Materials and reagents
Calcium chloride (CaCl2), sodium citrate (Na3Cit), sodium phosphate dibasic (Na2HPO4), N-hydroxysuccinimide (NHS) and sodium hydroxide (NaOH) were obtained from the Aladdin Reagent Co. (Shanghai, China). N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC•HCl), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine conjugated polyethylene glycol acid (DSPE-PEG-COOH) and amine-cyanine 5.5 (Cy5.5) were purchased from Sigma-Aldrich Trading Co., Ltd. (Shanghai, China). TP-10 was obtained from MedChemExpress (Shanghai, China). CTP (CSTSMLKAC) was supplied by QYAOBIO (Hubei, China). Deionized water (18.2 MΩ cm) was used throughout all the experiments. All reagents and chemicals were of analytical grade and used as received without further purification.
Preparation and characterization of TP-10@CaP-CTP nanoparticles
The CaP nanoparticles were synthesized according to previous work9,24. Briefly, 5 mL of CaCl2 (100 mM) and 5 mL of Na3Cit (500 mM) were mixed, followed by the addition of 1 mL of TP-10 in DMSO (0.5 mg/mL). Then, 5 mL of Na2HPO4 (150 mM) was added prior to shaking for 30 min. Subsequently, the pH of the solution was adjusted to approximately 12 by adding NaOH (1.0 M), and the mixture was stirred for 1.5 h. The product (TP-10@CaP) was dialyzed against 500 mL of deionized water (with a MW cut-off of 3500 Da) for 3 days (replacing the water with fresh water every 24 h) and collected for further use. Moreover, the CaP nanoparticles were prepared by the same methods except that TP-10 was not added. The loading capacity and EE of TP-10 were determined by HPLC–MS/MS, and the detection conditions were the same as those used for the in vitro drug release experiment, as presented in the Supplementary Information.
For CTP (CSTSMLKAC, Supplementary Fig. 1) modification, TP-10@CaP was initially modified with DSPE-PEG-COOH. Five milligrams of DSPE-PEG-COOH was predissolved in DMSO and then added to the TP-10@CaP dispersion; the resulting mixture was stirred for 24 h. The TP-10@CaP-DSPE-PEG nanoparticles were collected by centrifugation and redispersed in 5 mL of deionized water. Then, EDC (35 mg) was added to the TP-10@CaP-DSPE-PEG dispersion, followed by the addition of NHS (53 mg) with ultrasonication. The mixture was stirred for 24 h to activate the carboxylic groups. Afterwards, 4 mL of CTP (2 mg/mL) was added. After gentle stirring overnight, the product TP-10@CaP-CTP was collected by centrifugation and washed with deionized water and ethanol several times. The amount of CTP bound and EE were evaluated by a similar procedure and finally determined by a BCA assay (Beyotime, #P0012) with a low limit of detection of 0.23 mg/mL. Moreover, the CaP-CTP nanoparticles were prepared by the same process but TP-10@CaP was replaced with CaP nanoparticles. In addition, Cy5.5-labelled TP-10@CaP-CTP and TP-10@CaP nanoparticles were prepared through electrostatic adsorption. Briefly, 10 mL of a TP-10@CaP-CTP or TP-10@CaP dispersion (1 mg/mL) was mixed with 1 mL of an amine-Cy5.5 DMSO solution and stirred at room temperature overnight in the dark. The obtained Cy5.5-labelled TP-10@CaP-CTP and TP-10@CaP nanoparticles were collected by centrifugation, washed with deionized water and ethanol, and dispersed in PBS for further in vivo imaging.
Cell culture
Primary CMs and CFs were isolated from male adult mice. Primary NRVMs were isolated from male neonatal rats. M199 medium supplemented with 2% BSA, 1% ITS, 1% BDM, 1% CD lipids and 1% penicillin/streptomycin was utilized to culture the CMs. Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin was utilized to culture the CFs. AC16 cells were purchased from the Chinese Academy of Science Cell Bank (Shanghai, China). MLE-12 and MLFs were purchased from Shanghai Zhong Qiao Xin Zhou Biotechnology Co., Ltd. (Shanghai, China). AC16 and MLE-12 cells were cultured in DMEM supplemented with 10% foetal bovine serum and 1% penicillin/streptomycin. MLFs were cultured in mouse lung fibroblast complete culture medium (#PCM-M-06, Zhong Qiao Xin Zhou, Shanghai, China) supplemented with 5% foetal bovine serum, 5% IST and 1% penicillin/streptomycin. All cells were incubated in a humidified 37 °C, 5% CO2 culture incubator (Thermo Fisher Scientific, Waltham, MA, USA) for subsequent experiments.
