WO2016135364A1

WO2016135364A1 - Silk fibre with lateral light emission

Info

Publication number: WO2016135364A1
Application number: PCT/ES2016/070120
Authority: WO
Inventors: Aurelio Arenas Dalla Vecchia; Marta Rojo Martinez; Juan MUÑOZ MADRID; José Luis Cenis Anadon; Luis Meseguer Olmo; Salvador David Aznar Cervantes; Antonio Abel Lozano Perez
Original assignee: Universidad De Murcia
Priority date: 2015-02-27
Filing date: 2016-02-24
Publication date: 2016-09-01
Also published as: ES2534528A1; ES2534528B1

Abstract

The invention relates to a biocompatible and biodegradable device for emitting light, which acts on animal cells and tissues with biological and therapeutic effects. The fundamental element of the device consists in a silk fibroin fibre with a diameter of between 0.1 and 1.0 mm and a variable length, obtained from the silk gland of the silk worm. This fibre is traditionally known as silk filament and was historically used as suture and fishing lines. When the silk filament is illuminated on one end with conventional or laser light, it emits light laterally along various centimetres. This fibre can be implanted in human tissue without needing to to be removed, as a result of the biocompatible and biodegradable character thereof.

Description

DESCRIPTION

Silk fiber of lateral emission of light. Object of the invention

Electromagnetic radiation in the visible range has widely demonstrated biological effects. Through the application of light to the cells it is possible to stimulate its development, inhibit it, modify it or induce its apoptosis. The application of the light is preferably carried out through conventional silicon optical fibers. However, in a biological context, it would be preferable to use fibers based on organic polymers that have better biocompatibility and degradability qualities. It is also desirable that the fiber emits light laterally in those cases where the cell target has a longitudinal configuration.

In the present invention, the use of a fiber of organic origin is proposed, such as the silkworm granddaughter as a vector for the application of light in organic cells and tissues. This fiber has an exclusively protein composition, and is composed of a protein, the fibroin that constitutes the silkworm of the Bombyx morí silkworm. This fiber is highly biocompatible and degradable. Therefore, it can be implanted in the target tissue and in the chosen position, so that being illuminated at one end, it emits light that acts on the surrounding tissues. The grandmother is a fiber obtained by acidification and mechanical stretching of the sericigenic gland of the Bombyx mori worm. During the period between 1880 and 1950, it was widely used as a suture in surgery and fishing line, being manufactured exclusively in the Region of Murcia [MARDEN, L. Spain's Silkworm Gut. National Geographic Magazine July 1951, pages 100-108] and [HUMPHRIES, A. M. C. The Story of Silk and Silkworm Gut. Postgraduate Medical Journal. 1949, Vol. 25 (288), pages 483-488].

Technical sector The present invention is part of the field of new instruments, devices, tools or methods of biology and medicine.

State of the art

The application of light radiation has a wide range of effects on living cells and tissues. Some of these effects are positive for their proliferation and biological functioning, which has led to the development of therapies based on the application of light in the field of biomedicine. The set of therapies and applications derived from the exposure of light to cells and tissues is divided into three main sections: stimulation of cell proliferation using low intensity laser light (Low Level Laser Therapy, LLLT), activation or inhibition of neurons that they express opsins in their membrane (Optogenetics) and activation of photoactivatable molecules that emit reactive oxygen species inducing apoptosis in tumor cells (Photodynamic Therapy).

LLLT irradiation encompasses the use of laser lighting in the red and infrared light range, with a wavelength between 600 and 1,100 nm and an output power between 1 and 500 mW. This type of radiation can be continuous or pulsating, with a relatively low energy density (0.04 to 50 J / cm ² ). The light is directed to the target tissue, or to cell monolayers, with powers in the order of milliwatts. At low doses of energy density (2 J / cm ² ), LLLT stimulates cell proliferation, while at high doses (16 J / cm ² ) it acts as a suppressor. The LLLT transmits energy at low levels and does not emit heat, sound or vibrations. Irradiated tissues do not experience a significant increase in temperature, unlike what happens through the use of power lasers that can cut and vaporize them.

