COMPOSITION FOR LASER TISSUE SOLDERING

Abstract

A composition including a temperature sensitive biocompatible solder and at least one type of nanoparticles, characterized in that a first type of nanoparticles is a fluorescent nanothermometer, wherein the fluorescent nanothermometer exhibits an excitation maximum and temperature dependent emission spectrum each in the range of between 650 and 1350 nm. The composition can be used for laser tissue soldering.

Claims

1. Composition comprising a temperature sensitive biocompatible solder and at least one type of nanoparticles, wherein a first type of nanoparticles is a fluorescent nanothermometer, wherein the fluorescent nanothermometer exhibits an excitation maximum and a temperature dependent emission spectrum each in the range of between 650 and 1350 nm.

2. Composition according to claim 1, wherein the composition additionally comprises a photothermal agent.

3. Composition according to claim 1, wherein the fluorescent nanothermometers are selected from the group consisting of quantum dots, nanodiamonds, gold nanoclusters, hosted rare earth ions, upconverting nanoparticles, transition metal ions, luminescent organic dyes, luminescent organic polymers and luminescent gels.

4. Composition according to claim 2, wherein the plasmonic nanoparticles are selected from the group consisting of plasmonic metal nanoparticles, plasmonic carbon nanotubes, plasmonic organic nanoparticles, plasmonic conductive oxides and plasmonic conductive transition metal nitrides, hybrids, silica coated nanoparticles or mixtures thereof, wherein the metal is selected from the group consisting of Au, Al, Ag, Cu, Pt and Pd.

5. Composition according to claim 2, wherein the photothermal dyes are near-infrared absorbing dyes.

6. Composition according to claim 1, wherein comprising neodymium-doped BiVO.sub.4 as fluorescent nanothermometers and TiN and/or gold nanorods as plasmonic nanoparticles.

7. Composition according to claim 1, wherein the excitation maximum of the fluorescent nanothermometer differs from the absorption maximum of the plasmonic nanoparticle by at least 200 nm.

8. Composition according to claim 1, wherein the temperature sensitive solder solder is selected from the group consisting of serum albumin, chitosan, collagen, fibrinogen and gelatin or a mixture thereof.

9. Composition according to claim 8, wherein the temperature sensitive solder additionally comprises a biodegradable polymer selected from the group consisting of poly(L-lactic acid) (PLA), poly(glycolic acid), poly(L-lactic-co-glycolic acid) (PGA), poly([epsilon]-caprolactone) (PLGA) and polyortho esters, polyanhydrides or combinations thereof.

10. Composition according to claim 2, wherein the fluorescent nanothermometers and the plasmonic nanoparticles are present in a ratio of 1:1 to 5000:1 % by weight.

11. Composition according to claim 1, wherein the composition additionally comprises a therapeutic agent selected from the group consisting of antimicrobial agents, anti-inflammatory agents and angiogenic agents.

12. Composition according to claim 1, wherein the composition is formulated as paste, gel, strip, patch or viscous fluid having a viscosity of at least 2000 centipoises at 25 C.

13. Composition according to claim 1, wherein the composition is in lyophilised form.

14. Composition according to claim 1 for use in the treatment of biological tissue repair, wherein the tissue is selected from the group consisting of cornea, blood vessels, fascia, diaphragm, anastomosis of vessels and tissues, dura mater leaks, gastrointestinal tissues and nerves.

15. Composition according to claim 1 for use in the treatment of deep tissue wounds.

Description

FIGURES

[0056] FIGS. 1A and 1B show the temperature increase in the composition with different compositions (all containing BSA and gelatin as temperature sensitive solder) under laser light irradiation at 750 nm.

[0057] FIG. 2 shows a scheme of the laser tissue soldering setup.

[0058] FIG. 3 shows a calibration curve that relates temperature and spectra at different temperatures (through the Fluorescence Intensity Ratio FIR).

[0059] FIG. 4 shows a temperature during laser tissue soldering measured using a thermal camera (Reference temperature) and the fluorescent nanothermometers (Computed temperature) (using Nd-doped BiVO.sub.4 at 2.5 mg/ml and TiN at 0.02 mg/ml; BSA 0.250 g/ml and gelatin 0.065 g/ml solution dissolved in PBS (phosphate-buffered saline)).

[0060] FIG. 5 shows a typical stress-strain graph of a laser soldered tissue (BSA 60%, TiN at 1%.).

[0061] FIG. 6 shows histological images with and without the composition on top.

[0062] FIG. 7A shows the stability of the composition in digestive fluids.

[0063] FIG. 7B shows that the composition is able to avoid bacteria leakage from intestinal defects.

