1.1.

Introduction

The development of minimally invasive techniques holding the promise of reduced tissue pain and faster recovery is one of the leading trends in surgical medicine. In particular, the possibility to perform surgeries without sutures or staples is a valuable objective in view of minimally invasive clinical interventions.^1,2 One innovative approach for sutureless bonding entails the combination of laser light and an endogenous or exogenous optical absorber that may lie at the wound edges and mediate local generation of heat.³ The most relevant endogenous absorbers are water, melanin, and haemoglobin, which may be excited by infrared and visible light. In order to enhance the localization of light deposition, it proves advantageous to use an exogenous chromophore and light at frequencies overlapping its optical extinction bands within a window where the competition from the endogenous absorbers is poor.^1,2 Finally, the thermal modification of the tissue is intended to generate new bonds between the wound edges, thus resulting in its closure.³

1.2.

Current Laser Tissue Bonding Strategies

Nowadays, one of the preferred setups for sutureless laser-induced bonding involves the combination of near-infrared (NIR) light, which penetrates deep into the body, and an organic dye with optical extinction at the same frequencies.³ Among the NIR-absorbing dyes proposed for laser bonding, Indocyanine Green (ICG)⁴ has attracted very much interest from the medical community and is currently used to support a variety of surgical applications with a history of safety in humans.^5,6 In a typical laser-welding procedure, an aqueous solution of ICG is accurately spread between the tissue margins to be welded, while paying particular care to leave surrounding tissues unstained, thus avoiding their accidental absorption of laser light.⁷ Then the wound edges are approximated and laser welding is performed under a surgical microscope by means of a NIR-emitting laser device (such as a diode laser) usually equipped with a fiberoptic delivery system. The heat produced upon irradiation with a NIR laser drives a thermal reorganization of the main tissue components, which results into the immediate closure and prompt repair of the wound.^3,8–10 This strategy proves particularly appropriate for those applications where suturing and stapling may prevent a regular healing process due to foreign-body reaction, such as, for example, in lamellar or penetrating keratoplasty, or for the treatment of thin tissues such as the lens capsular bag.^11–13 Additional advantages include reduced operation times and a substantial simplification of the overall surgery. The above procedure may be customized for specific applications by varying the irradiation conditions, such as laser power, emission mode, treatment duration, and mutual arrangement between the fiber tip and tissue.¹⁴

In addition, different and functional formulations of ICG may be employed, including liquid, semisolid, and solid solutions.^1,2 For instance, the chromophore may be homogeneously blended with a protein or polymer dispersion (e.g., albumin, fibrin, collagen, hyaluronan, etc.) that is pasted between the margins of the wound and finally irradiated with a NIR laser.^15–19 On heating, the ICG formulation coagulates and seals the wound, thus acting as a solder.²⁰ When the adhesion is mediated by the topical interposition of soldering material, this bonding technique is more properly referred to as laser soldering.² While liquid solders generally suffer from a high mobility of the absorbing chromophore during the soldering procedure, solid formulations exhibit low pliability, both of which hamper the achievement of reproducible results.^2,21 In this context, the use of semisolid polymer formulations such as polycaprolactone (PCL) and chitosan in the form of flexible films and scaffolds is one hopeful alternative.^18,21–23 These materials, in addition to an intrinsic versatility in their fabrication process, hold the promise of good biocompatibility, biodegradability on a timescale of several days to months inside the body, and capacity to host functional chemicals, which attracts much interest for tissue repair and drug delivery purposes.^24,25 Moreover, some formulations exhibit antimicrobial and chemoactractant properties, which can support and accelerate the wound healing process.²⁶ In a recently proposed system, ICG molecules were loaded into silica nanoparticles, which are further dispersed with PCL and used in the form of semidry scaffolds.²⁷ This system proved advantageous in improving the control over the local dye concentration, which is important to ensure better heat dosimetry.

1.3.

