Materials

Poly(D-, L-lactic-co-glycolic) (PLGA, 50:50, viscosity 0.38 dL/g, Av. Mw 40,000) with an ester end group was purchased from Shandong Institute of Medical Instrument (Shandong, China). Rapamycin (Sirolimus, 98% purity), Methanol, acetonitrile (chromatographic grade), 10% sodium dodecyl sulfate (SDS) solution, and benzoyl peroxide were purchased from Macklin Biochemical Technology Co., Ltd. (Shanghai, China). The leadless pacemaker electrodes were provided by Shandong Rientech Medical Technology Co., Ltd. (Dezhou, China). Dichloromethane, ethanol absolute, and xylenes were supplied by Sinopharm Chemical Reagent Co., LTD. (Shanghai, China). Dulbecco's modified Eagle medium (DMEM), sterile phosphate-buffered saline (PBS), dimethyl sulfoxide (DMSO, ACS grade), penicillin–streptomycin, 0.25% trypsin, fetal bovine serum (FBS) was purchased from Thermo Fisher Scientific (Massachusetts, USA). The Cell counting Kit–8 was purchased from Proteintech Group, Inc. (Chicago, USA). Xylazine hydrochloride was purchased from Jilin Province Huamu Animal Health Products Co., LTD. (Jilin China). Methyl methacrylate and dibutyl phthalate were purchased from Shanghai Aladdin Biochemical Technology Co., LTD. (Shanghai, China). HE staining solution (kit) was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China).

Microbalance (MX5) was purchased from Meltlier-Toledo International Inc. (Ohio, USA). The vacuum-drying machine (DFZ) was bought from Lichen Instrument Technology Co., LTD. (Shanghai, China). The air bath thermostat oscillator (SHZ-82 A) was purchased from Shaoxing Supo Instrument Co., LTD. (Shaoxing, China). A digital ultrasonic cleaning machine was bought from Jeken Ultrasonic Equipment Co., LTD. (Dongguan, China). The HPLC instrument (LC-20 ADXR) was purchased from Shimadzu Corporation (Kyoto, Japan). Cell incubator and magnetic stirring were bought from Yiheng Scientific Instrument Co., Ltd. (Shanghai, China). The labeled instrument was purchased from BioTek Instruments, Inc. (Vermonte, USA). An electrochemical workstation was purchased from Ametek Inc. (Pennsylvania, USA). The embedding machine and the embedding machine cooling table were purchased from Rayto Life and Analytical Sciences Co., Ltd. (Shenzhen, China). Microtome was purchased from Leica (Wetzlar, Germany). Hard sectioning microtom was purchased from Xi'an Lanming Medical Technology Co., LTD (Xian, China).

NCTC clone 929 cells were bought from Rocell Life Science & Technology Co., Ltd. (Wuhan, China).

LPM electrode loaded RAPA–PLGA drug sustained-release system and morphological characterization

The RAPA–PLGA drug sustained-release system was put into the LPM electrode cavity after being manufactured using the organic solvent evaporation method. Science 200 mg of PLGA was weighed and added to 5 ml dichloromethane, and the mixture was further ultrasonically mixed with 300 mg of RAPA. The system was then constantly stirred for four hours using a magnetic stirring apparatus in the fuming cupboard in order to exclude volatile organic solvent fuming, further dehydration of the organic solvents,, the RAPA–PLGA drug sustained-release was injected into the LPM electrode cavity, and moved to the vacuum-drying oven for three days to exclude organic solvent completely. At this point, the RAPA–PLGA drug sustained-release system was loaded onto the LPM electrode; through the opening of the electrode cavity surface, contact between the drug sustained-release system and the local tissue can be achieved to play a role of drug sustained-release (Fig. 1A–C).

Fig. 1

Schematic of leadless pacemaker electrode tip architecture. A Schematic representation of leadless pacemaker, B The head of Leadless pacemaker, C The electrode cavity of leadless pacemaker

Three LPM electrodes with and without a drug sustained-release system loaded with RAPA–PLGA and were removed and vacuum-dried. An energy-dispersive spectrophotometer (EDS) was used to investigate the surface element distribution, and scanning electron microscopy (SEM) was used to examine the shape of LPM electrodes both before and after drug loading.

Determination of RAPADrawing of the standard curve of RAPA

10 mg of RAPA was dried under reduced pressure and precisely weighed to constant weight acetonitrile in a 100-volumetric flask. Acetonitrile was added to a constant volume of 100 ml. 100 μg⋅ml−1 RAPA standard sample stock solution was obtained after ultrasonic mixing. With the RAPA stock solution diluted to 10–100 μg⋅ml−1 (n ≥ 5) and 0.1–10 μg⋅ml−1 (n ≥ 5), high-performance liquid chromatography (HPLC) was used to quantify the absorption peak area. The concentration (C) served as the abscissa, the peak area of the sample (Y) as the ordinate, and the regression curve was created. The standard curve equation is generated using the linear regression equation.

