Abstract Background Liposuction is one of the most performed cosmetic surgery procedures. In a previously reported study, gold-nanoparticle (GNP) laser-assisted liposuction (NanoLipo) was shown to improve procedure parameters and outcomes in a porcine model. Objectives An ex vivo human liposuction model was developed to assess the ease, efficacy, and outcomes of NanoLipo, and to further explore its mechanism of action in facilitating liposuction. Methods NanoLipo was compared to a control without GNPs in sets of fresh, nonperfused, anatomically symmetric, matched tissue specimens from 12 patients. A subset of three experiments was performed under single-blinded conditions. Intraoperative assessments included lipoaspirate volume, percentage of free oil, ease of removal, and temperature rise. Specimens were palpated, visualized for evenness, and graded with and without skin. Postoperative assessment included viability staining of the lipoaspirate and remaining tissues. Microcomputed tomography was used to assess the distribution of infused GNPs within the tissues. Results NanoLipo consistently removed more adipose tissue with more liberated triglycerides compared to control. NanoLipo specimens were smoother, thinner, and had fewer and smaller irregularities. Infused solutions preferentially distributed between fibrous membranes and fat pearls. After NanoLipo, selective structural-tissue disruptions, indicated by loss of metabolic activity, were observed. Thus, NanoLipo likely creates a bimodal mechanism of action whereby fat lobules are dislodged from surrounding fibro-connective tissue, while lipolysis is simultaneously induced. Conclusions NanoLipo showed many advantages compared to control under blinded and nonblinded conditions. This technology may be promising in facilitating fat removal. Level of Evidence: 5 According to data gathered by The American Society for Aesthetic Plastic Surgery (ASAPS) suction-assisted lipectomy (SAL), or liposuction, is the most popular cosmetic surgery procedure, with over 400,000 procedures performed in 2016.1 However, the procedure is not perfect, and the largest reasons for dissatisfaction are contour irregularities and asymmetry, which can result from inadequate or excessive fat removal, residual skin laxity, or general practitioner inexperience.2 For example, Rohrich et al reported that satisfaction rates were 73% and 82% for patients who either gained or did not gain weight postsurgery, respectively.3 Broughton et al similarly reported that 79.7% of a cohort of 209 surveyed patients would elect to have the procedure again.4 These numbers leave some room for improvement, given that satisfaction rates as high as 98% have been reported for procedures such as breast augmentation.5 For these reasons, interest in less invasive alternatives to conventional liposuction, such as cryolipolysis,6 ultrasound-assisted liposuction,7 and laser-assisted liposuction8 has increased in recent years. Practitioners performing liposuction could benefit greatly if the procedure could be performed more quickly and less invasively, while more fat is removed, with more forgiving and even results, especially in the patients with more fibrous tissue. The objective of this study was to assess a technology called NanoLipo. NanoLipo involves gold nanoparticles (GNPs) added to standard tumescent fluid, injected into the targeted fat, followed by the application of externally applied near-infrared photothermal laser energy to create lipolysis and focused disruptions to fibro-connective tissues to assist in producing superior aesthetic results. Concepts were borrowed from photothermal therapy (PTT) for cancer that similarly involves the use of various agents capable of capturing optimally penetrating laser energy and creating focused heating to lethally damage cancerous tissues.9 Plasmonic GNPs were chosen for this application because it is one of the most extensively studied photothermal agents in oncology. However, a major distinction from cancer therapies is that in NanoLipo, GNPs are injected at concentrations orders of magnitude lower than those used in PTT (1.2 OD for NanoLipo vs 50 OD for PTT), since the goal is not complete tissue destruction.10 Another important distinction between NanoLipo and cancer therapies that rely on bulk heating is that the laser energy in NanoLipo is applied for strategically predetermined pulse durations and optimized power outputs, leveraging known heating relaxation times for the GNPs and affected target fat structures. NanoLipo is thereby able to specifically disrupt the fibroconnective tissues and lipocytes through selective photothermolysis.11,12 The ability of this novel photothermal-assisted liposuction procedure to produce superior outcomes in many aspects was previously reported in a porcine model.10 While this model is the best alternative to human skin and fat, porcine tissues still have significant biological, physiological, and mechanical differences from tissues of human origin.13 In this work, the first ex vivo human tissue liposuction model was developed, to the best of our knowledge, to successfully evaluate the limitations, benefits, and efficacy of the NanoLipo technology in improving liposuction before proceeding with clinical trials. METHODS Adipose Tissue Harvest and Preparation for NanoLipo Treatment The use of human tissues conformed to the principles of the Declaration of Helsinki, as well as all US federal guidelines and regulations.14 Patients voluntarily undergoing combined liposuction and abdominoplasty procedures at the private practices of participating plastic surgeons provided informed consent for the use of their tissues. Detailed demographics regarding