Reconstruction of Postinfarcted Cardiac Functions Through Injection of Tanshinone IIA@ Reactive Oxygen Species‐Sensitive Microspheres Encapsulated in a Thermoreversible Hydrogel

Myocardial damage resulting from acute myocardial infarction often leads to progressive heart failure and sudden death, highlighting the urgent clinical need for effective therapies. Recently, tanshinone IIA has been identified as a promising therapeutic agent for myocardial infarction. However, efficient delivery remains a major issue that limits clinical translation. To address this problem, an injectable thermosensitive poly (lactic acid‐co‐glycolic acid)‐block‐poly (ethylene glycol)‐block‐poly (lactic acid‐co‐glycolic acid) gel (PLGA‐PEG‐PLGA) system encapsulating tanshinone IIA‐loaded reactive oxygen species‐sensitive microspheres (Gel−MS/tanshinone IIA) has been designed and synthesized in this study. The thermosensitive hydrogel exhibits good mechanical properties after reaching body temperature. Microspheres initially immobilized by the gel exhibit excellent reactive oxygen species‐triggered release properties in a high‐reactive oxygen species environment after myocardial infarction onset. As a result, encapsulated tanshinone IIA is effectively released into the infarcted myocardium, where it exerts local anti‐pyroptotic and anti‐inflammatory effects. Importantly, the combined advantages of this technique contribute to the mitigation of left ventricular remodeling and the restoration of cardiac function following tanshinone IIA. Therefore, this novel, precision‐guided intra‐tissue therapeutic system allows for customized local release of tanshinone IIA, presenting a promising alternative treatment strategy aimed at inducing beneficial ventricular remodeling in the post‐infarct heart.


Introduction
Myocardial damage resulting from acute myocardial infarction (MI) often leads to adverse remodeling of the left ventricle (LV) and consequent progressive heart failure and is the leading cause of death worldwide. [1]In the early stages of MI, loss of Myocardial damage resulting from acute myocardial infarction often leads to progressive heart failure and sudden death, highlighting the urgent clinical need for effective therapies.Recently, tanshinone IIA has been identified as a promising therapeutic agent for myocardial infarction.However, efficient delivery remains a major issue that limits clinical translation.To address this problem, an injectable thermosensitive poly (lactic acid-co-glycolic acid)block-poly (ethylene glycol)-block-poly (lactic acid-co-glycolic acid) gel (PLGA-PEG-PLGA) system encapsulating tanshinone IIA-loaded reactive oxygen species-sensitive microspheres (GelÀMS/tanshinone IIA) has been designed and synthesized in this study.The thermosensitive hydrogel exhibits good mechanical properties after reaching body temperature.Microspheres initially immobilized by the gel exhibit excellent reactive oxygen speciestriggered release properties in a high-reactive oxygen species environment after myocardial infarction onset.As a result, encapsulated tanshinone IIA is effectively released into the infarcted myocardium, where it exerts local antipyroptotic and anti-inflammatory effects.Importantly, the combined advantages of this technique contribute to the mitigation of left ventricular remodeling and the restoration of cardiac function following tanshinone IIA.Therefore, this novel, precision-guided intra-tissue therapeutic system allows for customized local release of tanshinone IIA, presenting a promising alternative treatment strategy aimed at inducing beneficial ventricular remodeling in the post-infarct heart.
group has previously demonstrated that TIIA treatment suppresses the inhibition of pyroptosis, suppresses the inflammatory response, cardiomyocyte apoptosis, and fibrotic processes, and helps mitigate LV remodeling and restore cardiac function after experimental MI in rats. [15]However, it has been determined that during treatment, general intracoronary administration does not achieve sufficient drug accumulation at the injury site, as it "washes away" into the bloodstream very quickly. [16,17]To address this issue, developing a safe and more efficient delivery strategy for sustained and controlled release of TIIA becomes an essential factor.
