Transcript Document

NIRT: LIGAND NANODISPLAY FOR CELL INTERNALIZATION AND SUPERACTIVATION
Prabhas V. Moghe (PI)1, 2, Jean Schwarzbauer3, Thomas Tsakalakos4, Charles Roth2, David Talaga5
1Department
of Chemical and Biochemical Engineering, Rutgers University, Piscataway, NJ
2Department of Biomedical Engineering, Rutgers University, Piscataway, NJ
3Department of Molecular Biology, Princeton University, Princeton, NJ
4Department of Materials Science and Engineering, Rutgers University, Piscataway, NJ
5Department of Chemistry and Chemical Biology, Rutgers University, Piscataway, NJ
MOTIVATION: BARRIERS TO
SKIN MOTILITY DURING RE-EPITHELIALIZATION
Aim 1 is to fabricate organic nanocarriers functionalized with cell-adhesive ligand
fragments and investigate cellular motility on interfaces of nanocarriers as a function of
nanocarrier size and variable ligand loading. (Moghe, Schwarzbauer)
Aim 2 is to investigate the changes in the intracellular signaling pathways (via
gene microarrays and studies of selected protein signaling targets) activated by cellinternalizable particles functionalized with ligand fragments in relation to substrate
immobilized ligand-functionalized nanocarriers. (Roth, Schwarzbauer, Moghe)
Aim 3 is to investigate the modes and mechanisms of cytointernalization of
ligandnanocarriers via (a) molecular studies modulating endocytic uptake; (b) single
molecule imaging of intracellular trafficking kinetics of reporter ligand nanocarriers; and (c)
the use of new biosensors for nanoscale cytointernalization events. (Talaga, Tsakalakos,
Moghe)
Ligand-Nanoparticle
Trafficking
Aim 3
(Talaga, Co- PI)
Trafficking
Nanoscale Biosensing of
Cytointernalization
Aim 3
(Tsakolakos, Co- PI)
• Largest organ in the human body; protects it from water loss,
heat, and microorganisms
• For proper wound healing, need to re-establish the epidermal layer
via skin cell migration to close the wound site
• Defects in re-epithelialization processes can to lead deficient
Healing, chronic wounds, or in extreme cases morbidity and mortality
• Chronic wounds cost about $3 billion annually
• Diabetic foot ulcers affect 1 million in the U.S. (86,000 amputations)
• Manipulation of matrix components can be used to modify cellular
responses to improve wound repair
Cell Motility
Dynamics
Aim 1
(Moghe, PI)
Keratinocyte morphology is affected
by ligand display and nanoparticle
size, with smaller nanoparticles
demonstrating
a
more
motile
phenotype.
FNf-ANP 75 nm
35
FNf
FNf-ANP
FNf-ANP
FNf-ANP
FNf-ANP
30
25
20
ERK → MLCK →
Migration
Ligand Binding
10
10
5
5
0
0
0.4
1.5
2.5
3.4
Ligand Concentration ( g/cm )
2
Nanoparticle
Design
Aim 1
(Moghe, PI)
0
0.1
Nanotechnology has the potential to significantly amplify the functional roles of the currently known
repertoire of biomolecules. While the search for new biomolecules with therapeutic function continues,
the cell-specific exposure of established biomolecules is far from optimized to maximize the cellular
function of interest. Since cellular binding and subsequent interactions with extracellular biomolecules are
events occurring at the nanoscale, cellular engineering affords a fertile landscape for controlled
application of nanotechnology. To successfully exploit such opportunities, however, will require integrative
studies of the roles of molecules in dynamic systems. At Rutgers, we have discovered that fragments of
cell adhesion biomolecules like fibronectin, which has been widely studied for its effects on cell adhesion,
growth, migration, can elicit markedly altered cell responses, with consequences for internalization and
superactivation of cell functions, if displayed on a nanoscale. The substrate-based display of fragments
of adhesion molecules from nanoscale carriers affects receptor-mediated cell binding followed by the
internalization of the adhesion ligands. The concerted remodeling and internalization processes appear to
superactivate intracellular biochemical signaling pathways, leading to unprecedented levels of cell function
(e.g., cell motility; cell matrix assembly). Building further on this finding, this NIRT proposal has assembled
5 investigators with diverse backgrounds yet convergent expertise to probe two key questions:
1. How can the nanoscale display of biomolecular ligands be engineered to enable new
paradigms of activation of signaling processes within living cells?
