Mechanical underwater adhesive units for gentle substrates

Micro-computed tomography

We used a SkyScan 1173 micro-computed tomography unit (Bruker) with SkyScan 1173 software software program to scan particular person heads of varied remora species. The scanning parameters included a voltage vary of 51–130 kV, an amperage vary of 40–136 µA, an publicity time vary of 337–730 ms and a picture rotation vary of 0.06–0.07°. Slice reconstruction of the osteological construction of the remora suction disc was carried out utilizing NRecon (Micro Photonics) and rendered in Mimics 15.0 (Materialise). The lamella angle of the remoras, considered dorsally, was characterised utilizing Fusion 360 (Autodesk).

Fabrication of MUSAS

MUSAS are versatile to be fabricated utilizing a wide range of silicone rubbers and biodegradable lamella supplies. An in depth fabrication process is supplied in Supplementary Fig. 3. Unless in any other case specified, all testing of MUSAS was carried out utilizing MUSAS fabricated with Ecoflex 0030 elastomer and form reminiscence nitinol lamellae.

Universal mechanical testing

Universal tensile testing was carried out to measure the stiffness and adhesion efficiency of the specimens and supplies of curiosity, together with tissue and materials samples, euthanized remora, units and adhesives. A 5944 common testing system (Instron) with Bluehill V3.11 was used for the tensile take a look at. For each stiffness and adhesion examine, the tensile extension price was set to 30.0 mm min⁻¹, with a measurement interval of 20–100 ms for time, 1 × 10⁻⁵ N measurement accuracy for load, and 1 × 10⁻⁵ mm measurement accuracy for displacement. All take a look at specimens, together with units and adhesive supplies used within the adhesion take a look at, had been bonded to the highest of a double-stacked 0.5-mm-thick polyimide Kapton strip (McMaster-Carr) utilizing ultraviolet-cured 5055 silicone adhesive (Loctite). The Kapton strips had been secured with an Instron tensile grip (Supplementary Figs. 2 and 4). A preload of 0.5 N was utilized to all units and adhesive supplies earlier than take a look at. To guarantee a good comparability, the pre-adhesion pressurization for different adhesive supplies was for 3 min, whereas for MUSAS there was no extended pressurization. Soft substrates used within the mechanical adhesion testing had been freshly ready inert supplies or collected tissue (<1 h post-euthanasia) with out floor washout, tissue trimming or liquid removing. Soft substrates had been solely partially secured, with the 4 corners of the tissue squares glued to the holder to permit for pure sliding and dynamic morphing. All units and adhesive supplies used within the adhesion take a look at had the identical adhesion floor space of roughly 250 mm², which equals the adhesion floor space of MUSAS. The adhesion stress was calculated by dividing the drive by the adhesion floor space. Extended dialogue of the experiment set-up and measurement particulars will be present in Supplementary Text 2, Extended Data Fig. 2, and Supplementary Figs. 2 and 4–11.

Numerical simulation

Solid–fluid interactions between water, tissue and MUSAS

Finite component evaluation was carried out to characterize the hydrostatic differentiation of the adhesive disc of remoras. Commercial software program Abaqus 2021 (SIMULIA) was used for the examine. The remora disc phantom units had been assumed to be composed of Ecoflex 0030, with a density of 1.07 g cm⁻³, a Young’s modulus of 125 okPa and a Poisson’s ratio of 0.49 (ref. ⁴⁸). The bodily parameters of abdomen tissue embrace a density of 1.088 g cm⁻³, a Young’s modulus of 700 okPa and a Poisson’s ratio of 0.49 (ref. ¹⁰). We used coupled Eulerian–Lagrangian methods to mannequin the stable–fluid interactions between tissue, machine and water. Water was handled as a Newtonian laminar movement, with a density of 0.997 g cm⁻³ and a dynamic viscosity of 8.90 × 10⁻⁴ Pa s. The simulation was configured in order that the mimicry remora suction cups descended at a relentless pace of 0.3 mm s⁻¹ till they touched the abdomen tissue submerged in water. Solid–stable interactions had been modelled as laborious contact for regular behaviour, with tangential behaviour modelled utilizing a penalty technique with a friction coefficient of 0.02. Details of calculation of relative vacuum ratio V_r are laid out in Supplementary Information.

