Jet physics and theoretical model
To study the influence of jetting parameters (ampule pressure, jet diameter and jet velocity) on tissue deposition, a theoretical model was derived (Extended Data Fig. 1a and Supplementary Methods 1). The jetting apparatus used to investigate jet delivery into ex vivo tissue is described and characterized in Extended Data Fig. 1b–g. The main components of this apparatus were a handheld jetting device for generating jets, and a piezoelectric force transducer (9215 A with 5165A4KH10 LabAmp amplifier; Kistler Instrument) and software (Network Setup Wizard; Kistler Instrument) for measuring jetting impingement force (at 200 kHz sampling frequency). Based on previous literature and theory, variation of nozzle diameter and ampule pressure has the highest impact on penetration in soft tissue20,21,22. A subset of 4 exemplary jetting force profiles with a 257 µm nozzle is shown in Extended Data Fig. 1c. From these force profiles the experimental period of the jetting event, T (Extended Data Fig. 1d), the experimental steady-state jetting force, Fj,exp (Extended Data Fig. 1e), and the experimental steady-state jetting power, Pj (Extended Data Fig. 1f) were extracted. An expanded set of results, including all nozzle–pressure permutations, is shown in Extended Data Fig. 2a–g and the procedure is described in Supplementary Methods 2 and 3.
To examine the effect of fluid viscosity on jet performance the ampule pressure and diameter were fixed and viscosity was measured23,24,25 between 1 and 219 cPa. The system pressure factor dropped significantly—from a mean of 0.82 to a mean of 0.40 (Extended Data Fig. 2h), while no significant changes were observed for the CT contrast fluid (Extended Data Fig. 2i,j).
Porcine tissue excision and processing methods
Female Yorkshire pigs, 3–5 months old and weighing 35–70 kg were used. After euthanasia (procedure described below), a midline incision allowed access to the abdominal cavity. Stainless steel pean forceps were placed at the oral and aboral part of the organ of interest. The organ was then separated from the omentum and removed from the carcass with a scalpel. Precautions were taken to ensure that the tunica serosa and other important tissue layers were kept intact. After excision, the tissue was placed in a plastic bag kept on ice and shipped to the laboratory where ex vivo injections were performed on the same day.
Bulk sections of tissue ranging from 10 to 35 cm in length (depending on the organ) were extracted from the organs with Mayo scissors and placed into beakers filled with phosphate buffer solution (PBS), pH 7.4. For both the cheek and oesophagus, tissue from the entire organ was used. For the stomach, only tissue from the antrum/lower corpus was used. For the small intestine, only jejunal tissue at least 20 cm from the pylorus was used. For the colon and rectum, distal sections were used. An image of a bulk section of rectal tissue is shown in Extended Data Fig. 3b.
Smaller sections of tissue (approximately 4 × 4 cm, the final sample size) were extracted from the bulk with scissors. These samples were further cleaned in a separate wash beaker with PBS to remove any remaining chyme or debris. Finally, immediately before injection, the tissue samples were placed on a piece of foam in a 6.5 × 4.7 cm plastic dish. In total, the time between euthanasia of the animal and injection experiments was 2–3 h.
Ex vivo jet injection study with porcine tissue
Prior to each set of ex vivo experiments, the polycarbonate ampules were lubricated to improve sliding of the plunger. To accomplish this, the ampules were dipped in a mixture of 5% hexamethyldisiloxane, 95% deionized water solution, then left to dry overnight. Before each injection, the ampule was filled with 232 µl of an aqueous mixture containing 30% micro-CT contrast agent (Iomeron 350 mg ml−1; Bracco), 1% green tissue dye (WAK-HM-G-1/60, WAK-Chemie Medical) and 69% deionized water. The ampule was then mounted onto the handheld jetting device and the input pressure of the device was adjusted to the desired level with a digital pressure regulator. An example image of the device positioned for injection is depicted in Extended Data Fig. 3c,d. Finally, the triggering sleeve was actuated, causing the jetting injection event. Between two and eight replicates were performed for each pair of pressure-diameter inputs, with certain sets of shots performed on the same tissue sample (space permitting). All experimental points can be found in Supplementary Table 3.
