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Photodynamic Tissue Repair and Healing.
Michael R Hamblin,
Florencia Anatelli, John J Khadem,
Reza Dana, Tayyaba Hasan.
Wellman Center for Photomedicine
Massachusetts General Hospital
Harvard Medical School
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
Outline
Methods of wound closure
Tissue glues
Photodynamic laser activated tissue
glue (PLATG)
PLATG: in-vivo Experiment
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
What is PDT?
+
Oxygen
LIGHT
PS*
PS
Photosensitizer
= light activatable
chemical
Michael R Hamblin Ph. D.
Cell Destruction
Light Activated Tissue Regeneration and Therapy I
Wound closure
Sealing wounds and defects in tissues is a common shared step
among different medical specialties.
Gold standard closure techniques: Sutures and Staples.
These techniques present drawbacks:
Labor intensive.
Inflammation, Infection, foreign body response
Scarring. Uneven healing. Leaking.
Neither of this two techniques can
Easily close a defect in which the edges cannot be
evenly apposed
Be used in extremely sensitive tissues such as the
eyes
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
Alternatives to traditional methods….
Various tissue glues
Strength of adhesion
Non-Toxicity
Biodegradability
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
Alternatives to traditional methods….
Tissue sealants, adhesives and glues:
a) Cyanocrylates glues
b) Fibrin sealants
c) Gelatin-Resorcinol-Formol Glues
•
Light activated adhesives and bonding
technologies
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
Light Activated Tissue Adhesives
Hydrogels: polymerize into solid in the presence of
light (UV or blue)
Dye activated protein solders: protein in the solder
forms non-covalent bonds to the tissue protein
(collagen) by Photothermal mechanism
Photochemical Tissue bonding : Dye (Rose Bengal)
without exogenous proteins forms direct covalent
bonds between collagen surfaces.
PLATG: Photodynamic Laser Activated Tissue Glue
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
PLATG
Photoactive dye
Have a good quantum yield of excited singlet state
and triplet state.
Exogenous Protein (Albumin)
Very soluble to allow viscous formulation
Glue remain in place in tissues defects.
Proteins molecules very close: efficient proteinprotein cross linking
Light
Provides the energy for the chemical reaction.
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
PLATG: Underlying Mechanism?
Covalent Protein
Cross links
Tissue Protein
(collagen)
Photosensitizer
(Dye)
Michael R Hamblin Ph. D.
GLUE
Glue Protein
Light Activated Tissue Regeneration and Therapy I
PLATG versus photothermal
PLATG strength depends on fluence not irradiance
temperature does not rise
Photothermal strength depends on irradiance not fluence
temperature rises to > 60 deg C
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
PLATG: Previous Work
PS -PROTEIN
Rivoflavin-6-P
Fibrinogen
LIGHT
TISSUE
Argon Laser
(457-514 nm)
IN VIVO:
Rabbits eyes
Argon Laser
(457-514 nm)
EX VIVO:
Human
cadaveric eyes
Goins et al
J Cataract Refract Surg. 1997
Khadem et al,
J Clin Laser Med Surg. 2000
Chlorin(e6)
BSA
Khadem et al,
Invest Ophthal Vis Sci 1999
Michael R Hamblin Ph. D.
RESULTS
Strong Bonds
Human Protein
Viscosity
Strong bonds
BSA-Ce6
Conjugate +
unconjugated BSA)
Light Activated Tissue Regeneration and Therapy I
Ex vivo experiments
Investigate and compare gluing
properties in-vivo of albumin +
photosensitizer Chlorin(e6) and Janus
Green
Test the appropriate consistency and viscosity
of the glues.
Study different protein-dye molar ratios of both
glues
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
BSA-ce6 conjugate
1
H2C
H2C
absorbance
0.8
CH3
H3C
N
N
H H
N
N
660-nm laser
0.6
H
H
CH2
H2C
C
O
OH
0.4
CH3
CH3
H3C
CH3
H3C
COOH
COOH
1 DCC
2 NHS
3
N
N
H H
N
N
H3C
H
CH3
CH3
H
CH2
H2C
C
O
O
N
O
1
2
3
COOH
COOH
O
C h l ori ne6
H3C
CH3
Ce6-NHS e s te r
0.2
H3C
0
200
300 400
500 600
700 800
900
BSA
COOH
COOH NH
H
N
H C2 C O
C
N H
H H2
H N
CH3
N
CH2
CH3
BS A-ce6
wavelength (nm)
Good absorption peak at 660 nm
Good fluorescence & singlet oxygen quantum yields
Easy conjugation chemistry via carbodiimide activation of
COOH group
Readily available
Conjugates can be characterized by PAGE
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
Janus Green
N
H2
H3C C
+
N
H3C
C
H2
CH3
N
N
N
_
N
CH3
Cl
Janus Green B
3-Diethylamino-7-(4-dimethylaminophenylazo)-5-phenylphenazinium
Cationic phenazine azo-dye
Capable of Photodynamic reaction?