2. How can these processes be sensed via nanoscale platforms to yield cellular functional
endpoints with relevance to biotechnology and biomedicine?
0.2
0.3
0.4
0.5
0.6
Rate of Intensity Increase (AU/h)
a5 integrin
Green
30 nm ANC
50 nm ANC
75 nm ANC
100 nm ANC
Fnf
10
0.7
0.8
0
0
b1 integrin
Red
Whole length
Fibronectin
FNf-ANP
Recombinant Fragment of Fibronectin (FNf) for Engineered Cell Motility
5
10
Cell Migration (m/h)
Post-Internalization
Processing
Separate Integrins
Lysosome
Trafficking
To lysosome
Internalization
Clathrin
Mediated
Albumin Nanoparticle (ANP)
15
20
Cytoskeletal
Rearrangements
Cdc42, Rac
Integrin
Recycling
To leading
edge of cell
Migration ← Cdc42,
Rac← JNK
ERK → MLCK →
Migration
Ligand
Binding
Thinking Model: On dynamic interfaces, keratinocytes can sequester
and internalize the ligand-functionalized nanoparticles.
The
internalization increases with smaller sized carriers, leading to increased
rates of post-uptake processing and signaling within the cell.
Preliminary data: Integrin a5b1 clustering and diffusivity throughout the
Fn I 1-5
Fibrin
Heparin
OVERVIEW & ORGANIZATION OF THE NIRT PROJECT
15
Smaller sized nanoparticles can be taken up at faster rates than larger sized nanoparticles. Nanoparticle
size and mobility can amplify relevant signaling element expression, which could be leading to
superactivation of cell motility.
Nanocarrier
Nanodisplayed ligand
Highly clustered integrins &
Cytointernalizable Carriers
20
5
Albumin Nanoparticle (ANP) Fabrication
Mix
FNf-ANP 100 nm
25
30 nm
100 nm
0
Temperature and
pH treatment Denatured albumin
Albumin
FNf-ANP 75 nm
35
PBS
Ligand
FNf-ANP 50 nm
Nanoparticle Size and Mobility Modulates
Uptake Rate and Signaling
30
5
The assembled team is composed of five investigators:
P. Moghe, PI (Cellular Bioengineering; Nanoscale Cell-Interactive Materials);
Jean Schwarzbauer, Co-PI (Molecular Matrix Biology); Thomas Tsakalakos, Co-PI
(Nanostructured Inorganic Bio-Materials); Charles Roth, Co-PI (Molecular
Bioengineering); David Talaga, Co-PI (Single Molecule Spectroscopy & Biophysics)
FNf-ANP 30 nm
Cell migration kinetics is sensitively governed by ligand concentration, nanocarrier size, mobility and uptake
mechanisms, suggesting that nanoparticles must be internalized by cells to induce cell superactivation.
25
Ligand Design
Aim 1
(Schwarzbauer, Co-PI)
FNf Adsorbed
Substrate
10
1. The display of ligands on the nanoscale via albumin nanoparticles will promote integrin nanoclustering.
2. The substrate mobility of the nanoparticles will promote membrane-based ligand/integrin translocation
and possible internalization.
3. Integrin nanoclustering & internalization will activate cell signaling for enhanced cellular motility.
Ligand adsorbed surfaces
Non - or slightly clustered integrins
20
15
HYPOTHESES
vs.