LLLT can stimulate a variety of biological processes, which include cell growth, proliferation and differentiation. These effects are verified on a wide variety of cell types, including fibroblasts, endothelial cells, mesenchymal cells, keratinocytes, myoblasts, etc. However, the cellular mechanism through which the LLLT acts is not completely clarified. The generally admitted mechanism indicates that the energy of the laser radiation is absorbed by intracellular chromophores and converted to metabolic energy, since there is always a significant increase in the levels of ATP (Adenosine Triphosphate) in the irradiated cells. In turn, the increase in ATP increases the synthesis of proteins, DNA and the concentration of intracellular calcium. The joint effect is the activation of a series of cell cascades, acting in the direction of increased cell proliferation. This cellular stimulation translates, at the tissue level, into various effects such as the stimulation of scarring, collagen synthesis, peripheral nerve regeneration, bone tissue remodeling and repair, normalization of hormonal function, pain attenuation, stimulation of the release of endorphins and modulation of the immune system.

Optogenetics is a technology that is based on the use of membrane proteins of microbial origin, generically referred to as opsins. These proteins directly convert light into changes in the electrical potential on the sides of the cell membranes in which they are inserted. Microbial opsins respond to light by translocating ions through the membranes of the cells in which they are genetically expressed. In the case of neurons, this makes it possible to act externally on the activation or inactivation of action potentials in them, which allows the analysis and control of their function. Opsins must be previously integrated into the membrane of the cells on which they want to act, and this is done by genetic transformation of the cells with the genes that express opsins. There are many types of opsins and other photoactivatable proteins, but the most frequently used are the canalrodopsin-2 (ChR2) of the green algae C. reinhardtii that translocates cations within the neurons when activated with blue light, activating the electrical activity of the cell. In contrast, halorodopsin and archaeorodopsin pump chloride ions inside and cations outside the cells, respectively, under green or yellow light. This causes an inactivation of the electrical activity of the cell. The possibility of acting on the activation or inactivation of neurons by means of a pulse of laser light opens the possibility of repairing dysfunctions caused by degenerative processes in brain tissue.

Photodynamic Therapy is an antitumor therapy that is based on the localized or systemic administration of a non-cytotoxic photosensitizer compound. Upon exposure to a light source, the compound emits reactive oxygen species that induce apoptosis in tumor cells in contact with it. Most photosensitizing compounds have a heterocyclic ring structure similar to that of chlorophyll II or the heme group of hemoglobin. After the capture of photons by the compound, the light energy produces a chemical reaction in the presence of molecular oxygen that produces a singlet of oxygen or superoxide that induces cellular damage. Photosensitizer compounds can be divided into three categories in general: those based on porphyrin, those based on chlorophyll II and dyes. There is a wide variety of these compounds, which have proven effective and are authorized for clinical use. As for the light source necessary to activate the photosensitizer compounds, a variety of types have been used. It is possible to use conventional non-coherent light sources, or LEDs. However, the most common light source in Photodynamic Therapy is laser light. This light is monochromatic and coherent, with a specific wavelength, optimized for each photosensitizer compound.

The clinical applications of Photodynamic Therapy are very numerous. They can be found in the field of therapy of dermatological diseases, ophthalmic diseases, head and neck cancer, brain and lung tumors, cardiovascular disease and urological diseases.

For the performance of the described techniques it is essential to have a light source and transmit it to the target with great precision. Conventional and LED light bulbs have been used as the light source. However, laser light of specific wavelength and higher power is usually preferred. There are numerous types of laser light sources for the indicated applications. As for the conduction of light, lighting is used in some cases from outside the area to be treated. This is feasible for skin treatments or relatively superficial organs. For deeper targets, optical waveguides are preferred, and optical fibers endowed with lenses at their end that produce adequate focusing, and therefore, greater precision, of the ray of light applied.