Novel Tissue Bonding Strategies with Metal Nanochromophores

Despite the thorough research work that has been performed by several international groups in ex vivo models and in in vivo animal models,^2,11,28,29 only a few of the proposed approaches have reached the preclinical and clinical phases thus far.^11,12,30,31 The reason for this gap has to be found in a lack of standardization in the tissue bonding procedure, mainly related to the use of conventional organic chromophores and, in turn, to a poor control over the heat deposition, as mentioned above.

Notable issues ascribed to the use of exogenous chromophores of common use in surgical practices such as ICG include a dependence of their structural and optical response on their biochemical environment and temperature, and a rapid degradation over time.^4,32–34 In addition, these molecules suffer from limited light extinction efficiency, photobleaching, and possible phototoxicity (e.g., generation of reactive oxygen species).^4,35 With this background, there have been conceived recent proposals to replace these organic chromophores by metal³⁶ and composite metal-coated bead³⁷ nanoscale transducers, which hold the promise to extend the range of application of tissue bonding.^21,38 Similar to the organic dyes, these nanoparticles can be formulated both in aqueous suspensions and after dispersion in a suitable protein or biopolymer scaffold.^3,21 In the latter case, the biochemical matrix is activated by the photothermal conversion from the embedded chromophores, ultimately adhering to its adjacent tissues.¹

To the broad class of metal nanoparticles belong the so-called gold nanorods (cylindrical nanoparticles) and gold nanoshells (silica core, gold shell), which are the most efficient nanochromophores for photothermal applications in biomedicine disclosed thus far.^39,40 Typical extinction spectra of gold nanorods and gold nanoshells display characteristic features originating from collective electron oscillations or plasmon resonances and comprise a weaker band in the green (such as that found for gold nanospheres) and a fairly stronger band in the NIR.^41–43 The latter can be tuned throughout the NIR window in a controlled manner during the nanoparticles fabrication, by changing the aspect ratio (length divided by waist diameter) of gold nanorods or the core diameter to shell thickness ratio of gold nanoshells (Fig. 1).^40,43–45

Fig. 1

(a) Gold nanorods of different aspect ratios display different colors which correspond to different absorption spectra ($\text{bar}=100\mathrm{nm}$). Reproduced with permission from Ref. 46. (b) Optical tunability for nanoshells with a 60-nm silica core radius and gold shells 5, 7, 10, and 20 nm thick. The plasmon resonance red shifts with decreasing thickness of the gold shell (or increasing core to shell ratio). Reproduced with permission from Ref. 43.

In turn, upon excitation with radiation from a NIR laser, these plasmon resonances undergo excitation and relaxation mainly through nonradiative channels. The interplay of physical and chemical properties of gold nanorods and gold nanoshells gives a number of favourable features,^{21,40,41,46–49} such as: (1) possibility to modulate the geometrical and optical properties during fabrication; (2) a unique enhancement of the near field; (3) exceptional absorption (e.g., gold nanorods can provide absorption coefficients higher by up to five orders of magnitude than those of conventional absorbing dyes) and scattering (preferentially ascribed to gold nanoshells) efficiencies; (4) high stability in the body and thresholds before photothermal degradation; (5) broad versatility of conjugation with additional biochemical molecules including ligands, drugs, and genes; and (6) inertness in the body and negligible intrinsic cytotoxicity. For these reasons, these nanoparticles have been investigated as valuable contrast agents and sensitizers in a variety of diagnostic (e.g., optical and photoacoustic imaging and optical coherence tomography) and therapeutic (e.g., photothermal and photoacoustic microsurgery) applications^50,51 including laser bonding.

