WO2008053432A2

WO2008053432A2 - Chromophore formulations for use in the laser welding of biological tissues

Info

Publication number: WO2008053432A2
Application number: PCT/IB2007/054411
Authority: WO
Inventors: Roberto Pini; Francesca Rossi; Luca Menabuoni; Patrizia Chetoni; Susi Burgalassi; Daniela Monti; Silvia Tampucci
Original assignee: Consiglio Nazionale Delle Ricerche
Priority date: 2006-10-31
Filing date: 2007-10-31
Publication date: 2008-05-08
Also published as: WO2008053432A3; ITFI20060270A1

Abstract

The invention relates to chromophore formulations in semi-solid or solid form to be used for the colouring of biological tissues in order to absorb in a selective and localised manner the laser radiation in the processes of laser welding of biological tissues. These procedures are applied in surgical treatments for the suturing, healing and sealing of incisions and wounds in various types of tissue, such as the cornea, skin and mucous membranes.

Description

TITLE

CHROMOPHORE FORMULATIONS FOR USE IN THE LASER WELDING OF

BIOLOGICAL TISSUES DESCRIPTION Field of the invention

The present invention relates in general to the field of techniques of laser welding of biological tissues, more particularly for the suturing, healing and sealing of wounds and surgical incisions in biological tissues. More particularly the invention relates to formulation of biocompatible chromophores to be used for colouring the biological tissues in order to absorb, in a selective and localised manner, the laser radiation in the processes of laser welding of biological tissues. State of the art

Techniques of laser-induced welding of biological tissues have been proposed in order to achieve suturing of various types of tissue such as the cornea [F. Rossi, R. Pini, L. Menabuoni, R. Mencucci, U. Menchini, S. Ambrosini, G. Vannelli, "Experimental study on the healing process following laser welding of the cornea", Journal of Biomedical Optics 10, pp. 1-7 (2005)], the walls of blood vessels [A. Puca, A. Albanese, G. Esposito, G. Maira, G. Rossi, R. Pini, "An experimental study on laser-induced suturing of venous grafts in cerebral revascularization surgery" , in Photonic Therapeutics and Diagnostics, SPIE Vol. 5686, p. 276-281, Bellingham, WA, USA, (2005)] and the skin, nerve tissue, etc. [K. M. McNally-Heintzelman, "Laser Tissue Welding", Chap. 39 in Biomedical Photonics Handbook, pp. 39-1/39-45, T. Vo-Dinh, Ed., CRC Press, Boca Raton (2003) ] . These techniques are based on irradiation of the biological tissues with laser radiation, whose purpose is to induce a thermal effect in the tissue and activate some proteins of the extracellular matrix, such as for example collagen and elastin, thus producing the immediate adhesion of the edges of the wound. Laser irradiation of the tissues to be sutured is often associated with the application of an exogenous chromophore (for example a biocompatible dye) which exhibits an high optical absorption at the wavelength of the laser used. This chromophore is applied locally in the edges of the wound to be sutured and serves as a selective absorber of the laser radiation. With the application of the chromophore, the tissue welding is more controlled and localised, with a considerable reduction in the risk of thermal damage to the tissues treated, since, thanks to the high absorption of the coloured tissue, much lower energy per unit surface is required than that which would be necessary for welding without a chromophore.

Exogenous chromophores are currently used in an aqueous solution which must be prepared at the time of application in the tissue, in that the dye in solution is subject to deterioration in time. For example, in the case of use of the indocyanine green chromophore (hereinafter abbreviated to ICG) , it is known that this dye, dissolved in some solvents and exposed to the light, is subject to rapid photodegradation [see for example M. L. J. Landsman, G. Kwant, G. A. Mook, W. G. Zijlstra, "Light-absorbing properties, stability, and spectral stabilization of indocyanine green", J. Appl . Physiol. 40, pp. 575-583 (1976) ] . The problems of preparation of the solution of the dye at the time of application, in addition to the impracticality of the operations which require conditions of sterility for use on humans, include the fact that this preparation is subject to variations of some of its chemical and physical properties, such as the concentration (which determines the degree of optical absorption of the coloured tissue and hence the development of a local temperature under laser irradiation) or the density and viscosity (on which the ease of application in the wound to be sutured depends) . This can influence the repeatability of the welding technique and its efficacy in producing the tissue suture. Objects of the Invention