Poly (lactic acid-co-glycolic acid)-block-poly (ethylene glycol)block-poly (lactic acid-co-glycolic acid) (PLGA-PEG-PLGA) triblock polymers have been shown to form thermosensitive gels for certain drug delivery and tissue engineering applications. [18]It has been determined that drugs can be incorporated into the polymer solution at low temperatures to facilitate living tissue injection.When the mixture reached body temperature, the micelles automatically transformed into a viscous gel, which formed a depot and allowed sustained release of the drug in situ.On the other hand, it has also been reported that such a viscous gel provides good structural support for the weak myocardial wall and enhances compliance of the ventricular wall, thereby reducing ventricular remodeling.Moreover, it has been found that the abovementioned thermosensitive gel itself is not only biodegradable and biocompatible, but it is also easy to adjust the physicochemical properties during the manufacturing process, making it a safe and controllable drug delivery system. [18]revious studies have also confirmed that TIIA is released rapidly when directly encapsulated in a hydrogel. [19]22] Therefore, there is a real need to develop a more precisely controlled drug delivery system that can deliver doses tailored to ensure that the therapeutic effects are adequate to suit the severity of MI in individual patients.Interestingly, a number of previous studies have reported faster reactive oxygen species (ROS) production and accumulation in the infarcted myocardial area in patients with severe MI than in those with mild MI. [23,24] These observations support the theory that the presence of local myocardial ROS may contribute to MI treatment.It has been previously shown that the local delivery of ROS-sensitive materials can effectively promote the release of several tested drugs from the initially formed liposomes while neutralizing ROS production. [25][28] Therefore, the main objective of the current study was to develop an ROS-triggering system for customized local release of the main therapeutic component, TIIA, into the infarcted myocardium.
Here, we established an oil-in-water emulsion-solvent evaporation method that allows the efficient initial formation of a TIIA-encapsulated ROS-sensitive microsphere system (MS/TIIA), which facilitates high drug loading and controlled drug release.More precisely, a drugcontaining thermosensitive gel (Gel-MS/TIIA) was injected directly into the myocardium (Figure 1).This system exhibited several advantages: 1) controlled rapid in situ immobilization of microspheres to prevent unexpected particle diffusion, 2) initial structural support for weakened myocardial wall and enhanced ventricular wall compliance, 3) ultimate dissociation of the injected Gel-MS/TIIA units, after their contact with ROS-rich-containing infarcted myocardium that caused a subsequent release of TIIA in the "on-demand" manner, which exerts long-term anti-pyrolysis and anti-inflammatory effects.Thus, the collective actions of this therapeutic system achieve the purpose of alleviating pathological ventricular remodeling after MI.It is reported that thioketal bonds is a ROS-sensitive group, and materials containing thioketal bonds in the main chain can degraded upon abundant ROS and release the wrapped drug. [29,30]A polymer (PSSP) with ROS-sensitive thioketal bonds as repeat unit in the main chain was first synthesized, which synthetic routes was presented in the Figure S1, Supporting Information.The structures of the prodrugs were confirmed by 1 H-NMR (Bruker, AV-400, Switzerland) using DMSO-d6 as the solvent (Figure S2, Supporting Information).The emulsion-solvent evaporation method was employed to formulate TIIA-loaded PSSP MS. [18,28] Briefly, PSSP and TIIA were thoroughly dissolved in dichloromethane.After emulsification and solidification, MS/TIIA was filtered to obtain an enriched population smaller than 2 lm.In terms of the morphology of MS/TIIA (determined using SEM and TEM), the particles exhibited a smooth surface and a spherical shape with a homogeneous average size of ~1.3 lm.The DLE of MS/TIIA, determined using ultraviolet-visible (UV-Vis) spectroscopy, was ~50 wt.% (standard curve in Figure S3, Supporting Information).The triblock copolymer PLGA-PEG-PLGA gel prepared via the ring-opening polymerization (ROP) method exhibited an interconnected three-dimensional (3D) porous network structure.The GelÀMS/TIIA homogeneous suspension was generated by mixing MS/TIIA with PLGAÀPEGÀPLGA solution at 4 °C for 24 h and subsequently placed in a thermostat at 37 °C for 10 min.MS was uniformly dispersed on the surface and within the gel matrix (Figure 2a,b).Overall, GelÀMS/TIIA exhibited a well-structured and homogeneous framework with the effective encapsulation of TIIA.