A few papers reporting the successful application of gold nanorods and gold nanoshells for tissue bonding have appeared in the recent scientific literature. For instance, Gobin et al. have demonstrated the use of a solder composed of bovine serum albumin (BSA) and gold nanoshells ($\sim 110\mathrm{nm}$ diameter and $\sim 10\mathrm{nm}$ shell thickness) to successfully seal ex vivo muscular and in vivo skin tissue lesions.³⁷ These authors used continuous wave radiation generated by a diode laser emitting at 808 to 820 nm with a 14 to $20\mathrm{W}{\mathrm{cm}}^{-2}$ intensity to achieve successful fusion with tensile strength equal to that of native tissues. Similarly, Nourbakhsh and Khosroshahi⁵² realized the closure of 20-mm-long incisions performed on explanted skin samples mounted on an automatic programmable scanning system by using a diode laser (peak emission at 810 nm) running in continuous wave mode at an optimal intensity of $60\mathrm{W}{\mathrm{cm}}^{-2}$. Recently, our research group demonstrated the successful application of an aqueous suspension of gold nanorods for the direct welding of explanted lens capsular tissues.⁵³ Later substantial advancements have been obtained upon inclusion of gold nanorods into polysaccharide scaffolds.²¹ In these systems the sugar chains enwrap the nanoparticles, thus creating a protective barrier against the physiological environment and enhancing their stability, durability, effectiveness, and biocompatibility. Preliminary investigations consisted of in vivo laser closuring of full thickness incisions performed in the carotid artery wall by means of a 810-nm diode laser in conjunction with the topical application of a composite gel of hyaluronan and gold nanorods (Fig. 2).⁵⁴ Irradiation of the wounds with a continuous intensity of $40\mathrm{W}{\mathrm{cm}}^{-2}$ provided successful closure of their edges with sufficient strength to sustain blood pressure and ideal healing within a 30-day follow-up.

Fig. 2

Sequence of images recorded during the laser welding of a rabbit carotid artery with a gold nanorods/hyaluronan gel. (a) The artery is clamped and a 3-mm longitudinal incision is performed. (b) After application of the soldering formulation, the incision is treated with a 810-nm diode laser light. (c) Appearance of the sealed artery immediately after intervention.

A further optimization of tissue repair procedures with gold nanochromophores consisted of the use of flexible materials with enhanced handiness and stability with respect to liquid or soluble solders such as those mentioned above. We recently proposed the introduction of a novel biocompatible material consisting of chitosan films containing gold nanorods, which can be readily bonded to biological tissues by photothermal activation.⁵⁵ These films are resistant, pliable, and stable in physiological conditions over time, while the chitosan matrix provides excellent control over the distribution and stability against aggregation of the nanoparticles, which translates into a dependable optical response and photothermal conversion [Figs. 3(a) and 3(b)]. Effective and reproducible adhesion of these films onto explanted carotid artery samples was achieved by means of contiguous spots of laser irradiation realized by gently bringing an optical fiber tip into contact with the films and delivering 810-nm diode laser pulses of 100 ms duration and 100 mJ energy, corresponding to $140\mathrm{J}{\mathrm{cm}}^{-2}$ fluence at the outermost film surface [Figs. 3(c)–3(f)].⁵⁵

Fig. 3

(a) Comparison between the light extinction profiles of a hybrid gold/chitosan film (solid line) and the initial aqueous suspension of gold nanorods (dashed line). (b) SEM image of the gold nanorods distribution within a film sample ($\text{bar}=50\mathrm{\mu m}$). A good balance between gold nanorods and chitosan concentrations provided for a homogeneous dispersion of particles within the polymer medium with an optical absorption profile closely resembling that of isolated nanoparticles. (c) Scheme of laser bonding of a hybrid gold/chitosan film to an arterial tissue—1: the artery is clamped proximally and distally and a 5-mm longitudinal cut is performed by means of a sharp-point; 2 and 3: a hybrid gold/chitosan film is topically applied onto the lesion and rolled all over the artery and 4: laser welded into its final position. Laser bonding is obtained by keeping the fiber tip in contact with the outer layer of the film and delivering single laser pulses of 100 mJ, 100 ms. (d) Toluidine blue stain of a cross-sectional slice of a film/tissue adhesion ($\text{bar}=100\mathrm{\mu m}$). Comparison between TEM images of an untreated hybrid gold/chitosan film region (e) and of an adhesion region at the tissue/film interface (f) ($\text{bar}=500\mathrm{nm}$; $f=\text{film}$; $t=\text{arterial}\text{tissue}$; gold nanorods are evidenced by arrows). A homogeneous adhesion characterized by close interdigitation between the biopolymer matrix and the external arterial wall is evidenced.

