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Engineering Cell Interactions
     with Nanomaterials:
Synthetic and Bio-Inspired Ligands
         for Cell receptors

   ...
Outline
   Ligand-Receptor Interactions
    •   The biology of the interactions
    •   Roles of ligand-receptor interact...
Biological Receptor-Ligand
                 Interactions
Biological systems have evolved membrane-
spanning receptor molec...
Receptor Tyrosine Kinases




  Dimerization creates conformational changes transmitted to
                 the intracellu...
Model of EGF receptor dimerization
EGF-Receptor binding changes
from closed to open state, exposing
dimerization interface...
Pertuzamab, Iressa, Tarceva




 Iressa/Tarceva used
  for metastatic non-
small cell lung cancer
 after chemotherapy
Other Natural Ligand-Receptors Systems
    Stimulation of cell differentiation and proliferation
     •   Growth factors ...
Neurotransmitters and
Ligand-Gated Ion Channels




                    Freeman, Scott. Biological Science. Prentice Hall:...
Strategies in Engineering
        Ligand-Receptor Interactions
   Competitive binding
    •   Compete with ligands for re...
Nanoparticles and Synthetic Ligands

•Goal: Use nanoparticles conjugated with
molecules that mimic the natural ligand-rece...
Examples of Synthetic Nanoparticle-Ligand Systems
   RGD Peptides
       Three amino acids: Arginine-Glycine-Aspartic ac...
Examples of Synthetic Nanoparticle-Ligand
• Polyvalent Carbohydrate-Protein
  Interactions
      Multiple oligosaccharide...
Examples of Synthetic Nanoparticle-Ligand Systems
    •     Targeting via Folic Acid

                 Folic Acid (Vitami...
Antibiotics - Bacterial cell targets




Bacteria differ significantly from eukaryotic cells:
-no nuclear membrane
-no mito...
Antibiotics - Bacterial cell targets
Antibiotics

-chemotherapeutic agents designed to kill bacterial cells, fungi
or prot...
Penicillin, Vancomycin, Sulfa Drugs
Bacterial cell walls contain peptidoglycans, in gram positive
cells ~50% of the cell w...
Antibiotic Resistance/MRSA
Several modes of
antibiotic resistance exist
-cleavage or other
modifications of the drug, e.g....
Antibiotic Discovery and Development




                                                         (9)

Bacterial physiolog...
Synthetic Surfaces for
Ligand-Receptor Interaction Studies
   Restrict binding to a 2D surface
    •   Better spatio-temp...
The Immune Response




                Freeman, Scott. Biological Science. Prentice Hall: 2002
Immune Cell Activation

   Activation of T cells, B cells, thymocytes and
    natural killer cells
   Surface receptor o...
Study of the Immunological Synapse

   Currently available computer models
    •   Patterning arise from physical chemist...
internalization), (2) spatially pattern ligand-engaged       immunological studies.
             receptors, and/or (3) pre...
466 Immunological techniques
                                                     Results
   Physical pattern ligand
    ...
Results
   Synthetic ligand surfaces allow for prolonged
    period of stable ligand-receptor contacts
    •        T cel...
Immune Response Activation
   Synthetic ligands presenting surfaces
           Benefits
        •     Define composition, ...
Folate Targeting Drug-Delivery
   Trojan Horse Approach: drugs attached to folates move inside cells
    featuring folate...
Folate Receptor Mediated Endocytosis