Self-actuation of nitinol lamella of MUSAS

The self-actuation of nitinol lamellae in MUSAS in response to temperature stimuli was simulated utilizing the business software program COMSOL Multiphysics 6.2 (COMSOL). The Lagoudas phenomenological inelastic constitutive mannequin for SMAs (nitinol), together with the related materials properties, was carried out to simulate the section transformation of the SMAs⁴⁹.

Synthesis of supplies for in vitro adhesion characterization

Polyacrylamide–alginate powerful hydrogel

The powerful hydrogel was composed of alginate and polyacrylamide (pAAm) double networks (pAAm–alginate) crosslinked by quite a few dimethacrylate monomers. The synthesis was based mostly on a beforehand reported protocol¹⁴. Hydrogel fabrication was achieved by way of one-step aqueous free-radical polymerization. In transient, in a 50-ml tube, 30 ml phosphate buffer (100 mM, pH 7), 3.6 g acrylamide, 600 mg sodium alginate (medium viscosity), 1.3 mg N,N′-methylenebisacrylamide and 10 mg ammonium persulfate had been added and vortexed to type answer A (pAAm–alginate).

Calcium sulfate was added to deionized water and stirred to type a homogeneous suspension. Then, 5 ml pre-gel answer A was loaded right into a 5-ml syringe (diameter 12 mm), and 120 mg calcium sulfate and 29.4 mg N,N,N′,N′-tetramethylethylenediamine had been loaded into one other 5-ml syringe. The two syringes had been linked with a syringe connector and blended over ten occasions. Afterwards, the gel answer was poured right into a glass mould coated with a 3-mm-thick glass plate. After 12 h, the hydrogel was able to be faraway from the mould. In explicit, for exams leveraging powerful hydrogel as gentle substrates to guage adhesion efficiency of MUSAS (Fig. 3c–g), air bubbles had been manually launched in the course of the preparation process to create a porous and tough substrate floor.

NHS–EDC bridging polymers for powerful hydrogel

The bridging polymers, which embrace chitosan, polyallylamine, gelatin and polyethyleneimine, had been ready following a beforehand reported protocol¹⁴. Sulfated NHS and EDC had been used as coupling reagents. Right earlier than the adhesion of the powerful hydrogel to tissue surfaces, the bridging polymers and coupling reagents had been blended to realize a focus of 12 mg ml⁻¹ of each NHS and EDC within the bridging polymer options. A 250 μl blended answer was then smeared onto the floor of the powerful hydrogel, adopted by instant compression of the powerful hydrogel to the tissue surfaces for 3 min earlier than the adhesion take a look at.

Carbopol powerful hydrogel

To put together Carbopol powerful hydrogel, 100 mg Carbopol 971P was dissolved within the pAAm–alginate answer described within the pAAm–alginate powerful hydrogel protocol. The remainder of the preparation procedures had been similar to these used for the pAAm–alginate powerful hydrogel. A 3-min compression of the Carbopol powerful hydrogel to the tissue surfaces was utilized earlier than the adhesion take a look at.

SEBS thermoplastic elastomer

The SEBS substrate was ready by mixing 10 ml toluene with 4 g SEBS (Kraton G1645). After dissolution, the ink was homogenized utilizing a speedmixer (FlackTek 330) at 2,000 rpm for five min. The ink was then drop-cast onto chrome steel Petri dishes to realize a movie thickness of three mm. The substrate was dried in a fume hood for two h and cured at 60 °C for 1 h. After curing, the stretchable SEBS substrate was peeled off from the petri dish.