Experimental animals
Eight-week-old male C57BL/6 J mice were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). All animal experiments were performed according to the Guidelines for the Care and Use of Laboratory Animals published by the National Research Council (U.S.) Institute for Laboratory Animal Research and were approved by the Ethics Committee of Zhongshan Hospital, Fudan University, Shanghai, China (approval no. 2023-004). The mice were given free access to food and water and housed under an alternating 12 h light–dark cycle in a room with constant temperature (22 ± 1 °C) and 50% relative humidity. We used age-matched male mice in all animal experiments to avoid possible shielding of estrogen on heart failure. Both sexes of rats were used to isolate neonatal ventricular cardiomyocytes since it is difficult to separate male and female in neonates. The mice were euthanized in a carbon dioxide (CO2) chamber for experiments.
TAC mouse model
A heart failure mouse model was established by TAC-induced pressure overload according to previous studies23,46,47. Briefly, eight-week-old male C57BL/6 J mice were anaesthetized by continuous administration of inhaled isoflurane (2%) during surgery. Mice were subjected to endotracheal intubation via a 22-gauge plastic catheter, which was then connected to a ventilator. Afterwards, the left chest was opened, and blunt dissection was performed at the proximal portion of the sternum to access the thoracic aorta. A 27-gauge needle was placed on the transverse aorta between the innominate region and the left common carotid artery. Then, 6-0 silk sutures were used to ligate the transverse aorta with the needle. Immediately after the aorta was completely occluded, the needle was removed, and the thoracic cavity was closed. Sham mice underwent the same procedure without ligation. Aortic velocity peak pressure was determined by in vivo echocardiography, and mice with gradients greater than 30 mmHg were used.
Targeting ability of the nanoparticles in vivo
Four weeks after TAC or sham operation, the mice received intratracheal treatment with 50 μL of PBS, Cy5.5-labelled TP-10@CaP or TP-10@CaP-CTP. The mice were sacrificed at 0.5, 1, 3, 6, and 24 h to harvest heart tissue, which was then imaged with a Spectrum in vivo imaging system (IVIS) (VISQUE, InVivo Elite). Moreover, 1 h after inhalation or 3 h after I.V. injection, the hearts were harvested to generate frozen specimens, which were cut into 6 μm cryosections for immunofluorescence staining. In parallel, CMs were isolated from the mice and fixed for immunofluorescence staining.
Mouse inhalation treatment
For inhalation treatment, a high-pressure microsprayer aerosolizer (BioJane BioTech, cat. no.: BJ-PW-M) was used to deliver inhalable agents to TAC model mice. Briefly, the mice were anaesthetized by intraperitoneal injection of pentobarbital sodium (30 mg/kg) for nebulization. Before tracheal cannulation, the mouths of the mice were opened with a laryngoscope to visualize the porch of the trachea. The tip syringe of the high-pressure microsprayer was gently fed into the main trachea, and the aerosols were rapidly delivered.
In vitro TP-10 release from TP-10@CaP and TP-10@CaP-CTP
The in vitro release of TP-10 was measured using the dialysis bag diffusion method. 1 mg of TP-10@CaP and TP-10@CaP-CTP was dispersed in 5 mL of PBS and then incubated in dialysis bags (MWCO = 8–14k). Then, the dialysis bag was immersed in 10 mL of buffer at different pH values (7.4, 6.5 and 5.5) under stirring (300 rpm). Then, 1 mL of media was collected at given time intervals for further analysis and replaced with 1 mL of fresh buffer. The amount of released TP-10 was determined by using HPLC-ESI-MS/MS (EXPEC 5250, Agilent Technologies, Santa Clara, US). The chromatographic conditions are as follow: stationary phase is a reversed phase HPLC-column (ZORBAX RRHD Eclipse XDB-C18, 50 mm × 2.1 mm, 1.8 μm; Agilent Technologies, USA), a mixture of triethylamine-glacial acetic acid buffer solution (pH 3.5) and acetonitrile (87:12, v/v) served as mobile phase and the flow rate was fixed at 0.6 mL/min. For this established method, the limits of detection (LODs) of TP-10 were estimated as the minimum concentration determined with a signal-to-noise ratio of 3 and the limit of quantitation (LOQ) values taken by signal-to-noise ratio of 10. The LOD and LOQ were determined to be 2.5 and 8 ng/mL, respectively.