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
Preparation of the glues:
Bovine serum albumin: 50%w/w - 7.4 mM Solution
Chlorine6 (Conjugate) and Janus Green (Mix)
1:1 ; 1:0.5 ; 1:0.33 ; 1:0.25 Protein/Dye
2
absorbance
1.5
BSA-ce6 + BSA
BSA + JG
1
665-nm laser
0.5
0
200
Michael R Hamblin Ph. D.
300
400
500
600
w ave le ngt h (nm )
700
800
Light Activated Tissue Regeneration and Therapy I
Preliminary Ex vivo Studies
Murine Skin Strips
50-100 ml of Glue
665-nm 1W diode
Laser:
Irradiance: 181 mW/cm2
Fluence: 32.5 J/cm2 –
109 J/cm2
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
Tensiometer
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
Breaking strength
4
Ce6 1/1
Ce6 2/1
Ce6 3/1
Ce6 4/1
3
2
1
0
-1
JG 1/1
JG 2/1
JG 3/1
JG 4/1
3
breaking force (N)
breaking force (N)
4
2
1
0
-1
-2
0
50
100
Time (s)
Michael R Hamblin Ph. D.
150
200
0
5
10
15
20
25
30
35
40
Time (sec)
Light Activated Tissue Regeneration and Therapy I
Preliminary Ex Vivo Study: Conclusions
1:033 P/D ratios
Michael R Hamblin Ph. D.
strongest adhesion
Ce6= 2.08N (kg.m/s2)
JG= 3.43N
JG glue stronger adhesion than
Ce6 glue.
The strength of adhesion:
directly proportional to
delivered fluence:
PHOTOCHEMICAL MECHANISM
Light Activated Tissue Regeneration and Therapy I
Demonstrate photochemical mechanism?
3.5
breaking strength (N)
3
2.5
2
1.5
1
0.5
0
D2O
Michael R Hamblin Ph. D.
H2O
H2O/NaN3
Light Activated Tissue Regeneration and Therapy I
PLATG: In-vivo Study
Rat model of penetrating corneal wound
Sprague Dawley rats
(350g)
One eye of each rat
operated
6 mm incision in center of
cornea
CONTROL GROUP
(n=10)
Procedure ended here
Khadem JJ, Martino M, Anatelli F, Dana MR, Hamblin MR.
Healing of perforating rat corneal incisions closed with photodynamic laser-activated tissue glue.
Lasers Surg Med, in press, 2004
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
PLATG in rat eyes
Experimental Group
Glue : approx. 50-100 ml
1:0.33 ratio P/D
BSA - Ce6 (n=10)
BSA + JG (n=10)
Light: 665 nm diode laser
Irradiance: 600 mW/cm2
Fluence: 71.5 J/cm2
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
PLATG in rat eyes
Control and Experiment groups
Clinical follow up
Sacrificed at day 1, week 1 and week 2
following the treatment
Eyes were enucleated and randomly divided into
two groups to evaluate:
Strength of adhesion :
Leaking pressure by inserting
needle connected to sphygmomanometer
Inflammation/Toxicity: Histopathology
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
Results:
Control
ce6
JG
600
Bursting pressure (PSI)
Leaking pressure
500
** †
400
*
300
200
100
0
1 day
1 week
2 weeks
* p < 0.01 vs control, ** p < 0.001 vs control, † p < 0.05 vs ce6
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
Results:
Michael R Hamblin Ph. D.
Histopathology findings
Light Activated Tissue Regeneration and Therapy I
Control: Day 1
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
JG: day 1
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
Ce6: Week 1
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
Control: week 2
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
JG: week 2
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
Conclusions:
Strong bonds:
JG glue showed greater adhesive strength
than Chlorin(e6) glue at day one.
Both glues formed stronger bonds than
control at day one.
Glues are biodegradable and temporary
Low irradiance (<200 mW/cm2)
Low risk of thermal damage to the tissue
Both glues induced less inflammatory
response compare to control
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
Remaining questions…
Can Oxygen-dependent photochemical process
that generates COVALENT crosslinks between
albumin and collagen be demonstrated?
Can it be demonstrated that JG worked by a
Photodynamic mechanism?
Can PLATG form tissue seals without harming
the surround living cells?
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
Future Work…
Compare PLATG with Gold standard method to
close corneal incision in living rats.
Study PLATG Effectiveness in other tissues…
Involving load bearing and stress (strong
bonds)
Irregular surfaces difficult to seal (space filling
glue)
Non-thermal where living cells are involved
(Carticel)
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I
Acknowledgements
John J Khadem, MD
Reza Dana, MD
Tayyaba Hasan, PhD
Florencia
Anatelli, MD
Funding: NIH (R01 CA/AI838801 to M. R. Hamblin).
Michael R Hamblin Ph. D.
Light Activated Tissue Regeneration and Therapy I