In vivo testing

All swine research had been permitted by and carried out in accordance with the Committee on Animal Care on the Massachusetts Institute of Technology. All fish research had been permitted by and carried out in accordance with the Institutional Animal Care and Use Committee of Boston College. Additional particulars and prolonged dialogue will be present in Supplementary Text 6.

Fabrication of MUSAS fish tag with a temperature sensor

A ProtoLaser R4 laser cutter (LPKF) was used to sample the highest and backside 35-μm-thick copper claddings of an RT/duroid 6010.2LM laminate (Rogers), which contains a 0.635-mm-thick ceramic–PTFE composite dielectric core. The laser was then employed to ablate a by way of gap via the substrate, establishing {an electrical} connection to the highest copper layer utilizing a soldered 32 AWG feedthrough wire. The antenna geometry, measuring 12 mm × 6 mm, was laser-cut from the patterned RT/duroid laminate. A Magnus S3 tag chip (Axzon) was mounted onto the bottom airplane with a non-conductive ultraviolet-cured epoxy adhesive. Chip-to-antenna interconnections had been made by way of thermosonic gold ball bonding, strengthened with silver conductive paste and cured at 65 °C for 40 min utilizing a C174740 Mech-El MEI Marpet 1204B wire bonder. A superstrate with hatched high and backside copper claddings on an RT/duroid 6010.2LM core was then fabricated and affixed to the highest floor of the antenna with an adhesive layer. Finally, the assembled antenna was packaged onto the remora machine by underfilling and encapsulating the underside and sides with 5055 UV Curing Silicone Adhesive (Henkel Loctite) epoxy resin. See Supplementary Information for particulars of in vitro and in vivo exams on a tilapia mannequin.

Fabrication and analysis of MUSAS impedance biosensor for detecting gastroesophageal reflux

The impedance sensor was laser-cut on single-sided versatile copper laminate (Pulsar Professional fx FR4 5 mil ½-oz copper). Each of the 13 traces had been 75 μm huge and 75 μm aside from one another. Alignment of the reduce was carried out with digicam on a R4 laser (LPKF). Parylene coating was then carried out on a PDS 2010 Labcoater (Special Coating System), to forestall pointless shortcut of the impedance sensor. A Kapton tape masks (0.03-mm thickness, McMaster-Carr) was reduce with the R4 laser to cowl the impedance sensor traces in the course of the parylene-coating process. After completion of the parylene coating, the Kapton tape masks was peeled off. To enhance the electrical conductivity and sensitivity of the traces of the impedance sensor, electron beam evaporation was carried out to deposit gold on the copper traces. The deposition process included coating a 10-nm adhesion layer of titanium after which 200-nm gold and was carried out on EE-4 E-beam evaporator (Denton).

For impedance measurement of tissue, we used a ISX-3 Impedance Analyzer (Sciospec) geared up with Sciospec software program v2.0.8 for four-point impedance measurements. The measurements spanned frequencies between 100 Hz and 1 MHz. For the in vivo examine, MUSAS impedance biosensors had been positioned by way of an over tube into the oesophagus of anaesthetized feminine Yorkshire pigs (70–95 kg) and self-adhered via the contraction of the oesophagus. A gastroesophageal reflux mannequin was created by utilizing an endoscope to periodically spray gastric fluid, obtained from the pig itself, into the oesophagus.

Fabrication of gastric resident dosage types for sustained launch of CAB

The polycaprolactone (PCL) matrices containing CAB had been ready with soften mixing. The drug stability of the matrices was confirmed in earlier analysis⁵⁰. Specifically, PCL and CAB had been weighed in a 10-ml glass vial. The vial and a destructive mould for the PCL patch had been then heated on a warmth plate (Thermo Scientific) to 75 °C. The matrices had been melted and nicely blended earlier than transferring to the mould. After cooling to room temperature, the PCL–CAB patches had been adhered onto fabricated MUSAS with ultraviolet-cured epoxy (Henkel Loctite).