   Plasma membrane invaginates
   Drug complex is contained within an intracell...
Dendrimer Targeted Drug Delivery
   Drugs can be coupled to the dendrimer in two ways:
           Hydrophobic drugs can ...
Human Trials and Future Research
   Academic success of FA targeting spurred creation of
    “Endocyte” biotech company f...
Conclusion
   Receptor-ligand binding is a complex interaction
    governing numerous biological processes
   Nanomateri...
Image Sources
(1) Wikipedia
(2) http://www.pharmer.org/files/images/Penicillin%20VK%
    20500mg.jpg
(3) Wikipedia
(4) htt...
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  1. 1. Engineering Cell Interactions with Nanomaterials: Synthetic and Bio-Inspired Ligands for Cell receptors INBT Journal Club Tania Chan Jacob Koskimaki Patrick Stahl February 18, 2008
  2. 2. Outline  Ligand-Receptor Interactions • The biology of the interactions • Roles of ligand-receptor interactions in the body  Synthetic Ligands • Nanoparticle-ligands system  Case Studies • Antibiotics • Synthetic ligand surfaces for T cell activation studies • Folate targeting for drug delivery
  3. 3. Biological Receptor-Ligand Interactions Biological systems have evolved membrane- spanning receptor molecules that can detect a signal outside the cell -information passed to responsive components inside the cell -second messengers -tyrosine kinases -G protein coupled receptors Images adapted from Dr. Jon Lorsch, Johns Hopkins University School of Medicine
  4. 4. Receptor Tyrosine Kinases Dimerization creates conformational changes transmitted to the intracellular kinase domain. -phosphates are transferred from ATPs to tyrosine residues -cross-phosphorylation -activation of down-stream second messenger cascades
  5. 5. Model of EGF receptor dimerization EGF-Receptor binding changes from closed to open state, exposing dimerization interface (1) ErbB2 form of the EGF receptor in the constitutively open form -over-expression in aggressive breast tumors -clinical use, Pertuzamab binds dimerization interface
  6. 6. Pertuzamab, Iressa, Tarceva Iressa/Tarceva used for metastatic non- small cell lung cancer after chemotherapy
  7. 7. Other Natural Ligand-Receptors Systems  Stimulation of cell differentiation and proliferation • Growth factors and receptor tyrosine kinase  Protein transduction and delivery • Polypeptide ligands and transmembrane receptors  Chemical reactions catalysts • Enzymes  Signals transduction • Neurotransmitters and ligand-gated ion channels
  8. 8. Neurotransmitters and Ligand-Gated Ion Channels Freeman, Scott. Biological Science. Prentice Hall: 2002
  9. 9. Strategies in Engineering Ligand-Receptor Interactions  Competitive binding • Compete with ligands for receptor  Noncompetitive binding • Binds to somewhere other than the binding site, renders receptor inactive  Uncompetitive/Mixed binding • Binds to and inactivate ligand- receptor complex Nelson, D., Cox, M. Lehninger Principles of Biochemistry 3rd Ed. Worth Publishers: 2000
  10. 10. Nanoparticles and Synthetic Ligands •Goal: Use nanoparticles conjugated with molecules that mimic the natural ligand-receptor binding. •Motivation: Synthetic ligands provide selective targeting while the nanoparticles provide imaging or drug delivery capability. •Uses: Targeting of diseased cells with anticancer, antibacterial, antiviral drugs etc. while limiting unwanted delivery to healthy cells
  11. 11. Examples of Synthetic Nanoparticle-Ligand Systems  RGD Peptides  Three amino acids: Arginine-Glycine-Aspartic acid  Part of recognition sequence for integrin binding to promote cell attachment  Bind to αvβ3 integrin cell receptor overexpressed in endothelial cells of tumors.  RGD conjugated to nanoparticles containing chemotherapy drugs are preferentially internalized by tumor cells over other organs  Glycomimetics  Cell surface carbohydrates from glycoproteins and glycolipids play a large role in recognition sites  Selective binding between the membrane protein (lectin) and the oligosaccharide chain (carbohydrate that functions as a ligand)  Glycomimetics entails designing sugar-conjugated drugs that will target cells possessing glycoreceptors on the cell membrane  The lectins of some cells will not only bind to the ligand, but also internalize them, providing an opportunity for sugar-mediated drug delivery inside the cell  Example: Use of galactose-conjugates that selectively target hepatic (liver) cells and can bring antiparasitic and antiviral drugs where needed