In vivo biodistribution of TP-10@CaP-CTP
To quantify the amount of TP-10 delivered to the myocardium and different organs, TAC model mice following 1 h TP-10@CaP or TP-10@CaP-CTP treatment through inhalation approach were sacrificed. The different organs including heart, liver, spleen, lung, and kidney were collected, washed and then homogenized in 1.0 mL of lysis buffer with superfine homogenizer. Then, 200 μL of tissue lysate was mixed with Triton X-100 under vortexing, following added 1.5 mL of the extraction solution (HCl–IPA). After incubated at −20 °C overnight, the samples were centrifugated and the amount of TP-10 was quantified by HPLC-MS/MS according to the chromatographic conditions described above.
Adult mouse CMs (AMCMs) isolation
Primary CMs were isolated according to a previously published protocol48. Briefly, after sacrifice, EDTA buffer was injected into the right ventricle in situ. The ascending aorta was clamped, and the heart was then transferred to a dish containing EDTA buffer. Next, the left ventricle was perfused ex vivo using EDTA buffer, which was replaced with perfusion buffer when the thrombus was completely removed. Afterward, collagenase buffer containing collagenase II (Worthington, USA), collagenase IV (Worthington, USA) and protease XIV (Sigma-Aldrich, Singapore) was injected into the left ventricle until the digestion was completed. Then, the heart was dissociated by pipetting, and digestion was terminated with stop buffer. The isolated cells were filtered with a 100 μm pore-size strainer and then allowed to settle by gravity for 20 min. After filtration, CM pellets were sequentially resuspended in three intermediate calcium reintroduction buffers to gradually restore the calcium concentration to physiological levels. Then, the precipitates were collected. resuspended in culture medium, and cultured in a humidified 37 °C, 5% CO2 culture incubator for subsequent experiments.
Adult mouse CFs isolation
When the heart was dissociated and filtered with the 100 μm pore-size strainer, the CFs were found in the supernatant fraction. After 20 min of gravity settling, the supernatant was collected, centrifuged at 300 × g for 5 min, resuspended in fibroblast media, and plated in 6 cm dishes. The cell media was changed after 2 h of culture.
Neonatal rat ventricular myocytes (NRVMs) isolation
NRVMs were isolated from 1- to 3-day-old Sprague-Dawley rats (Vital River Laboratory Animal Technology Co., Ltd, Beijing, China) using a cardiomyocyte isolation kit (Miltenyi Biotec, #130-098-373) as previously described46. The isolated cells were cultured at 37 °C in a humidified 5% CO2 incubator for 2 h. Then, collected the supernatant which containing cardiomyocytes and transferred into another culture dish with Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin/streptomycin (Gibco) for 24 h. NRVMs were then subjected to subsequent experiments.
Measurement of intracellular cAMP and cGMP levels
CMs were stimulated with phenylephrine (100 μM) or vehicle for 24 h, following by the treatment of 25 μg/mL CaP-CTP, TP-10@CaP, TP-10@CaP-CTP or vehicle for 1 h. The cAMP and cGMP levels were measured using cAMP and cGMP ELISA kits (Cayman, Ann Arbor, MI, USA) according to the manufacturer’s protocol. Briefly, CMs were lysed with 1 mL HCl (0.1 M) for every 35 cm2 surface area, and then the lysates were centrifuged at 1000 × g for 10 min. The supernatants were collected and diluted 1:2 with dilation buffer. The samples and reagents were added to 96-well plates and incubated at 4 °C overnight, followed by five washes with wash buffer. Ellman’s reagent was added to the plate and incubated at room temperature for 2 h in the dark. The plate absorbance was measured at 420 nm using a microplate reader.
Intracellular cAMP and cGMP levels in CFs were measured with a similar procedure. Briefly, CFs were stimulated with TGF-β (10 ng/mL) or vehicle for 24 h, following by the treatment of 25 μg/mL CaP-CTP, TP-10@CaP, TP-10@CaP-CTP or vehicle for 1 h. The subsequent steps were the same as those mentioned above.
Boyden chamber migration assay
A Boyden chamber migration assay was performed to evaluate the migration of adult mouse CFs (8 μm pore size; Corning, Acton, MA, USA). Briefly, after 24 h of serum starvation, CFs were added to the upper chambers and cultured in serum-free DMEM. DMEM supplemented with 10% FBS and 1% P/S was added to the lower chambers. Then, the CFs were pretreated with 25 μg/mL CaP-CTP, TP-10@CaP, TP-10@CaP-CTP, TP-10@CaP-CTP with 1 μM PKG inhibitor DT-2 (MedChemExpress, Shanghai, China) or vehicle for 2 h, followed by treatment with TGF-β (10 ng/mL) or vehicle. After 24 h of incubation at 37 °C, the cells on the upper surfaces were removed with cotton swabs, and migrated cells on the lower surfaces were fixed and stained with a 0.1% crystal violet solution. For each Boyden chamber, the total cell numbers in six random fields were determined and analyzed.