Optical waveguides are physical structures that guide electromagnetic waves in the optical spectrum. The most common types include optical fibers and rectangular guides. The optical waveguides may be printed on implantable microfluidic devices, but more commonly, the laser light is delivered to the target by means of a silicon optical fiber. This type of fiber is efficient and easy to handle. However, for applications in living tissues and organs, it is preferable to have waveguides and optical fibers that are biocompatible, to avoid foreign body reactions in highly sensitive organs. It is also convenient that these optical devices are biodegradable, to prevent their removal once the treatment has been performed. Due to these requirements, alternative materials to standard silicon fiber have been actively sought, and optical waveguides based on biopolymers have been developed. This development of guides made of polymeric materials has been included in various patents, in the USA. and Europe, among which the following stand out:

• Patent 1 - US 8195021 B2 Biopolymer optical waveguide and method of manufacturing the same.

· Patent 2- US 2010/0065784 A1 Electroactive biopolymer optical and electro-optical devices and method of manufacturing the same.

• Patent 3- EP 2612751 A2 Method of manufacturing a biopolymer optical waveguide. The polymeric materials that are proposed are polymers of organic origin, such as silk fibroin, chitosan, collagen, gelatin, agarose, starch, chitin, cellulose and combinations thereof. Of all the biopolymers indicated in the aforementioned patents, silk fibroin is the most efficient in terms of optical properties and mechanical strength. Silk is a protein secretion of many arthropod species, with very diverse functions. The best known, due to its use as a textile fiber for centuries, is the silk produced by the Bombyx Mori Lepidoptera, known as the silkworm. This silk is composed of two proteins: fibroin, of a fibrous nature that forms a continuous fiber, and sericin, a globular and adhesive protein that surrounds fibroin and allows a three-dimensional structure such as the cocoon to pupate. In turn, fibroin is a protein that combines peptide domains in beta sheet, composed of repetitions of Gly-Ala-Gly-Ala-Gly-Ala, which give it great mechanical resistance, with domains in alpha helix that give it flexibility. Fibroin has been revealed in the last decade as a high-performance biomaterial for the manufacture of stem cell growth frameworks in Tissue Engineering applications. This is because of the great biocompatibility of fibroin, which does not generate inflammation or foreign body reaction, once inserted into animal tissues. It is biodegradable, with a degradation rate that can be modulated, and has a remarkable mechanical resistance. The use of fibroin as a biomaterial requires its prior solubilization to form an aqueous solution from which various configurations such as films, gels, porous three-dimensional sponges, electro-spun meshes, etc. are manufactured. In these structures, stem cells are sown and subsequently differentiated to form diverse tissues. Silk fibroin has optical properties, such as high transparency (98%), in addition to being very suitable for the manufacture of optical devices intended for use in a biological environment. As an example, the fibroin films used to manufacture supports for epithelial and corneal stromal growth can be cited. In the use of silk fibroin as an optical waveguide, described and protected by the aforementioned patents, it is based on a liquid solution of previously solubilized fibroin that is extruded through a nozzle in a polymerizing bath, typically methanol. The polymerized aqueous fibroin filament lacks mechanical strength. Because of this, it is typically applied on a surface that supports it. Subsequently, it is coated with another polymer of a suitable index of refraction, which allows the confinement of the light inside the fiber to the distal end thereof. This way of manufacturing the fiber allows it to be incorporated into printed microfluidic devices, but does not make it possible to use it as an independent, resistant and implantable fiber. Therefore, it is necessary to look for other configurations of a silk fiber that is resistant, implantable, and capable of transmitting light. The grandmother is a fibroin fiber formed from the two sericigen glands of the silkworm. To manufacture it , proceed to the extraction live glands from the larva of 5 ^or state, acidification by immersion in a bath of acetic acid and manual stretching. This stretching produces a phase change from the liquid fibroin in the micellar state inside the glands to a solid and resistant fiber where the fibroin is in a molecular configuration of beta sheet. The resulting fiber has a thickness between 100 and 1000 microns and a length between 30 and 40 centimeters. This fiber was widely used from the mid-nineteenth century as a suture in surgery and as a fishing line. This industry disappeared completely towards the 1950s, due to the appearance of nylon and other artificial fibers that perform the same function at a lower cost. The grandmother was produced exclusively in the Region of Murcia, exporting almost all the production to England. Description of the invention