  12. 12. Examples of Synthetic Nanoparticle-Ligand • Polyvalent Carbohydrate-Protein Interactions  Multiple oligosaccharides (ligands) of one biological entity bind with multiple proteins (cell receptors) on one cell.  Monovalent oligosaccharides cannot as effectively compete for cell receptors with natural polyvalent carbohydrates.  Thoma et al. synthesize glycodendrimers that comprise several oligosaccharide end groups that can bind in concert to polyvalent cell receptors. Figure that compares monovalent and polyvalent  These glycodendrimers have been shown synthetic ligand binding to cell polyvalent cell to inhibit polyvalent cell receptor process in receptor. vitro and in vivo. [Thoma, G. et al. (2005). Chemistry 12, 99-117]
  13. 13. Examples of Synthetic Nanoparticle-Ligand Systems • Targeting via Folic Acid  Folic Acid (Vitamin B9) is necessary for essential cell functions.  Folate conjugates enter cells through the folate receptor, which is overexpressed in many cancer cells that require folic acid in order to proliferate quickly. • Lipoproteins as Nanoparticles (K. Jain. Nanoparticles as Targeting Ligands, TRENDS in Biotechnology. 2006.) •Protein-Lipid complexes function to deliver fats to cells in the body by displaying charged groups of protein outward and carrying hydrophobic fats internally to shield them from water. •By conjugating folic acid and peptides to these lipoproteins, one can alter the route of the lipoproteins from normal lipoprotein cell receptors to tumor cells. [Zheng, G. et al. (2005) PNAS. 102; 17757-17762]
  14. 14. Antibiotics - Bacterial cell targets Bacteria differ significantly from eukaryotic cells: -no nuclear membrane -no mitochondria, microtubules or true cytoplasmic organelles -cell division by fission rather than mitosis -the bacterial cell wall is made of a rigid peptidoglycan layer, contains muramic acid, which is not found in eukaryotes Bruce Alberts, Alexander Johnson, Julian Lewis, Martin Raff, Keith Roberts, Peter Walter, Molecular Biology of the Cell, 4th Edition
  15. 15. Antibiotics - Bacterial cell targets Antibiotics -chemotherapeutic agents designed to kill bacterial cells, fungi or protozoa -first produced by living organisms (penicillin), now produced by chemical synthesis (sulfa drugs) -many are very small molecules with low molecular weights (~2000 Da) (Wikipedia) -often have low toxicity in mammals, unlike previous treatments for infections (arsenic) -efficacy dependent on lots of factors: severity of infection, delivery of drug, ability of drug to destroy bacteria, resistance
  16. 16. Penicillin, Vancomycin, Sulfa Drugs Bacterial cell walls contain peptidoglycans, in gram positive cells ~50% of the cell weight, in gram negative ~1-2%. L-Ala 1 | L-Glu | (2) (3) (4) DAP 3 | D-Ala 4 | D-Ala 5 (5) (6)
  17. 17. Antibiotic Resistance/MRSA Several modes of antibiotic resistance exist -cleavage or other modifications of the drug, e.g. Beta-lactamases (7) -resistance can occur by a change in the drug target – Streptomycin resistance results from a change in the ribosomal RNA sequence so that the drug can no longer bind to the ribosome -Vancomycin resistance results from changing the D-ala:D-ala peptidoglycan (8) target of Vancomycin to D-ala-D-lactate
  18. 18. Antibiotic Discovery and Development (9) Bacterial physiology/genetics extremely limited during the 1940s-1960s -1st bacterial genome sequenced 1995 -high throughput technologies -random screening campaigns of soil and marine samples (platensimycin)
  19. 19. Synthetic Surfaces for Ligand-Receptor Interaction Studies  Restrict binding to a 2D surface • Better spatio-temporal resolution of binding events  Adjustable interaction parameters • Control ligand density, composition • Control spatial distribution and mobility of ligands  Example application: T-cell activation studies
  20. 20. The Immune Response Freeman, Scott. Biological Science. Prentice Hall: 2002
  21. 21. Immune Cell Activation  Activation of T cells, B cells, thymocytes and natural killer cells  Surface receptor on one cell bind ligand on a second cell  Binding triggers formation of immunological synapse and activates cells • Contact zone rich in signaling and adhesion receptors • Little understanding of this activation process
  22. 22. Study of the Immunological Synapse  Currently available computer models • Patterning arise from physical chemistry of receptor binding between nearby membranes  Needs for synthetic ligand-receptor system • Arrest transient signaling event • Spatially pattern ligand-engaged receptors