Rheological Properties
To investigate the gelation properties, the rheological characteristics of the gels with and without TIIA or MS/TIIA were evaluated.G 0 represents the stiffness and G″ refers to the viscosity of the sample.Gel-TIIA and Gel-MS/TIIA were fluid with low viscosity at 4 °C and shear modulus increased rapidly with temperatures of up to 17.85 °C and 18.21 °C, respectively, representing the corresponding sol-gel transition temperatures.Subsequently, as the temperature increased, the value of G 0 was greater than that of G″, confirming the formation of the gel.Meanwhile, the maximum G 0 value for GelÀMS/TIIA was >400 Pa, indicating that the formulation stiffness of the in-situ drug release system was satisfactory (Figure 2c).Gel-MS/TIIA presented as an orange solution at 4 °C, while a hydrogel was formed at 37 °C (Figure 2d), indicating that when the drug was injected immediately after removal from the 4 °C refrigerator, the hydrogel formulation could still be conveniently administered through a 30 G insulin needle (Figure 2e) (Video S1, Supporting Information).Overall, GelÀMS/TIIA exhibited favorable syringeability, stiffness, and gelation capacity, thereby validating the feasibility of intramyocardial injection.After the temperaturesensitive hydrogel reaches body temperature, its stiffness increases, thus providing structural support for the weakened myocardial wall after MI and improving compliance of the ventricular wall. [31,32]

Drug Release Profile
To validate the efficient drug release of the hydrogel system, samples were obtained periodically over 14 days for UV-vis spectroscopic analysis.Overall, 54.2%, 2.3%, 34.0%, and 27.7% of TIIA was  released from Gel-TIIA, Gel-MS/TIIA + PBS, Gel-MS/TIIA + 0.5 mM H 2 O 2 , and Gel-MS/TIIA + 0.25 mM H 2 O 2 , respectively, within the first 48 h.Compared with Gel-TIIA, TIIA was released from the Gel-MS system at a more stable rate with a significantly reduced burst frequency (p < 0.05).For Gel-TIIA, the initial burst release was followed by diffusion once it contacted the release medium.However, for the GelÀMS/TIIA system, the rapid release of the drug dispersed in the gel was retarded by the outer ROS-sensitive microspheres, thereby preventing burst release.Degradation of the gel and ROS-sensitive microspheres in a high redox environment induced slow and controllable release. [33,34]TIIA release was accelerated in the presence of increasing H 2 O 2 concentrations, and the drug release efficiency increased.Conversely, Gel-MS/TIIA without H 2 O 2 showed an extremely slow drug release (Figure 2f).The in vitro erosion and degradation data were consistent with the experimental drug release data (Figure 2g).mM degradation rates of Gel-TIIA were mM fastest, followed by Gel-MS/ TIIA + 0.5 mM H 2 O 2 , Gel-MS/TIIA + 0.25 mM H 2 O 2 , and Gel-MS/ TIIA + PBS.The presence of ROS accelerated the rate and degree of burst release.Therefore, the MS-encapsulating gel strategy used in this study appears to be suitable for sustained drug delivery and to achieve ROS-triggered "on-demand" release according to the level of ROS produced based on the severity of MI.  (Figure 3b-I) and the left anterior descending (LAD) coronary artery was ligated to create a LV infarction, other than the sham group that underwent LAD threading as previously described (Figure 3b-II).After ligation, 100 lL of each drug, including saline, TIIA, Gel-MS, Gel-TIIA, or Gel-TIIA/MS, was injected into the border and center regions of the infarct (Figure 3b-III, IV), followed by surgical closure of the incision in the chest of rats (Video S1, Supporting Information).All rats were anesthetized and evaluated with echocardiography 2 days after ligation to measure the infarct size in terms of the percentage of scar area to the LV free wall area.Only rats with infarction of >25% of the LV free wall were included (n = 10 in each group).Fourteen days after drug injection, the chest of the rats was re-opened to collect blood samples and heart tissue (Figure 3b-V).