Computed tomography scanning (Phoenix Nanotom M micro-CT; GE Inspection Technologies) was used to evaluate each sample. The stage and detector were positioned at travel distances of 130 mm and 300 mm from the X-ray source, respectively. Background detector calibrations were performed before each scan series. Scans were performed at a tube voltage of 100 kV, radiation intensity of 100 μA and a target scan time of 6 min.
All scans were performed 10–15 min after injection. After each scan, the tissue samples were placed in plastic histology cartridges (Extended Data Fig. 3e) and stored in 10% formalin solution. Although it was not possible to maintain the original structure of the depot with this storage method due to diffusion and dissolution, the use of green tissue dye in the payload fluid dye enabled us to later identify the tissue layer(s) in which the depot resided. The entire injection and scanning procedure is depicted in Extended Data Fig. 3a.
Volumetric diffusion, standoff distance and jetting angle ex vivo studies
Using the methods described above, we also performed a set of studies to determine the impact of volumetric diffusion within tissue, standoff between nozzle and tissue, and angle of incidence between the jet and tissue on delivery characteristics.
To evaluate the effect of diffusion on the apparent volume of fluid in tissue after injection we performed a set of calibration experiments and found that the apparent volume of fluid in tissue increased at a rate of 0.5 ± 0.1% per min (95% confidence interval). Results from this diffusion study are shown in Extended Data Fig. 3g. GraphPad Prism (GraphPad Software) was used to calculate the linear rate of change of volume with 95% confidence (the intercept was set at 0, 0). Furthermore, the upper error boundary from this analysis was used to make conservative corrections to VDE estimates in subsequent analyses. Photos and diagrams showing the injection, scanning and segmentation processes are shown in Extended Data Fig. 3b–f. Additional scans from jejunal and stomach tissue are shown in Extended Data Fig. 4b–e. Next, each set of injections was semantically categorized as luminal, submucosal or intraperitoneal based on where most of the micro-CT scanned fluid resided on average.
The results of the nozzle-to-tissue standoff and angle study are shown in Extended Data Fig. 3h,i. For the angle study, a constant standoff of 5 mm was chosen.
Histological imaging of samples from ex vivo injection studies
Tissue samples were fixed in 10% formalin solution for a minimum of 24 h. They were then processed in an ASP300S fully enclosed tissue processor (Leica Biosystems), embedded in paraffin wax and cut on a microtome in 4 µm thick sections. From there, sections were mounted on glass slides and stained with haematoxylin and eosin. Finally, the slides were scanned on a NanoZoomer S60 Digital Slide scanner (Hamamatsu Photonics) at 40×. Results from this process can be seen in Fig. 2b and Extended Data Fig. 5.
Processing of volumetric data from ex vivo injection studies
Acquisition, processing and reconstruction of images were performed with Phoenix Datos|x (GE Measurement & Control). Examples of processed scans are shown in Fig. 3a and Extended Data Fig. 4b–e. Once the scanning and reconstruction was complete, a segmentation of regions of the contrast fluid that were on top of, beneath and contained inside the tissue was performed. A simplified representation of the segmentation process is shown in Extended Data Fig. 3f. A standard Student’s t-distribution was used to determine 95% confidence intervals for the submucosal volume and VDE. Statistical analysis was applied only after all other quantitative analyses—including segmentation and diffusion correction—were complete. Further details on the processing of volumetric data are provided in Supplementary Methods 4.