The present invention relates to a device designed to apply light from a laser light source to surface or deep tissues of living beings' organs. Unlike the systems used so far with artificial light guides (optical glass or plastic fibers), in this invention the light application element consists of a stretch of fiber obtained from the grandmother, extracted from the processing of the sericigen gland of a silkworm and which is used as a light guide. The little daughter, who is constituted essentially by fibroin, it has a high transparency and has the quality of emitting light through its lateral surface along it, the light that is applied at one of its ends. This achieves a more extensive area of application of light radiation than that obtained with an artificial optical fiber emitting light only at its end. In addition, this light guide has the advantage that it is constituted by a biocompatible protein, which allows its reabsorption by the tissues in which it is implanted. In addition, the grandmother has a high mechanical tensile strength (up to 5 kilograms of tensile strength), with values much higher than those of the fibers obtained by the extrusion procedures described in the patents mentioned above. This constitutes a considerable advantage over existing fibroin optical guidance devices, since the strength of the fiber allows the use of a support to not be required. This allows absolute flexibility in relation to the implantation of the fiber for the performance of its function, as well as in regard to the connection to the light source. This property of the grandmother empowers it as an ideal element to make superficial or deep insertions in living tissues and apply light to them, through it. In addition, the surface of the daughter presents the chemical reactivity of fibroin, characterized by the presence on its surface of amino acids with amino and carboxyl reactive groups. By chemical activation of the same, it is possible to bind to the grandmother molecules of various types that can increase their functionality, for example, enzymatic activities or drugs that are activated by the action of light. It is also possible to coat the daughter with conductive polymers (polypyrrole, PAÑI, etc.), or conductive carbon compounds (graphene, carbon nanotubes, etc.), or metals such as silver or copper, which give it conductivity, allowing the fiber constitute an optical and electrical guide (optoelectrode).

Description of the content of the figures

FIG 1. Drawing showing the system formed by the light source, the artificial guide and the grandmother connected by the assembly tube. FIG 2. Photograph showing the cellular proliferation of L929 mouse fibroblasts around the grandmother when red light is applied through it in a culture.

Reference List

one . - Light source.

2. - Artificial light guide.

3. - Little daughter.

4. - Hollow assembly tube

Description of a preferred embodiment of the invention

The construction of the complete system of fiber of lateral emission of light begins with the manufacture of the grandmother. For this, the larvae are anesthetized by keeping them at 4 ° C. The head of the worm is sectioned with a blade, the two seric glands are removed, washed in water and placed in a 2% acetic acid solution bath for two minutes. Then the glands are manually stretched by holding them at their ends, until they reach a length between 40-50 cm. In this way, a translucent fiber of approximately 0.5 mm in diameter is achieved, covered by cell debris and sericin that is removed by washing with water. Finally, the clean fiber is dried and cut to the required length.

Subsequently, one of the ends of the grandmother is sectioned with a 90 ° blade and the sectioned surface is mechanically polished, for example, with very fine sandpaper.

The same cutting and polishing operation of a section of an artificial light guide (fiber optic glass or plastic fiber) having the same diameter as the obtained grandmother and whose length is suitable for handling the system is also performed.

A section of about 2 cm of hollow assembly tube 4 (extracted from a hypodermic needle, for example) of internal diameter equal to that of the grandmother 3 is cut and to the artificial guide 2 and the polished ends, one of the granddaughter and one of the artificial light guide, are introduced therein until the surfaces thereof make contact therein (see FIG 1). Next, the other end of the artificial guide is connected to a light source 1. This source is preferably laser light whose color or wavelength is appropriate in the specific application to be addressed. The other end of the is the one that will be used to apply the light in the area of the selected fabric.

The dosage of times and intensities in the application of the light by means of this system will depend on the results that one wishes to obtain, in addition to being necessary to take into account the results of previous tests carried out for each case of tissue and situation.

Examples:

Example 1. Stimulatory effect of laser light on cell proliferation.

The grandmother is inserted into a tissue to stimulate the growth of native cells [ALGHAMDI, K. M., KUMAR, A., & MOUSSA, N. A. Low-level laser therapy: a useful technique for enhancing the proliferation of various cultured cells. Lasers in Medical Science. 2012, Vol. 27 (1), pages 237-249], [BASSO, FG, PAN SAN I, TN, TURRIONI, APS, BAGNATO, VS, HEBLING, J., & DE SOUZA COSTA, CA In vitro wound healing improvement by low-level laser therapy application in cultured gingival fibroblasts. International Journal of Dentistry. 2012], or cells added in that tissue, once we illuminate the daughter by its end. This is applicable to the manufacture of a suture that, illuminated at its end, promotes a more active proliferation of fibroblasts, improving healing. It can also be placed linearly along a nerve to stimulate neuronal growth and accelerate its repair.

