Novel Living Tissue Culture Stylus for the Interrogation of Bioactive Nanostructures

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Novel Living Tissue Culture Stylus for the
Interrogation of Bioactive Nanostructures
Brij M. Moudgil Department of Materials Science
and Engineering and Particle Engineering Research
Center University of Florida, Gainesville, FL
32611 Florida Research Consortium, Tech
Transfer Conference May 18-19, Orlando, FL
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Scientific Team
Dr. Brij M. Moudgil - Distinguished Professor
and Alumni Professor of Materials Science and
Engineering, Director of the Particle Engineering
Research Center (PERC), Member of the National
Academy of Engineering Dr. Veena B. Antony
Cheif of the Division of Pulmonary Critcal Care
Medicine, College of Medicine, UF Dr. Yakov
Rabinovich AFM Expert, Research Professor,
PERC Dr. Mohammed A. Kamal Assistant
Professor, Division of Pulmonary Critical Care
Medicine, UF Scott C. Brown PhD Candidate
Department of Materials Science Engineering and
PERC
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Background Motivation

• Emerging evidence that certain nanostructures
  exhibit unique bioactivity

• Estimated that the world market for medical
  nanotechnology will surpass 3 billion within the
  next 4 years (Theta Reports, 2004)

• Current approaches to isolating bioactive
  nanostructures are low thoroughput cost
  intensive

No existing rapid screening technologies for
screening nanostructures in biosystems
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The Concept

• Nanostructure length-scales similar to those of
  biomolecules binding principles are
  fundamentally the same

2 Possibilities

• Adhesion (binding)
• No Adhesion

- change in gene expression likely -
nanostructures impact cells

Nanostructure Cell Interfacial Contact
No apparent change in bioactivity
Adhesion between cells and nanostructure can
predict unusual bio-activity
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Screening Approach
Nanostructure-Cell Interfacial contact
NEXT Nanostructure, Cell line, or Solution
Condition
No
Adhesion?
Yes
Scale-up Structure
Standard biological Assays to identify
consequence (protein / gene expression, etc.)
Optimization Fundamental Studies Database
Creation Engineering Stratagies
New Products!
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The Atomic Force Microscope
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Modification of AFM Force Sensors
Standard Cantilever tips 10 60 nm
Diameter
Colloidal Probes Particle Attached to
Cantilever 1 500 µm Diameter
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The Technology
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Technology Bioactive Nanostructure Screening

• High throughput screening technology for
  isolating uniquely interactive nanostructure-cell
  pairs under given physiological conditions
• Reusable Nanostructured Array chips
• Tissue Culture Force probes can be optimized for
  nearly any adherent cell culture
• Sensitivity can be adjusted by choice of
  cantilever

• NOVELTY
• Shotgun approach to identifying bioactive
  nanostructures
• Innovative diagnostic tool
• - Utilizes adhesive events between engineered
  nanostructures and living confluent adherent
  cells to predict unusual bioactivity

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Technology Status
Novel Living Tissue Culture Stylus for the
Interrogation of Bioactive Nanostructures US
Patent Application Serial 60/334,158
Proof of Concept Experiments In Progress
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Preliminary Evidence Pull-off Force Profiles
Cell-Cell ECM-ECM Glass-ECM Glass-Cell ECM-Cell
pH 7.4 HBSS
ECM-ECM
ECM
Force sensors used to measure Adhesive Integrin
binding Interactions between Mesothelial Cells
and Extra-Cellular Matrix (ECM) Surface
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Differential Adhesion to Calcium Oxalate Crystals
Periodic Nanoscale Surface Features
1.5 nm RMS
nm
nm
Interaction between kidney epithelial cells
retrieved from different organ sites with Calcium
Oxalate Crystals in artificial urine.
LPCK Cells No Adhesion MDCK Cells
Adhesion (0.5 nN)
Good Correlation with Kidney Stone Formation
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Advantages Over the
State-Of-The-Art

• Current Strategies
• Conventional Biological Assays
• - Large (mm or larger) nanostructured area
  required
• - Tests typically take days to weeks to perform
• - Cell encounter nanostructures in a free
  unnatural state
• Single-Molecule Probe Techniques
• - Requires interacting cell receptors to be
  known (many have not been identified
  expression can change with physiological
  environment)
• - Contributions from participation of accessory
  molecules and lateral mobility of binding
  molecules lost (non-physiological)

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Advantages Over the
State-Of-The-Art (contd.)

• Tissue Culture Stylus / Nanostructure Array
  Approach
• Area of Nanostructures reduced to a few microns
  (cost effective)
• High thoroughput method (efficient, seconds to
  minutes per measurement, multiple structures in
  single experiment)
• Interaction between nanostructures and adherent,
  differentiated, biologically significant tissue
  cultures measured
• Receptors do not need to be known
• Physiological measurements performed accessory
  molecules present, lateral mobility of surface
  molecules available

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Potential Commercial Impact

• Potential Products Opportunities
• Novel Scaffold Materials for Tissue Engineering
  
• - Surfaces for programmed differentiation
  (stem-cells others)
• - Surfaces for selective attachment of cells
• Cell-Separation Devices
• Nanostructured interfaces for bio-electrical
  interfacing
• Structured surfaces for Drug Targeting
• Advanced tissue culture surfaces
• Nano-surface architechtures enhanced biodevice
  compatibility
• Anti-Fouling Surfaces S
• Many More.

Proprietary Nano-Bio Toolbox!!!
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Target Industries

• Medicine/Biomaterials
• (scaffolds, smart device surfaces (e.g.
  programmed differentiations and integration,
  surfaces for artificial signal transduction,
  nanostructure therapeutics)
• Pharmaceutical
• (smart delivery surfaces, passive surfaces for
  long-term drug delivery (anti-fouling,
  therapeutic surfaces)
• Life Sciences
• (advanced culture surfaces, cell separation
  technologies, programmed differentiation)
• Analytical Equipment
• (nanostructure identification kits,
  nanostructure interaction databanks, bio-sensors
  etc.)

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What is Next?

• Optimization of Force Sensors for Enhanced
  Sensitivity and Reliability
• Analysis of longer term biological
  endpointsconfirmation of screening strategy.
• Develop Standard Protocols for Assessing
  Nanostructures with Cellular Probes
• Development of fully automated screening
  technologies (scale-up)
• Begin Compiling a Database of Cell-Nanostructure
  binding pairs

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Point of Contact
Brij M. Moudgil, Director, PERC
(bmoudgil_at_erc.ufl.edu) OR Victor
Jackson, PERC Associate Director for Industrial
Collaboration Technology Transfer
(vjackson_at_erc.ufl.edu) 205 Particle Science and
Technology Bldg, PO Box 116135 Gainesville, FL-
32611-6135 USA [phone] phone (352)
846-1196 fax
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Acknowledgments
The authors acknowledge the financial support of
the Particle Engineering Research Center (PERC)
at the University of Florida, The National
Science Foundation (NSF) (Grant EEC-94-02989),
and the Industrial Partners of the PERC for
support of this research. Any opinions, findings
and conclusions or recommendations expressed in
this material are those of the author(s) and do
not necessarily reflect those of the National
Science Foundation.