In Vitro XDROP® Evaluation

To assess the properties of XDROP®, in vitro studies were conducted to evaluate several parameters, including the swelling coefficient, weight-averaged molecular weight, mechanical properties, injectability, and hydrogel morphology. Alginate samples were performed thrice to obtain the average value for each in vitro test. For swelling coefficient, the pre-weighed dry hydrogels were immersed in distilled water at 37 ℃, and their weight change was monitored at different intervals until the hydrogels showed complete dissolution. The swelling coefficient was defined as follows:

$$\mathrm\left(\mathrm\right)=[(\mathrm-\mathrm)/\mathrm]*100$$

The weight-averaged molecular weight distribution of alginate hydrogel was measured on an aqueous gel permeation chromatography, GPC (H20, Waters) system.

For mechanical properties, rheological experiments were conducted in an Anton Paar MCR 302 rheometer using a cone-and-plate geometry with a diameter of 25 mm. The sample was applied to the plate. The test parameters of the mechanical property were γ = 0.5%, f = 1 Hz, and T = 20 ℃.

EndoWings® Design Features

The EndoWings® catheter system was built with the assistance of Deke MedTech. It comprised three components: a guiding catheter (GC), an injection catheter (IC), and a stabilizer. The system layout and a schematic illustration of catheter manipulation are shown in Fig. 1A–C and Supplemental video.

Fig. 1

Illustrates the design and performance of the EndoWings® in vivo. A In this panel, ① is the 18-F guiding catheter (GC) and its controller (enclosed in a yellow frame). ② is the injection catheter (IC), specifically designed with a dedicated controller (enclosed in a green frame) to deliver therapeutic agents to the target anatomical site. The needle’s advancement and retraction are facilitated through a rotating wheel (enclosed in a blue frame), and the depth can be accurately specified via a distance monitoring window. The proximal end of the IC is connected to a syringe containing the treatment material and contrast medium. The stabilizer ③ enables the precise manipulation of the catheter and maintains stable endomyocardial contact during the procedure. Its primary function is to reduce the need for an interventional cardiologist to manually hold the catheter, allowing more accurate and consistent catheter movements. B This panel shows the IC, which features a double-lumen microcatheter with an outer diameter of 0.97 mm and an inner diameter of 0.81 mm and is equipped with a 25-gauge retractable needle. The design of this dual lumen microcatheter features a concentric circular structure with a side hole (SH) at the distal end of the inner lumen, facilitating the injection of contrast agent. C This section demonstrates the multiple orientation capacity of EndoWings®. The distal part of the GC can be oriented anteriorly or posteriorly. The distal portion of the IC is manipulated using a dedicated controller that enables deflection (straightening and bending) and steering (anterior to posterior) functionalities. Once the distal end of the catheter is directed toward the target endocardial site, the distal portion of the IC can be advanced to the LV wall. D Under fluoroscopy, the manipulated catheter tip can reach from the anterior to the posterior wall of the left ventricle and from the apical to the midventricular region, achieving consecutive circumferential LV-mid wall injections (enclosed in dots line frame). E The short axis of the echocardiogram clearly shows the relationship between the catheter and the wall of the left ventricle. F the deposition of the XDROP® (white arrows) in the myocardium. G Macroscopic evaluation shows the distribution of the injection site within the endocardium. H Six months post-injection, a gross anatomical examination reveals the retention of XDROP® (black arrows) in the myocardium. I Hematoxylin and eosin staining of the heart tissue showed slight multinucleate giant cell infiltration (black arrow) in the alginate hydrogel area (triangle) and no abnormalities in the surrounding myocardium (asterisk) 6 months after the procedure. J Masson trichrome staining showing encapsulation of the hydrogel (triangle) by fibrosis (black arrow). ALPM, anterolateral papillary muscle; LV, left ventricle; PMPM, posterior media papillary muscle; RV, right ventricle. SH, side hole

Injectability and Compatibility

The force used to inject the XDROP® via the EndoWings® was measured by the universal testing machine (UTM4103, SUNS, China). The test rate was 30 mm/min, equivalent to 1 mL/100 s. The maximum force per injection was recorded.

In vitro simulation tests were designed to measure the changes in mechanical properties of the XDROP® before and after delivery through the EndoWings®, including the change in swelling coefficient and the ratio of loss modulus to storage modulus (G''/G'). Scanning electron microscopy (SEM) examined the microstructure of the XDROP® before and after delivery through the EndoWings®.