patients were purposely not obtained to achieve complete patient privacy. Patients were selected by the involved plastic surgeons on the condition that sufficient adipose tissue could be extracted for the proposed studies. Fitzpatrick skin type was initially used as an exclusion criterium but was later eliminated due to the paucity of available tissue specimens. An institutional review board was not used for this study because experiments were conducted on discarded tissue and did not involve direct experimentation on human patients.15,16 During the timeframe from August 2015 to March 2017, 12 bilaterally symmetric tissue flaps containing skin and a sufficient amount of adipose tissue were surgically removed from the lower abdomen, lateral flank, and sometimes extending to the area above the buttocks of patients, and were provided, without identification, to the researchers (Table 1). All tissues were excised and tested on the same day and in most cases within a few hours to ensure tissue viability and similarity to in situ tissues. Table 1. Physical Characteristics of Specimens Tested Experiment # Thickness Fitzpatrick type 1 3-5 cm I 2 4-6 cm III 3a 3-5 cm II 3b 3-5 cm II 4 2-3 cm III 5 4-6 cm I 6 3-5 cm II 7 2-3 cm II 8 3-4 cm VI 9 3-4 cm II 10 4-6 cm II 11 3-5 cm II 12 3-5 cm II Experiment # Thickness Fitzpatrick type 1 3-5 cm I 2 4-6 cm III 3a 3-5 cm II 3b 3-5 cm II 4 2-3 cm III 5 4-6 cm I 6 3-5 cm II 7 2-3 cm II 8 3-4 cm VI 9 3-4 cm II 10 4-6 cm II 11 3-5 cm II 12 3-5 cm II View Large Table 1. Physical Characteristics of Specimens Tested Experiment # Thickness Fitzpatrick type 1 3-5 cm I 2 4-6 cm III 3a 3-5 cm II 3b 3-5 cm II 4 2-3 cm III 5 4-6 cm I 6 3-5 cm II 7 2-3 cm II 8 3-4 cm VI 9 3-4 cm II 10 4-6 cm II 11 3-5 cm II 12 3-5 cm II Experiment # Thickness Fitzpatrick type 1 3-5 cm I 2 4-6 cm III 3a 3-5 cm II 3b 3-5 cm II 4 2-3 cm III 5 4-6 cm I 6 3-5 cm II 7 2-3 cm II 8 3-4 cm VI 9 3-4 cm II 10 4-6 cm II 11 3-5 cm II 12 3-5 cm II View Large Samples were typically between 3 cm to 6 cm thick, 10to 20 cm wide, and up to 30 to 36 cm in length per bilaterally symmetric sample when received. Except for one sample, a Fitzpatrick skin type VI, all samples were type III or less. A treatment zone of approximately 4 cm × 7 cm was identified and marked in symmetrical areas selected for lack of stretch marks, hair, and scars, with matched even consistency, texture, and pigmentation of the skin. A larger nontreatment region of 2 to 3 cm, at minimum, was included on all sides of the treatment zone to minimize loss of infused tumescent solutions out of the exposed edges, to ensure even vacuum and application of the laser, and to serve as handles for the specimen during liposuction while keeping the tumescent and liposuction cannula tips in the treatment area. All samples were immediately postprocessed and analyzed after treatment. NanoLipo Procedure In our model, NanoLipo involved the infusion of 40 cc of GNP-containing phosphate buffered saline (PBS) solution injected into the interstitial spaces of adipose tissue in the targeted treatment zone using standardized tumescent and instrumentation techniques. The control contained no GNPs in the tumescent solution. The GNPs were surface functionalized with a passivating poly(ethylene-glycol) (PEG, 5 kDa) polymer coating to prevent clumping and to improve tolerability by biological tissues (NanoSpectra BioSciences, Inc., Houston, TX).17 The stock 50 optical density (OD) GNPs provided was diluted with PBS to down to 1.2 OD before infusion into tissue specimens. GNP size, aspect ratio, and other physical, and optical properties were confirmed to be as indicated by the manufacturer. A tumescent infiltration protocol was developed and instituted to maximize the even disbursement of the GNP within the treatment zone. A 2 mm × 130 mm multiport infusion cannula was inserted parallel to and approximately 1 to 3 cm below the skin surface, approximately the maximum depth of light penetration.18 The cannula was inserted fully into and through the tissues to the distal edge of the treatment zone. The tumescent or GNP solution was introduced into the tissues only during withdrawal of the cannula to the proximal edge of the treatment zone, injecting approximately 1 cc per pass with 10 passes per side. This process was repeated as multiple and separate introductions from all four sides for a total tumescent infusion of 40 cc. Every attempt was made to infuse the solutions evenly while manually locating and digitally guiding the cannula within the specimen. For the laser energy application, the 4 cm × 7 cm tumesced treatment zone was exposed to the laser through the skin in a predetermined application pattern to maximize the even distribution of the laser energy within the treatment zone. A commercially available 800 nm pulsed diode laser (Lumenis LightSheer DUETT, Lumenis Ltd. Yokneam, Israel) was used for all studies. The laser was set to generate passes comprised of three consecutive pulses of 30 ms duration, at an energy density of 6 J/cm2 (46 J/pulse; 138 J/pass) through its 22 mm by 35 mm exposure area. A total of 12 consecutive effective passes were administered, for a total of ~1650 J of energy per treatment area. Mild vacuum, a built-in function of the machine, was applied during the laser treatment. The skin surface temperature was monitored periodically with a thermal camera (FLIR E50; FLIR Systems, Wilsonville, OR). A cool dampened towel (~10°C) was placed over the treated region every two passes for ten seconds, or more often if needed, to cool the skin surface. A thermocouple was periodically placed approximately 2 cm parallel to and below the skin surface, through one of the exposed edges