Drug Biodistribution
To establish the biodistribution of in situ injected Gel-MS/TIIA, cyanine 7.5 (Cy 7.5) maleimide was loaded into the gel and Gel-MS for visualization.Immediately after injection of Gel-Cy 7.5 and Gel-MS/Cy 7.5, blue dye was distributed over the entire apex of the heart.Subsequently, the biodistribution patterns of Gel-Cy 7.5 and Gel-MS/Cy 7.5, after administration, were examined.Compared with Gel-Cy 7.5, gelencapsulated ROS-responsive microspheres significantly enhanced the retention of Cy 7.5 in the heart on days 7 and 14 after injection (Figure 3c,d).Drug-loaded ROS-sensitive microspheres in this system were immobilized by the gel and exhibited excellent local retention characteristics, enabling precision-guided drug release and retention within an effective administration time in vivo.More importantly, we assessed the retention time of TIIA after intramyocardial injection (Figure S4, Supporting Information).The drug was completely washed away within 1 min, and its local concentration was quite low (Figure 3e).

Gel-MS/TIIA Improves Cardiac Function and Ameliorates Fibrosis
To investigate the therapeutic effects of Gel-MS/TIIA on morphological and functional changes post-MI, echocardiography was performed 7 and 14 days after LAD ligation.In terms of morphology, both the left ventricular end-diastolic dimension (LVEDD) and left ventricular endsystolic dimension (LVESD) were significantly enlarged in the MI group (p < 0.05).Notably, LVEDD and LVESD of rats treated with Gel-MS/ TIIA were significantly reduced on days 7 and 14 after LAD ligation compared to those in the MI group (p < 0.05, Figure 4a).In terms of functional changes, the left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), and cardiac output (CO) of the rats in the MI group were significantly lower than those in the sham group (p < 0.05, Figure 4a).The deterioration of LVEF, LVFS, and CO in the group treated with Gel-MS/TIIA was reduced at days 7 and 14 after LAD ligation (Figure 4a, Tables S1 and S2, Supporting Information).These echocardiographic data suggest that Gel-MS/TIIA can significantly mitigate cardiac dysfunction in post-infarct rats.
To further evaluate the histological changes in the heart after Gel-MS/TIIA treatment, we performed hematoxylin and eosin (HE) staining of samples collected at the end of the experiment.Necrotic muscle fibers in the infarct area were replaced with loose fibrous connective tissue and the structure of the infarcted area was loosened (Figure 4b).The capillaries were dilated, and the surviving cardiomyocytes were scattered at the edge of the infarction area.These histological characteristics suggest hypertrophy and fibroblast proliferation in the post-MI heart (Figure 4b).Notably, these changes were partially reversed in the Gel-MS/TIIA group.Statistical analyses of the left heart cavity and thickness of the ventricular wall in the infarct zone were also performed.Compared to the sham group, the left heart cavity area in the MI group was significantly enlarged, and the thickness of the ventricular wall was significantly reduced (Figure 4d,e).In the group treated with Gel-MS/ TIIA, the left heart cavity and LV wall thickness were markedly improved compared with those in the MI group (Figure 4d,e).
Masson staining was performed to observe the deposition of myocardial collagen fibers.Compared to the sham group, severe fibrosis was evident in the heart tissues of the MI group.A large area of collagen fiber deposition was observed in the infarct area (mainly including type I and II collagen fibers), which was significantly reduced in the Gel-MS/TIIA treatment group (Figure 4c).The percentages of collagen fiber areas were 37.72%, 20.05%, 18.74%, and 9.75% in the MI, Gel-MS, Gel-TIIA, and Gel-MS/TIIA groups, respectively (Fig- ure 4c,f).Remarkably, the infarct size was significantly decreased in the Gel-MS/TIIA-treated group compared with the Gel-MS and untreated MI groups (24.75% vs 38.7% 44.81%, p < 0.05) (Figure 4g).In parallel, echocardiographic data verified that there were no significant differences in the LVEDD, LVESD, LVEF, LVFS, or CO with intramyocardial injection of Gel-MS or TIIA (5.8 mg/100 lL) compared with MI group (p > 0.05, Table S3 and S4, Supporting Information).In addition, HE and Masson staining showed no significant differences in the characteristics of the left heart cavity area, ventricular wall thickness, or percentage of collagen fiber area between the TIIA and MI groups (p > 0.05).Our collective findings clearly demonstrated that Gel-MS/ TIIA significantly reduced infarct size and restrained adverse LV remodeling in rats with MI.