The Ecoflex matrices containing CAB had been instantly ready by mixing CAB with Ecoflex 0030 for moulding the suction cup of MUSAS.

In vitro and in vivo analysis of pharmacokinetics of CAB

In vitro analysis of the drug launch of CAB was carried out in a launch medium of simulated gastric fluid containing 5% w/v Tween 20 surfactant (Thermo Scientific). The drug-loaded MUSAS had been positioned in 10 ml of the discharge medium in a 37 °C incubator (New Brunswick Innova 44/44R) shaking at 250 rpm. At 2 h, 6 h, 1 day, 2 days, 3 days, 5 days and seven days, 1 ml of the medium was sampled and saved at −20 °C till high-performance liquid chromatography (HPLC) evaluation, as described later. During every sampling, the remaining launch medium was changed with contemporary medium.

In vivo pharmacokinetics had been carried out in feminine Yorkshire pigs (55–95 kg) in an unblinded style. MUSAS loaded with 40% CAB within the PCL matrices had been both dropped via an over tube or endoscopically positioned within the abdomen of anaesthetized Yorkshire pigs, which had been fitted with ear catheters. The pigs had been fed and monitored day by day within the morning and afternoon with a laboratory mini-pig grower food regimen, 5081, together with noon snacks of fruit and veggies. At 2 h, 6 h, 1 day, 2 days, 3 days, 5 days and seven days, 5 ml of blood was sampled by way of the ear catheter. The blood samples had been centrifuged for serum separation at 4,000 rpm (Eppendorf 5810r) for 10 min after which saved at −80 °C till bioanalysis, as described later.

Synthesis and in vitro characterization of mRNA nanoparticles

Messenger-RNA-loaded lipid nanoparticles (LNPs) had been made utilizing a typical four-component lipid combination. Specifically, ethanol-based options of SM102, 1,2-distearoyl-sn-glycero-3-phosphocholine, ldl cholesterol and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[(methoxy(polyethylene glycol)−2000) (ammonium salt) were mixed to achieve a molar ratio of 50:10:38.5:1.5. Firefly luciferase mRNA (Trilink) was dissolved in 10 mM citrate buffer, pH 3. The lipid solution was mixed with mRNA solution at a volume ratio 1:3 to achieve a SM102/mRNA weight ratio of 12.86. The LNPs were placed on ice for 10 min to complete the complexation. For measuring the activity of fresh LNPs, the LNPs were diluted in appropriate media and added to the cells. For measuring the activity of freeze-thawed LNPs, the LNPs were diluted with 200 mg ml⁻¹ sucrose in 10 mM citrate buffer at a volume ratio of 1:1 and incubated at 4 C for 1 h. The LNPs were then frozen at −20 °C for 1 h. The LNPs were then thawed, diluted with complete media and added to the cells.

In vitro studies were conducted with primary human oral epithelial cells (Celprogen). Cells were seeded in a 96-well plate overnight. On the next day, LNPs (fresh, freeze-thawed with sucrose solution and freeze-thawed without sucrose solution) were added to the cells to achieve an mRNA concentration of 1 μg ml⁻¹. The cells were incubated with the LNPs overnight. Transfection efficiency was measured using the SteadyGlo assay using the manufacturer’s recommendations.

Administration of mRNA nanoparticles to swine

The mRNA LNPs were prepared as described for the in vitro studies. The firefly-luciferase-mRNA-loaded LNPs diluted in sucrose solution were transferred into the MUSAS and frozen at −20 °C for 1 h. Each MUSAS was loaded with 12.5 μg of mRNA. The MUSAS was applied to the pig buccal and pharynx manually with surgical forceps 8 h to 24 h before euthanasia. Immediately after the pig was euthanized, the site of administration and control were collected and placed in cold DMEM media (Thermo Fisher Scientific) containing 10% fetal bovine serum. Within 30 min of pig euthanasia, the tissue was immersed in 0.3 mg ml⁻¹ potassium luciferin solution (Gold Biotechnology) in PBS without calcium and magnesium. For increased diffusion of the substrate, luciferin solution was injected into the swine oesophageal tissue after submersion and before imaging. Bioluminescence was captured over a 30-min span with an in vivo imaging system (PerkinElmer) to capture bioluminescence, via LivingImage software 4.8.2 (PerkinElmer). The luminescent images were taken using Field of View D, automatic exposure time, medium binning, F/Stop = 1, and taking images every minute.