Axial endoscopic device prototype MiDeAxEndo
The MiDeAxEndo prototype can deploy a therapeutic dose via the working channel of an endoscope. A schematic of all its components is shown in Extended Data Fig. 6a. The MiDeAxEndo system consists of a nitrogen pressure tank, a pressure controller, an air-tight 18 ml polycarbonate drug reservoir with an internal plunger separating pressurized gas from the drug product, a high-speed valve operated by a microcontroller and 2.8 m of polyether-ether-ketone (PEEK) tubing capped with a computer numerical control (CNC) machined PEEK adapter and stainless steel nozzle. Polytetrafluoroethylene (PTFE) sealing tape was used at all junctions to ensure air-tight connections. The stainless steel nozzle was assessed to have a 254 µm diameter with scanning electron microscopy. Jetting is conducted by loading the reservoir and tubing with the drug product, applying the desired pressure, and then opening the high-speed valve to initiate jetting. Operating valve times were characterized for each jetting pressure such that 200 µl was ejected. In Fig. 4a,b, valve times were 120 ms, 97 ms, 77 ms, 75 ms and 50 ms for the 3.5 bar, 5.0 bar, 9.4 bar, 11.3 bar and 24.5 bar jetting pressures, respectively.
Radial endoscopic device prototype MiDeRadEndo
In this study, a spring-loaded drug delivery device with a radially oriented jetting nozzle was utilized (Extended Data Fig. 6b). Activation of the device was achieved through a pneumatic tube, which also served as a tether for holding the device in place. The pneumatic tube was threaded through the endoscopic working channel for simultaneous delivery and positioning. The device, capable of ejecting a drug volume of 188 µl, was designed with an inflatable bag, featuring a diameter of 32 mm, to ensure optimal device-tissue proximity. Further details on the operation, design and assembly of MiDeRadEndo are provided in Supplementary Methods 5.
Autonomous intestinal device prototype MiDeRadAuto
The devices were used with the minor assistance from an endoscope. It is triggered via a dissolvable ‘polymer pellet’ that holds a detent pin captive. When the polymer pellet dissolves, the detent pin is released, allowing the spring to pressurize the ampule. The device is placed directly into the duodenum with an endoscope and allowed to trigger and pass without further assistance.
The nominal ampule volume and diameter for MiDeRadAuto are 200 µl and 6 mm, respectively. The stainless steel spring used in the device has an initial force of 70 N, resulting in corrected ampule pressures of 14 ± 1.5 bar (n = 48). The nozzles are oriented radially, and its diameter is 240 ± 10 µm (95% confidence, n = 8). The overall footprint of the device is depicted in Extended Data Fig. 6c–h. The device is made of machined polyoxymethylene (POM), PEEK and common metals. The piston seals are made from NBR-70. A photo of this device is shown in Fig. 1h and a section view is shown in Fig. 4f. Diagrams of MiDeRadAuto before and after assembly are shown in Extended Data Fig. 6c–h, and each of the assembly steps are described in Supplementary Methods 6.
Autonomous gastric device prototype MiDeAxAuto
The autonomous axial prototype (MiDeAxAuto) can deploy a therapeutic dose to the stomach via direct oral ingestion. The device relies on a sugar plug-based triggering mechanism which passively degrades in the stomach. The sugar plug is located directly in front of the nozzle orifice, so the payload fluid—which is constantly pressurized—cannot escape from the chamber. To prevent the payload fluid from degrading the sugar plug, the plug is separated from the orifice exit by a thin polymeric burst membrane. Thus, when the sugar plug dissolves enough so that it can no longer support the pressure exerted by the payload fluid, the burst membrane ruptures and the payload exits the device as a columnar jet. A diagram of the triggering mechanism is shown in Extended Data Fig. 9a.
As the stomach is a cavernous organ, we needed to implement an axial localization mechanism to align the jet. To achieve this, we drew inspiration from the methods used by Abramson et al.4 in their self-orientating system. MiDeAxAuto is 10.8 mm in diameter and 11.8 mm in height, with a centre of mass 3.5 mm above the bottom face. The diameter of the nozzle orifice is 298 ± 10 µm (95% confidence, n = 3). The nominal ampule volume and diameter are 80 µl and 7.9 mm, respectively. These ampule dimensions, combined with the pressure requirements for delivery in the stomach, mandated use of a relatively strong spring. We found that any sufficiently strong coil or disk spring’s mass interfered with the device’s self-orientation properties, so we decided to use compressed CO2 at its saturation pressure of 60 bar instead. Assuming a pressure factor of 70%, we deemed the aforementioned nozzle diameter to be most appropriate per our heat map results in Fig. 3c.
