Conductive nanocomposite hydrogel and mesenchymal stem cells for the treatment of myocardial infarction and non-invasive monitoring via PET/CT | Journal of Nanobiotechnology


Characterization of the injectable nanocomposite hydrogel

Decide 2A reveals the zeta potentials of GNR, SN, and GNR@SN composite dispersions [1% (w/v) SN, 0.3% (w/v) GNR]. TEM image of the GNR@SN composite dispersions demonstrates GNRs stabilized in SN with good dispersity (Fig. 2B). Subsequent, phosphate buffered saline (PBS) was added to the GNR@SN composite dispersion or naked GNR dispersion. GNR@SN composite dispersion didn’t have agglomerated particles (Fig. 2B), whereas the naked GNR dispersion had agglomerated particles [25, 26].

Fig. 2
figure 2

Characterization of the nanoparticles and nanocomposite injectable hydrogel. A Zeta potential of silicate nanoplatelets (SNs), gold nanorods (GNRs), and GNR@SN. B TEM image of GNR@SN (Scale bar: 50 nm). The insert is GNR@SN mixed with PBS after 1 day of resting. C The sol–gel transformation of GNR@SN/Gel. D SEM images of SN/Gel and GNR@SN/Gel (Scale bar: 50 μm). E Swelling ratio of SN/Gel and GNR@SN/Gel. F Dry gel weight low cost of SN/Gel and GNR@SN/Gel. G Temperature dependence of the storage modulus (G′) and loss modulus (G″) of SN/Gel and GNR@SN/Gel. H BSA adsorption verify of SN/Gel and GNR@SN/Gel. I Conductivities of varied samples

An inverted verify tube experiment was used to measure the part transition temperature of the hydrogel. A polymer of PLGA2000-PEG3400-PLGA2000 (characterization outcomes are confirmed in Additional file 1: Fig. S1) didn’t sort a sol–gel transition and never utilizing a crosslinker; nonetheless, when the crosslinker SNs was added to formulate SN/Gel [3% (w/v) PLGA2000-PEG3400-PLGA2000, 1% (w/v) SNs], the important transition temperature was 31 °C, the hydrogel precursor transformed proper right into a gel at 37 °C, with a flowable sol part at 28 °C (Additional file 1: Fig. S2). When the GNR@SN composite dispersion was added to create GNR@SN/Gel [3% (w/v) PLGA2000-PEG3400-PLGA2000, 1% (w/v) SN, 0.3% (w/v) GNR], the important transition temperature was 27 °C, the hydrogel precursor transformed proper right into a gel at 37 °C, with a flowable steady part at 25 °C (Fig. 2C).

The SEM image reveals that every SN/Gel and GNR@SN/Gel formed a 3D network-like development, GNR@SN/Gel appears to be denser than SN/Gel (Fig. 2D). Changes throughout the hydrogel crosslink density and porosity can alter plenty of completely different hydrogel properties, e.g., modulus and diffusivity [27, 28]. GNR@SN/Gel exhibited poor swelling properties (Fig. 2E), a slower degradation payment (Fig. 2F) and better mechanical properties than SN/Gel (Fig. 2G). The GNR@SN/Gel has a lower important transition temperature than the SN/Gel (Additional file 1: Fig. S2), which is per the rheological verify outcomes (Fig. 2G).

On account of PEG, PLGA, and GNRs are biologically inert to bioactive protein adsorption/retention and cell proliferation, the presence of SNs can enhance cell adhesion and proliferation on the gel flooring [21]. In experiments on the adsorption of pure substances from the hydrogel, GNR@SN/Gel adsorbed bovine albumin (BSA) additional slowly than SN/Gel; nonetheless, there was no distinction throughout the full amount of BSA adsorbed by GNR@SN/Gel and SN/Gel at 48 h (Fig. 2H). It’s attainable that the lower porosity of GNR@SN/Gel affected the diffusivity of BSA from the reply to the hydrogel; nonetheless, we didn’t observe an impression of GNR on the adsorption functionality of SN.

Lastly, we examined the conductivity of samples (Fig. 2I). The conductivity of GNR@SN/Gel [(6.19 ± 0.07) × 10−3 S/cm] is significantly elevated than that of SN/Gel [(4.92 ± 0.03) × 10−3 S/cm, P < 0.0001], every exceeding that of pure myocardial tissue. Nonetheless the conductivity of SN powder was solely 9.70 ± 0.04 × 10–6 when squeezed into blocks at 30 MPa.