Wound healing scratch assay
CFs were seeded into 24-well plates and cultured in DMEM supplemented with 10% FBS and 1% P/S. When the cells reached ~100% confluence, they were serum starved for 24 h, and scratches were made with a 200 µL pipette tip. Then, the cells were treated with 25 μg/mL CaP-CTP, TP-10@CaP, TP-10@CaP-CTP, TP-10@CaP-CTP with 1 μM DT-2 or vehicle for 2 h, followed by 10 ng/mL TGF-β or vehicle. Wound closure was monitored for 24 h.
CCK-8 assay
CF proliferation was evaluated by using the CCK-8 cell counting kit according to the manufacturer’s instructions. Briefly, CFs were seeded in 96-well plates and cultured in DMEM supplemented with 10% FBS and 1% P/S overnight. Then, the CFs were serum starved for 24 h. Next, the CFs were pretreated with 25 µg/mL CaP-CTP, TP-10@CaP, TP-10@ CaP-CTP, TP-10@CaP-CTP with 1 μM DT-2 or vehicle for 2 h, followed by treatment with TGF-β (10 ng/mL) or vehicle. After 24 h of treatment, CCK-8 reagent (10 μL) was added to each well, and the plates were incubated at 37 °C. The absorbance was measured at 450 nm using a microplate reader.
Shortening/relengthening assay of ADCMs
The mechanical properties of the ADCMs were evaluated by a Softedge MyoCam system (IonOptix Corporation, Milton, MA, USA)49. Briefly, after isolation from the adult mouse, CMs were placed in a chamber under an inverted microscope (Olympus, IX-70) in contractile buffer (containing 135 mM NaCl, 10 mM HEPES, 1.0 mM MgCl2, 1.0 mM CaCl2, 10 mM glucose and 4.0 mM KCl (pH = 7.4)). Then, the CMs were electrically stimulated at 0.5 Hz, and relevant parameters were recorded, including resting cell length, peak shortening (PS), maximal velocity of shortening ( + dL/dt), maximal velocity of relengthening (−dL/dt), time-to-PS (TPS), and time-to-90% relengthening (TR90). The parameters were analyzed by IonOptix Softedge software.
Echocardiography
Echocardiography was performed on TAC model and sham mice at 0, 2, 4, 6, 8 weeks by an expert laboratory technician who was blinded to the mouse information. The mice were anesthetized via continuous administration of inhaled 2% isoflurane. Echocardiography was performed on anesthetized mice using a multimode small animal ultrasound imaging system (Vevo 3100, FUJIFILM Visual Sonics, Canada). M-mode echocardiography on the short-axis was used to assess cardiac systolic function. The E/A ratio and aortic blood flow velocity were determined to evaluate mouse cardiac diastolic function. Pressure gradients were determined at 1 week post TAC operation.
Lung wet/dry (W/D) ratio
After mouse sacrifice, the bilateral fresh lung tissues were harvested and weighed (wet weight). Then, the lung tissues were incubated in an oven at 60 °C for 48 h and weighed (dry weight).
Bronchoalveolar lavage fluid (BALF) analysis
One milliliter of PBS was instilled into the trachea and flushed three times to collect BALF. The supernatant was stored after centrifugation (400 g, 10 min). To evaluate alveolar-capillary permeability in the lungs, the total protein level of the BALF was determined by the BCA Protein Assay Kit (Beyotime, #P0012). Inflammatory cytokine levels in the BALF were determined by enzyme-linked immunosorbent assay (ELISA).
RNA extraction and quantitative RT‒PCR
Total RNA was extracted using TRIzol (Invitrogen) and reverse-transcribed to cDNA using the PrimeScript™ RT Reagent Kit (cat. no. DRR037A, Takara, Japan). Gene expression levels were measured by mixing target primers with SYBR Green master mix (Yeasen, #11201ES03), and the reactions were performed with Bio-Rad’s CFX96 (Bio-Rad). Each reaction was performed in triplicate. The primers were purchased from Tsingke Biological Technology (Shanghai, China) and are shown in Supplementary Table 2.
