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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