of the tissue, to measure the internal bulk temperature. The same preliposuction treatment procedures were administered to both the Nanolipo and the control group, with the only difference being the presence or absence of GNPs in the infusion solution, respectively. To perform the liposuction, a standard fat harvesting/transfer multiport cannula (Tulip Medical, San Diego, CA) was connected to a commercially available liposuction vacuum pump (Grams, Newport Beach, CA) and set to 28 mmHg. Liposuction was first performed in the control, then in the NanoLipo specimen. Liposuction was performed for a defined amount of time (30 sec-1 min), and varied depending on the initial amount of aspirate from the first sample from the control group so that the target volume of about 40 cc would be harvested and be about the same as the amount used during tumescent infiltration. The time to suction the 40 cc for the control group was then used for the NanoLipo group. The liposuction procedure was performed similarly to the tumescent solution infiltration procedure, with suctioning from all four sides into the treatment zone as evenly as possible and with similar effort. For the last three specimens, practitioner 1, who had conducted the previous experiments under nonblinded conditions, performed both the NanoLipo and control pretreatments. Practitioner 2, an experienced board certified plastic surgeon with his primary practice in liposuction surgery, performed the liposuction procedures and made clinical determinations under blinded conditions between the NanoLipo and control specimens, regarding ease of liposuction, amount and speed of lipoaspirate harvest, and evenness of fat removal. Digital photographs detailing the protocol for this ex vivo human tissue liposuction model can be found in Supplemental Figure 1. Posttreatment Assessments The criteria used to assess clinical treatment outcomes in the tissue remaining after liposuction were manual palpation and visual inspection. During manual palpation, the practitioner applied gentle digital pressure on the skin over the treatment zone to assess for the amount and evenness of fat removal. A comparison of evenness and any lumps or unexpected divots was made between each NanoLipo treated specimen and its bilaterally matched control. A pinch test was subsequently performed to further assess for the smoothness and evenness within the treatment region. After these initial assessments, the skin was surgically removed from the subcutaneous fat with a scalpel blade, much like harvesting a full-thickness skin graft, leaving only the liposuctioned fatty tissues behind. Inspection, palpation, visual determinations, and rank ordering of clinical criteria were completed using before and after skin-removal assessments by practitioner 2. Digital photographs of the specimens and underlying adipose tissue were taken to document the achieved results. Surgical forceps were used to stretch and manipulate the post-liposuction specimens to aid in the inspection and visualization of the treatment outcomes. Lipoaspirate was collected directly into conical centrifugal tubes within the suction canister and gently centrifuged at 1000 RPM for 5 min to standardize the settling and separation of three distinct volume fractions. The top fraction contains the liberated triglyceride oils, the middle layer contains intact adipose tissue and cells with trapped free oil, and the bottom layer contains the tumescent fluid, blood cells, plasma, and stem cells. The middle layer was isolated and treated with collagenase type I (powder, from Clostridium histolyticum) to digest the connective tissue and liberate the adipocytes and any remaining oil. The released fat cells were then stained with a Calcein/Ethidium Homodimer live/dead assay to assess cell viability. This digestion and staining was performed by following standard protocols provided by the supplier (all reagents and kits purchased from Thermo Fisher Scientific, Waltham, MA). Optical microscopy was used to image the cells and tissues. In a related experiment, the viability of intact adipose tissue remaining after liposuction was assessed with nitroblue tetrazolium chloride (NBTC) staining, as previously reported.11,19 Briefly, NBTC is a dye that stains for mitochondrial activity such that metabolically active tissue will be stained a deep-blue color while photothermal-induced reduction of metabolic activity would lead to loss of staining. A thin tissue slice approximately 1.0 cm thick bisecting the treatment region along the vertical laser beam axis was isolated, stained, and photographed. To assess the distribution of infused GNP solution, a micro-computed tomography scanner (µCT, Bruker Skyscan 1076, Kontich, Belgium) was used to image adipose tissue specimens that were either injected or not injected with solution. A calibration curve involving air, adipose tissue, saline, and various concentrations of GNPs was constructed to help identify the structures imaged. Imaging was performed at 18 μm isotropic voxel size at an electrical potential of 50 kVp and current of 200 μA using a 0.5 mm aluminum filter with a beam hardening correction algorithm applied during image reconstructions. RESULTS Assessment of Treatment Outcomes Under Non-Blinded Conditions During the tumescent step, no differences were seen between the NanoLipo and the control saline only groups. During the laser treatment step, there was a minimal rise in the skin temperature of approximately 10°C in the control group after the complete 12 passes, while in the NanoLipo group, the skin surface temperature typically rose quickly after only a few passes (Figure 1A). The most significant heating was observed at