Nano Materials For Potential Cardiovascular and Other Biomedical Applications
Nano Materials For Potential Cardiovascular and Other Biomedical Applications
Nano Materials For Potential Cardiovascular and Other Biomedical Applications
Sungho Jin Professor of Materials Science University of California, San Diego Outline Introduction Nanotechnology for medicine using forefront new materials --- Nano Particles --- Nano Needles --- Nanostructured Substrates Potential Applications for Cardiovascular Research and Therapeutics Summary
Types of nanomaterials being studied for medical applications 1. Nano Particles --- Iron oxide mangetic nanoparticles --- Au nanoparticles --- Other shapes, e.g., core-shell coated, rod shape, hollow geometry, composites with quantum dot, etc. 2. Nano Needles --- Carbon nanotubes --- Sharp probes for ion channel study --- Nano-pipettes for single cell manipulations 3. Nanostructured Substrates --- Porous substrates --- Protruding arrays of soft or hard pillars Most of these nanomaterials are being fabricated and their medical applications being investigated at UCSD.
(e.g., superparamagnetic Feoxide) --- actively investigated for targeted cancer treatment (magnetic hyperthermia), stem cell sorting & manipulations, guided drug delivery, gene therapy, DNA analysis, and medical diagnosis (magnetic MRI). Potential use of nanotubes/nanowires for nanoscale probing of cell behavior, biosensing, therapeutics, etc. Quantum dots for bio imaging, genome sequencing, etc. Au nanoparticles for imaging, photothermal cancer treatment (by laser). These are all very rapidly advancing fields. Many opportunities exist to design/develop diagnostic or therapeutic techniques, and instrumentations using these nanotechnologies.
Nanoparticles -- can be designed to target specific cells or biomolecules with the ability to reach those targets by crossing tissue and membrane barriers. Multifunctional nanoparticles may combine a targeting agent specific for a protein biomarker, with conjugated agents for imaging, therapy, and reporting efficacy Magnetic core can provide mobility, imaging (MRI), and local temperature rise if needed.
Magnetic Nanoparticles
Fe-oxide nanoparticles have been used as magnetic recording media (audio or video tape, disk, etc.) Fe3O4(magnetite) or -Fe2O3 (maghemite) 6-12 nm diameter particles --- Need to be superparamagnetic to avoid agglomeration. Biocompatible. Magnetic nanoparticles --- actively investigated for targeted cancer treatment (magnetic hyperthermia), stem cell sorting & manipulations, guided drug delivery, gene therapy, DNA analysis, and medical diagnosis (magnetic MRI). Biomagnetics --- Potentially a huge field for science and applications of magnetism and magnetic materials!
Different types of magnetic materials ---Magnetic Properties can be altered by chemistry, geometry and processing.
Soft Magnetic
2-3 nm
5-6 nm
12-15 nm
10 nm
(a)
(b)
CdSe/ZnS qdots conjugation onto the magnetic nanoparticles. (a) Schematic illustration, (b) TEM micrograph showing the satellite conjugation of qdots on iron oxice particle. The scale bar is 5 nm.[From Wang et al, Nano Lett, 4, 409-413 (2004). ] --- Possibly configured and applied for combined medical applications of bio-imaging + magnetic MRI + magnetic hyperthermia treatment of cancer.
Crystalline CdS
Biotech applications --- Magnetic nanoparticles can be utilized as a localized, intra-cellular source of Programmable heat (by applied AC magnetic field) --- Active R&D projects in progress worldwide.
Temperature rise induced by remote magnetic field (100 KHz used) --- In a liquid containing various percentage of magnetic nanoparticles. --- ~10 nm diameter Fe3O4 particles.
(a)
target
(b)
0.15 %
200 400 600 800 1000 1200 1400 Time of AC Magnetic Heating [sec]
Nano-Bio Tech Biomedical Devices for Cancer Treatment Highly interdisciplinary (materials, devices, physics, chemistry, biology,
biomedical engineering, electronics, mechanical engineering, manufacturing ). Example --- Magnetic Hyperthermia Cancer Treatment using Targeted Magn. Nanoparticles (Jordan et al., Dept. of Radia. Oncology, Virchow Univ., Germany). [Progress in Clinical Trial]
Control Systems
Carbon Nanotubes, Sharp Probes, etc. Fabricated at UCSD for Bio Applications
(a)
Extremely sharp AFM Probe (~1 nm tip) --- For ion channel conductivity study
(b)
2 m
1 m
10 nm nm 10
(b)
(c)
Substrate
Magnetic Particles for Targeted Drug Delivery (e.g., gene therapy, cancer chemotherapy applications)
Magnetofection: enhancing and targeting gene delivery by magnetic force in vitro and in vivo, F. Scherer et al., Nature/Gene Therapy 9(2), 102-109 (2002).