Viability of MSCs loaded contained within the conductive hydrogel

Transfer cytometry analysis revealed that the extracted MSCs had extreme purity after three generations of custom (Additional file 1: Fig. S3). The biocompatibility of the hydrogel was determined by the viability assay of the encapsulated cells. MSCs have been readily encapsulated in SN/Gel and GNR@SN/Gel by the use of thermal gelation, 98.6% ± 1.2% and 98.8% ± 1.1% of the cells have been alive inside 30 min of gel formation (Fig. 3A), respectively, with no important distinction between two groups. Cells encapsulated in SN/Gel and GNR@SN/Gel exhibited regular proliferation over 14 days, the proportion of keep cells was 90.06% ± 4.74% for the GNR@SN group and 91.00% ± 0.39% for the SN/Gel group on day 14 (Fig. 3B). No important distinction was observed microscopically (Fig. 3C).

Fig. 3
figure 3

Viability of mesenchymal stem cells (MSCs) loaded contained within the hydrogels. A Dwell/lifeless assay of the MSCs encapsulated in SN/Gel and GNR@SN/Gel at 30 min and a pair of weeks. Residing cells have been stained with calcein AM (inexperienced) and lifeless cells have been stained with PI (purple) (Scale bar: 50 µm). B Quantitative analysis of the cell viability. C Microscopic calculations of encapsulated cells in hydrogels on Day 14

Monitoring of injection location and myocardial viability by PET after remedy

Thirty-six rats (n = 6 per group) have been divided into 6 random groups. Rats throughout the Sham group underwent sham surgical process with out MI operation. The remaining 5 groups underwent MI operation and purchased intramyocardial injection on the infarct space: saline alone (Saline group), PLGA-PEG-PLGA hydrogel precursor with SNs (SN/Gel group), MSCs-encapsulated PLGA-PEG-PLGA hydrogel precursor with SNs (MSC/SN/Gel group), GNRs stabilized on the SNs with PLGA-PEG-PLGA hydrogel precursor (GNR@SN/Gel group), and MSCs-encapsulated PLGA-PEG-PLGA hydrogel precursor with the GNRs stabilized on the SNs (MSC/GNR@SN/Gel group). Left ventricles have been divided into 17 segments in step with the AHA 17-segment model (Fig. 4A).

Fig. 4
figure 4

Non-invasive monitoring remedy and efficacy analysis by PET imaging. A Left ventricle is cut up into 17 segments in step with the AHA 17-segment model. B PET imaging of 68Ga-loaded GNR@SN/Gel hydrogel with PBS in vitro. C Space of hydrogel injection into myocardium visualized by 68GaCl3 PET. D Advisor 17-segment polar map by the use of 18F-FDG PET imaging of myocardial viability in each group after 1 day and 4 weeks. E Quantification of non-viable myocardium house as a share of full left ventricular house; F Comparability of non-viable myocardium between groups. *P < 0.05 and **P < 0.01, ***P < 0.001, ****P < 0.0001, ns > 0.05

First, a launch verify of the 68Ga-loaded hydrogel was carried out. As confirmed in Additional file 1: Fig. S4, GNR@SN/Gel hydrogel launched 4.73% ± 1.65% 68Ga3+ after 1 h, in vitro PET imaging of 68Ga-loaded GNR@SN/Gel hydrogel reveals very low train throughout the PBS (Fig. 4B), demonstrating tight 68Ga3+ binding to the hydrogel, and it’s attainable to visualise the hydrogel injected into the middle in vivo by loading 68Ga3+ (Fig. 4C). The PET imaging of the hydrogel in rats was processed to accumulate a 17-segment polar map. The realm the place the hydrogel injected was appropriate with the world of non-viable myocardium induced by MI operation (Fig. 4C, D).

Subsequent, 18F-FDG-PET imaging was carried out to research the impression of MSCs blended with GNR@SN/Gel on the myocardial viability. As confirmed in Fig. 4D, 18F-FDG uptake throughout the myocardium of Sham group was uniform in addition to low uptake on the apex part, which was nonetheless increased than 40% of the most effective price of your complete coronary coronary heart uptake, indicating no non-viable myocardium. On the first day after MI operation, the realm percentages of non-viable myocardium have been 15.95 ± 1.97, 13.45 ± 5.82, 8.12 ± 3.24, 4.75 ± 2.73, 4.68 ± 2.89 throughout the Saline group, SN/Gel group, MSC/SN/Gel group, GNR@SN/Gel group and MSC/GNR@SN/Gel group, respectively. The non-viable myocardium house of MSC/GNR@SN/Gel group and GNR@SN/Gel group was significantly lower than completely different groups (P < 0.05), nonetheless, no important deference was found between GNR@SN/Gel group and MSC/GNR@SN/Gel group (P = 0.9664). These outcomes level out that GNR@SN/Gel and MSC/GNR@SN/Gel had a defending impression on the myocardial viability at an early stage.