Chromatographic conditions

WondaSil-C18 column (4.6 mm × 150 mm, 5 μm, S/N:6L5503-22), Mobile phase:methanol:water (v/v) = 75:25 [19], Detection wavelength: 277 nm, Velocity: 1 ml⋅min−1, column temperature: 50 ℃, Injection volume: 20 μL.

Method specificity

Taking the RAPA standard solution, respectively, the extracted solution of LPM electrode loaded with RAPA–PLGA drug sustained-release system, and blank 0.3% SDS solution 20 μL, using the methods shown in 2.3.2 sample, record chromatograph chart to examine the specificity of the detection method.

Precision, accuracy, and method recovery

Respectively prepared mass concentration of 50 μg⋅ml−1, 5 μg⋅ml−1 and 0.5 μg⋅ml−1 RAPA high, medium, and low are three kinds of reference substance solutions. The chromatographic conditions shown in 2.3.2 were used for sample injection analysis. Intra-day precision (intra-RSD) and accuracy were calculated by repeated measurement three times within one day, and inter-day precision (inter-RSD) and accuracy were calculated for three consecutive days. To figure out the RAPA content, the measured peak area was inserted into the standard curve. The computed value was then divided by the known content to determine the required recovery rate.

Stability of RAPA in the release medium

Three samples of 0.3% SDS solution with RAPA mass concentration of 5 μg⋅ml−1 were prepared and placed at (37 ± 1) ℃, the chromatographic conditions as shown in 2.3.2 were used for sample analysis at 1 day, 3 days, 5 days, and 8 days, respectively. In order to determine the RAPA content and recovery rate, the measured peak area was substituted into the standard curve, and the stability of RAPA in a 0.3% SDS solution was examined.

Drug-loading rate and Encapsulation efficiency determination

Three aliquots of 5 mg RAPA–PLGA drug sustained-release systems prepared at different time points were individually solubilized with 20 mL acetonitrile under vortexing for complete dissolution. The solution was placed in an air bath with a constant temperature oscillator at 37 ± 1 ℃, shaken at 100r⋅min−1 for 6 h, mixed by ultrasound for 30 min, filtered by 0.22 μm filter membrane, and injected for analysis according to the chromatographic conditions shown in 2.3.2. The peak area was recorded and replaced into the linear regression equation to calculate the drug content of RAPA. DL rate and EE were calculated according to the following Eqs. (1 and 2).

$$\text() =\frac-\text}-\text}\times 100$$

(1)

$$}\left( \% \right) = \frac} - }}}}} \times 100$$

(2)

RAPA content loaded in the LPM electrode

The microelectronic balance MX5 was used to weigh the mass of 32 LPM electrodes loaded with the RAPA-PLGA drug sustained-release system before and after drug loading to determine the actual mass of the RAPA–PLGA drug sustained-release system. According to the determination of the DL rate calculated in 2.5, RAPA theory content in the electrode was figured out by Eq. (3) where RAPAthrory is the theoretical content of RAPA in the electrode, Mafter is the mass of electrode after loaded RAPA-PLGA, Mbefore is the mass of electrode before loaded RAPA-PLGA.

$$}_}}} = \, \left( }_}}} - }_}}} } \right)*}\left( \% \right)$$

(3)

The in vitro release of LPM electrode loaded with RAPA-PLGA drug sustained-release system

Three LPM electrodes loaded with RAPA–PLGA drug sustained-release system were used, and 5 ml of 0.3% SDS solution with pH of about 7.4 was used as the release medium of rapamycin (SDS surfactant was added to the water to regulate the drug release rate and meet the blood pH use environment). The in vitro release system of each group was placed in an air bath with a a constant temperature of 37℃ and oscillator; frequency of (75 ± 15) r⋅min−1. The release medium was replaced with a fresh medium at predetermined time intervals of (1, 3, 5, 8, 15, 22, 30, 37, 41, and 44 days) following sample extraction. RAPA content was determined using the chromatographic conditions described in 2.3.2. The cumulative release rate was calculated from Eqs. (4 and 5), where Qn is the accumulative drug release mass, Vi is the volume of the withdrawn medium, Ci is the drug concentration in the release medium at each time point, and W is the total drug content of the release sample.

$$}_}} \sum\limits_^} - 1}} V_ }$$

(4)

$$}\left( \% \right) \, = }_}} /} \times 00$$

(5)

Cell inhibition effect of LPM electrode loaded with RAPA–PLGA drug sustained-release system in vitro