Gene therapy ---Correcting defective genes responsible for disease by inserting a normal gene to replace a non-functional gene, or repair. Associate gene vectors with superparamagnetic nanoparticles and targeted gene delivery by application of a magnetic field. (Coating of magnetic particles with colloidal chemistry and attach DNAs). Enhanced efficacy by several hundred-fold, reduction of the duration of gene delivery time to minutes. High transduction efficiency observed in vitro was reproduced in vivo with magnetic field-guided local transfection (in the gastrointestinal tract and in blood vessels). Magnetofection provides a novel tool for high-throughput gene screening in vitro and can help to overcome fundamental limitations to gene therapy in vivo. Cardiovascular disorders Many cardiovascular diseases are excellent targets for somatic gene therapy as drug therapy may have reached a plateau in its effectiveness. Use of gene transfer approaches for long-term control of the pathophysiological conditions being explored by various groups. Gene vectors (such as adeno-associated viruses (AAV)) are currently available that can efficiently transduce cells of the myocardium & vasculature. Magnetic vectors may also be explored.
1. Remote pacemaker. 2. Tissue engineering for growth of cardio-myocyte on cyclically moving substrate. 3. Endothelialization of coronary stent surface to minimize Late Stent Thrombosis
Current pace maker systems use wire connection between heart and the implanted, battery-operated control circuit system. Lead wires --- involve complicated surgery going through blood vessels, with a risk of foreign body rejection on lead wires. Substantial risks associated with leaving failed and unused leads in place --- Increased likelihood of infection or blood clot formation around the old and entangled pacing leads, and the leads can restrict the operation of the heart valves and hinder the implantation of new leads in the heart, etc. Pacemaker lead explantation(removal) --- complicated, time-consuming procedure with significant risk.
RETO CANDINAS, M.D.; FIRAT DURU, M.D.; et al , Mayo Clin Proc. 1999;74:120-125 1999
Galvanis experiment in 1790 -Muscle contraction from bi-metallic rod contacting frog leg nerves
Small wire electrode implanted --- Minimal chance of infection Highly locallized stimulation treatment minimizes side effects in unwanted regions
New Magnetic Actuator Alloy Wires --- Fast magnetization reversal & large induced voltage (V d/dt) New Sensor Alloys
B B
Wiegand wire
New alloys Fe20Cr4Ni Fe6Ni Fe-10Si-15B (amorphous) HC 10~20 2~10 0.1~10 4MS 15kG 20kG 14kG
Hard shell + soft core Complicated processing Expensive High output voltage
uniform structure easy to process & reproducible low cost (no cobalt) high output voltage & wide pulse lower drive field
Broken nerve
Remote electrode
Implanted voltage-generating wire electrode (e.g., helically magnetic Fe-6%Ni) is transcutaneously actuated by remote magnetic field --- To induce muscle movement or to stimulate for nerve regeneration.
(b) Use helically anisotropic magnetic wire which generates end-to-end voltage
Fig. 3. Two approaches to supply remotely actuated voltage impulses to implanted sensor wires for heart pacing.
(a)
M
Pulse field magnetized (e.g. 1 msec) 0
H
Hc
500 nm
10
(a)
38.0
Fe-20Cr-4Ni D=0.05 cm, L=3.7 cm N=1000 turns coil f= 1000 Hz
(b)
Fe-20Cr-4Ni D=0.05 cm, L=3.7 cm N=1000 turns coil Hmax = 200 Oe
37.5 2.0
Volts
Volts
1.5 1.0
0.5
0 0 50 100 150 200 250 300
Fig. 4. Induced voltage impulse in the solenoid surrounding a square loop magnetic wire of Fe-20Cr-4Ni alloy as a function of (a) amplitude of applied AC magnetic field, (b) frequency of magnetic field applied.