On the 28 day after MI operation, the realm percentages of non-viable myocardium have been 16.79 ± 1.95, 17.28 ± 7.52, 11.14 ± 3.07, 9.13 ± 4.76, 5.47 ± 2.70 throughout the Saline group, SN/Gel group, MSC/SN/Gel group, GNR@SN/Gel group and MSC/GNR@SN/Gel group, respectively. GNR@SN/Gel group and SN/Gel group didn’t current a sustained defending impression on myocardial viability, whereas MSC/SN/Gel group and MSC/GNR@SN/Gel group confirmed an inhibitory impression on the unfold of non-viable myocardium. The non-viable myocardium house of MSC/GNR@SN/Gel group was significantly lower than completely different groups.

Paired t-test was carried out to match the realm of non-viable myocardium of each group on day 1 and day 28. The outcomes confirmed Saline group, SN/Gel group and GNR@SN/Gel group have been statistically completely completely different (P = 0.029, P = 0.013), whereas no statistical distinction in MSC/GNR@SN/Gel group and MSC/SN/Gel group (P = 0.082, P = 0.353). The non-viable myocardium house throughout the MSC/SN/Gel group was significantly greater than that in MSC/GNR@SN/Gel group, indicating that the MSC/GNR@SN/Gel group had the easiest impression of inhibiting the enlargement of non-viable myocardium, and throughout the completely different groups, the inhibition of non-viable myocardium enlargement was weaker or didn’t ultimate as long as MSC/GNR@SN/Gel group (Fig. 4E).

Evaluation of coronary coronary heart carry out by echocardiography

Left ventricular (LV) carry out have been measured by echocardiography to judge the therapeutic impression of MSC-loaded GNR@SN/Gel in rat MI fashions at postoperative Day 28 (Fig. 5A).

Fig. 5
figure 5

Cardiac carry out evaluated by echocardiography at postoperative Day 28. A Advisor echocardiography images of varied groups. B Left ventricle ejection fraction (EF). C Fractional shortening (FS). D The left ventricular inside diameter at end-systole (LVIDs). E The left ventricular inside diameter at end-diastole (LVIDd). *P < 0.05 and **P < 0.01, ***P < 0.001, ****P < 0.0001

In distinction with the Sham group, all MI rats had decreased LV contractile carry out and elevated LV inside diameter. The ejection fraction (EF) and fractional shortening (FS) of all MI rat groups have been so much lower than Sham group (all P < 0.05). Whereas EF and FS throughout the MSCs/GNR@SN/Gel group have been the most effective amongst MI rats (Fig. 5B, C). The left ventricular inside diameter at end-systole (LVIDs) and left ventricular inside diameter at end-diastole (LVIDd) of all MI rat groups have been so much elevated than Sham group (all P < 0.05). The MSCs/GNR@SN/Gel group demonstrated the smallest improve throughout the LVIDs in distinction with the other MI group (Fig. 5D, P < 0.05). LVIDd in MSC/GNR@SN/Gel group and MSC/SN/Gel group was so much lower than completely different MI groups (Fig. 5E, P < 0.05), nevertheless no important distinction in LVIDd was observed between the MSC/GNR@SN/Gel group and MSC/SN/Gel group (Fig. 5E, P > 0.05).

Evaluation of fibrosis by Masson trichrome staining and Sirius purple staining

MI normally leads to excessive degradation of the extracellular matrix (ECM) and excessive collagen deposition, which is a vital perform in cardiac transforming [29]. Cardiac transforming was observed using Masson trichrome staining. Infarcted areas exhibited completely completely different ranges of fibrotic tissue in quite a few groups. Basically probably the most excessive diploma of myocardial fibrosis was present throughout the Saline group, whereas the MSC/GNR@SN/Gel group had minimal fibrosis (Fig. 6A). The collagen amount fraction and LV wall thickness have been calculated using Masson staining for quantitative comparability. Every the MSC/SN/Gel group and GNR@SN/Gel group confirmed a giant low cost in collagen amount fraction and important improve in LV wall in distinction with the SN/Gel group. As anticipated, coadministration of the conductive injectable hydrogel and MSCs throughout the MSC/GNR@SN/Gel group contributed to the easiest inhibition profile in opposition to cardiac transforming (Fig. 6A–C).

