:  Spherical  50-100 nm in diameter  Composed of a mix of phospholipids and cholesterol  Coated with polyethylene glycol mRNA Vaccine  Viruses that infect humans are typically:  Spherical or filamentous  20-300 nm in diameter  Composed of a protein capsid that protects nucleic acids  Possibly coated with a lipid envelope XR Li, XH Cheng et al. J Ovarian Res. 15:96 (2022). S Daniel, Z Kis et al. Trends Biotechno. 40(10):1213-1228 (2022). DDS-INNATE IMMUNE INTERACTIONS  DDS CAN BE ENGINEERED TO EVADE OR HARNESS THE INNATE IMMUNE SYSTEM Key Points for Today: 1. Selection of appropriate DDS properties can bias particles towards or against immune recognition 2. This can be used to promote delivery to specific organs and/or cells and bias response to therapy F Zahednezhad, M Saadat, et al. J Control Rel. 305:194-209 (2019). EVADING THE INNATE IMMUNE SYSTEM  FIRST IMMUNOLOGICAL BARRIER: COMPLEMENT Key Points for Today: 1. The complement system functions very efficiently on surfaces (e.g, lipid membranes) 2. Release of C3a and C5a lead to further activation of the innate immune system and can cause anaphylactoid reactions 3. There are numerous regulators of the complement pathway that can prevent deposition of C3 and/or release of C3a/C5a including Factors H and I J Giang, MAJ Seelen, et al. Front Immunol. 9:639 (2018).  COMPLEMENT-ASSOCIATED DDS TOXICITIES  Complement deposits on DDS via several mechanisms (properidin, antibody, PRR)  Release of anaphylatoxins (C3a, C5a) can promote a significant response termed Complement Activation Related PseudoAllergy (CARPA)  Most severe symptoms are cardiovascular in nature, including systemic hypotension, pulmonary hypertension, edema, elevated thromboxane B2, etc.  Slow infusions appear to reduce the NM La-Beck, MR Islam, et al. Front Immunol. 11:603039 (2021). severity of CARPA  ENGINEERING DDS TO ELIMINATE CARPA  Attachment of a natural complement regulator (Factor I) to liposomes is a strategy to reduce complement-associated side effects  Factor I:  Reduces C3 deposition on liposomes  Reduces release of C3a and C5a in plasma  Eliminates liposome-induced cerebral hypoperfusion Z Wang, ED Hood, et al. Adv Mater. 34(8):e2107070 (2022).  MACROPHAGES ELIMINATE DDS FROM CIRCULATION  Macrophages in contact with the bloodstream are the primary elimination site for DDS  We refer to this as the reticuloendothelial system (RES)  Kupffer cells in the liver take a large fraction (>90%) of injected DDS  Spleen, lungs, and bone marrow also represent sites of elimination  Significant effort has been paid to evading macrophage-dependent W Ngo, S Ahed, et al. Adv Drug Deliv Rev. 185:114238 (2022). elimination  POLYETHYLENE GLYCOL (PEG) SLOWS ELIMINATION  Grafting PEG onto the surface of liposomes helps evade recognition by the innate immune system  One key mechanism is slowing of opsonization – reduced complement activation  We refer to these as ‘stealth’ liposomes RK Jani, KM Gohil. Int J Pharm Sci Drug Res.10(5):386-393 (2018).  EFFECTS OF PEGYLATION ON DDS BEHAVIOR  PEGylation significantly prolongs pharmacokinetics of liposomal drugs Doxil: PEGylated liposome  PEGylation reduces uptake in clearance organs, Myocet: Non-PEGylated liposome particularly spleen and liver  PEGylation reduces the amount of protein that opsonizes nanoparticles  PEGylation alters the content of the protein corona  However, PEGylation may induce an adaptive immune response… R Luo, Y Li, et al. Int J Pharm. 519(1-2):1-10 (2017). K Partikel, R Korte, et al. Eur J Pharm Biopharm. 141:70-80 (2019).  SELF-MIMICRY TO EVADE THE INNATE IMMUNE SYSTEM  Cells display CD47 on their surface as a ‘don’t eat me’ signal to macrophages via SIRPα binding  Nanoparticles displaying CD47 (or a fragment thereof) on their surface may behave similarly  CD47 may be a viable alternative to PEG for prolonging A Veillette, theImmunol J Chen. Trends half-life of DDS. 39(3):173-184 (2018). NG Sosale, II Ivanovska, et al. Mol Ther Methods Clin Dev. 3:16080 (2016). HARNESSING THE INNATE IMMUNE SYSTEM  NEUTROPHIL MARGINATION  Neutrophils transiently associate with the vessel wall in organs including the lungs in a process termed margination  ~50% of the intravascular pool of neutrophils are in this marginated pool  Local inflammation and injury can lead to dramatic increases in this pool A Dahdah, J Johnson, et al. Front Cell Dev Biol. 10:795784 (2022). C Summers, SM Rankin et al. Trends Immunol. 