Ha=200 Oe Ha=20 Oe
Remote Pace Maker (with pulse rate sensor + voltage impulse generator)
(a)
Heart with implanted electrode needle Remote pace generator (e.g., magnetic pulse field train generator) Sensor outside the body for heart beat rate/ intensity sensing (piezoelectric, strain gauge, or pressure sensor) Chest and shoulder belts to hold pace generator in place, and to keep heart beat sensor in skin contact
(b)
Implanted pulse rate sensor either on the electrode or a separate piece (solenoid device, piezoelectric, strain gauge, pressure sensor, etc.) with RF signal emitting systems Remote pace generator RF signal sensor/ analyzer to detect irregular heart beats and initiate remote pacing (e.g., by activating magnetic field) Implanted electrode piece
RF signals
TiO2 nanotubes
Flat polished Ti
Rotating cantilever
Cross-sectional TEM of Aligned TiO2 Nanotubes (top view) Nanotube wall Spacing between nanotubes (~10 nm thick)
TiO2 nanotubes
Ti base
500 nm
100 nm
Effect of Nanotube Surface Layer on Osteoblast Cell --- Significantly Accelerated Growth by ~ 300 400% Number of Adhered Cells, 102/cm2 1000 800 600 400 200 0
On bare implant metal (such as Ti) On amorphous TiO2 nanotubes On crystalline TiO2 nanotubes
50
60
Flat Ti
TiO2 nanotube
# of cells
Nanotube
50 40 30 20 10 0
Nanotube
Glass
}
Agitated
TiO2 Nanotube
Surface type
3. Prevention of Late Stent Thrombosis (LST): A Cellular Approach (by Karla Brammer (GSR))
http://www.smh.com/img/hv_institute/stent_eluting.jpg
Issue --- There is a clinical problem of late stent thrombosis (blood clotting, blockage of blood vessel, heart attack, etc.) due to a lack of endothelialization of the inner stent wall, where the stent fails to be fully integrated into the vessel wall. Proposed Solution --- A possible novel solution to the clinical problem of late stent thrombosis is to pre-endothelialize the stent with the patients own vascular endothelial cells prior to stent implanting. (Perhaps preventing restenosis, thus eliminating the need for drug-eluding stents?). To test this hypothesis --- Culture endothelial cells on conventional biocompatible, metallic, implant material with different surfaces (flat vs nanostructured). Look for --- attachment, adhesion, and spreading characteristics of endothelial cells on the different surfaces Determine --- the endothelialization of cells on the surfaces for potential use in stent applications. Some in vivo animal tests may follow.
7/9/07
(a)
(b)
Stent crosssection
(c)
(d)
Artery vessel
Lumen
Fig. 7
Ti substrate
Figure 3. Biocompatible Ti chips with vertically aligned TiO2 nanotubes. Previous work with an osteoblast cell line proves that the pronounced topological feature and increased surface areas plays a significant role in the adhesion/propagation of osteoblast cells. The TiO2 nanotubes allow for filopodia of growing cells to actually go into the nanotube pores, producing a type of interlocking cell structure. Large surface areas, small pores, gaps between nanotubes are thought to facilitate long term survival and function of primary hepatocytes Super Hepatocyteswith enhanced functionality -- 3-D configuration and albumin secretion function better than real liver! There are pathways for fluid present between the nanotubes. Use of TiO2 nanotube surface is now being explored for pre-endothelialization experiments.
Experimental Procedure
Plate 120,000 primary bovine aorta endothelial cells in 1ml media per each one well of a 12-well plate containing a 1.27cm x 1.27cm square Ti or TiO2 nanotube chip. Preformed a flouroscein diacetate (FDA) stain of viability to observe morphology/spreading of cells at time points 6, 12, 24, and 48 hours after plating
6 hours 12 hours 24 hours 48 hours 6 hours 12 hours 24 hours 48 hours
VS.
Pure Ti
TiO2 Nanotube
Flat Ti substrate
FDA(flouroscein diacetate) viability of Bovine Aorta Endothelial Cells (BAECs) on Ti flat vs TiO2 nanotube substrates after 24 and 48 hours of culture were compared. --- An aggregated, disrupted, and possibly detached status of cells on the Ti surface was seen. --- A flat monolayer of cells on the TiO2 surface similar to natural endothelium were observed.