the skin surface in nonstudy pilot experiments for the NanoLipo group when towel cooling was not performed, with temperatures exceeding 40°C or more at local hot spots matching the pattern of the diodes in the laser aperture after each pass that would quickly equilibrate with the surrounding tissue. The heating was significantly mitigated with towel cooling, although the hot spots were still observed. In the control group, skin surface temperature never exceeded 40°C regardless of whether towel cooling was performed or not. Additionally, thermocouple readings for internal bulk temperature for the NanoLipo group barely exceeded 30°C at the end of 12 passes, while the internal bulk temperature for the control group was unchanged from the starting temperature. Figure 1. View largeDownload slide (A) Heating profiles of tissue specimens for NanoLipo and control pretreatments, measure externally and internally for NanoLipo, and externally only for control. Both NanoLipo internal measurements and control skin surface measurements showed mild heating, while NanoLipo external measurements showed the most heating. (B) After centrifugation, the liberated triglyceride phase accounted for nearly 50% of total lipoaspirated tissue volume for the NanoLipo group (left tube, indicated by the arrow), while almost no liberated triglyceride was observed in the control (right tube). (C) Calcein/ethidium homodimer live/dead assay; tissue fluorescing green from the calcein dye shows that the majority of the tissue is viable, while minimal red staining, indicative of dead cells, was observed. Figure 1. View largeDownload slide (A) Heating profiles of tissue specimens for NanoLipo and control pretreatments, measure externally and internally for NanoLipo, and externally only for control. Both NanoLipo internal measurements and control skin surface measurements showed mild heating, while NanoLipo external measurements showed the most heating. (B) After centrifugation, the liberated triglyceride phase accounted for nearly 50% of total lipoaspirated tissue volume for the NanoLipo group (left tube, indicated by the arrow), while almost no liberated triglyceride was observed in the control (right tube). (C) Calcein/ethidium homodimer live/dead assay; tissue fluorescing green from the calcein dye shows that the majority of the tissue is viable, while minimal red staining, indicative of dead cells, was observed. During the liposuction step, in general, the NanoLipo treated specimen appeared to be better liposuctioned and more evenly reduced than the controls. In thicker samples, the practitioners most often found a clinical difference in the ease with which the suction cannula moved to remove the fat for the NanoLipo group, as well as an increased rate at which fat was removed compared to the control. Practitioner 1, a nonplastic surgeon researcher, on average rated the NanoLipo and control procedures 5/10 and 7/10 in terms of difficulty, respectively, on a scale of 1 to 10 with one being very easy and ten being very difficult. Practitioner 2, a trained plastic surgeon, on average rated the NanoLipo and control procedures 4/10 and 5/10 in terms of difficulty, respectively, on the same grading scale. Both practitioners noted that the improvement in the ease of suction was markedly greater for more fibrous specimens. The immediate appearance of the NanoLipo treatment area was the presence of a clearly defined depressed region approximately matching the shape and size of the 4 cm × 7 cm laser treatment area (Figure 2A, dashed rectangle). This feature was only occasionally present, and to a much lesser extent, in the control specimens (Figure 2B). In some thinner specimens, nearly all of the adipose tissue within the treatment zone was removed for the NanoLipo group, resulting in a full-thickness void spanning most of the sample (Supplemental Figure 2). Meanwhile, in the thicker specimens, large indentations directly beneath where the laser was applied, could be observed. (Supplemental Figure 3). In the control treatments, this full thickness tissue removal was not seen and the indentations were always to a lesser extent. When visually examined from the deep side (turned up-side-down), or when the skin was surgically removed from the specimens, the NanoLipo treated specimens were more uniform, smoother, and had proportionally more fibrous tissue strands exposed, with fewer lumps and smaller, and much finer residual fat lobules compared to control, as can be clearly seen in Figure 2C and D, respectively. Figure 2. View largeDownload slide NanoLipo procedure results in the removal of visibly more fat, indicated by (A) a clearly visible depressed region within the underlying adipose tissue matching the shape and size of the laser aperture (dotted rectangle). (B) The same feature is not present in the control (saline + laser + liposuction) treated specimen. (C) Proportionally more fibrous tissue is observed in the NanoLipo treated specimen with very few fat lobules remaining, while (D) many fat lobules adhering tightly to the connective tissue membranes are observed for the control specimens. Arrows in Figures 2C and 2D indicate lobules of fat. Figure 2. View largeDownload slide NanoLipo procedure results in the removal of visibly more fat, indicated by (A) a clearly visible depressed region within the underlying adipose tissue matching the shape and size of the laser aperture (dotted rectangle). (B) The same feature is not present in the control (saline + laser + liposuction) treated specimen. (C) Proportionally more fibrous tissue is observed in the NanoLipo treated specimen with very few fat lobules remaining, while (D) many fat lobules adhering tightly to the