Imaging

Profilometry

Sample surface roughness and three-dimensional reconstructions were quantitatively determined by an optical VK-X3000 profilometer (Keyence) using the ×5 and ×10 lens. The ring and lens lighting were used together and set to maximum intensity. To compare the smoothness across the specimens, the surface roughness parameter S_a (areal average roughness) was evaluated across representative fields of view using the included VK viewer software v2.2.0.135 (Keyence).

Confocal microscopy

Confocal microscopy to measure hydrostatic differentiation of MUSAS’ underwater adhesion

A near-infrared fluorescent dye solution, Sulfo-Cyanine5.5 (Cy5.5) (Thermo Fisher Scientific), was prepared at a concentration of 0.01 mg ml⁻¹ (40 ml) to stain 1 ml of water spread onto a microscopic glass slide for confocal microscopy. Confocal microscope imaging was performed on MUSAS before and after adhesion to the water-rich glass slide, using a 635-nm laser line with a measuring depth of 400 µm, with a FV1200 Laser Scanning Confocal Microscope (Olympus).

Firefly-luciferase-mRNA transfection visualization in pharyngeal tissue using MUSAS via immunofluorescence confocal microscopy

Fixed oesophageal pig tissues transfected for firefly luciferase expression with MUSAS along with untransfected controls were stained using DAPI (Thermo Fisher Scientific), as well as firefly luciferase polyclonal antibody primary antibody (Thermo Fisher Scientific) with a 1:2,000 dilution ratio, conjugated with goat anti-rabbit IgG (H+L) cross-adsorbed, Alexa Fluor 647 secondary antibody (Thermo Fisher Scientific) with a 1:500 dilution ratio. Five immunohistochemistry (IHC) slides per block of pig oesophageal tissue were prepared for imaging. Fluorescence images were taken with a FV1200 Laser Scanning Confocal Microscope (Olympus) with two channels: DAPI (405-nm laser line) and AlexaFluor647 (635-nm laser line). Images were taken using a ×10 objective. All images were processed using Fiji (Image J 1.54) software.

Scanning electron microscopy

Before scanning electron microscopy, all samples were mounted inside a copper vise and then subjected to vacuum and liquid nitrogen inside the electron microscope vacuum cryo manipulation tool (Leica). Using the portable temperature-controlled vacuum arm, each cryogenic sample was transferred to the ACE 600 high-vacuum sputter coater (Leica), which cryo-fractured a fresh cross-section and sputter-coated approximately 10 nm of platinum. This conductive coating prevented excessive surface charging artefacts during scanning electron microscopy imaging. Using the same portable vacuum arm, each cryogenic sample was transferred to the Gemini 360 SEC scanning electron microscope (Zeiss). While under high vacuum, using the secondary electron detector, low-voltage imaging (2–3 kV) was used to prevent damage from electron bombardment while providing high surface detail. Typical imaging conditions would also entail a probe current near 2 nA and a working distance between 6 mm and 8 mm.

Fluorescence microscopy

An ex vivo study was performed to evaluate the bioavailability of a nanoparticle formulation delivered through MUSAS; 215 µl of fluorescent polystyrene nanoparticles were prepared with FluoSpheres Polystyrene Microspheres (Thermo Fisher Scientific) and stored at −80 °C. The fluorescent nanoparticles were then subcutaneously injected, smeared with a pipette or delivered via MUSAS to freshly collected porcine oesophagus tissue. The oesophagus tissues, including a negative control, were resected, rapidly frozen in OCT gel (Agar Scientific) using liquid nitrogen and sectioned using a cryostat microtome. Subsequently, the sectioned tissues were imaged using an EVOS fluorescence microscope (Life Technologies) with excitation and emission wavelengths of 580 nm and 605 nm, respectively.