Figure 2. SEM micrographs of BAECs on flat Ti (a,c) vs TiO2 (b,d) surfaces after 2 hours of culture. Loose, detached filipodia are present on the Ti substrate (arrow) while filipodia are able to probe and survey (arrows) on the TiO2 surface as they are even seen protruding into the pronounced features of the nanotubes (white dashed circles).
(a)
(b)
500 nm
500 nm
Figure 3. SEM micrographs after 6 hours of incubation. (a) BAECs are deficient in ECM material on the flat Ti controls compared to (b) where a coarse ECM material is deposited on and filled in the nanotubes on the TiO2 surface.
Polystyrene
Flat Ti
TiO2 Nanotubes
50 m
50 m
Figure 4. Immunofluorescent images of cytoskeletal actin for BAECs after 24 hours of culture. The lower magnification images show that the nanotube surface increases cell to cell interactions. The higher magnification images reveal that the nanotube surfaces trigger more organized, prominent lamellipodia for increased cell locomotion.
Polystyrene
Flat Ti
TiO2 Nanotubes
50 m 50 m
Figure 5. Immunofluorescent images of cytoskeletal actin for BAECs after 48 hours of culture. The BAECs on the nanotube surface are more elongated and mobile. The nanotube surface generates greater cell communication, interaction, and movement where BAECs on flat controls are spread wider with more stationary morphology. Unstable and broken cell extensions are seen on the Ti surfaces.
* **
Figure 6. Analysis of BAEC morphology on polystyrene tissue culture plates, flat Ti, and TiO2 nanotubes.
Polystyrene Ti
TiO2 Nanotube
The spreading area and minor/major axis ratio are calculated from at least 100 cells.
**
Polystyrene Ti
TiO2 Nanotube
Figure 7. Immunofluorescent staining of vinculin at focal adhesions. (a) Cells on flat Ti and (b) Cells on TiO2 nanotubes.
--- In the endothelium, nitric oxide (NOx) is continuously synthesized as an important vasodilator (vascular endothelium relaxation factor) which inhibits platelet aggregation. --- On the other hand, the release of endothelin-1 counteracts NOx and is a vasoconstrictor that promotes platelet aggregation.
NOx/Endothelin-1 Ratio
NOx/Endothelin-1 ratio of endothelial cells on different culture substrates. --Higher ratio on TiO2 nanotubes indicates a reduced platelet aggregation and risk of LST.
TiO2 Nanotube
Comparative time-dependent migrational assay for bovine aortic endothelial cells on different culture substrates
Cells behind the moving front
Polystyrene
100 m
Polystyrene, flat Ti controls, vs TiO2 nanotubes with 50, 70, 100 nm diameter. The red dotted line indicates the start line of the cell migration.
}
Flat Ti
}
24 hours migration
The cells on the 100nm diameter TiO2 nanotube surface have traveled the greatest distance after 24 hours and are much more elongated (indicating a migrational morphology with enhanced movement and a higher mobility rate compared to smaller sized nanotube surfaces and flat Ti controls).
Before migration
12 hours migration
Primary bovine aorta endothelial cells were able to interact more efficiently with the TiO2 nanotube surface with enhanced cellular migration and functioning as compared to a flat Ti surface. The nanotubes also stimulated an increase in ECM deposition and more organized lamellipodia on the nanotube surface. The TiO2 nanotubes increased the functionality of the cells by substantially increasing the NOx/endothelin-1 ratio in the media. The nanotube structure with enhanced endothelialization, much increased extra cellular matrix formation, and substantially raised level of nitric oxide-endothelin ratio, may be useful as a vascular stent material with a reduced probability of late stent thrombosis.
Figure 8. NOx/Endothelin-1 ratio with respect to the polystyrene tissue culture dish.
Summary
TiO2 nanotubes adherent on Ti surface --- introduced to provide large-surface area, nanoscale base microstructure and cavity configuration, which produces mechanically locking structure for improved cell adhesion. The TiO2 nanotubes significantly enhanced the adhesion and growth of osteoblast cells (in vitro) by ~300 400% as compared to non-nanostructure surface. In vivo rabbit implant tests indicate a much enhanced osseointegration of new bones on TiO2 nanotube implant surface, with ~x6 improved adhesion strength than conventional sand-blasted Ti implanted surface. The TiO2 nanotubes enhance endothelial cell migration and nitric oxide (NOx) formation as an vasodilator, which can be useful for inhibiting platelet aggregation and LST.