[0064] FIG. 7C shows that the composition increases the burst pressure if applied on sutures.

[0065] FIG. 7D shows the bond strength of the composition.

[0066] FIG. 8A shows lasering with a two-laser setup.

[0067] FIG. 8B shows the performance of the composition on a surface-near wound and on a deep tissue wound.

EXAMPLES

Preparation of the Composition

[0068] First, the fluorescent nanothermometers need to be prepared. Luminescent Nd-doped BiVO.sub.4 particles were prepared by flame spray pyrolysis (FSP). Bismuth (III) nitrate pentahydrate (Bi(NO.sub.3).sub.3*5H.sub.2O) and stoichiometric amounts of vanadium (ammonium metavanadate, NH.sub.4VO.sub.3) were dissolved separately in a 2:1 volumetric mixture of 2-ethylhexanoic acid and acetic anhydride under magnetic stirring for two hours at 100 C. The Nd.sup.3+ is added by dissolving neodymium nitrate hexahydrate (Nd(NO.sub.3)3*6H.sub.2O). The Nd.sup.3+ concentration was defined as atomic fraction (at %) of the total metal ion concentration resulting in the formula Bi.sub.0.99Nd.sub.0.01VO.sub.4, which has been shown to lead to the highest luminescence intensity. The total metal concentration was kept constant at 0.4 M. All chemicals were supplied by Sigma-Aldrich. The resulting precursor solution was fed at a constant rate (8 ml/min) through a nozzle and dispersed by 3 l/min of oxygen resulting in a fine spray that was ignited and sustained by a surrounding premixed oxygen/methane (1.5/3.2 l/min) flamelet. The particles were collected on a glass microfibre filter (Whatman GF) with the aid of a vacuum pump (Busch Mink MM 1202 AV). The collected particles were sieved (standard mesh size of 250) and annealed for 6 h at 600 C. (Carbolite Gero 30-3000 C.).

[0069] When the nanothermometers are ready, the fabrication of the composition can begin. Various final concentrations of BSA (Sigma-Aldrich, lyophilised powder, 96%), gelatin (Sigma-Aldrich, from Porcine Skin, type-A, strength 300), BiVO.sub.4 and TiN (PlasmaChem, 20 nm) can be selected. In this case the final concentrations (w/v) are BSA 25%, Gelatin 6.5%, Nd-doped BiVO.sub.4 0.2%, and TiN 0.003%. The composition is made from four starting solutions. A solution of 50% BSA was prepared by dissolving BSA in PBS. The dissolution speed can be increased using a mixer. A solution of 26% gelatin was prepared by adding gelatin to PBS. The solution was mixed and heated in a heated shaker (LLG Labware, uniTHERMIX 1) at 400 rpm and 60 C. until the solution was completely dissolved (usually in less than 30 min). A 1% solution of Nd-doped BiVO.sub.4 in water and a 0.1% solution of TiN were prepared and put in an ultrasonicator. Then, 0.5 ml of 26% gelatin were brought to 42 C. and 1.224 mL of 50% BSA were added to it. The mixture was vortexed for about 10 seconds and put in the shaker at 42 C. Then, 0.349 ml of Nd-doped BiVO.sub.4 solution were added and the obtained solution quickly vortexed for a few seconds. The process was repeated using instead of the BiVO.sub.4 solution 52.4 l of TiN solution. Before the solution cooled down, it was poured into the desired mould, making sure that no bubbles were present. The composition was put in the fridge until gelation.

[0070] The presented protocol can be used to prepare compositions with other concentrations.

[0071] For example, compositions comprising only fluorescent nanothermometers can be created by using distilled water instead of solutions comprising plasmonic nanoparticles.

Results and Discussion

Heating of the Composition

[0072] The temperature that can be reached by shining a laser on the composition depends on the optical properties of the solder, mainly given by the nanothermometers and in particular by the plasmonic nanoparticles that are added. Whether a certain mix of nanoparticles can be used for laser tissue soldering depends on its ability to convert laser light into heat efficiently.

[0073] Three different compositions were used in order to investigate the temperature reached during soldering: two with either only TiN or only Nd-doped BiVO.sub.4, and one with both types of nanoparticles (same concentrations described in the Materials section). A composition comprising 25% BSA, 6.5% Gelatin, 0.3% Nd-doped BiVO.sub.4, and 0.01% GNR with a thickness of 0.5 mm was used. A 750 nm multimode CW laser (Laser Century, RLM750TA) is coupled to a fibre and used with a collimator. Laser power of 0.8860.001 W was used. The laser was shined diagonally, leading to an elliptical beam spot size of 1.51.0 cm (area of 2.36 cm.sup.2). The temperature is measured using a thermal camera (Fluke Ti110).

