Engineering Cell Interactions with Nanomaterials:

Synthetic and Bio-Inspired Ligands for Cell receptors

INBT Journal Club

Tania ChanJacob Koskimaki

Patrick Stahl

February 18, 2008

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

Outline

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

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

Model of EGF receptor dimerizationEGF-Receptor binding changes from closed to open state, exposing dimerization interface

ErbB2 form of the EGF receptor in the constitutively open form

-over-expression in aggressive breast tumors-clinical use, Pertuzamab binds dimerization interface

(1)

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 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

Neurotransmitters and Ligand-Gated Ion Channels

Freeman, Scott. Biological Science. Prentice Hall: 2002

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

Strategies in Engineering Ligand-Receptor Interactions

Nelson, D., Cox, M. Lehninger Principles of Biochemistry 3rd Ed. Worth Publishers: 2000

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

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

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.

These glycodendrimers have been shown to inhibit polyvalent cell receptor process in vitro and in vivo.

Figure that compares monovalent and polyvalent synthetic ligand binding to cell polyvalent cell

receptor.

[Thoma, G. et al. (2005). Chemistry 12, 99-117]

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.

•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]

• Lipoproteins as Nanoparticles (K. Jain. Nanoparticles as Targeting Ligands, TRENDS in Biotechnology. 2006.)

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

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

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|

DAP 3|

D-Ala 4|

D-Ala 5

(2) (3) (4)

(5) (6)

Antibiotic Resistance/MRSA

Several modes of antibiotic resistance exist-cleavage or other modifications of the drug, e.g. Beta-lactamases

-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 target of Vancomycin to D-ala-D-lactate

(7)

(8)

Antibiotic Discovery and Development

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)

(9)

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

Synthetic Surfaces for Ligand-Receptor Interaction Studies

The Immune Response

Freeman, Scott. Biological Science. Prentice Hall: 2002

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

Immune Cell Activation

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

Study of the Immunological Synapse

Strategies to Create Synthetic Surfaces for Ligand-Receptor Interaction

events (e.g. ligand engagement followed by receptorinternalization), (2) spatially pattern ligand-engagedreceptors, and/or (3) present activation signals in discretesites, allowing motile immune cells the possibility ofdisengaging from activating ligands. Recently, severalstrategies for patterning ligands on surfaces have beenapplied to studies of immune cell triggering (Table 1).These include patterning of supported lipid bilayers bystandard photolithographic techniques, patterning gridsof discrete supported lipid bilayer patches using electronbeam (e-beam) lithography, patterning discrete patchesof immobilized ligand using e-beam lithography, or creat-ing microscale patterns of ligands using a photolitho-graphic approach designed for multicomponent proteinpatterning. Each of these strategies provides access to adifferent length scale of resolution and different maxi-mum surface areas that can be patterned, and ranges fromsimple bench-top processes (‘biophotolithography’methods) to complex nanofabrication techniques requir-ing clean room manufacturing facilities (e-beammethods). Most of these approaches allow the creationof patterns with multiple regions of distinct composition,but only the approaches utilizing supported lipid bilayershave been demonstrated as a way to introduce planardiffusional mobility to ligands bound to the surface pat-terns. For comparison, characteristics of microcontactprinting using poly(dimethyl siloxane) (PDMS) rubberstamps and dip-pen nanolithography (utilizing an atomicforce microscope) are also listed in Table 1 — twotechniques widely used for patterning biomolecules thatmay be of interest for future studies in immunology. Notethat this list of approaches is by no means exhaustive andonly serves to illustrate the range of possible methods,

with emphasis on those techniques already applied inimmunological studies.