31(8):318-324 (2010).  PATTERN RECOGNITION BY MARGINATED NEUTROPHILS  Under inflammatory conditions, marginated neutrophils are primed to respond to further danger  Following induction of inflammation in mice using lipopolysaccharide (TLR4 signal), the pulmonary marginated neutrophil pool responds to specific patterns  Nanomaterials displaying agglutinated protein on the surface have high levels of lung uptake  This process was complement-dependent JW Myerson, PN Patel, et al. Nat Nanotechnol. 17(1):86-97 (2022).  ISCHEMIC STROKE: NOT JUST LOSS OF BLOOD FLOW  The initial insult in ischemic stroke is vessel occlusion, typically by a blood clot  Primary treatment must focus on reperfusion (tPA, mechanical thrombectomy)  Secondary damage due to oxidative stress and inflammation can persist for weeks after stroke BCV Campbell, DA De Silva, et al. Nat Rev Dis Primers. 5, 70 (2019). Z Zhou, J Lu, et al. Pharmacol Ther. 191, 23-42 (2018).  THE BLOOD-BRAIN BARRIER IN STROKE  The blood-brain barrier exhibits time-dependent increases in permeability following ischemic stroke  Inflammatory processes in the acute and subacute phases include endothelial activation  This includes upregulation of cell adhesion molecules that S Bernardo-Castro, JA Sousa, et al. Front Neurol. 11, 594672 (2020). play a role in immune cell recruitment  CELLS INVOLVED IN POST-STROKE INFLAMMATION  Endothelial activation and chemokine release promotes migration of immune cells into the brain following stroke  These cells can promote tissue repair or exacerbate damage based on their phenotype  Shifting macrophages from M1 to M2 could promote tissue repair  Immune cells can be recruited from circulating pools or from lymphoid tissues AM Planas. Stroke. 49(9), 2261-2267 (2018).  DISTAL ORGANS RESPOND TO DANGER SIGNALS  Following local injury, chemokines, cytokines, and DAMPs emanate from the brain to recruit immune cells  The first organ these mediators encounter is the lungs, which contain a large number of intravascular leukocytes (marginated neutrophils)  Signaling between danger signals and marginated leukocytes primes them for response to the brain injury J Nong, PM Glassman, et al. ACS Nano. 17(14), 13121-13136 (2023).  TARGETING MIGRATING LEUKOCYTES ICAM CD45 **** ***  Coating nanoparticles with 300 ** 3 **** 80 ** * 0.6 * antibodies binding to Lung Uptake (%ID/g) Brain Uptake (%ID/g) Lung Uptake (%ID/g) Brain Uptake (%ID/g) 60 200 2 0.4 leukocytes promotes: 40 100 1 20 0.2  Rapid uptake and elimination 0 0 0 0.0 from lungs Lungs Brain Lungs Brain 100  Steady delivery to the ns % of Recovered Cells Endothelial Cells 80 Leukocytes inflamed brain That Are NC+ 60 40 ✱✱✱✱  Essentially all of the 20 nanoparticles in the brain are 0 Lung (2 Hours) Brain (24 Hours) found in monocytes and neutrophils  Selective targeting of migrating innate immune cells! J Nong, PM Glassman, et al. ACS Nano. 17(14), 13121-13136 (2023).  TARGETING MIGRATING LEUKOCYTES TO TREAT INFLAMMATION **** 1.5 **** % Albumin Leakage 1.0 *** 0.5 0.0 Naive Sham TNF-  Brain inflammation is characterized by edema (fluid buildup), which can be tracked using albumin accumulation  Liposomes were loaded with a corticosteroid (dexamethasone) and targeted to leukocytes  Treatment effectively reversed edema and polarized macrophages to the anti-inflammatory M2 phenotype J Nong, PM Glassman, et al. ACS Nano. 17(14), 13121-13136 (2023). SUMMARY  The innate immune system (both proteins and cells) plays a critical role in the pharmacokinetics and toxicities of drug delivery systems  Nanoparticles can be engineered to evade the innate immune system using several approaches  Complement inhibitors – prevention of anaphylaxis and macrophage uptake  PEGylation – evasion from opsonization  Self-mimicry – ‘don’t eat me signals’ to macrophages  Under inflammatory conditions, nanoparticles can be engineered to harness the innate immune response for selective delivery  Marginated neutrophils – pattern response to agglutinated protein  Endothelial cell activation – selective delivery in stroke  Leukocyte migration – hitchhiking to sites of injury