Bioanalytics

In vitro HPLC

Dissolution samples in simulated gastric fluid were directly analysed by HPLC and ultraviolet detection on a 1260 Infinity system (Agilent Technologies). Samples were injected at a volume of 2 µl onto an Agilent EC-C18 Poroshell column (3.0 × 50 mm, 2.7 µm particle size d_p) held at 25 °C. The mobile phase consisted of 0.1% formic acid in water (v + v, A) and acetonitrile (B), pumped at 800 µl min⁻¹ with a gradient programme of: 0 min, 5% B; 8 min, 60% B; 8.1 min, 95% B, over 10 min and with an equilibration time of 2 min. Eluite was quantified with a diode array detector at CAB’s local absorption maximum at 258 nm in the ultraviolet region at 5 Hz.

In vivo liquid chromatography–mass spectrometry

Porcine serum samples were prepared via protein precipitation at a 1:3 volume ratio of serum to acetonitrile with bictegravir or verapamil as an internal standard. The liquid chromatography–mass spectrometry method used was validated according to FDA recommendations, on high-performance liquid chromatograph’s coupled to Agilent 6495 triple quadrupole mass spectrometers in positive mode. Concentrations were calculated based on the linear regression of CAB response relative to internal standard response.

Specifically, samples were injected at a volume of 10 µl onto the same column using the same mobile phase above, but without temperature control. The gradient programme used a 400 µl min⁻¹ flow rate with 0 min, 5% B; 0.5 min, 5% B; 4 min, 95% B, with a run time of 5 min and equilibration time of 2 min. The Agilent Jet Stream source used the following parameters: gas temperature, 200 °C; gas flow, 14 l min⁻¹; nebulizer pressure, 20 psi; sheath gas temperature, 250 °C; sheath gas flow, 11 l min⁻¹; capillary voltage, 3,000 V; nozzle voltage, 1,500 V; high radiofrequency, 150 V; low radiofrequency, 60 V. The same transitions for CAB above were used, except both at 38-V collision energy. Verapamil was used as an internal standard, quantified with the transition from 455.1 m/z to 303.1 m/z at 35 V, and qualified with the transition from 455.1 m/z to 303.1 m/z at 40 V.

Histology fixation

Unless specified, histology samples were fixed with 10% formalin for 24 h and then stored in 70% ethanol before embedded in paraffin for histopathological analysis.

Statistical quantification and analysis

Statistical quantification and analysis were performed via Prism 9.3 (GraphPad). All error bars represent standard deviation (s.d.), except in pharmacokinetics studies conducted on independent Yorkshire pigs, where standard error of the mean (s.e.m.) was used to measure the population variability. For box plots, the box represents the median and the Q1 and Q3 quartiles (25% and 75%), and whiskers extend to the maximum and minimum values. For violin plots, the density distribution is calculated using the often-used Gaussian kernel density estimator, with dashed and dotted lines representing the median and the Q1 and Q3 quartiles (25% and 75%), respectively. The theoretically optimal Gaussian kernel density estimator, which minimizes the mean integrated squared error, is calculated with a bandwidth given by (1.06widehat{sigma }{n}^{-1/5}), where (widehat{sigma }) is the standard deviation and n is the sample size. Student t-test and analysis of variance (F-test) were performed to compare differences between two groups, and among three or more groups, with details defined in the legends of relevant figures. We chose several P values to systematically evaluate the statistical significance, including P ≤ 0.05 (*) as the entry level of significance, P ≤ 0.01 (**) for highly significant, P ≤ 0.001 (***) and P ≤ 0.0001 (****) for extremely significant. The number of independent experiments of replicates and definition of significance level are further elaborated in each figure, figure caption and relevant methods section where statistical quantification and analysis was performed.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.