The general object of the present invention is to provide biocompatible exogenous chromophore formulations to be used in the laser welding of biological tissues in order to enhance absorption of laser radiation by the tissues coloured with these chromophores and ensure a repeatable, selective and homogeneously distributed effect in the tissues to be treated by the laser irradiation. Achievement of this object is essential for making clinical application of the laser welding technique safe, standardizable and effective in inducing immediate sealing of the wounds, without thermal damage to the surrounding tissue. The biological tissues of interest for these healing techniques are for example the cornea, blood vessel walls, skin and mucous membranes.

A particular object of the present invention is to provide a formulation of a biocompatible dye for the welding of corneal tissue with a diode laser which emits at the wavelength of 810 nm, capable of forming a vehicle for the chromophore characterised by high viscosity and/or density which facilitates its possible insertion in the incision . - A -

A further object of the present invention is to provide a chromophore formulation of the type mentioned above which is capable of developing marked colouring of the walls of the incision, which remains after washing and provides laser radiation absorption such as to induce efficient laser welding of the incision.

Another particular object of the present invention is to provide a chromophore formulation of the type mentioned above which has sufficient chemical stability to allow its preparation in times prior to clinical use and guarantees the accuracy of the concentration of the chromophore for the purposes required by the laser treatment .

It is still a particular object of the present invention is to provide a high concentration chromophore formulation with of the type mentioned above which allows the transfer through contact of the chromophore to the wall of the incision to be sutured.

A further particular object of the present invention is to provide a chromophore formulation of the type mentioned above which, in the case of tissues having a layered structure such as for example the skin, allows the walls of the incision to be coloured differentially in order to compensate the different degree of optical absorption of the laser radiation shown by the various tissue layers and therefore make thermal development along the depth of the incision more even.

Summary of the invention

These objects are achieved with the chromophore formulation for use in laser welding of biological tissues whose feature consists of the fact of being made available in a semi-solid or solid form. According to an aspect of the present invention a semi-solid chromophore solution is obtained from a highly super-saturated chromophore solution which appears in the form of a gel characterised by high viscosity and optical absorption peak at approximately 700 nm; the formulation is applied in a corneal incision and made to remain there for a preset time, it colours the walls of the same incision, before being washed away, in such a way to colour the incision walls by diffusion in the corneal tissue, with a concentration much lower than that of the original super-saturated formulation corresponding to an absorption spectrum exhibiting a new absorption peak around 810 nm, at the wavelength of emission of the diode laser used. According to another aspect of the present invention a semi-solid chromophore formula is composed of the chromophore and other biocompatible materials such as polyvinyl alcohol or sodium hyaluronate, having a rheological behaviour such as to facilitate its insertion in the incision to be welded.

According to a further aspect of the present invention a solid chromophore formula is formed by the chromophore mixed with a biocompatible polymeric material so as to form a film or a matrix incorporating the chromophore at a high concentration and which can be used as an insert in the tissue incision to colour the walls of the incision through contact thanks to the controlled release of the chromophore, also allowing its removal before laser welding. According to another aspect of the present invention a solid chromophore solution of the aforesaid type has the structure of a film or a matrix formed in several superimposed layers with growing chromophore concentration and suitable for being inserted in the incision to be welded so that the deeper layers of the incision are more coloured. Brief description of the drawings

The features and advantages of the preparations of exogenous chromophores to be used for the suturing, healing and sealing of biological tissues, according to the invention, will be made apparent by the following description of embodiments given by way of non-limiting examples with reference to the accompanying drawings, in which :

Figure 1 shows the spectra of optical absorption of a saturated aqueous solution and of super-saturated solutions of the ICG chromophore;

Figure 2 shows the spectra of optical absorption of non-saturated aqueous solutions of the ICG chromophore;

Figure 3 shows the spectrum of optical absorption of corneal tissue after it has been coloured with a formulation of the ICG chromophore according to the invention;

Figure 4 schematically illustrates the phase of lateral irradiation of an incision in the corneal tissue whose faces have been coloured with the chromophore; Figure 5 shows by way of an example the calculated map of the thermal distribution developed during the laser welding of an incision produced in a tissue transparent to radiation of the diode laser at 810 nm, such as the cornea, in conditions of lateral irradiation (indicated by the arrows) , after the interior of the incision has been coloured evenly with ICG;

Figure 6 shows, by way of an example, the map of the thermal distribution which develops in the skin during irradiation with a diode laser, whose beam (indicated by the arrow) comes from above in a parallel direction to the axis of the incision (indicated by the dotted line) , after the interior of the incision has been coloured evenly with ICG; .