ELISA
Serum and BALF inflammatory cytokine levels were determined by ELISA according to the manufacturer’s instructions. The levels of IL-1β were measured using a mouse IL-1β ELISA kit (WELLBI, #EM30300M). The levels of IL-6 were measured using a mouse IL-6 ELISA kit (WELLBI, #EM30325M). The levels of TNF-α were measured using a Mouse TNF-α ELISA kit (WELLBI, #EM30536M). The levels of ANP were measured by a Mouse ANP ELISA kit (WELLBI, #EM30655M). The levels of Complement C3 were measured by a Mouse Complement C3 ELISA kit (WELLBI, #EM30760M). The levels of Complement C3b were measured by a Mouse Complement C3b ELISA kit (Biomatik, #EKC36654). The levels of Complement C3c were measured by a Mouse Complement C3c ELISA kit (Yojanbio, #YJ-E-94812Q). All of the absorbance values were measured using a microplate reader.
Immunofluorescence staining
Frozen heart sections were fixed with acetone for 10 min at −20 °C. After washing three times with PBS, the sections were blocked with 5% bovine serum albumin (BSA) for 1 h at room temperature and then incubated with primary antibodies overnight at 4 °C. Afterward, the sections were washed with PBS three times and incubated with the corresponding fluorescence-labelled secondary antibodies for 1 h at room temperature. After four washes with PBS, the sections were counterstained with Antifade Mounting Medium with 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) (Beyotime, #P0131). For cell immunofluorescence staining, a similar procedure was performed, except the cell samples were fixed with 4% paraformaldehyde (PFA) (Beyotime, #P0099) for 10 min and permeabilized with 0.5% Triton X-100 (Beyotime, #P0096) for 10 min at room temperature. Images were captured using a confocal microscope (Olympus FV3000, Japan). The primary antibodies that were used were as follows: antibodies against cardiac troponin T (#ab209813, 1: 3000 for cardiomyocytes), antibodies against Col1α1 (#ab270993, 1:2000), antibodies against Vimentin (#ab24525, 1:300) and cardiac troponin T (#ab8295, 1: 2000 for myocardial tissue) were obtained from Abcam (Cambridge, UK); antibodies against α-smooth muscle actin (#19245 T, 1:500) were purchased from Cell Signaling Technology (MA, USA); antibodies against Discoidin domain receptor 2 (#sc-81707, 1:50) was purchased from Santa Cruz Biotechnology (Dallas, USA); antibodies against CD31 (#DIA-310, 1:200)was purchased from BIOZOL (Eching, Germany); antibodies against Phalloidin (#40736ES75, 1:1000) was purchased from Yeasen (Shanghai, China).
Western blot analysis
Cells were lysed in lysis buffer containing RIPA buffer (Beyotime, #P0013C), PMSF, protease inhibitors, and phosphatase inhibitors. The heart tissues were mixed with the abovementioned lysis buffer and ground in a homogenizer (Servicebio, #KZ-5F-3D) according to the manufacturer’s instructions. Then, the lysates were centrifuged at 14,000 × g (Thermo Fisher Scientific, 75004250) for 20 min at 4 °C, and the supernatants were collected for Western blotting. The protein concentrations were determined by BCA assay. Equal amounts of protein were added to a sodium dodecyl sulfate-polyacrylamide gel and separated by electrophoresis. Then, the proteins were transferred to PVDF membranes. After blocking with 5% BSA for 1 h at room temperature, the membranes were incubated with primary antibodies at 4 °C overnight. Next, the membrane was washed three times with TBST and incubated with the HRP-conjugated secondary antibodies for 1 h at room temperature. Afterward, specific bands were imaged using a Bio-Rad detection system (Bio-Rad Laboratories, Hercules, CA, USA). The images were analyzed using ImageJ. The primary antibodies that were used were as follows: antibodies against AMPKα (#2532, 1: 1000), phospho-AMPKα (#2535, 1: 1000), and α-smooth muscle actin (#19245 T, 1: 1000) were obtained from Cell Signaling Technology (MA, USA); antibodies against Col1α1 (#ab270993, 1:1000) were purchased from Abcam (Cambridge, UK).
Statistical analysis and software
All the data are presented as the mean ± SEM. Two-tailed Student’s t tests were performed to compare two groups and identify differences. For multiple group comparisons, one-way ANOVA was conducted, followed by Bonferroni’s multiple comparisons test. A P value < 0.05 was considered to indicate statistical significance. All data analyses were performed with GraphPad Prism 8.0 software (San Diego, CA, USA).
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
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