  23. 23. internalization), (2) spatially pattern ligand-engaged immunological studies. receptors, and/or (3) present activation signals in discrete sites, allowing motile immune cells the possibility of Recently, two strategies specifically developed for studies disengaging from activating ligands. Recently, several of immune cell activation have been reported, which are strategies for patterning ligands on surfaces have been based on tethering proteins or small-molecule ligands to applied to studies of immune cell triggering (Table 1). surfaces via short (e.g. 1–4 nm) flexible polymer chains, as Strategies to Create Synthetic Surfaces These include patterning of supported lipid bilayers by illustrated in Figure 1. Conceptually, these patterned standard photolithographic techniques, patterning grids surfaces are used to fix the physical display of a ligand of discrete supported lipid bilayer patches using electron that would normally be present in a soluble or particulate beam Patterned lithography, tools to study ligand recognition and or to model a cell–cellTcontact by aDoh and Huang 465 (e-beam) surfaces as patterning discrete patches form, synapse formation by cells Irvine, cell–surface of immobilized ligand using e-beam lithography, or creat- contact (Figure 1a). The first of these methods grew out of for Ligand-Receptor Interaction ing microscale patterns of ligands using a photolitho- graphic approach designed for multicomponent protein patterning. Each of these strategies provides access to a Figure 1 different length scale of resolution and different maxi- studies of IgE-sensitized mast cell triggering by haptens [20,21]. Supported lipid bilayers bearing immobilized dinitrophenyl (DNP) groups were patterned on silicon surfaces in order to visualize the clustering of Fc receptors mum surface areas that can be patterned, and ranges from and signaling proteins following mast cell contact with simple bench-top processes (‘biophotolithography’ microscale ligand patches. Cells seeded onto these sur- methods) to complex nanofabrication techniques requir- faces differentially clustered inner-leaflet and outer-leaf- ing clean room manufacturing facilities (e-beam let plasma membrane components to hapten ‘islands’ methods). Most of these approaches allow the creation [21]. To create smaller ligand-bearing domains that of patterns with multiple regions of distinct composition, would allow events following the engagement of only a but only the approaches utilizing supported lipid bilayers handful of receptors to be tracked, a patterning strategy have been demonstrated as a way to introduce planar based on advanced lithography and self-assembled mono- diffusional mobility to ligands bound to the surface pat- layers (SAMs) was developed (Figure 1b) [22]. Gold terns. For comparison, characteristics of microcontact ‘islands’ with diameters as small as 45 nm were fabricated printing using poly(dimethyl siloxane) (PDMS) rubber on silicon substrates using electron beam (e-beam) litho- stamps and dip-pen nanolithography (utilizing an atomic graphy. Short alkyl chains bearing hapten groups on one force microscope) are also listed in Table 1 — two end and thiols on the other were adsorbed onto the techniques widely used for patterning biomolecules that patterned gold spots, forming gold-thiol bonds and pack- may be of interest for future studies in immunology. Note ing laterally to form ligand-presenting SAMs on each gold that this list of approaches is by no means exhaustive and site (Figure 1b). The density of ligand in this approach only serves to illustrate the range of possible methods, is readily controlled by mixing ligand-functionalized Table 1 Characteristics of representative methods used to create patterns of biological ligands on surfaces Patterning approach Smallest Typical Multiple Ligand References feature size patterned area regions of mobility? typically distinct fabricated composition possible? Photoresist-patterned 1 mm cm 2 Yes Yes [20,21] supported lipid bilayers e-Beam lithography- 50 nm 0.01–0.25 mm 2 ? No [22] defined SAMs (Figure 1B) e-Beam lithography- 50 nm 0.01–0.25 mm 2 Yes Yes [38,43] defined supported lipid bilayers Biophotolithography (Figure 1C) 1 mm cm 2 Yes ? [27,28,29] PDMS microcontact 1 mm cm 2 Yes Yes, with [17,44] printing lipid ‘inks’ Dip-pen nanolithography 50 nm 500 mm2* Yes Yes, with [45–47] and scanning probe lipid lithography ‘inks’ First four entries (eight lines) of the table body indicate strategies already applied in studies of immune cells. * ‘Massively parallel’ dip-pen nanolithography under development allows access to pattern sizes up to cm2 [47]. Current Opinion in Immunology 2007, 19:463–469 www.sciencedirect.com