Gel-MS/TIIA Decreases the ROS Level and Apoptosis in Cardiomyocytes
Myocardial cells in the post-MI heart are ischemic and hypoxic, which results in the production of large quantities of ROS. [35][38] To monitor the ROS levels in the post-MI heart, we measured the ROS content in the myocardial tissue via staining.Additionally, the levels of ROS-related factors, such as malondialdehyde (MDA), superoxide dismutase (SOD), and glutathione (GSH), were determined using enzyme-linked immunosorbent assay (ELISA).Notably, the levels of ROS in the myocardial tissue were significantly higher in the MI group than in the sham group.In contrast, the Gel-MS/TIIA group displayed a marked reduction in ROS relative to the MI group (Figure 5a).Similarly, ELISA revealed a significant increase in the levels of oxidative stress factors, including MDA, SOD, and GSH, in the myocardial tissue in the MI group, which was markedly reduced in the group treated with Gel-MS/TIIA (Figure 5b).Furthermore, the TdT-mediated dUTP nick-end labeling (TUNEL) test, which measures apoptosis, showed excessive cardiomyocyte apoptosis in the heart tissue of rats in the MI group compared to the sham group.Notably, cell apoptosis was significantly inhibited in the Gel-MS/TIIA treatment group compared with that in the MI group (Figure 5c).Moreover, both Gel-MS and Gel-TIIA treatment groups showed decreased ROS Energy Environ.Mater.2024, 7, e12555 production and apoptosis compared with the MI group, but to a lesser extent than the Gel-MS/TIIA-treated group.The free radical scavenging activity of Gel-MS/TIIA suggested that the TIIA-loaded hydrogels could also be used to scavenge harmful radicals in infarcted tissue to minimize the critical microenvironment following MI.

Gel-MS/TIIA Decreases Cell Pyroptosis and Inhibits Inflammation
Pyroptosis is a novel type of programmed cell death accompanied by the release of a large number of pro-inflammatory factors.Myocardial  9 of 13 cells initiate pyroptosis and inflammatory response in the presence of myocardial ischemia and hypoxia.[44] In view of earlier findings, we determined the expression of typical pyroptosis marker proteins and downstream pro-inflammatory factors to assess the anti-pyroptosis and antiinflammatory effects of Gel-MS/TIIA and clarify the underlying mechanisms in post-MI hearts.
Immunofluorescence staining was performed to evaluate the expression of the pyroptosis marker proteins N-terminal gasdermin D (N-GSDMD) and gasdermin E (GSDME).Heart sections from the MI group contained a large number of pyroptosis marker proteins.Conversely, sections from the Gel-MS/TIIA treatment group showed suppressed protein expression (Figure 6a).Western blotting was used to determine the protein expression of the pyroptosis gene, N-GSDMD, and its downstream inflammatory factors, NOD-like receptor protein 3 (NLRP-3) and cysteinyl aspartate specific proteinase-1 (caspase-1).Compared with the sham group, the expression of pyroptosis and inflammatory proteins was significantly increased in the MI group, and the group treated with Gel-MS/TIIA showed significantly inhibited expression compared with the MI group (Figure 6b).
In addition, immunohistochemical (IHC) staining was performed to observe the expression of inflammatory factors NLRP-3, interleukin-1 (IL-1b), interleukin-18 (IL-18), and tumor necrosis factor-a (TNF-a).Consistently, the expression of inflammatory factors was significantly increased in the MI group compared to the sham group, whereas that in the Gel-MS/TIIA group was markedly inhibited relative to the MI group (Figure 6c).ELISA was subsequently used for quantitative analysis of the expression levels of the above inflammatory factors, which revealed patterns similar to those obtained with IHC staining (Figure 6d).