Recently, two strategies specifically developed for studiesof immune cell activation have been reported, which arebased on tethering proteins or small-molecule ligands tosurfaces via short (e.g. 1–4 nm) flexible polymer chains, asillustrated in Figure 1. Conceptually, these patternedsurfaces are used to fix the physical display of a ligandthat would normally be present in a soluble or particulateform, or to model a cell–cell contact by a cell–surfacecontact (Figure 1a). The first of thesemethods grew out ofstudies of IgE-sensitized mast cell triggering by haptens[20,21!]. Supported lipid bilayers bearing immobilizeddinitrophenyl (DNP) groups were patterned on siliconsurfaces in order to visualize the clustering of Fc receptorsand signaling proteins following mast cell contact withmicroscale ligand patches. Cells seeded onto these sur-faces differentially clustered inner-leaflet and outer-leaf-let plasma membrane components to hapten ‘islands’[21!]. To create smaller ligand-bearing domains thatwould allow events following the engagement of only ahandful of receptors to be tracked, a patterning strategybased on advanced lithography and self-assembled mono-layers (SAMs) was developed (Figure 1b) [22!!]. Gold‘islands’ with diameters as small as 45 nm were fabricatedon silicon substrates using electron beam (e-beam) litho-graphy. Short alkyl chains bearing hapten groups on oneend and thiols on the other were adsorbed onto thepatterned gold spots, forming gold-thiol bonds and pack-ing laterally to form ligand-presenting SAMs on each goldsite (Figure 1b). The density of ligand in this approachis readily controlled by mixing ligand-functionalized

464 Immunological techniques

Table 1

Characteristics of representative methods used to create patterns of biological ligands on surfaces

Patterning approach Smallestfeature sizetypicallyfabricated

Typicalpatterned area

Multipleregions ofdistinctcompositionpossible?

Ligandmobility?

References

Photoresist-patternedsupported lipid bilayers

"1 mm >cm2 Yes Yes [20,21!]

e-Beam lithography-defined SAMs (Figure 1B)

"50 nm "0.01–0.25 mm2 ? No [22!!]

e-Beam lithography-defined supported lipidbilayers

"50 nm "0.01–0.25 mm2 Yes Yes [38!!,43]

Biophotolithography (Figure 1C) "1 mm >cm2 Yes ? [27!,28,29!!]PDMS microcontactprinting

"1 mm "cm2 Yes Yes, withlipid‘inks’

[17,44]

Dip-pen nanolithographyand scanning probelithography

"50 nm "500 mm2* Yes Yes, withlipid‘inks’

[45–47]

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

Patterned surfaces as tools to study ligand recognition and synapse formation by T cells Irvine, Doh and Huang 465

Figure 1

Patterning surfaces to study receptor–ligand interactions governing immune cell activation. (a) Conceptual view of using a cell–synthetic surfacecontact to model a cell–cell contact, illustrated here for the case of immunological synapse formation by T cells. Ligands normally presented on thesurface of an APC are patterned into regular arrays on a surface. Cells seeded onto the surface react to the APC ligands in a manner determined in partby the physical pattern of the ligands displayed on the surface. (b) Submicron patterning of ligands of surfaces using electron beam lithography andself-assembled monolayers, following the approach of Senaratne et al. [22!!]. A photoresist coated on a silicon substrate is patterned by an electronbeam, and then over-coated with a thin ("10 nm thick) layer of gold. The remaining resist is then dissolved away, leaving behind gold ‘islands’ in adefined pattern. Alkanethiols bearing dinitrophenyl ligands (red diamonds) or passive endgroups (blue circles) are finally chemisorbed to the gold sites,forming self-assembled monolayers. (c) Photolithographic patterning of multicomponent ligand patterns, after the approach of Doh and Irvine[27!,29!!]. A biotinylated copolymer resist is exposed through a photomask to UV light and washed with PBS to expose defined binding sites for ligandon the surface. Ligand is immobilized in the UV-exposed sites via streptavidin (SAv), and then the background remaining resist is removed to allow

www.sciencedirect.com Current Opinion in Immunology 2007, 19:463–469

Results Physical pattern ligand

template aggregation of receptors

Physical appopsition of receptors affect signaling

• Truncated ligands lead to weaker cell proliferation

Downstream changes in T cell dynamics and cytokine production

• Affects T cell stop signals

• Secrete 10X fewer effectors

alkanethiols with nonfunctionalized thiols, and in prin-ciple many different chemical groups could be incorpor-ated, allowing more complex ligands or complete proteinsto be coupled with the patterned sites after self-assembly.This approach for surface patterning was used to visualizeclusters of IgE/FcR complexes and phosphorylated sig-naling proteins in mast cells following contact with pat-terned surface arrays of submicron patches of DNP ligand[22!!]. The ability to precisely localize ligands in sub-micrometer domains for subsequent visualization byfluorescence microscopy, particularly if combined withrecently developed high resolution/low noise imagingstrategies based on total internal reflection fluorescencemicroscopy [23,24!,25,26], may allow the dynamics of thevery first signaling events following receptor clustering tobe followed during immune cell activation.