  24. 24. 466 Immunological techniques Results  Physical pattern ligand template aggregationgroupsthiols, andincorpor- ciple many different chemical of could be alkanethiols with nonfunctionalized in prin- Figure 2 ated, allowing more complex ligands or complete proteins receptors approachwithsurface patterning was used to visualize to be coupled This for the patterned sites after self-assembly. clusters of IgE/FcR complexes and phosphorylated sig- naling proteins in mast cells following contact with pat-  Physical appopsition of terned surface arrays of submicron patches of DNP ligand [22]. The ability to precisely localize ligands in sub- receptors affect signaling micrometer domains for subsequent visualization by fluorescence microscopy, particularly if combined with recently developed high resolution/low noise imaging strategies based on total internal reflection fluorescence • Truncated ligands lead to microscopy [23,24,25,26], may allow the dynamics of the very first signaling events following receptor clustering to weaker cell proliferation be followed during immune cell activation. The approach just described allows extremely high-resol- ution patterns to be created, but e-beam lithography is a  Downstream changes in T cell serial process (an electron beam is rastered over the surface, creating one feature at a time) and is practically dynamics and cytokine incapable of being applied to create patterns over large (e.g. millimeters or centimeters) length scales at present. An alternative strategy based on traditional photolitho- production graphy can be used to create multicomponent protein patterns over large surface areas [27,28]. In this ‘biopho- Templating immunological synapse formation using tethered ligand patterned surfaces. Surfaces bearing microscale patterns of anti-CD3 tolithography’ approach, a biotinylated polymer is used as surrounded by ICAM-1 were created, similar to the schematic arrays both a photoresist (a coating to selectively block the shown in Figure 1A. Shown are fluorescence images of immunostained T • Affects T cell stop signals surface until exposed to light at a selected wavelength) and a ligand-binding coating (Figure 1c). Via a two-step cells interacting with three different geometries of anti-CD3 ‘activation sites’ 20 min after seeding on surfaces: (a) focal, (b) multifocal, or (c) annular. Top panels show brightfield images overlaid with anti-CD3 site ‘lift off’ process, a ‘foreground’ and ‘background’ can be fluorescence Irvine DJ, Doh J, and Huang B, “Patterned surfaces as tools (blue). Bottom and lower panels show fluorescence patterned with two different types of biotinylated protein Secrete 10X fewer effectors overlays of TCR, PKC-u, and LFA-1 immunostainingformation by T to study ligand recognition and synapse as marked. Scale • bound to the surface via streptavidin intermediates. This bars, 5 mm. Parts (B) and (C) adapted from [29], 19 463-469 (2007). cells,” Current Opinion in Immunology, copyright 2006, National Academy of Sciences, U.S.A. system is limited to patterning features 2 mm in diameter or larger (using simple benchtop photolitho- graphic methods), but allows the creation of segregated
  25. 25. Results  Synthetic ligand surfaces allow for prolonged period of stable ligand-receptor contacts • T cells arrest only on focal ligand patterns • T cells arrest for up to 20 hours till cell division Patterned surfaces as tools to study ligand recognition and synapse formation by T cells Irvine, Doh and Huang 467 • Newly divided daughter cells migrated through Figure 3 ligand sites Irvine DJ, Doh J, and Huang B, “Patterned surfaces as tools T cell responses modulated by microscale surface patterns of anti-CD3 and ICAM-1. Surfaces bearing microscaleligand recognition and synapse formation by T to study patterns of anti-CD3 in either circular or annular geometries were fabricated, and the dynamics of T cells seeded onto these surfaces were tracked by time-lapse microscopy. 19 463-469 (2007). cells,” Current Opinion in Immunology, T cells stop and center themselves on focal patches of anti-CD3 (a, often until cell division occurs as shown here), but fail to completely arrest on annular microscale patterns of anti-CD3 (b). Elapsed times: (a) hour:min:s and (b) min:s. Adapted from [29], copyright 2006 National Academy of Sciences, U.S.A.