Cardiomyocyte necrosis, inflammatory cell infiltration, and ECM degradation result in thinning of the ventricular wall and dilation of the heart. [35,45]However, the efficacy of current therapeutic strategies for MI remain limited unfavorable, at least partly, due to low retention of the drugs in the infarcted heart zone. [46]Localized administration of anti-inflammatory agents benefited patients suffering from MI by repressing or modulating the inflammatory response of the MI region, thus accelerating the repair of impaired tissues. [47]Therefore, the use of injectable hydrogel-encapsulated agents to improve global cardiac function and favorable therapeutic efficacy has recently attracted considerable attention. [48,49]Thermoresponsive poly(L-lactic acid)-poly (ethylene glycol) gel injection could limit the infarction size and mitigate post-infarction LV remodeling in a rat model. [50]The p-p conjugation-containing conductive injectable polymer hydrogel significantly increased the LV ejection fraction, reduced the fibrosis area, and increased vessel density. [51]PEG hydrogels are widely used in cardiac tissue engineering.However, the bioinert PEG hydrogel could not inhibit adverse LV expansion, suggesting that treatment with this bioactive hydrogel alone is not recommended for functional recovery of postinfarcted cardiac functions.
In this study, we designed and synthesized a Gel-MS/TIIA drug delivery system to inhibit pathological changes after MI.Owing to its physicochemical properties, the PLGAÀPEGÀPLGA triblock is suitable for convenient living tissue injection at 4 °C, and automatically transforms into a viscous gel at 37 °C.These results validated the successful application of this hydrogel system administered via in situ injection in a post-MI rat model.The mechanical properties of the hydrogel could support myocardial tissue, limit infarct expansion, improve ischemic border zone function, and ameliorate LV remodeling after MI onset.
Furthermore, hydrogel-encapsulated ROS-responsive release microspheres could trigger on-demand release of TIIA on the one hand, while consuming excessive ROS generated in post-MI heart and improving the local microenvironment.
Moreover, our design realizes a high TIIA loading efficiency in hydrogels and a stable and sustainable release behavior.Consequently, long-term inhibition of oxidative stress and inflammation factors in locally infarcted myocardium was observed by immunofluorescence staining and western blot results (Figures 5 and 6).Therefore, Gel-MS/ TIIA might potentially provide a minimally invasive therapeutic method aimed at attenuating fresh infarct expansion and directly providing structural support for a favorable cardiac repair response and prevention of adverse ventricular remodeling.

Conclusion
In this study, a novel injectable Gel-MS/TIIA system consisting of an insitu thermosensitive PLGA-PEG-PLGA gel and TIIA-loaded ROS-sensitive microspheres was fabricated.The thermosensitive hydrogel exhibited good mechanical properties after reaching the body temperature.Moreover, ROS-sensitive microspheres within this system can be immobilized by a thermo-sensitive gel and eventually released after its interaction and partial degradation, stimulated by ROS accumulation in the MI-afflicted myocardium.We have established that TIIA is eventually released from GelÀMS/TIIA and exerts effective antioxidative, antipyrolysis, and anti-inflammatory effects on the infarcted myocardium.The collective therapeutic properties of our novel system demonstrate effective mitigation of LV remodeling and a consequent remarkable improvement in cardiac functions of the infarcted heart, strongly encouraging its utility as a potential future option for clinical MI therapy.