In order to both maintain self-orientation and safely contain the gas pressure, we chose to machine the bottom piece of the MiDeAxAuto from brass and its top piece from 7075 aluminium. The piston is made from POM, the seals from silicone rubber and the burst-film from 25 µm thick fluorinated ethylene propylene (FEP). A photo of this device is shown in Fig. 1i and a section view is shown in Fig. 4i. Diagrams of MiDeAxAuto before and after assembly are shown in Extended Data Fig. 7, and each of the assembly steps are described in Supplementary Methods 7.
Force profile studies with MiDeRadAuto and MiDeAxAuto
The aim of the force profile studies was to determine whether the devices we had fabricated produced sufficiently strong jets to deliver therapeutics. A Kistler 9215 A piezoelectric force transducer was again used to measure jetting force. Because MiDeAxEndo and MiDeRadAuto have identical ampules, springs and nozzles, we decided that it was only necessary to test the latter of the two devices. Diagrams of setups for force profile testing of MiDeRadAuto and MiDeAxAuto devices and their associated results are shown in Extended Data Fig. 8. Further details are provided in Supplementary Methods 8.
Formulation of therapeutic payloads
For the MiDeAxEndo and MiDeRadAuto studies—including controls—we used an insulin payload solution (pH 7.4) with the following concentrations: 244.2 µM insulin analogue (Novo Nordisk), 8.05 mM sodium phosphate dibasic, 1.96 mM potassium dihydrogen phosphate and 140 mM sodium chloride. This solution (200 µl) was added to the device, resulting in an insulin dose of 0.28 mg (8 U). For the siRNA, a payload solution with a concentration of 170 mg ml−1 in 10 mM phosphate buffer at pH 7.4 was used. The solutions were stored at 4 °C until they were used.
For the MiDeAxAuto studies—including controls—we used an insulin payload solution (7 < pH < 8) which was tailored based on the animal’s weight. To make this solution, between 10 and 20 mg of human insulin powder (Novo Nordisk) was weighed, and the exact mass noted. The insulin was then added into a 2 ml vial, followed (successively) by the following excipients: 600 µl 0.1 M sodium hydroxide, 0.5 mg PF68 (Sigma-Aldrich), 12.6 mg HEPES (Sigma-Aldrich), 300 µl 0.1 M hydrochloric acid and 100 µl deionized water. This solution was then diluted with deionized water in a separate vial based on the weight of the animal (35–80 kg) and payload volume of the device (80 µl) to achieve a dose of 0.25 U kg−1.
In vivo pharmacokinetic exposure studies
All in vivo pharmacokinetic studies were performed either at MIT or Novo Nordisk’s animal facilities by trained veterinary technicians and complied with relevant ethical regulations on animal research. Our procedures were reviewed and approved by review boards at each respective site (Committee on Animal Care at MIT and the Animal Experiment Inspectorate, Ministry of Justice, Denmark). For MiDeAxAuto, MiDeRadAuto and MiDeAxEndo (exclusively small intestine delivery) studies, female LYD (crossbred Landrace, Yorkshire and Duroc) pigs (body weight 50–70 kg; Novo Nordisk) and female Yorkshire pigs (body weight 35–80 kg; Tufts University, USA) were used, and for the gastric MiDeAxEndo study, female Beagle dogs, from 10 months old and weighing 8–13 kg were used. Pigs were placed on a liquid diet up to two days before each study and fasted overnight with the aim of reducing the amount of chyme and food debris in the gastrointestinal tract.