connective tissue membranes are observed for the control specimens. Arrows in Figures 2C and 2D indicate lobules of fat. After the lipoaspirate was centrifuged, three distinct volume fractions were observed. The free fatty triglyceride oil volume fraction was situated at the top in the NanoLipo treated lipoaspirate. In some cases, it accounted for nearly half of the total lipoaspirate tissue and oil volume (not including the aqueous phase), while this fraction was practically nonexistent in the control lipoaspirate (Figure 1B). Liberated triglycerides were consistently observed for NanoLipo treated specimens, as clearly seen in the liposuction tubing in Video 1. Liberated triglycerides in the NanoLipo treated specimen were even observed for the single high-Fitzpatrick skin type specimen that was tested (Supplemental Figure 4). Adipocytes isolated from the middle fatty tissue volume fraction were viable, as indicated by the green staining imparted to the cells (Figure 1C). Video 1. Watch now at https://academic.oup.com/asj/article-lookup/doi/10.1093/asj/sjy027 Video 1. Watch now at https://academic.oup.com/asj/article-lookup/doi/10.1093/asj/sjy027 Close Assessment of Treatment Outcomes Under Single-Blind Conditions Clinical assessment of the NanoLipo vs the control treated specimens was undertaken. In a single-blind series with three consecutive cases, practitioner 2 noted that one of the specimens was markedly thinner and had significantly fewer lumps after suctioning. After skin removal, the specimen that was thinner was also much smoother within the treated region. Figure 3 displays a representative comparison between two blinded specimens from one set of tissues. As shown, the NanoLipo specimen is smooth, even, and has smaller fatty lobules (Figure 3A) while the control specimen has many large fat lobules and pearls clearly visible throughout the region (Figure 3B). A demarcation (dotted line) that matched well with the edge of the treatment zone was observed in the NanoLipo treated specimen, beyond which distinctive large fat pearls similar to those found throughout the control specimen are clearly observed (arrows). Practitioner 2 noted that the first blinded specimens (NanoLipo) was marginally easier to suction than the second (control) and that the former (NanoLipo) had regions where the difficulty of suction was significantly reduced. Although practitioner 2 indicated that he would not have been comfortable making a determination based on the ease of suction alone, he was able to identify the NanoLipo treated specimen correctly based on the obvious visual and physical differences after suctioning. Figure 3. View largeDownload slide Smoothness within the treatment zone was used as a key marker to distinguish the NanoLipo treated specimen from control specimens under blinded conditions. Digital photographs for one set of blinded experiments showing adipose tissue after liposuction and skin removal are shown. (A) NanoLipo treated specimen was even in appearance and was absent of fat lobules, whereas in (B) large and lumpy fat lobules are clearly visible and can be felt throughout the control specimen. Near the edge of the NanoLipo specimen outside of the treatment zone (below the dotted line in A.), several large fat pearls are clearly visible (arrow). Figure 3. View largeDownload slide Smoothness within the treatment zone was used as a key marker to distinguish the NanoLipo treated specimen from control specimens under blinded conditions. Digital photographs for one set of blinded experiments showing adipose tissue after liposuction and skin removal are shown. (A) NanoLipo treated specimen was even in appearance and was absent of fat lobules, whereas in (B) large and lumpy fat lobules are clearly visible and can be felt throughout the control specimen. Near the edge of the NanoLipo specimen outside of the treatment zone (below the dotted line in A.), several large fat pearls are clearly visible (arrow). NanoLipo Mechanism Investigations Upon infusion into adipose tissue, photothermal solution expanded the interstitial spaces between the connective tissue membranes separating pearls of fat. Figure 4A, C, and E, and B, D, and F, respectively, show the sagittal, transverse, and coronal views of µCT images of tissue specimens that were or were not infused with GNP photothermal solution. The relatively darker spaces represent air, the relatively grey spaces represent fat-rich regions, and the relatively white spaces represent water-rich regions. Scarpa’s fascia can be seen in the transverse and sagittal views as a relatively thicker white line parallel to the skin surface. In Figure 4A, C, and E, connective tissue regions have been expanded by infused solution while the same connective tissue separating pearls of fat in the uninjected control specimen remains collapsed. The expanded regions were, in some cases, up to 2 mm in size. Consistent with the observed distribution of photothermal solution, there was a loss of staining in some regions of the NanoLipo treated specimen (Figure 5A, arrows), whereas the whole specimen was stained a deep-blue color for the control specimen. Figure 4. View largeDownload slide Micro computer-aided tomography images of the distribution of infused AuNR solution within adipose tissue compared to noninjected control. (A, B) The transverse views, (C, D) coronal views, and (E, F) sagittal views of injected and noninjected adipose tissue specimens, respectively. The relatively darker spaces represent air, the relatively grey spaces represent fat-rich regions, and the relatively white spaces represent water-rich regions of adipose tissue, such as connective tissue membranes. Infused solution preferentially