Preparation of thermoreversible hydrogel-encapsulating TIIA@ROS-sensitive microspheres (Gel-MS/TIIA): Preparation of TIIA@ROS-sensitive microspheres (MS/TIIA)-2, 2 0 -Diselanediylbis (ethan-1-ol) (DSB; 0.20 mmol, 49.8 mg) and L-lysine diisocyanate (LDI; 0.21 mmol, 47.5 mg) were dissolved in anhydrous DMF (dimethylformamide) (5 mL) and stirred at 50°C for 24 h.The mixture was Energy Environ.Mater.2024, 7, e12555 dialyzed (MWCO of dialysis bag, 10 kDa) and freeze-dried under reduced pressure to obtain the PSSP.The synthetic routes for PSSP are presented in the Figure S1, Supporting Information.The structures of the prodrugs were confirmed via 1 H-NMR (Bruker, AV-400, Switzerland) using d-DMSO and CDCl 3 as the solvents (Figure S2, Supporting Information).The synthetic PSSP and 1 H-NMR verification information is provided in the Supporting Information.Briefly, PSSP (100 mg) and TIIA (50 mg) were dissolved in 2.0 mL DCM.After complete dissolution, the solution was added dropwise to 20.0 mL of a 1.5% (w/v) PVA aqueous solution and homogenized using a high-speed shearing machine (BME100LX, Wei Yu Mechanical and Electrical Manufacturing Co., Ltd., China) at 3000 rpm for 3 min.The emulsion formed was immediately poured into 100 mL of a 0.5% (w/ v) PVA aqueous solution and stirred using a magnetic stirrer for 6 h until MS solidification.MS was collected via centrifugation at 1000 g for 5 min, washed with distilled water three times to remove PVA and untrapped drugs, and freezedried until further use.The amount of TIIA was determined by ultraviolet-visible (UV-vis) spectrometry (UV-1800, Shimadzu, Kyoto, Japan) at 452 nm, and the drug-loading content (DLC) was calculated based on the following formula: DLC (%) = W TIIA/W MS/BUP 9 100%, where W TIIA represents the weight of TIIA in MS/TIIA and W MS/TIIA is the weight of MS/TIIA.
Preparation of thermoreversible hydrogel-encapsulated ROS-sensitive microspheres (GelÀMS/TIIA)-The triblock copolymer PLGA-PEG-PLGA was prepared via the ring-opening polymerization (ROP) of L-LA and GA, with PEG and Sn (Oct) 2 as the macroinitiator and catalyst, respectively.The mass percentage of the PLGA copolymer in the thermosensitive gel was 20 wt.%, as described previously.The sol-gel transition temperature of the PLGA polymer is related to its concentration.Upon increasing the polymer concentration from 15 to 25 wt.%, the sol-gel temperature decreased from 29 °C to 24 °C.Accordingly, PLGA-PEG-PLGA at a concentration of 20 wt.% was established as suitable for in situ drug delivery based on its solution state at room temperature (25 °C) and rapid alteration to the gel state at body temperature (37 °C).TIIA-containing PLGA-PEG-PLGA gel (GelÀTIIA) and gel microspheres (GelÀMS/TIIA) were prepared by dissolving the PLGA copolymer and TIIA or MS/TIIA in phosphate-buffered saline (PBS).Specifically, 120 mg MS/TIIA (containing 58 mg TIIA) was added to 1 mL Gel-MS/TIIA.In the GelÀTIIA system, 120 mg MS/TIIA was replaced with 58 mg TIIA.Samples were stirred at 4 °C for 24 h to obtain homogeneous suspensions.Subsequently, the suspensions were placed in a thermostat at 37 °C for 10 min for the formation of GelÀTIIA and GelÀMS/TIIA.
SEM and TEM characterization-The morphologies of TIIA, MS/TIIA, and GelÀMS/TIIA were examined using scanning electron microscopy (SEM; SEM ZEISS Gemini 300) and transmission electron microscopy (TEM; FEI Talos F200X).Samples of TIIA, MS/TIIA, and GelÀMS/TIIA were prepared as described above, stirred for 24 h at 4 °C, and placed in a thermostat at 37°C for 10 min for gel formation, followed by freezing in liquid nitrogen for 30 s and lyophilization.All drug preparations were visualized by scanning electron aid of SEM and TEM.
Rheology analysis-The rheological behavior of the PLGA-PEG-PLGA copolymer solution with and without TIIA and MS/TIIA was investigated using an MCR 302 rheometer (Anton Paar, Graz, Austria).The temperature was set to 5-60 °C, with increments of 0.5 °C min À1 .Premixed samples (350.0 lL) were added to a 25.0 mm parallel plate and allowed to stand for 5 min for structural recovery before the addition of sufficient silicone oil to cover the free surface and limit dehydration.The storage modulus (G 0 ) and loss modulus (G″) were measured at strain and frequency of 1% and 1 Hz, respectively.