Although beyond the focus of this paper, it is worth mentioning a different nanoparticle-inspired tissue bonding approach, which is based on magnetic instead of laser stimulation. This was recently proposed by Bregy et al.⁵⁶ with the final aim of reducing the effects of the inhomogeneities in the solder absorption and of increasing the radiation penetration through the tissue. The use of an albumin solder enriched with spherical superparamagnetic iron oxide nanoparticles (SPIONs) of 15 nm diameter allowed these authors to weld explanted blood vessels on application of 170-m radio waves⁵⁶ with strength comparable to that obtained with conventional suturing.

2.1.

Tissue Bonding with Highly Stabilized Gold Nanochromophores

The positive reception of gold nanochromophores for important photothermal applications, such as the laser hyperthermia of cancer, is backed by their excellent photostability, which outclasses that of organic dyes. However, tissue bonding may require unusually high laser fluences (well above $10\mathrm{W}{\mathrm{cm}}^{-2}$), which may challenge the shape durability of these nanoparticles. For instance, already well below the melting point of bulk gold, gold nanorods tend to transform into stable gold nanospheres on laser excitation or thermal bathing,^57,58 which may result from surface diffusion^59,60 and immediately translate into a loss of plasmon resonances and light extinction in the NIR window. Figure 4(a) displays the appearance of a chitosan film loaded with gold nanorods and irradiated through its central spot under conditions that may be required for tissue bonding. Already after a few seconds, this spot is seen to undergo a visible change in color from purple to green, which reflects the bleaching of the gold nanochromophores (i.e., their inhibition to further laser absorption). Similar dynamics have been reported for the gold layer surrounding the silica core of gold nanoshells as well, which was shown to degrade into byproducts such as small gold nanospheres with a large size distribution and little capacity to absorb NIR light.⁶¹

Fig. 4

(a) $A\sim 1\mathrm{cm}$ diameter gold nanorods/chitosan film after exposure to $18\mathrm{W}{\mathrm{cm}}^{-2}$ 810 nm laser light, which is typical for laser bonding. Note the change in color at the 2-mm-diameter central spot under irradiation. (b) Representative TEM micrograph of silica-coated gold nanorods, which may benefit from additional stability.

A possible solution may come from surface modification of the particles with a rigid inorganic or organic coating, in an attempt to impart enhanced stability and a mechanical constraint against photothermal reshaping.⁴¹ For instance, deposition of a silica shell around gold nanorods [e.g., Fig. 4(b)] was shown to substantially delay the deformation of gold nanorods in front of NIR laser pulses in the second-to-nanosecond timescales.^62–64 Similarly, a carbon or polymethylmethacrylate coating was reported to significantly improve the photostability of gold nanoparticles.^65,66 The possibility to sustain high local temperatures is expected to enable better stability of the photothermal conversion and thus increase the overall reproducibility of tissue bonding. In addition, highly stable nanochromophores shall be less prone to laser-induced decomposition into small fragments,⁶⁶ whose presence may be undesirable in vivo. In fact, previous cytotoxicity studies provided evidence for cell death by necrosis or apoptosis when using clusters of few gold atoms around 1.4 nm in size,^46,67 which become negligible for gold nanoparticles of larger size.^46,68–70

2.2.

Enhanced Tissue Bonding with Patterned Nanocomposites

Further optimization of the tissue bonding technology may be achieved by the use of patterned nanocomposites. By this phrase we intend the fabrication of well-defined and functional particle geometries and distributions inside a composite formulation.^71,72 One example of patterned nanocomposites includes matrices with a gradient of optical absorbance along their length. These shall find application as laser-activated inserts inside an accidental wound or surgical incision. Indeed, the deeper the nanochromophore inside the cut, the larger the absorbance that is needed in order to produce an effective photothermal response, which is due to the light attenuation from the upper layers.⁷³ While the fabrication of durable graduated inserts with organic dyes remains a challenge,²³ this may be achieved for instance by subjecting gold nanorods embedded into a polymer strip to a suitable temperature gradient in order to modulate the particle’s transformation along its length and so the spectrum of plasmon resonances.⁶⁵

2.3.