disperses between and expands into the interstitial spaces between fat pearls (A, C, E), while the interstitial spaces between connective tissue of noninjected adipose tissue specimens remain collapsed in their native state (B, D, F). Scarpa’s fascia can be seen in the transverse and sagittal views as a thicker white line parallel to the skin surface. Figure 4. View largeDownload slide Micro computer-aided tomography images of the distribution of infused AuNR solution within adipose tissue compared to noninjected control. (A, B) The transverse views, (C, D) coronal views, and (E, F) sagittal views of injected and noninjected adipose tissue specimens, respectively. The relatively darker spaces represent air, the relatively grey spaces represent fat-rich regions, and the relatively white spaces represent water-rich regions of adipose tissue, such as connective tissue membranes. Infused solution preferentially disperses between and expands into the interstitial spaces between fat pearls (A, C, E), while the interstitial spaces between connective tissue of noninjected adipose tissue specimens remain collapsed in their native state (B, D, F). Scarpa’s fascia can be seen in the transverse and sagittal views as a thicker white line parallel to the skin surface. Figure 5. View largeDownload slide Absence of nitroblue tetrazolium staining (arrows) shows selective thermal damage of connective tissue membranes at the fat pearl level in adipose tissue due to photothermal inactivation of intracellular reductases, and is consistent with the way AuNR solution distributes within iinterstitial spaces of adipose tissue. Specimens were subjected to (A) NanoLipo or (B) control treatment, vertically bisected with a surgical scalpel through the optical exposure axis to isolate thin slices approximately 0.5 cm thick, and stained. Complete staining of the control specimen, as well as staining of the overlying skin and deeper adipose tissue regions in the NanoLipo treated specimen, demonstrates that these tissues were unaffected and that photothermal damage was selectively restricted to the fibrous connective tissue membranes at the pearl level. Figure 5. View largeDownload slide Absence of nitroblue tetrazolium staining (arrows) shows selective thermal damage of connective tissue membranes at the fat pearl level in adipose tissue due to photothermal inactivation of intracellular reductases, and is consistent with the way AuNR solution distributes within iinterstitial spaces of adipose tissue. Specimens were subjected to (A) NanoLipo or (B) control treatment, vertically bisected with a surgical scalpel through the optical exposure axis to isolate thin slices approximately 0.5 cm thick, and stained. Complete staining of the control specimen, as well as staining of the overlying skin and deeper adipose tissue regions in the NanoLipo treated specimen, demonstrates that these tissues were unaffected and that photothermal damage was selectively restricted to the fibrous connective tissue membranes at the pearl level. DISCUSSION We previously reported the ability of NanoLipo to produce aesthetically superior results compared to saline in our porcine model.10 These promising results motivated us to develop an ex vivo human liposuction model to investigate whether NanoLipo could be successfully used as an adjunct for fat removal in human tissue specimens before proceeding with human clinical trials. One of the distinctive findings in the current study was the presence of liberated triglycerides in the lipoaspirate of NanoLipo treated specimens before any postprocessing (Figure 2B, left). We speculate that a specific form of the photothermal effect, called selective photothermolysis (SPT), may be the mechanism responsible.12,20 SPT, which has been applied extensively in the context of laser hair removal, where high energy lasers at 800 nm are similarly applied in a pulsatile manner to target for melanin in hair, a protein that imparts the ability to absorb 800 nm light,21 posits that this manner of exposure can achieve selective and precise heating of pigmented structures based on their geometry without significant heating of the surroundings. For example, a 3 ms pulse of light would be able to preferentially heat spherical targets with diameters 100 µm or smaller, while a 300 ms pulse can heat structures up to 1 mm.20 Incidentally, adipocytes, as well as some of the fibrous membrane support structures in adipose tissue, are in this size regime in their native state. Thus, they may be readily targeted by this selective photothermal phenomenon in the context of photothermal-assisted liposuction. The first observation in support of this mechanism is the lack of significant bulk heating in the adipose tissue of the NanoLipo treated group, which can be expected during selective photothermolysis. The most significant heating for both the NanoLipo and control specimens was observed at the skin surface. For the heating profile in Figure 1A, the thermal camera was specifically aimed at the local hot-spots in the pattern generated by the laser aperture, which were typically at least 10°C hotter, or more, than the surrounding tissue. Although the laser applicator does not have a cooling capability, this inhomogeneous heating was manageable when a damp towel was used to immediately cool the skin surface after laser exposure to quickly thermal equilibrate the hot-spots with the surrounding tissue. A laser with a chilled applicator may be used to mitigate the heating of the skin surface instead of a damp towel. These results are consistent with what is historically regarded as the greatest limitation in laser-based biomedical applications (ie, the limited penetration