You can also make a flat braid of sequins that form a surface or structure (scaffold) on which to sow cells. After lighting At one end of the fibers that constitute the braid, the structure will emit light that will stimulate the growth of the cells seeded therein.

Example 2. Use of laser light in Photodynamic Therapy.

There are photoactivatable molecules (coumarins, feoforbide, etc.) that when illuminated with laser light emit singles of oxygen that are highly oxidizing. This oxidation induces cell apoptosis and when these cells are part of a tumor, the cells are removed, leading to antitumor therapy [HENDERSON, BW, WALDOW, SM, MANG, TS, POTTER, WR, MALONE, PB, & DOUGHERTY, TJ Tumor destruction and kinetics of tumor cell death in two experimental mouse tumors following photodynamic therapy. Cancer Research 1985, Vol. 45 (2), pages 572-576], [HOPPER, C. Photodynamic therapy: a clinical reality in the treatment of cancer. The Lancet Oncology. 2000. Vol. 1 (4), pages 212-219]. The photoactivatable molecules are not toxic in the absence of light, therefore, they can be administered locally or systemically so that they have access to the vicinity of the tumor cells. Therefore, if the little daughter is inserted into a tumor, following the administration of a photoactivatable molecule, the light provided by it produces activation of the molecule that in turn will trigger the apoptosis of the tumor cells. This application is especially suitable for acting on tumors located in organic tubular structures, given the linear distribution of the lumen of the grandmother.

On the other hand, a grandmother with photoactivatable molecules can also be functionalized and implanted in a tumor. This done, when applying light to the daughter, the cells in contact with it go into apoptosis and disappear.

Example 3. Use of laser light in photochemical suture. The same phenomenon described above works by activating a photoactivatable molecule in contact with collagen with light. The oxidative species produced induce crosslinking in the collagen fibers, strongly joining two separate pieces of it. This process is known as Photochemical Tissue Bonding, [CHAN, BP, KOCHEVAR, IE, & REDMOND, RW Enhancement of porcine skin graft adherence using a light-activated process. Journal of Surgical Research. 2002, Vol. 108 (1), pages 77-84], [JOHNSON, TS, O'NEILL, A. C, MOTARJEM, PM, AMANN, C, NGUYEN, T., RANDOLPH, MA, & REDMOND, RW Photochemical tissue bonding: a promising technique for peripheral nerve repair. Journal of Surgical Research. 2007, Vol. 143 (2), pages 224-229].

Therefore, using as a suture a functionalized daughter with a photoactivable molecule (rose flare with green light, for example), once illuminated produces a cross-linking between the collagen of the edges of the wound, accelerating healing.

Example 4: Use of laser light in Optogenetics

Optogenetics is a technology that is based on inserting in the brain neurons that have been transformed with membrane proteins (opsins) to respond to blue laser light by opening the ion channels. Therefore, a pulse of laser light allows to activate or deactivate these neurons, inducing or stopping an action potential, [CARDIN, JA, CARLÉN, M., MELETIS, K., KNOBLICH, U., ZHANG, F., DEISSEROTH, K., & MOORE, CI Targeted optogenetic stimulation and recording of neurons in vivo using cell-type-specific expression of Channelrhodopsin-2. Nature Protocols 2010, Vol. 5 (2), pages 247-254], [ZHANG, F., GRADINARU, V., ADAMANTIDIS, AR, DURAND, R., AIRAN, RD, DE LECEA, L, & DEISSEROTH, K. Optogenetic interrogation of neural circuits: technology for probing mammalian brain structures. Nature Protocols 2010, Vol. 5 (3), pages 439-456]. Therefore, if the biocompatible daughter is implanted in brain tissue and illuminated with that laser light, specific neurons are activated. If you are interested in focusing the light on a point, use the daughter-daughter coated with a biocompatible polymer with an adequate index of refraction so that the light is emitted only at the end. If you want to stimulate a wide area of neural tissue, with a linear distribution, the grandmother is used in its native configuration. It has also been possible to coat the grandmother with a thin layer of conductive polymer, graphene or an electrically conductive metal that allows the transmission of a useful electrical signal, either to electrically excite tissue areas, or to capture and record bioelectric signals produced by cells during a neurophysiological intervention in the area of neural tissue to study.