Figure 7 shows, by way of an example, the map of the thermal distribution of the skin in identical irradiation conditions to those of Figure 6, yet providing a different technique of colouring of the walls of the incision, obtained with a solid insert with uneven distribution of the dye .

Detailed description of the invention

A semi-solid or solid chromophore formulation according to the present invention can be used depending on the features of the biological tissue to be treated and the methods through which the laser welding is performed.

The chromophore used in the present invention is indocyanine green (ICG), which is the most widely common one for this type of application. However it is clear that any other type of biocompatible dye with equivalent chemical and physical features and properties may be used as an alternative. For example in the present invention the commercially available IC-GREEN pharmaceutical form, produced by Akorn, Buffalo, IL, USA, was used as ICG chromophore .

Equivalent results are obtained using the chromophore ICG-PULSION, PULSION Medical System AG, Monaco (Germany) . As is known to a person skilled in the art, an aqueous solution of ICG has an optical absorption spectrum characterised by two peaks around 700 and 780 nm respectively. The relative intensity of these peaks changes with the concentration of the solution, as shown in Figures 1 and 2, which give respectively examples of absorption spectra of solutions with a different degree of super-saturation and non-saturated solutions of ICG.

More particularly Figure 1 shows the spectra of optical absorption of the saturated solution of the ICG chromophore, corresponding to the lowest curve and with a concentration of 0.5 x 10^~2 % w/w (weight of chromophore/weight of solvent) and of four solutions with different degrees of super-saturation, of which the highest one corresponds to the concentration of 7.0xl0^~2 % w/w. These curves show that the principal maximum of the absorption falls in these cases at around 700 nm. Figure 2 instead shows the spectra of optical absorption of unsaturated solutions of the ICG chromophore for six different values of the chromophore concentration between 16.3 x 10^~4 % w/w of the uppermost curve and 1.1 x 10^~4 % w/w of the lowest curve. These curves show that in the case of unsaturated solutions the principal maximum of the absorption falls at around 780 nm.

For the purposes of the present invention and according to a first embodiment thereof, a highly supersaturated concentration of ICG is used at a concentration between 5 and 10%, preferably around 7.0% w/w, to be used in the welding of corneal tissue, performed with AlGaAs diode laser with emission at 810 nm. Such a high concentration of ICG creates a highly dense gel which is suitable for being easily inserted and coated around a corneal incision by means of a small spatula or a front chamber cannula.

The gel is made to remain inside the incision for a time of 1-5 minutes, more particularly 2-3 minutes, in order to colour the walls. The corneal incision is then washed with plenty of sterile water to remove the excess gel. In this way the tissue is coloured through diffusion of the dyeing solution in the tissue. With the values indicated above of concentration and of the time the solution remains in the incision, typically a diffusion of the dye by approximately 100 microns is obtained in the corneal tissue at the walls of the incision. The spectrum of absorption of the tissue coloured in this way, shown in Figure 3, is similar to that of a non-saturated solution, i.e. with the absorption maximum at the second peak at the greater wavelengths. It should however be noted that the bond with the corneal collagen has the effect of moving this peak from the 780 nm of the non-saturated aqueous solution to the 810 nm, which coincide with the row of emission of the AlGaAs diode laser, indicated by the dotted line in Figure 3, making this device perfectly adequate for the performance of the corneal welding technique.

After having coloured in this way the faces of the incision, they are subjected to laser treatment by the method of lateral irradiation via optical fibre, since the corneal tissue is transparent at the wavelength of the diode laser, as shown schematically in Figure 4, where 1 and 2 denote the optical fibres, 3 the cornea seen in section and 4 the corneal incision to be welded.