Real-Time Temperature Monitoring by Optoacoustic Tomography during Tissue Bonding

Subtle temperature dynamics inside the tissue critically affect the closure of a wound.^74–76 Indeed, the temperature increase needs to be high enough to trigger particular thermal processes^2,3 yet without inducing irreversible thermal damage, with a narrow window of optimal conditions.^77,78 Nowadays commercially available systems that enable a real-time, noninvasive monitoring of temperature dynamics in the clinical practice are based on infrared detectors such as thermocameras, which provide the spatial distribution and temporal evolution of temperature with very decent resolution. Unfortunately, this approach only provides a superficial temperature detection, which prevents fundamental insight into the temperature distribution inside the target. To overcome this restriction, analytical models based on the solution of the bioheat equation in a two- or three-dimensional domain are used to approximately track the temperature evolution deep inside the tissue.^3,75 However, use of these models is very delicate, due to the great complexity of biological tissues with their heterogeneous composition and subtle dissipative pathways. Valuable alternatives are represented by magnetic resonance imaging (MRI)⁷⁹ and ultrasound imaging,⁸⁰ which provide for a direct real-time monitoring of the temperature rise within biological tissues subject to photothermal treatments without the need for numerical postprocessing. However, while MRI does not hold much promise to become a routine tool for monitoring thermal effects deep in the body,^81,82 efficient equipment for ultrasonic detection of temperature in the clinical practice is not yet available.⁸³

A recent and powerful alternative is nowadays represented by optoacoustic tomography (OAT), also called photoacoustic tomography (PAT), which is based on photoacoustic conversion (i.e., the generation of pressure waves and emission of ultrasound on absorption of light energy, preferably under thermal and stress confinement conditions).⁸⁴ OAT/PAT combines optical contrast based on absorption of NIR optical energy and detection of resulting acoustic pressure propagating as ultrasonic waves, resulting in a much higher lateral and depth resolution than pure ultrasonography and higher sensitivity with respect to MRI.^85–87 Due to the inherent optical contrast between cancerous and healthy tissue, OAT/PAT has originally found application in identifying cancerous lesions.^88–90 More recently, OAT/PAT has been proposed to measure temperature profiles by tracking thermally induced changes in thermoelastic properties and photoacoustic signal amplitudes (Fig. 5).^91–94 In this context, gold nanoparticles have been proposed as effective contrast agents for image-guided therapy based on photoacoustic imaging.^90,95 Once irradiated with pulsed laser light at low average power, these particles act as very local heat sources with temperature rapidly fading away with distance,⁹⁶ thus capable of triggering the photoacoustic conversion without interfering much with the overall tissue temperature. At present OAT/PAT represents a promising candidate to precisely monitor temperature changes during chromophore-enhanced photothermal therapies.⁹¹ We anticipate a future involvement of OAT/PAT in the real-time guiding of photothermal tissue bonding with nanochromophores or nanocomposites. The possibility to simultaneously implement both techniques by the use of the same particles and with an endoscope will provide further benefits for extreme surgeries including the laser welding of gastrointestinal, urological, or intracranial tracts.

Fig. 5

(a) Photoacoustic (PA) signal and actual temperature of a source sample (diluted black ink solution with ${\mu }_a=30{\mathrm{cm}}^{-1}$) as the heated sample was allowed to come to room temperature. The linear relationship between temperature and PA signal proves potential for real-time temperature sensing, (b) PA signal versus temperature from (a). The green line is a linear fit with an $R_2$ of 0.98. (c) PA signal and actual temperature of an initially cold sample warming up to room temperature, (d) PA signal versus temperature from (c). Again, the green line is a linear fit with an $R_2$ of 0.98. Reproduced with permission from Ref. 92.

2.4.