25
20
HOW: Nanodisplay of engineered biorelevant fibronectin fragments to cells via albumin
nanoparticles
Integrin
30 nm
50 nm
75 nm
100 nm
15
30
GOAL: Enhance cellular activity and dynamics via nanoscale
engineered ligand presentation
Passively Adsorbed
Immobilized
Clathrin inhibitor
Caveolin inhibitor
30
15
Cell Speed
 m/h)
Migration ← Cdc42, Rac←
JNK
Signaling
Aim 2
(Roth, Co- PI) (Gene)
(Schwarzbauer, Co-PI, Moghe, PI)
(Protein)
FNf
Nanoscale Display in Dynamic Interfaces Increases Cell Migration
0
Internalization
FNf-ANP 100 nm
Cell Speed
( m/h)
BROADER IMPACTS
-- Synergies with T32 Postdoctoral Training Program on Tissue Engineering/Biomaterials
Science
--Collaboration with New Jersey Center for Biomaterials and Department of Defense
--Synergies with IGERT on Biointerfaces (www.igert.rutgers.edu, PI: P. Moghe)
-Diversity Infrastructure: NE-AGEP; Institute for Teaching & Mentoring;
-Leadership in Diversity Award--Outreach to RISE/ISURF Undergraduate Program. Princeton U., RWJMS Medical School
--Translational Potential for Anti-Aging Therapies; Foundations for Ctr for Nanomedicine
Keratinocyte Morphology
F-Actin Organization is
Modulated by Nanodisplay and
ANP Size
Cell Speed (m/h)
-- Combination of Dynamic Approach to Cell Adhesion and Internalization of
Nanoparticles Accelerates Activation of Cell Motility
-- Interdisciplinary Team leveraging expertise in cellular bioengineering,
matrix molecular biology, molecular bioengineering, single particle tracking
and analysis, and inorganic nanoparticles for sensing/trafficking
SPECIFIC AIMS OF THE NIRT PROJECT
JNK Activity
INTELLECTUAL MERIT
FNf-ANP 50 nm
FNf-ANP 30 nm
Synergy
RGD
Fn III 12-14 Fn I 10-12
Heparin
Fibrin
NH2
SS
Gelatin
Collagen
cell membrane is enhanced when keratinocytes are presented with FNfANP’s.
Further studies are underway to examine the integrin and
endosomal dynamics.
Cell Binding Domain
Fn III 9-10
COOH
Variant
Fragment
Fragment Encompasses III 9-10 Domains of Fibronectin:
• 10th type III domain – contains the RGD tripeptide adhesion motif,
• 9th type III domain – contributes to integrin binding and signaling.
• Fragment directs binding of α5β1 integrin, required for cell migration and initiation of fibronectin
matrix deposition.
Main et al. Cell, 1992. 71:671. Altroff et al. J. Biol. Chem., 2003. 278:491. Schwarzbauer et al. Curr. Op. Cell Bio. 1999. 11:622.
Albumin Nanoparticle Functionalization with Fibronectin Fragment
• ANP’s and FNf are separately incubated with N-succinimidyl 3-(2-pyridyldithio)propionate (SPDP), which
forms an amide linkage between the cross-linker and the ANP’s and the FNf.
• Dithiothreitol (DTT) is added to the FNf to form a free sulfhydryl group.
• The FNf and ANP’s are incubated together, which induces the formation of a disulfide bond between the
FNf and the ANP (FNf-ANP).
FNf-ANP’s are passively adsorbed on substrates for cell presentation
CONCLUSIONS
• Ligand presentation from internalizable nanoparticles can activate significantly higher levels of cell
migration than those obtained via conventional ligand presentation.
• The endocytosis of ligands via Clathrin mediated mechanisms is key to the coactivation of cell
migration.
• Cell speed is correlated to uptake, suggesting that larger sized carries could be rate limiting during
migration on these interfaces.
FUTURE PLANS
• Elucidation of intracellular activation mechanisms and ANP trafficking kinetics (D. Talaga, S. Corbett);
• Magnetostrictive biosensors for cytointernalization kinetics (Tech. Univ, Denmark));
• Decoration of ANPs in 3-D scaffolds; In Vivo Implantation for Study of Wound Healing (H. Hsia).
PUBLICATIONS
Sharma RI, Pereira M, Schwarzbauer JE, Moghe PV. Biomaterials. 27(19):3589-98 (2006).
Pereira M, Sharma RI, Schwazbauer JE, Moghe PV, Tissue Engineering 13: 567-578 (2007).
Sharma RI, Shreiber DI, Moghe PV, Tissue Engineering, 2007 (In Review)
Rossi MP, Langkowski B, Uhrich KE, Moghe PV, Nanoletters 2007 (In Preparation)
Sharma RI, Schwarzbauer JE, Moghe PV. Nature Nanotechnology 2007 (In Preparation).
ACKNOWLEDGMENTS (GRAD STUDENTS, POSTDOCS)
Dr. Marian Pereira, Dr. Ram I. Sharma, Dr. María Pía Rossi, Vanesa Figueroa-Tañón