depth of light into tissue).18 Even wavelengths of light in the middle of the biological optical window around 800 nm, which was specifically chosen for this application, can penetrate only a few centimeters into tissue at maximum. Nevertheless, the light that does successfully penetrate the skin seems to be sufficient in producing a photothermal effect, as evidenced by the presence of liberated triglyceride even in the specimen with the highest Fitzpatrick skin type, which would have the lowest amount of light energy irradiating the GNPs within the fatty tissues among all of the tested specimens (Supplemental Figure 4). Another clue for the mechanism of NanoLipo may be the way that infused GNP solution distributes within adipose tissue. Adipose tissue is organized into several hierarchical levels.22 At the lowest level are the adipocytes, which are packed into lobules that are further nested within larger structures, called pearls. Many pearls packed together to form a section, such as the superficial or deep subcutaneous adipose tissue sections that are separated by a thick membrane called Scarpa’s fascia. In Figure 4, µCT images showed that the infused GNP fluid preferentially distributed between and expanded the interstitial spaces between fat pearls. This result is expected, since it would be unfavorable for the hydrophilic GNP fluid to be well mixed with the highly hydrophobic fat regions of adipose tissue. Upon exposure to external pulses of light through the skin, these photothermal fluid-filled compartments are heated up preferentially, which is supported by the loss of metabolic activity shown by NBTC staining precisely at this structural level (Figure 5A). Additionally, the deep blue color expressed throughout the remainder of the NanoLipo treated specimen indicates that full-thickness fat loss does not occur and that selective photothermal effects occurs only where the agents are deposited, while staining in the entirety of the control specimen (Figure 5B) indicates that the laser is well tolerated. These results taken together suggest a mechanism by which thermal stress imparted to the connective tissue network surrounding and intertwined within adipose tissue weakens those structures, while also acting to disrupt the nested lipocytes themselves, thus facilitating the removal of both the fat and parts of the fibrous tissue support structure during liposuction. Depending on the extent of heating of the connective tissue membranes, shortening of tissue fibers through mild heating, as opposed to significant destruction of the fibrous networks through excessive heating, may be desirable, and may lead to a vertical skin shrinkage effect,23-25 which is considered an appealing outcome in the context of aesthetic surgery. DiBernardo et al showed that focal collagen change and dermal inflammatory response were found in many samples without epidermal injury if the specimen’s temperature remained below 47°C, while epidermal and dermal injury occurred above this temperature, with skin blistering occurring about 58°C.25 In this study, extra care was taken to prevent the temperature from exceeding the recommended safety threshold of approximately 42°C for an extended period of time25 and thus no obvious skin tightening was noted, although it would be interesting to investigate the skin tightening capabilities of NanoLipo by allowing the temperature to rise to the levels necessary to induce skin tightening. In addition to selective heating of the fibrous membranes, photothermal heating imparted to the tissue likely extends beyond this tissue level through conduction, and may induce thermal lipolysis. We believe that it is this lipolysis that is responsible for the difference in liberated triglycerides we observed between the NanoLipo and the control.26 This specific mechanism we propose is consistent with all of the observations presented in this study (ie, the presence of liberated triglyceride and the lack of large fat pearls accompanied by the thinner and smoother appearance for the NanoLipo treated specimens). Furthermore, these observations, including the temperatures reached and morphological changes, are consistent with what has been observed for the effects of nonfocused ultrasound on adipose tissue.27 The photothermal phenomenon, which has been studied extensively in the context of cancer therapy,9 has only been sparsely explored in humans or human derived specimens. In one study, Tuchin et al stained thin human adipose tissue sections with a photothermal dye, indocyanine green, and exposed the specimen to over three hours of light exposure. The continuous and long-term exposure eventually resulted in “destructive engineering” of adipose tissue, but certainly cannot be applied realistically or practically in the context of liposuction.28 Another study involves the investigation of a 1060 nm pulsed Nd:YAG laser administered through the tip of fiber optic cable attached to a liposuction cannula, for laser lipolysis through photothermal heating of resident water in tissues.8 The 1-dimensional point-source nature of the laser aperture does not produce uniform heating when it is used to cannulate adipose tissue. Another limiting factor of this method is that the resonance wavelength is tuned to optical absorption of water molecules in tissue, which means that significant care must be taken not to overheat the targeted or nontargeted tissues, and thus, the effect of practitioner experience on the treatment outcome is amplified further. Therefore, although this technology has been approved by the FDA and marketed as SmartLipo (Cynosure, Westford, MA),29 it has not gained widespread popularity among plastic surgeons due to