Tracking of Tissue Regeneration Based on the Optical Resonances of Gold Particles

A key concept to take full advantage of gold nanoparticles for tissue bonding is the tunability of their optical properties, as it was already mentioned in Sec. 1.3 and Fig. 1, which makes these new chromophores particularly attractive for theranostic applications.⁹⁷ In order to expand this point, Fig. 6 displays a collection of simulations of the light absorption (uppermost curves) and scattering (lowermost curves) cross sections for prolate elliptical gold nanoparticles in water with parameters representative of typical gold nanorods. As is shown, the two contributions of light extinction (scattering and absorption) of the particles can be modulated in amplitude and position throughout the visible NIR region by changing their size (volume) and shape (aspect ratio). As a rule of thumb, smaller nanoparticles are ideal for OAT/PAT and photothermal treatment procedures as light absorption dominates, while larger nanoparticles exhibit higher scattering efficiency, which may be preferable for specific optical imaging applications including optical coherence tomography (OCT).

Fig. 6

Simulations obtained from Gans dipolar approximation,^98–100 and implemented with the analytical gold dielectric function given by Etchegoin et al.¹⁰¹, which describe the light absorption (upper curves) and scattering (lower curves) cross sections from prolate elliptical gold nanoparticles with different shape (a) and size (b). (a) Variation of the optical properties of gold nanoparticles with the same volume $V=1000{\mathrm{nm}}^3$ and different length/diameter ratios (i.e., aspect ratio [AR]) from 2.5 to 5 in steps of 0.5 from left to right. The plasmon band in the NIR window progressively gains intensity and red-shifts with AR. (b) Effect of a constant $\mathrm{AR}=4$ and different volumes from $1000{\mathrm{nm}}^3$ to $5000{\mathrm{nm}}^3$ in steps of $1000{\mathrm{nm}}^3$ from the low to the high scattering bands (the absorption bands scale with V in Gans models and so the absorption profiles overlap with each other in the normalization given in this figure). The principal bias is an increase of the ratio of light scattering to absorption with volume.

OCT provides noninvasive imaging of living tissues with penetration depth in the low millimeter range and spatial resolution in the low micron range, which is superior to standard clinical methods of noninvasive imaging such as ultrasonography and MRI.¹⁰² Highly scattering gold nanoshells (119 nm diameter, 12 nm shell thickness) have been shown to enhance OCT contrast in vivo.⁵¹ In this example, both imaging and photothermal therapy have been performed with a single particle formulation designed for both scattering and absorption.¹⁰³ Different OCT modalities are based on differential absorption or backscattering albedo (the ratio of backscattering to total extinction), which offer the advantage of producing contrast in tissues with high turbidity.¹⁰⁴ In this context it is the absorption properties of plasmonic nanoparticles that are concerned and should prevail in comparison with the case of the conventional backscattering OCT modality.

The twofold functionality of gold nanoparticles as both photothermal transducers to trigger tissue repair and contrast labels for biomedical diagnosis is well suited to provide for breakthrough insight into tissue bonding applications. Resonant imaging based on gold nanoparticles dispersed at the weld site will be useful to track the progress of the wound healing process in the period that follows the tissue bonding intervention. The low diffusivity of gold particles through the tissue matrix with respect to that of organic chromophores (e.g., $\sim {10}^{-8}{\mathrm{cm}}^2{\mathrm{s}}^{-1}$ for 100-nm gold nanoparticles¹⁰⁵ as compared with $\sim {10}^{-6}{\mathrm{cm}}^2{\mathrm{s}}^{-1}$ for ICG¹⁰⁶ through connective tissues) implies a long-term permanence of the former in situ, while the latter are cleaned from the body within two to three days, which generates intense interest for their use for tissue monitoring during the follow-up period. This tendency can be amplified by increasing the nanoparticles size.¹⁰⁵ Previous studies have demonstrated the presence of 40-nm gold nanoparticles interdispersed within the extracellular matrix up to at least 30 days after a tissue bonding intervention (Fig. 7).^21,54 In addition, the use of functionalized particles capable of labeling different cell-surface factors or extracellular matrix biomarkers is expected to confer specificity to a particular biophysical or biochemical response occurring during tissue regeneration such as fibroblast migration, collagen deposition, and self-organization, and provide information on the evolution of these processes.^107,108

Fig. 7

TEM micrograph ($4.2\times 3.0{\mathrm{\mu m}}^2$) of a closured rabbit carotid artery after a 30-day follow up from laser bonding with gold nanochromophores.