The Cell Counting Kit-8 (CCK-8) assay was utilized to evaluate the cell inhibition effect of the LPM electrode loaded with the RAPA–PLGA drug sustained-release system on NCTC clone 929 cells. The experimental groups were as follows: the LPM loaded with RAPA–PLGA group (one LPM electrode loaded with RAPA–PLGA drug sustained-release system in 5 ml complete medium (DMEM medium with 10% FBS and 1% penicillin–streptomycin) with 0.2% dimethyl sulfoxide (DMSO), the LPM blank electrode group (one LPM blank electrode in 5 ml complete medium with 0.2% DMSO), positive control (0.5 ml DMSO in 4.5 ml complete medium), negative control (1 g high-density polyethylene in 5 ml complete medium), and blank control (complete medium only). Samples from these different groups were extracted and stored in a 4 ℃ refrigerator. The extracts collected on the first, third, and seventh days and then co-cultured with cells to assess the in vitro cell inhibition effect of the drug-loaded electrodes. For cell culture, the NCTC clone 929 cells were cultured in a complete medium and placed in a 37 ℃ incubator with 5% CO2. Cells in the logarithmic growth phase were detached using 0.125% trypsin to create a single-cell suspension with a cell density of 4 × 104 cells⋅ml−1. These cells were then seeded onto a 96-well plate with 100 μL per well. After 24 h of culture and reaching 90% confluence, the previous medium was removed, and the appropriate extract was added according to the respective group. Following a 48-h co-culture, a complete medium with CCK-8 reagent was added to each well. After 1–4 h of incubation, the optical density (OD) at 450 nm was measured using an enzyme-linked instrument and compared across groups. Cell viability (%) was calculated using the formula provided, where As represents the experimental well (medium containing cells and tested substances, CCK-8), Ab is the blank well (medium containing CCK-8 without cells or tested substances). Ac is the control well (medium containing cells and CCK-8 without the test substance).

$$}\left( \% \right)\, = \,\left( }_}} - }_}} } \right)/(}_}} - }_}} )\, \times \,100\%$$

(7)

In vivo experiments of electrodes loaded with RAPA-PLGAAnimals and groups

Kunming mice with SPF grade were selected for the in vivo experiment, which was purchased from Jinan Pengyue Experimental Animal Breeding Co., LTD., qualification certificate number: SCXK (Lu) 20,220,006, a total of 6 mice, 12 weeks old, half male and half female, weight 30 ± 5 g. The experimental animals were raised in Shandong Anzhong Medical Equipment Inspection and Testing Co., LTD., license certificate number SYXK (Lu) 20,230,010. The experiment was approved by the Animal Ethics Committee Ethics Review No. (04) 2023 of Shandong Anzhong Medical Device Inspection and Testing Co., LTD. The mice were randomly divided into two groups: the experimental group was implanted with LPM electrodes loaded with RAPA–PLGA drug sustained-release system, and the control group was implanted with blank LPM electrodes, with 3 mice in each group.

Implantation of LPM electrode and determination of local bioelectrical impedance

The implantation experiment was carried out after 2 weeks of adaptive feeding. Since the mice were anesthetized with xylazine hydrochloride, the electrodes were loaded with the RAPA–PLGA drug sustained-release system, and blank electrodes were implanted into the muscle tissue of the legs of the corresponding mice. An electrochemical workstation was used to measure the local bioelectrical impedance among them. WE + SE is the first electrode, and the second electrode is CE + RE. The electrode probe head adopts two stainless steel conductive wires with a diameter of 1 mm, and the middle one is fixed with non-conductive polyamide so that the spacing between the two electrode needles remains 0.8 cm (Fig. 4B). After implantation of two months, mice were anesthetized with xylazine hydrochloride, and the skin at the electrode implantation site was cut open. Then, electrode probes were pierced in on either side of the electrodes implanted, with a depth of 0.5 cm. The Versa Studio software was used to measure the, spectral range (30 kHz–1000 kHz) frequencies to measure and record the impedance spectra corresponding for subsequent analysis.

Sample collection and hematoxylin–eosin (H&E) staining

After the measurement of the lung’s local bioelectrical impedance of different electrodes, muscle tissue samples containing electrodes and internal organs, such as liver, heart, spleen, lung, and kidney, were collected. Specimens were fixed with tissue fixative wax for 48 h and dehydrated. The organ specimens were infiltrated and embedded with paraffin, and the local tissues at the electrode implantation site were infiltrated and embedded with methyl methacrylate, dibutyl phthalate, and benzoyl peroxide. Pathological sections from the above-embedded samples were made, and the tissue pathology status was observed through H&E staining.

Statistical analysis

SPSS 25.0 and GraphPad Prism 8 software were used for statistical analysis. Numerical variables with normal distribution were described as (\(\overline}}\)  ± S), and categorical variables were described as the number of cases (n) and its percentage (%). The relationship between RAPA concentration and peak area was analyzed using simple linear regression, and R2 ≥ 0.99 indicates a good linear relationship. The difference between LPM loaded with RAPA–PLGA drug sustained-release system and other groups was analyzed by one-way ANOVA, one-way ANOVA analyzed the other groups. The LSD-t test was used for pairwise comparisons with a statistical difference. p < 0.05 was considered as statistical significance.