[0074] The aforementioned experiment was repeated with GNR (Gold NanoRods) instead of TiN using a composition comprising 25% BSA, 6.5% Gelatin, 0.3% Nd-doped BiVO.sub.4 and 0.01% GNR and a 1064 nm multimode CW laser (Ventus 1064, Laser Quantum Ltd) instead of the 750 nm laser. In this case the laser had a power of 1.29 W and a spot size of 0.40.3 cm.

Results and Discussion

[0075] The measurements of temperature increase for the various compositions are shown in FIG. 1. The increase in temperature from the sample with only Nd-doped BiVO.sub.4 is very low, while the temperature increase in the samples that contain TiN is much more pronounced. The increase in temperature for the compositions with the mix of nanoparticles is higher than the one for the compositions with only one type of nanoparticle. Not only that, but it is also higher than the sum of the temperature increases of the two other compositions. This can be attributed to the increased scattering coefficient given by Nd-doped BiVO.sub.4 that allows more light to be absorbed by TiN. The same applies when using GNR (Gold NanoRods) instead of TiN.

Temperature Measurements through Fluorescent Nanothermometers

[0076] In order to retrieve temperature information from the fluorescent nanothermometers, they need to be calibrated. The nanothermometer powder was placed on a heating plate. A thermal camera is used for reference temperature measurement. The fluorescent spectrum is acquired by a spectrometer (Ocean Insight, STS-NIR). The signal is collected by a collimator and goes to the spectrometer through an optical fibre. A bandpass filter (Thorlabs, FB750-10) is placed in front of the laser source and two longpass filters at 780 nm (Edmund Optics) are placed in front of the spectrometer. The same setup (without the heating plate in the bottom) is also used for the soldering process. A scheme of the setup is shown in FIG. 2. A laser beam (1) comes out of a fibre collimator and bandpass filter and hits the sample (2). The light emitted by the sample passes through a longpass filter (3) and is finally focussed by a collimator (4) into a fibre connected to a spectrometer.

[0077] A calibration curve (see FIG. 3) is obtained from the spectra at different temperatures, using the same method explained by Gschwend et al. (P. M. Gschwend, F. H. L. Starsich, R. C. Keitel, and S. E. Pratsinis, Nd.sup.3+-Doped BiVO.sup.4 luminescent nanothermometers of high sensitivity, Chem. Commun., vol. 55, no. 50, pp. 7147-7150, 2019). In order to validate the effectiveness of the temperature measurements, the temperature during laser tissue soldering has been measured using both a thermal camera and the nanothermometers (see FIG. 4), using 10 seconds as integration time for the spectrometer.

Tensile Strength

[0078] Preliminary data on the soldering strength have been taken using a solder that contained only TiN. A universal testing machine (Zwick) was used to measure the tensile strength of the samples. Pig small intestine was retrieved from a local slaughterhouse, cleaned with distilled water and frozen. Samples of around 54 cm.sup.2 were cut. Another side-to-side cut was placed in the middle of the sample, parallel to the short side. The composition 25% BSA, 6.5% Gelatin, 0.3% Nd-doped BiVO.sub.4 and 0.01% TiN was placed on this cut and the soldering process was carried out: laser light with a circular spot size of 0.8 cm.sup.2 and intensity of 2.5 W/cm.sup.2 was shined on the solder and the sample moved when a temperature of 80 C. was measured with a thermal camera. The tensile properties of the samples were measured soon after. Tensile strengths of about 5 N were measured (a typical stress-strain graph is shown in FIG. 5).

Water-Filtered Near Infrared Light

[0079] Water-filtered near infrared light (wIRA), which has higher penetration in tissue due to the elimination of water-absorbed wavelengths, can also be used for soldering. Porcine small intestine and pig liver were retrieved from a local slaughterhouse the same day of the experiment. Samples were cut into pieced of the desired shape using a surgical scissor or a scalpel. Pieces of intestine were kept at low temperature and hydrated spraying PBS when necessary. A composition containing TiN and BiVO.sub.4:Nd (25% BSA, 6.5% Gelatin, 0.3% Nd-doped BiVO.sub.4 and 0.01% TiN) with a thickness of 0.5 mm was placed on the tissue. Hydrosun 750, a medically approved device that produces wIRA illumination, was used as a source of illumination at 5 cm from the samples. Illumination of 4 min was carried out. Histological samples were prepared, stained, and imaged according to standard practice. Histological images show no visible damage under wIRA irradiation for both intestine and liver tissues, with or without the solder paste on top. Moreover, histological images (FIG. 6) show good adhesion of the paste to the tissue, hinting to a strong adhesion.