The approach just described allows extremely high-resol-ution patterns to be created, but e-beam lithography is aserial process (an electron beam is rastered over thesurface, creating one feature at a time) and is practicallyincapable of being applied to create patterns over large(e.g. millimeters or centimeters) length scales at present.An alternative strategy based on traditional photolitho-graphy can be used to create multicomponent proteinpatterns over large surface areas [27!,28]. In this ‘biopho-tolithography’ approach, a biotinylated polymer is used asboth a photoresist (a coating to selectively block thesurface until exposed to light at a selected wavelength)and a ligand-binding coating (Figure 1c). Via a two-step‘lift off’ process, a ‘foreground’ and ‘background’ can bepatterned with two different types of biotinylated proteinbound to the surface via streptavidin intermediates. Thissystem is limited to patterning features "2 mm indiameter or larger (using simple benchtop photolitho-graphic methods), but allows the creation of segregatedligand patterns covering large (cm2) culture substrates[29!!]. This approach has been used to create patterns ofadhesion proteins and TCR ligand for the study of T celltriggering, as discussed below.

Modulating T cell dynamics and synapseformation with patterned surfacesThe mast cell studies described above demonstrated thatwhen cells interact with surface-immobilized ligands, thephysical pattern of the tethered ligand can template notonly the positioning of cognate cell surface receptors butalso intracellular signaling components that directly orindirectly associate with the receptors [20,21!,22!!]. In asimilar manner, patterned surfaces composed of anti-CD3(a surrogate ligand for the TCR) spots surrounded bytethered intercellular adhesion molecule-1 (ICAM-1,a ligand for the key T cell integrin lymphocyte

function-associated antigen-1 (LFA-1)) (Figure 1a and c)were shown to template synapse formation by T cellscontacting these patterns (Figure 2) [29!!]. TCRs andPKC-u accumulated in clusters following the pattern ofanti-CD3when the TCR ligand was presented in ‘focal’ or‘multifocal’ patterns (Figure 2a and b, respectively). Onannular anti-CD3 sites, however, PKC-u accumulationsexhibited multiple morphologies: a fraction of T cellscentered over annular activation sites exhibited an annularpattern of PKC-u (Figure 2c), while other cells exhibitedtwo distinct focal clusters of PKC-u ormade partial contactwith the annulus and displayed a single focal cluster ofPKC-u at the point of contact with the TCR ligand. Theability of surface-bound ligands to pattern receptor cluster-ing as seen in this studymay also be a useful tool to explorehow physical apposition of receptors may enhance orsuppress signaling. For example, recent data has shown

466 Immunological techniques

(Figure 1 Legend Continued) ‘backfilling’ with a second ligand type, creating two-component patterns of surface-tethered anti-CD3 and ICAM-1.Part (c) adapted from [29!!], copyright 2006, National Academy of Sciences, U.S.A.

Figure 2

Templating immunological synapse formation using tethered ligandpatterned surfaces. Surfaces bearing microscale patterns of anti-CD3surrounded by ICAM-1 were created, similar to the schematic arraysshown in Figure 1A. Shown are fluorescence images of immunostained Tcells interacting with three different geometries of anti-CD3 ‘activationsites’ 20 min after seeding on surfaces: (a) focal, (b) multifocal, or (c)annular. Top panels show brightfield images overlaid with anti-CD3 sitefluorescence (blue). Bottom and lower panels show fluorescenceoverlays of TCR, PKC-u, and LFA-1 immunostaining as marked. Scalebars, 5 mm. Parts (B) and (C) adapted from [29!!], copyright 2006,National Academy of Sciences, U.S.A.

Current Opinion in Immunology 2007, 19:463–469 www.sciencedirect.com

Irvine DJ, Doh J, and Huang B, “Patterned surfaces as tools to study ligand recognition and synapse formation by T cells,” Current Opinion in Immunology, 19 463-469 (2007).