Fig. 6
figure 6

Evaluation of fibrosis by Masson trichrome staining and Sirius purple staining at postoperative Day 28 and quantitative analysis. A Advisor images of coronary coronary heart sections stained with Masson’s trichrome and Sirius purple (Scale bars of coronary coronary heart cross sections of Masson’s trichrome staining: 1 mm, enlarged images of infarcted house: 100 µm; Sirius purple: 100 µm). In Masson staining, collagen was stained blue; in Sirius purple staining, the purple and yellow tissues observed have been considered type I collagen, whereas the inexperienced tissues observed have been considered type III collagen. Quantitative analysis of the B ventricular wall thickness, C collagen content material materials, and D collagen I/III ratio in quite a few groups. *P < 0.05 and **P < 0.01, ***P < 0.001

Scar tissue consists of numerous elements, amongst which type I collagen is tough, whereas type III collagen has good elasticity [30]. Collagen I/III ratio can mirror the passive enlargement functionality of scar tissue. Sirius purple staining was carried out to take a look at collagen I and collagen III, the MSCs-encapsulated injectable hydrogel group (MSC/SN/Gel group, MSC/GNR@SN/Gel group) confirmed a very low collagen I/III ratio (Fig. 6A, D).

Analysis of myocardial tissue electric-contraction coupling, revascularization and apoptosis

The development of the electric-contraction coupling throughout the infarcted myocardium was analyzed by immunofluorescence staining. CX43 mediates electrical signaling between cardiomyocytes and capabilities by assembling into gap junctions between cells [31], α-actinin is probably going one of many key myocardial skeleton proteins. Every proteins are downregulated in response to myocardial hurt.

In all MI groups, CX43 protein and α-actinin expression decreased most throughout the Saline group, whereas MSC/GNR@SN/Gel group exhibited the most effective expression of CX43 (Fig. 7A). Furthermore, CX43 protein throughout the Saline group localized totally on the periphery of the nucleus, whereas remedy of every MSC/SN/Gel and GNR@SN/Gel improved the purposeful localization of CX43 (Fig. 7A), and the MSC/GNR@SN/Gel group achieved the easiest remedy outcomes, suggesting improved electromechanical coupling after remedy of MSC/GNR@SN/Gel.

Fig. 7
figure 7

Analysis of myocardial tissue electric-contraction coupling, revascularization and apoptosis at postoperative Day 28. A Advisor immunofluorescence images co-stained by CX43 (inexperienced) and α-actinin (purple) (Scale bars: 50 μm, enlarged images: 20 µm); CD31 (inexperienced) and α-SMA (purple), (Scale bars: 50 μm); TUNEL (inexperienced) and DAPI (blue) (Scale bars: 200 μm). Quantitative analysis of the B neovessels throughout the infarct areas and C cardiomyocyte apoptosis by TUNEL constructive cells in common myocardium adjoining to severely fibrotic tissue. *P < 0.05 and **P < 0.01, ***P < 0.001, ****P < 0.0001

Revascularization of myocardial constructions restores blood present to the ischemic myocardium. To guage the strategy of angiogenesis in ischemic myocardium, immunofluorescent staining for CD31 and alpha-smooth muscle actin (α-SMA), which collectively perform markers of mature neovessels, have been carried out. Decide 7A, B reveal completely completely different ranges of angiogenesis have been observed in all MI groups, other than the Sham group on account of angiogenesis doesn’t occur in healthful myocardium. The Saline group confirmed a small amount of angiogenesis because of tissue hypoxia, and the SN/Gel group confirmed a slight improve in distinction with the Saline group. Angiogenesis throughout the GNR@SN/Gel group confirmed a giant improve in distinction with the SN/Gel group, which coincides with plenty of animal experiments using conductive provides to take care of MI [32, 33]. MSC/SN/Gel group confirmed the easiest angiogenic impression, adopted by MSCs/GNR@SN/Gel group.

Myocardial apoptosis was assessed by the TUNEL assay and the positioning of cardiomyocyte apoptosis was observed in common myocardium adjoining to severely fibrotic tissue. This experiment represents the apoptosis of myocardium introduced on by excessive compensatory contraction. The Sham group didn’t endure MI operation, and almost no apoptosis was observed throughout the ventricular cardiomyocytes (Fig. 7A). Decide 7A, C revealed that the compensatory contract after MI operation introduced on additional apoptosis of cardiomyocytes throughout the Saline group than completely different groups, and the MSC/GNR@SN/Gel remedy significantly alleviated apoptosis of cardiomyocytes.



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