Example 5. Drug release Within the broad field of controlled and targeted drug release there is a strategy consisting of encapsulating drugs in liposomes in whose wall a photoactivable compound is integrated. Under a pulse of light, this component activates, breaks the liposome walls and releases its therapeutic load [ÁLVAREZ-LORENZO, C, BROMBERG, L, & CONCHEIRO, A. Light-sensitive Intelligent Drug Delivery Systems. Photochemistry and Photobiology. 2009, Vol. 85 (4), pages 848-860], [YANG, X., LIU, X., LIU, Z., PU, F., REN, J., & QU, X. Near-lnfrared Light -Triggered, Targeted Drug Delivery to Cancer Cells by Aptamer Gated Nanovehicles. Advanced Materials 2012, Vol. 24 (21), pages 2890-2895]. Using the granddaughter in conjunction with this type of liposomes, fixed on its surface, when emitting light by the grandmother, the therapeutic load of the liposomes fixed to its surface is released.

Claims

one . Lateral light emission fiber comprising: - a stretch of silk fiber (3) produced from the chemical and mechanical processing of the silkworm sericigen gland,

- a section of artificial fiber optics (2),

- a coupling of the previous fibers at one end of each of them,

- a light source connected to the free end of the artificial fiber (1).

2. Lateral light emission fiber, according to the preceding claim, characterized in that the silk fiber is obtained by longitudinal traction of the sericigenic gland of the previously acidified worm.

3. Lateral light emission fiber according to previous claims, characterized in that the cross section of the silkworm fiber has a diameter between 0.1 mm and 1.0 mm.

4. Lateral light emission fiber, according to previous claims, characterized in that the silk fiber is extracted from a silkworm of any of the races of the Bombyx Mori Lepidoptera insect.

5. Lateral light emission fiber, according to previous claims, characterized in that the artificial fiber is made of glass or plastic.

6. Lateral light emission fiber characterized in that the two fibers are connected at one of their ends by inserting them into a hollow tube with an inside diameter equal to the diameter of the fiber section.

7. Lateral light emission fiber, according to previous claims, characterized in that the silk fiber is covered, partially or in its entire length, with conductive polymers (polypyrrole, PAÑI, etc.), or compounds

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REPLACEMENT SHEET (RULE 26) conductive carbon (graphene, carbon nanotubes, etc.), or metals such as silver or copper.

8. Use of the lateral light-emitting fiber described according to claims 1-6, to stimulate the growth of native cells or added cells in a tissue.

9. Use of the fiber of lateral emission of light, according to claims 1-6, for suturing a wound in which fibroblast proliferation is promoted.

10. Use of the lateral light emission fiber, according to claims 1-6, to braid a fiber mesh on which to sow cells and favor their growth.

eleven . Use of the lateral light emission fiber according to claims 1-6, to eliminate tumor cells by induction of cellular apoptosis.

12. Use of the lateral light emission fiber, according to claims 1-6, to eliminate tumor cells by combining the illumination of the grandmother with the administration of a photoactivatable molecule.

13. Use of the lateral light emission fiber, according to claims 1-6, to produce functionalized grandmother sutures with photoactivatable molecules.

14. Use of the fiber of lateral emission of light, according to claims 1-6, to activate specific neurons by induction or suppression of the action potential.

15. Use of the fiber of lateral emission of light, according to claims 1-6, to activate the release of analgesics and other drugs by functionalization of flakes with loaded and photoactivatable liposomes.

fifteen

REPLACEMENT SHEET (RULE 26)

16. Use of the lateral light emission fiber, according to claims 1-7, to transmit electrical signals, during a neurophysiological intervention, with the intervened biological zone.

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REPLACEMENT SHEET (RULE 26)