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

• Newly divided daughter cells migrated through ligand sites

Results

that APCs expressing a truncated version of CD80 lackingits cytoplasmic tail elicit weaker proliferation than cellsexpressingwild-typeCD80. In the immunological synapseof T cells interacting with APCs bearing the truncatedCD80, this costimulatory ligand colocalized with TCR,while wild-type CD80 was observed to segregate fromTCRs in the T–APC interface [30]. The selective coloca-lization or segregation of ligands for these receptors onpatterned surfaces would allow the importance of recep-tor–receptor interactions to be analyzed in a systematicmanner.

Changes in T cell synapse assembly elicited by patternedsurfaces were accompanied by changes inT cell dynamicsand cytokine production [29!!]. T cells encountering highdensities of anti-CD3 patterned in ‘focal’ sites completelyarrested their migration for 10–20 hours (Figure 3a), whileT cells contacting annular patterns of anti-CD3 appearedto receive incomplete ‘stop’ signals, and in some casescontinuously precessed/repolarized around the annulus ofactivating ligand (Figure 3b). In addition, T cells con-tacting annular patterns secreted "10-fold lower totalamounts of the effector cytokine interferon-g. Thus,differences in ligand presentation even on micron lengthscales, significantly larger than clusters of TCR observedduring the early moments of T cell triggering on sup-ported lipid bilayers [23,24!,25], appear to influence Tcell responses to TCR ligand.

Observing immunological synapse resolutionwith patterned surfacesOn contact with antigen, motile T cells halt, repolarizetoward the presenting cell, and assemble an immunologi-

cal synapse [31,32]. However, the life of the T cell doesnot end with APC contact and synapse formation: in vivoimaging of T cell activation in lymph nodes using two-photon microscopy has revealed prolonged periods ofstable T–APC contacts, followed by a resumption of Tcell motility [33,34]; brief serial contacts between T cellsand multiple APCs at the earliest stages of T cell priminghave also been reported in some experimental settings[35,36]. While resolution of the IS and disengagement ofT cells from APCs may play a critical role in regulating Tcell priming (by controlling the duration of T cell stimu-lation), study of the end-stages of T cell priming in livecell–cell couples is technically problematic due to pro-blems of convection/cell migration in vitro and the infre-quency of these events in vivo. Patterned surface modelsmay provide a valuable tool for examining the late stagesof T cell priming and events following disengagement ofT cells from APCs, since the ‘APC’ in this case isimmobile and keeps the responding T cell in a fixedlocation. Using the patterned anti-CD3/ICAM-1 surfacesystem, it was shown that T cells arrested on anti-CD3spots will in some instances remain in contact with asingle activation site until cell division occurs, up to 20hours later (Figure 3a) [29!!]. Intriguingly, newly divideddaughter cells migrated through nearby anti-CD3 siteswithout changes in their trajectory or morphology; this‘ignorance’ of activation sites persisted for at least threehours. These observations may correlate with the rapidmigration and brief contacts between T cells and APCsobserved at late times in vivo 24–48 hours after the initialmeeting of T cells with antigen-bearing APCs [33,35,36].The reductionist nature of the patterned surface systemsuggests that antigen ignorance following cell division

Patterned surfaces as tools to study ligand recognition and synapse formation by T cells Irvine, Doh and Huang 467

Figure 3

T cell responses modulated by microscale surface patterns of anti-CD3 and ICAM-1. Surfaces bearing microscale patterns of anti-CD3 in eithercircular or annular geometries were fabricated, and the dynamics of T cells seeded onto these surfaces were tracked by time-lapse microscopy.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 onannular microscale patterns of anti-CD3 (b). Elapsed times: (a) hour:min:s and (b) min:s. Adapted from [29!!], copyright 2006 National Academy ofSciences, U.S.A.

www.sciencedirect.com Current Opinion in Immunology 2007, 19:463–469

Irvine DJ, Doh J, and Huang B, “Patterned surfaces as tools to study ligand recognition and synapse formation by T cells,” Current Opinion in Immunology, 19 463-469 (2007).

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

Immune Response Activation

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

Typical Design of pteroate-drug conjugate1. pteroate ligand: when linked to glutamic acid forms folate acid moiety2. Linker: reduces steric hindrance, provides favorable functional groups3. 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 bond4. Drug used for chemotherapy

C. Leamon and J. Reddy (2004). Folate-Targeted Chemotherapy.

Advanced Drug Delivery Reviews 56, 1127-1141.

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

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

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.

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

Conclusion

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