On the day of the study, anaesthesia was induced either with propofol intravenously (5 ml and supplemented as needed) or with a mixture of Telazol (tiletamine/zolazepam; 4–6 mg kg−1) and xylazine (2–4 mg kg−1) intramuscularly. Immediately after sedation, animals were transferred to an operating room where they were intubated and immediately provided with isoflurane (1.5–3% mixture with oxygen). The isoflurane was used throughout the entire duration of the study to maintain anaesthetization. During the study, vital signs were continuously monitored and noted at least every 15 min. Vital signs included breathing rate, end tidal CO2, oxygen saturation (SpO2) level in blood and pulse rate. The maximum period of anaesthetization was 4 h for non-terminal procedures and 8 h for terminal procedures. Whether a study was terminal was decided in advance and depended on external factors such as the age and weight of the animal.
During studies in which a device was deployed, a 100 cm over-tube (McMaster-Carr Tygon PVC tubing, 5/8 inch internal diameter, 13/16 inch outer diameter) was inserted into the oesophagus. This over-tube made it easier to transfer devices to and from the target location. The over-tube was removed immediately after device deployment was completed. For subcutaneous control studies, we used a 1 ml syringe with a hypodermic needle to administer the same dose as with the jet device to the subcutis on the animal’s belly or neck. For intraluminal or intragastric control studies, we used a Carr–Locke needle and endoscope to administer the same dose to the target organ.
Blood sampling was performed via a central line placed either in the ear (for non-terminal studies) or the femoral vein (for terminal studies). Though sampling frequency and duration varied depending on the test site and type of study, in general, samples were extracted at least every 15 min for the first 2 h and then at least every hour for up to 8 h. In the case of non-terminal studies, blood samples were drawn from the ear-catheter after the animal was recovered. Each blood sample was extracted from the catheter with a 3 ml syringe, then stored on ice in tubes pre-coated with EDTA. After collection, samples were centrifuged for 10 min at 1,500g. From there, 500–600 µl of plasma from each sample was extracted with pipettes and deposited into 750 µl Micronic tubes (Micronic). Plasma from each time point was stored in up to three separate aliquots at −80 °C until bioanalysis was performed.
In the case of terminal studies, pigs were euthanised with 80–100 mg kg−1 pentobarbital sodium intravenously or via an intra-cardiac injection. In all cases—before euthanasia—full anaesthetization was verified by the absence of pain-responsive reflexes (for example through a limb withdrawal test).
For the inactive GLP1 analogue tablet control study, 8 healthy male Beagle dogs (2.8–3.5 years old, 9.9–14.5 kg) were fasted overnight for ≥18 h, before receiving a single tablet orally the next morning along with 10 ml of tap water. Tablets contained 2.65 mg active pharmaceutical ingredient, 101 mg sodium N-[8-(2-hydroxybenzoyl) aminocaprylate] (SNAC), 66.7 mg nicotinamide, and 0.8 mg magnesium stearate. To reduce inter-individual variance among dogs, all dogs received a subcutaneous glucagon injection (3.2 nmol kg−1) 10 min prior to the tablet administration. Approximately 0.8 ml of whole blood was drawn into EDTA-coated tubes at the time points 5, 10, 15, 20, 30, 45 min, and 1, 1.5, 2, 4, 7, and 10 h, respectively, including one baseline sample immediately before dosing. 100 μl plasma from each sample was transferred into Micronic tubes and subsequently centrifuged at 4,000 rpm for 4 min at 4 °C.
In vivo deployment of MiDeAxEndo, MiDeRadEndo and MiDeRadAuto
All three devices were assembled and filled with the therapeutic payload within 10 min of deployment. The MiDeAxEndo device was then threaded through the endoscope’s channel and inserted into the over-tube. To reach the small intestine, the endoscope was further inserted into the pylorus and advanced 10–20 cm into the small intestines. During in vivo endoscopic operations, the jetting nozzle of the MiDeAxEndo device was positioned orthogonal to the tissue and secured in place by activating the elevator on the endoscope (to prevent MiDeAxEndo from recoil during the jetting event). At this point, the device was triggered and blood sample collection was initiated. After jetting, MiDeAxEndo was held in place for 10 s, then removed to allow for optical observation of the delivery site. The device was then withdrawn from the animal, disassembled, cleaned with isopropyl alcohol or soapy water and stored for future use. Results from MiDeAxEndo studies can be found in Fig. 4a,b for small intestine and stomach depositions, respectively.