XDROP® Aggregation Experiment

Swine EDTA plasma was activated by adding 25 μL of physiological saline containing CaCl2 (0.5 mol/L) to each milliliter of plasma [16]. Two PVC tubes (30 cm) with an inner diameter of 3 mm were used, and the circulation speed of the peristaltic pump was set at 64 mL/min to simulate the flow of human blood (approximately 15 cm/s). Three milliliters of XDROP® alginate hydrogel was added to 100 mL of simulated blood fluid, and the hydrogel was uniformly dispersed under low-speed magnetic stirring. A peristaltic pump circulated the entire solution containing the hydrogel for 1 h, 12 h, and 24 h. PVC tubes were wrapped with a polyetheretherketone (PEEK) filter (pore size 115 × 145 μm, similar to TriGUARD 3 cerebral protection device) to capture the potential embolic particles. The number of hydrogel clot particles in the filter was recorded and counted using a microscope (VMS322, Henglei Guangdian Technology Co., Ltd, Hangzhou, China).

Animal Experiment Design

The Animal Care and Use Committee of Xijing Hospital approved the animal experiments and protocols. Nine swine subjects (weight 78–90 kg) were divided into two groups. One group (n = 5) was sacrificed immediately after injection to evaluate the feasibility and safety of TEAi; the other group (n = 4) was sacrificed at 6 months to investigate the retention of XDROP® in the myocardium and alginate embolization to other organs.

The feasibility was defined as the ability of the EndoWings® to inject XDROP® into a specific LV endocardial area (from the apex to the mid-LV wall and from the anterior to the posterior wall) and achieve a consecutive circumferential mid-LV wall injection pattern. Safety was evaluated based on animal mortality and adverse events. In chronic animals, neurological events were recorded, and electrocardiographic monitoring was performed before, during, and after the procedure to assess conduction abnormalities and arrhythmias.

In Vivo Transcatheter Endomyocardial Alginate-Hydrogel Injection

The swine subject was sedated and placed on mechanical ventilation. Electrocardiograms were monitored throughout the procedure to detect arrhythmias. The left thoracotomy was performed, and the pericardial sac was removed to place the echo probe near the heart. An abdominal incision was made to expose the descending aorta. A needle was used to puncture the aorta and insert a 6-F sheath. A 0.035-inch guidewire (GW) was delivered into the LV, and the GC was advanced over the GW into the LV. The IC was inserted into the central lumen of the GC. Catheter manipulation was guided by echocardiography. The interventional cardiologist controlled the catheter and confirmed the tip position using echocardiography. The tip reached the LV wall and was detected by echocardiography in the presence of a transient premature ventricular beat. A retractable 25-gauge needle was extended approximately 5 mm into the myocardium and used to administer each injection lasting 30 s. The successful intramyocardial injection was marked by the appearance of a bright signal perpendicular to the needle tip relative to the LV wall. After the procedure completion, the catheter was removed, and the incisions in the chest and abdomen were closed.

Echocardiography

In the acute group, which was euthanized post-operatively, transthoracic echocardiography was performed within 24 h before the procedure (baseline echocardiogram) and immediately after the operation. For the chronic group under follow-up, echocardiographic evaluations were conducted within 24 h before the procedure (baseline echocardiogram) and at 6 months post-injection. Echocardiographic assessments were performed using a commercial echocardiography system (ACUSON Cypress™ System), including ejection fraction (EF), end-diastolic diameter (EDD), end-systolic diameter (ESD), and the ratio of early to late diastolic transmitral flow velocity (E/A ratio).

Gross and Histological Examinations

Animals in the acute-phase group were euthanized immediately after the injection. The hearts were extracted and dissected for direct visual confirmation of the injection sites. To evaluate chronic outcomes, the animals were sacrificed at 6 months. The LV was divided into slices along the short axis to assess alginate hydrogel retention. Histological analysis was performed on 0.5-mm-thick heart tissue slices stained with hematoxylin–eosin (H&E) and Masson’s trichrome to assess inflammation and fibrosis using light microscopy. Additionally, other organs were sectioned into 0.5-mm pieces for safety evaluation and examined by H&E staining.

Statistical Analysis

Normally distributed data are presented as mean ± standard deviation and others as median and interquartile range. Normality was tested with the Kolmogorov–Smirnov test. A two-tailed paired t-test or independent sample t-test was used for normally distributed data to assess differences between groups or time points. The Mann–Whitney U test or Wilcoxon signed-rank test was used for data not following a normal distribution. Categorical variables were described as n (%). p < 0.05 was considered to be statistically significant. Statistical analysis was performed with IBM SPSS Statistics for Windows, version 28.0 (IBM Corp., Armonk, NY, USA).