Another possible embodiment of a semi-solid chromophore formulation according to the present invention is prepared by dissolving in water biocompatible polymers such as polyvinyl alcohol, hyaluronic acid or its salts, semi-synthetic derivatives of cellulose, polymers of a natural origin (for example chitosans, xanthan gum, tamarind gum, alginic acid, pectins) in concentrations which vary from 0.5 to 15% w/w, and then adding ICG in a concentration which can be between 0.5 and 10.0% w/w and any other substances which stabilise the formulation (for example antioxidants, antibacterial and isotonizing agents and pH modulators) . For example the commercial product Erkol® W48/20, Celanese Chemicals, TX, U.S.A., can be used as polyvinyl alcohol. In addition to properties of density which facilitate its insertion in the wound to be sutured, the semi-solid formulation of ICG prepared in this way are able to provide greater stability in respect of chemical deterioration compared to the aqueous solution currently used. For example the residual ICG percentage two months after preparation can be 40% for the semi-solid formula with 12% w/w polyvinyl alcohol, against the 4% which can be found in the aqueous solution.

In another embodiment of the invention a solid formulation of the chromophore is provided. A preferred embodiment consists in the preparation of a transparent and flexible film with thickness of 30-60 micrometres incorporating the chromophore. This film can be prepared through slow evaporation at 37 ⁰C, away from the light in a Petri dish, of an aqueous solution of a polymer, for example tamarind gum (for example the commercial product TSP®, Farmigea, Italy) at a concentration between 1.0 and 4.0% w/w and of ICG chromophore at a concentration between 0.01-0.3% w/w. Possible other stabilising substances can be added, such as antioxidants, antibacterial agents and plasticizers (for example glycerol in a concentration between 0.01-0.025% w/w) . This solid formulation exhibits a very high stability of the chromophore, thanks to which a concentration of 80-90% of ICG two months after the film preparation is still ensured.

In the use, a strip of the film corresponding to the internal surface of the face of the incision to be welded can be cut from the film and then easily inserted in the incision. The film is kept there for 1-3 minutes so as to colour the faces of the incision through contact and can easily be removed without losing density.

A variation of the solid formulation described above consists of a multilayer film in which each layer is characterised by a different content of ICG. In the preferred embodiment the content of ICG rises from an external layer to the opposite external layer. A multilayer film of this type can be obtained by evaporating successive layers of polymeric solution containing ICG at different concentrations. Uneven colouring of the film can be advantageously used to obtain differential colouring of the incision, for example in the skin, where the various layers are characterised by different optical absorption at the wavelength of the laser radiation used for the welding.

Figures 5, 6 and 7 schematically show some simulations of the thermal development induced by laser radiation in various types of tissue, in which incisions were made perpendicular to the surface, to be welded with a laser technique. The cases refer in particular to tissues such as cornea and skin, and to different methods of colouring and of laser irradiation, which describe real application cases of use of the formulation according to the invention.

If in fact the tissue is transparent at the wavelength of the laser, as in the case of the cornea with respect to the 810 nm diode laser, it is possible to irradiate the tissue area by aiming the light radiation through the tissue itself. It is therefore possible to illuminate evenly both walls of the incision, for example according to the method of lateral irradiation shown in Figure 4, if the position and geometry of the incision allow it. This enables an homogeneous heating to be obtained along the entire depth of the incision, as shown in Figure 5, in which it can be seen that the heating is homogenously and symmetrically distributed along the axis of the incision, indicated by the dotted line.

On the other hand, if the tissue is not perfectly transparent and therefore dampens the laser radiation according to the depth of penetration of the latter, or in any case has differential absorption in its component layers, such as for example in the skin, wherein the epidermis absorbs more than the dermis at 810 nm, the only possible method of irradiation of the wound is from above, i.e. at the interface between the free surface of the wound and the external environment. Figure 6 schematically illustrates this method of irradiation. The laser light beam coming from above is indicated by an arrow and is aimed orthogonally at the incision in the direction indicated by the dotted line. In this case, in general, a surface heating effect occurs which does not reach the depth of the incision, making welding inefficient or exposing the tissue to the risk of damage from laser over- treatment. The condition is in fact typical in which thermal damage to the surface of the tissue has necessarily to be produced, in an attempt to obtain, in the depth of the incision, a sufficient heating for producing tissue welding. In the cases of partially transparent tissues, according to the present invention, it is instead possible to achieve even heating of the tissue if a chromophore with variable optical absorption is used. This can be obtained using the multilayer film with ICG concentration gradient according to the embodiment described above, thanks to which it is possible to colour the incision so as to increase optical absorption with depth. Figure 7 exemplifies this condition in the skin, wherein the production of colouring which increases proportionally with the depth of the incision has been considered. Consequently the effect of heating in the incision is much more even compared to the case of Figure 6.