The MiDeRadEndo was attached to the front of the endoscope via a rigid pneumatic tube which was fed through the working channel of the endoscope and allowed the operator to extend and retract the device as needed. As with MiDeAxEndo, an over-tube was used to reach the small intestine. To secure the device in place and establish its proximity to the tissue, the attached bag was inflated via a pneumatic tube threaded through the second working channel. A pressure of 25 mbar was applied for inflation. After optical confirmation of successful inflation, the device was activated to deliver the intended drug dosage, followed by deflation of the bag using negative pressure and retraction of the device.
The MiDeRadAuto device was gripped with endoscopic forceps and inserted into the intestine directly. The MiDeRadAuto device was then released from the endoscope, the endoscope retracted from the animal and the animal went through emergence from anaesthesia. Once the animal was fully awake the device actuated autonomously. X-rays were recorded 4 h after dosing to inspect the device’s spring and thereby determine whether it had triggered. All devices (n = 7) successfully activated in the small intestine. The above study was executed successfully seven times with MiDeRadAuto and an insulin analogue payload. Results from the MiDeRadAuto studies can be found in Fig. 4g.
To establish that there is no systemic exposure when peptides are delivered to the lumen of the small intestine (no disruption to the mucosal barrier) a negative control was performed. Eight female LYD pigs weighing 50–70 kg were anaesthetized according to the same anaesthetic protocol listed above. Once under deep and stable anaesthesia, an endoscope was navigated to the proximal small intestine of each animal. When in location, a primed tubing was fed through the working channel of the endoscope and 50 nmol (200 µl of 250 µM solution) of an insulin analogue was delivered to the lumen via a syringe attached to the primed tubing. The tubing was primed with the liquid insulin solution for 30 min before the procedure to mitigate any leeching of insulin into the tubing material during dosing. Thereafter the tubing was flushed with fresh insulin before each dosing. After dosing, the animals were recovered, and blood samples were taken for the next 4 h. The samples confirmed that there was no plasma exposure for each of the eight animals.
In vivo deployment of MiDeAxAuto
MiDeAxAuto devices were filled and pressurized within 10 min of deployment. The device was then dropped into the over-tube and advanced into the stomach with an endoscope. Once inside the stomach, the endoscope was used to monitor the device. An endoscopic image of a MiDeAxAuto in the stomach is shown in Fig. 4k. The device was continuously monitored until triggering occurred, at which point the time was marked and blood sampling was initiated. When triggering occurs, the device visibly jumps (because of recoil). After triggering, the device was retrieved with a Roth Net Standard Retriever (Steris). The device was then disassembled, and actuation of the piston was confirmed through inspection. Finally, the device parts were cleaned with isopropyl alcohol and stored for future use. The above study was executed successfully three times with MiDeAxAuto. Results from the MiDeAxAuto studies can be found in Fig. 4j.
For negative control studies with the MiDeAxAuto, we repeated the original procedure and dosage, except instead of using CO2 for pressurization, we used a weak spring (which generated pressures no more than 0.5 bar). All the above control studies were successfully executed three times.
Bioanalysis of blood samples
Blood samples from animal experiments were analysed for human insulin or insulin analogue in plasma using an AlphaLISA assay (Perkin Elmer), for the GLP1 analogue in plasma using LC-MS and for siRNA in plasma using the Meso Scale Discovery platform. Further details are provided in Supplementary Methods 14 for the human insulin and insulin analogue assay and in Supplementary Methods 15 for the siRNA quantification.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