the debatable improvement in results over traditional liposuction techniques, with the added potential for thermal injury. NanoLipo, being a technology that also relies on laser-assisted heating of tissues, could face similar challenges during adoption. However, NanoLipo fundamentally overcome the shortcomings of a point-source heating mechanism by applying the light energy in a controlled, 2-dimensional, and discontinuous nature over the target area that is infused with GNPs, making it more efficient and results in more uniform and even heating of the adipose tissue, as was demonstrated in this study. The next step towards translation of this technology is to further substantiate the outcomes of this study clinically. For example, since this study was performed solely on excised human tissue samples that had been separated from the patient, no medical complications were observed, while it would be critical to ensure that this procedure does not result in any real medical complications in actual patients. In terms of experimental complications, occasionally, infused solution would flow out of the sides of the specimens or diffuse above and below the plane of injection. Thus, as we stated previously, significant attention was paid towards infusing the solution in a planar fashion (ie, keeping the points of insertion within the same plane). Nevertheless, we anticipate this problem to be mitigated in actual adipose tissue compartments, since there would not be any exposed tissues surfaces. Finally, because NanoLipo involves the use of exogenous photothermal nanomaterials, one critical question that was unanswered at the time of the porcine study was whether the injected nanoparticles induced any potentially harmful side effects. We have since conducted a six-month biodistribution and biocompatibility study to assess any toxicity concerns based on the finding that the majority of nanoparticles are eliminated from the bodies of pigs, and that the minuscule amounts of residual particles exhibit no observable signs of toxicity in our porcine model and with other studies,30 suggesting that NanoLipo is safe to be tested clinically. Ultimately, the outcome of liposuction surgery depends, first and foremost, on the provider experience, skill, and training.2 Other factors, such as patient tissue history, including repeat procedures, scarring in the treatment area, or the location on the body,31 as well as the amount of time and care taken, and the amount of tissue to be removed, all play important roles. However, even for highly trained plastic surgeons, significant time, effort, and attention to detail are needed to carefully suction the tough to treat fibrous or large areas to produce aesthetically pleasing results. Many plastic surgeons opt to perform two or more rounds of suctioning within the same session, with latter rounds involving the use of finer cannulae to extract the tough fat residing within especially fibrous pockets of connective tissue that lead to the formation of lumps and bumps posttreatment if left unaddressed.22 In the current study, the practitioners were allotted at most only a few minutes to perform the simulated liposuction without multiple rounds of suctioning using finer cannulae. We believe and have substantiated with our data that NanoLipo treatment was able to generate superior outcomes compared to the control. If these results can be extrapolated to larger treatment sizes in clinical settings, we believe the photothermal pretreatment process of NanoLipo applied to liposuction may be a useful tool for liposuction practitioners. CONCLUSION Our goal in this study was to investigate NanoLipo primarily as a technique to hasten, facilitate, and improve the liposuction procedure and outcomes. Based on the observed results, we have shown that photothermal pretreatment of adipose tissue is effective in assisting the more rapid and efficient removal of fat while producing aesthetically superior outcomes compared to conventional suction assisted lipectomy in excised human tissue specimens. We believe that with this current data in hand, our next step is to evaluate NanoLipo in human clinical pilot studies while examining its short-term and long-term indications, advantages, results, and potential complications. Supplementary Material This article contains supplementary material located online at www.aestheticsurgeryjournal.com. Disclosures The authors declared no potential conflicts of interest with respect to the research, authorship, and publication of this article. Funding NIH R01EY024134 and Air Force Office of Scientific Research (AFOSR) FA9550-15-1-0273, in part, supported labor efforts (eg, author stipends), the purchase of equipment, and acquisition of general laboratory supplies. eLux Medical, Inc., in part, supported labor efforts and the purchase of gold nanoparticle solutions for the described studies. NIH PO1 AG007996 supported operation of the Bruker Skyscan micro-Computed Tomography scanner. Acknowledgments The authors would like to thank the private practices of Mark M. Mofid and William and Jeffrey Umansky for providing some of the tissue specimens used for this work. NanoLipo is currently being investigated as a potential product for the company eLux Medical Inc; in its current state, NanoLipo is simply a name given to the technology we describe in the manuscript and is not a registered trademark. REFERENCES 1. Cosmetic surgery national data bank statistics. Aesthet Surg J . 2017; 37( suppl 2): 1- 29. 2. Dixit VV, Wagh MS. 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Aesthetic Surgery Journal – Oxford University Press
Published: Feb 3, 2018
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