Variations and modifications to the present invention can be appreciated by a review of the description given above. These changes and additions which may be made can be considered as coming within the scope and spirit of the invention as specified in the following claims .

Claims

1. Chromophore formulations for use in laser welding of biological tissues, characterised in that they are in a semi-solid or solid form.

2. Chromophore formulations according to claim 1, wherein said semi-solid form consists of a highly dense gel formed by a super-saturated aqueous solution of chromophore at a concentration between 5 and 10% w/w.

3. Chromophore formulations according to claim 2, wherein said concentration is 7% w/w.

4. Chromophore formulations according to claim 1, wherein said semi-solid form consists of a highly dense gel formed by an aqueous solution of a biocompatible polymer at a concentration between 0.5 and 15% w/w and chromophore at a concentration between 0.5 and 15% w/w.

5. Chromophore formulations according to claim 1, wherein said solid form consists of a film or matrix incorporating the chromophore obtained through slow evaporation from an aqueous solution of a biocompatible polymer at a concentration between 1 and 4% w/w and chromophore at a concentration between 0.01 and 0.025% w/w.

6. Chromophore formulations according to claim 5, wherein said film or matrix is of the multilayer type with a chromophore concentration that can vary evenly from one layer to the next one.

7. Chromophore formulations according to claims 4, 5 or 6, wherein said biocompatible polymer is chosen from polyvinyl alcohol, hyaluronic acid and its salts, semisynthetic derivatives of cellulose and polymers of a natural origin such as chitosans, xanthan gum, tamarind gum, alginic acid and pectins.

8. Chromophore formulations according to claim 4, wherein said biocompatible polymer is polyvinyl alcohol.

9. Chromophore formulations according to claim 5, wherein biocompatible polymer is tamarind gum.

10. Chromophore formulations according to any one of the previous claims, wherein indocyanine green (ICG) is used as chromophore.

11. Method for producing a gel containing a biocompatible chromophore for use in the laser welding of biological tissues, characterised in that it comprises the steps of: - dissolving in water an amount of said chromophore such as to reach a concentration between 5 and 10 % w/w., preferably approximately 7.0 % w/w.

12. Method for producing a gel containing a biocompatible chromophore for use in the laser welding of biological tissues, characterised in that it comprises the steps of:

- dissolving in water a biocompatible polymer to a concentration between 0.5 and 15% w/w,

- adding said chromophore to the polymer solution to a concentration between 0.5 and 10.0% w/w.

13. Method for producing a transparent and flexible solid support containing a biocompatible chromophore for use in the laser welding of biological tissues, characterised in that it comprises the steps of:

- preparing an aqueous solution of a biocompatible polymer at a concentration between 1.0 and 4.0 % w/w,

- adding said chromophore to the polymer solution at a concentration between 0.01 and 0.3 % w/w,

- slowly evaporating the solvent away from the light on a flat support so as to form a transparent film with thickness between 30 and 60 micrometres.

14. Method according to claim 13, wherein polymer solutions are prepared at different chromophore concentrations that are evaporated successively one on the other to form a multilayer film, each layer being characterised by a chromophore concentration which increases or decreases with continuity in relation to the underlying layer.

15. Method according to any one of claims 11 to 14, wherein said biocompatible chromophore is ICG.

16. Method according to any one of claims 11 to 15, wherein said biocompatible polymer is chosen from polyvinyl alcohol, hyaluronic acid and its salts, semisynthetic derivatives of cellulose and polymers of a natural origin such as chitosans, xanthan gum, tamarind gum, alginic acid and pectins.