Stability in Digestive Fluids

[0080] Soldered and unsoldered pastes (of the composition 25% BSA, 6.5% Gelatin, 0.3% Nd-doped BiVO.sub.4 and 0.01% TiN) were immersed in PBS or fasted state simulated intestinal fluid (FaSSIF) for up to seven days. Weight measurements showed that the composition is resistant to digestive fluids (FIG. 7a).

Bacteria Leakage

[0081] Porcine small intestine and pig liver were retrieved from a local slaughterhouse the same day of the experiment. Three categories of samples were prepared: intact, defected and soldered. For defected and soldered samples, a cut of 5 mm was created and for the soldered samples soldering was performed using a composition with TiN and BiVO.sub.4:Nd (25% BSA, 6.5% Gelatin, 0.3% Nd-doped BiVO.sub.4 and 0.01% TiN). All intestines were placed in flasks containing 100 mL of LB broth. 6 mL of broth containing E. Coli modified to express green Fluorescent Protein were placed inside the intestines. Absorbance at 492nm of the surrounding broth was measured by a plate reader (Infinite F200 PRO, Tecan) at different times. Triplicates were used for all categories. Three measurements of absorption were performed for each sample. Absorbance of soldered and intact intestine are in good agreement with each other, indicating the effectiveness of soldering against bacterial leakage (FIG. 7B).

Influence on Burst Pressure

[0082] Porcine small intestine was retrieved from a local slaughterhouse the same day of the experiment. To measure the burst pressure the intestine was cut open and flat and a cut of 1 cm parallel to the intestine length was created with surgical scissors. The intestines were sutured using polypropylene sutures (Ethicon Prolene 5-0, the standard type and thickness of suture used for small bowel anastomoses in clinical practice). The intestines were soldered using a composition with TiN and BiVO:Nd (25% BSA, 6.5% Gelatin, 0.3% Nd-doped BiVO.sub.4 and 0.01% TiN) and a 750 nm laser. The intestine was mounted on a 3D-printed burst pressure setup. A pump (Lambda VIT-FIT) was used to fill the intestines with DI water with a rate of 1 ml/min and a pressure sensor (ABPDANV060PGSA3, Honeywell) was used to measure the burst pressure. Triplicates were used for each sample group. FIG. 7C shows that the composition according to the present invention results in an increasing of the burst pressure after being applied on sutures.

Bond Strength

[0083] Porcine small intestine and pig liver were retrieved from a local slaughterhouse the same day of the experiment. A water-based gravimetric tensile strength setup was used for tensile strength measurements. Intestine samples are cut into pieces with half cm width and an additional full-width cut was placed in the middle. Samples were soldered with solder pastes of various BSA, Gelatin and TiN concentrations within the defined ranges and using the 750 nm laser for up to 4 minutes at full or variable power. Soldering was performed either in surface-near wound configuration, with the paste on top of the wound, or in deep tissue wound configuration, with the paste below the wound. FIG. 7D shows that the composition according to the present invention is able to form strong bonds.

Two-Laser Setup

[0084] Soldering can be performed using one single laser, in the case of TiN, or with a two-laser setup, as in the case of GNR. Using GNR with a two-laser system allows for decoupling of heating and temperature measurement (see FIG. 8A). A 750 nm laser can be used for temperature measurement without increasing the temperature of the solder significantly. A 1064 nm laser can be used to heat the solder paste to the desired temperature.

Surface-Near Wound/Deep Tissue Wound

[0085] The solder performance based was investigated in two different configurations; soldering of i) a surface-near wound (e.g. solder paste on top of the wound) and ii) a deep tissue wound (e.g. solder paste below the wound), and benchmarked against the state-of-the-art temperature measurements using a thermal camera. After validation of the nanothermometry method in comparison to reference temperature, soldering performance was assessed. The composition is able to reach the necessary target temperatures in both its formulations and in both configurations. While in the surface-near configurations the temperatures measured by the thermal camera and the nanothermometry are in good agreement, in the deep tissue configuration the thermal camera dangerously underestimates the temperatures reached during soldering (FIG. 8B). The composition according to the present invention ensures heating only in the regions of the photothermal agent (i.e. the composition) and nanothermometry accurately measures the temperature exclusively of the most relevant area, namely the fluorescent nanothermometer in the two compositions. Thus, the compositions according to the present invention can be used for soldering beyond superficial wounds, for example, for deep tissue wounds.