  26. 26. Immune Response Activation  Synthetic ligands presenting surfaces  Benefits • Define composition, quantity and physical arrangement of ligands • Template for receptor migrations  Limitations • Limited ligand mobility  Future work • Introduce diffusional mobility to ligands • Study receptor-ligands binding of different systems
  27. 27. Folate Targeting Drug-Delivery  Trojan Horse Approach: drugs attached to folates move inside cells featuring folate receptors (FR)  Vitamin folic acid has very high affinity (KD ~ 10-10 M) for FR  High specificity low doses still effective yet lower toxicity  Folate Conjugates enter cell through receptor-mediated endocytosis  Released into cytosol instead of transport to lysosomes:  Avoids enzymes that could inactivate the drug C. Leamon and J. Reddy (2004). Folate-Targeted Chemotherapy. Advanced Drug Delivery Reviews 56, 1127-1141. Typical Design of pteroate-drug conjugate 1. pteroate ligand: when linked to glutamic acid forms folate acid moiety 2. Linker: reduces steric hindrance, provides favorable functional groups 3. Cleavable bond: might be used to separate drug from ligand after endocytosis in order to release drug in its original active form. Often use an acid sensitive group such as disulfide bond 4. Drug used for chemotherapy
  28. 28. Folate Receptor Mediated Endocytosis  Plasma membrane invaginates  Drug complex is contained within an intracellular vesicle (endosome)  pH of vesicle drops to ~5 due to proton pumps on endosome membrane  Acidification protonates carboxyl groups on FR that changes its conformation and releases the Folate-drug conjugate  Drug enters the cytoplasm/nucleus and begins its work  Endosome and its FR return to recombine with cell membrane
  29. 29. Dendrimer Targeted Drug Delivery  Drugs can be coupled to the dendrimer in two ways:  Hydrophobic drugs can be complexed within the hydrophobic interior  Methotrexate (MTX) anticancer drug readily released in solution of phosphate buffered saline (PBS)  Covalently attached to the dendrimer surface  MTX remains attached to dendrimer in PBS [A. Patri et al. (2005) Targeted drug delivery with dendrimers: Comparison of the release kinetics of covalently conjugated drug and non-covalent drug inclusion complex. Advanced drug delivery reviews. 57, 2203-2214.] •Modify the surface of the PAMAM dendrimer with folic acid (~5 per dendrimer) •Target KB tumor cells that over-express FR •Folic acid targeted dendrimer specifically kills cells with FR thru intracellular delivery by receptor- mediated endocytosis. •Drug remains attached to dendrimer until after endocytosis, it is not prematurely released  reduces toxicity to non-cancerous cells •After FA-conjugated dendrimer is internalized, hydrolysis frees the drug from the dendrimer. •At 0.1 µM concentration of drug complex, ~70% cells undergo necrosis •Control experiments with free folic acid or low FR expressing cells confirm active targeting
  30. 30. Human Trials and Future Research  Academic success of FA targeting spurred creation of “Endocyte” biotech company for clinical development of folate-targeted medicines  Conducted human trials with In-DPTA-folate (radioimaging agent) intravenous injection  Benign ovarian cyst does not uptake folate  Malignant ovarian tumor does show large uptake of folate- conjugate  Uptake in kidneys due to FR expression on the apical membrane of proximal tubules  Potential problem for folate therapeutics in humans may be difficulty in penetrating solid tumors  Reduce size of folate-drug complex  Use more potent drugs that are effective even at the low doses that penetrate a tumor  Next step is to conduct human trials for folate-targeted anticancer drug delivery Y. Lu and P. Low. Folate-mediated delivery of macromolecular anticancer therapeutic agents. Advanced Drug Delivery Reviews. 54 (2002) 675-692.
  31. 31. Conclusion  Receptor-ligand binding is a complex interaction governing numerous biological processes  Nanomaterials are used to mimic natural ligands and bind to receptors  Diverse selection of synthetic ligands made of various materials for different applications • Pharmaceutical agents • Drug delivery • Study of biological processes
  32. 32. Image Sources (1) Wikipedia (2) http://www.pharmer.org/files/images/Penicillin%20VK% 20500mg.jpg (3) Wikipedia (4) http://www.clemson.edu/caah/history/FacultyPages/PamMack/ lec122sts/penicillin.jpg (5) Wikipedia (6) Wikipedia (7) http://www.hud.ac.uk/sas/staffprofiles/sappapl_research.php (8) Wikipedia (9) Current Opinion in Microbiology Volume 7, Issue 5, October 2004, Pages 445-450
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