Evaluation of in vitro release, erosion and degradation properties-To establish the release properties of TIIA from GelÀTIIA and GelÀMS/TIIA, 0.5 g PLGAÀPEGÀPLGA copolymer solution with TIIA or MS/TIIA was placed in a cylindrical vial with a diameter of 16 mm within a water bath at 37°C until the formation of GelÀTIIA and GelÀMS/TIIA, respectively.Next, PBS (3.0 mL) with or with 0.25 mM or 0.5 mM H 2 O 2 was slowly added to the surface of GelÀTIIA or Gel-MS/TIIA.The vials were maintained in a 37 °C shaking incubator at 70 rpm.PBS was collected at predetermined intervals and fresh buffer was added to the samples for further measurements.The concentration of TIIA released into PBS was determined by measuring the absorption at 452 nm.To evaluate the erosion and degradation properties of GelÀMS/TIIA and GelÀTIIA, 3.0 mL) without or with H 2 O 2 (0.25 mM, 0.5 mM) was slowly added to the surfaces of GelÀMS/TIIA and GelÀTIIA in cylindrical vials, and the samples placed in a 37 °C shaking incubator at 70 rpm.The PBS was removed, and the remaining formulations were weighed every other day.
Establishment of a MI model in rats and drug administration via intramyocardial injection-Adult male Sprague-Dawley rats (10-12 weeks old, 200-250 g) were used for the experiments.All experiments were approved by the Committee on Ethics on Animal Experiments at Guangdong Provincial Hospital of Chinese Medicine and were performed according to the principles of the American Physiological Society.Rats were randomized into five groups: 1) Sham (LAD threading with no ligation and 100 lL saline injection), 2) MI (LAD ligation and 100 lL saline injection), 3) Gel-MS (LAD ligation and 100 lL Gel-MS injection), 4) Gel-TIIA (LAD ligation and Gel-TIIA injection; 5.8 mg TIIA in 100 lL gel), 5) Gel-TIIA/MS (LAD ligation and Gel-TIIA/MS injection; 5.8 mg TIIA in 100 lL Gel-TIIA/MS).Next, the rats were anesthetized via intraperitoneal administration of sodium pentobarbital (50 mg kg À1 ), intubated, and mechanically ventilated using a rodent respirator.The heart was exposed, and the left anterior descending coronary artery was ligated to create a left ventricular (LV) infarction.After ligation, 100 lL of each drug was injected into the border and center regions of the infarct (four injections, 25 lL per region), followed by surgical closure of the chest wound.All animals were transferred to constant-temperature pads and allowed to recover from surgery.After in situ injection of the drugs for 14 days, blood and heart tissue samples were collected.
Tissue distribution-For in vivo fluorescence imaging, cyanine 7.5 (Cy 7.5) maleimide, Cy 7.5, was loaded into Gel or Gel-MS for visualization during injection.After 1 min, 7, and 14 days of injection of Cy 7.5, Gel-Cy 7.5, and Gel-MS/Cy 7.5, rats were sacrificed, hearts excised, and the biodistribution of Cy 7.5, Gel-Cy 7.5, and Gel-MS/Cy 7.5, were examined via fluorescence imaging using an IVIS Lumina III Imaging System (Caliper, USA) with an excitation filter of 670 nm and an emission filter of 700-800 nm.
Echocardiography-Rats were anesthetized with 2% isoflurane/oxygen inhalation and placed in a supine position on a constant-temperature pad.LV function was assessed using a Vevo 770 high-resolution digital ultrasound imaging system (Primetech Inc.) and 18 MHz scanning head (MS 200) with two-dimensional guided M-mode echocardiographic measurements at baseline and 1 and 2 weeks after ligation.LV dimensions, including the left ventricular end-systolic dimension (LVESD) and left ventricular end-diastolic dimension (LVEDD), were obtained via M-mode tracings, and left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), and cardiac output (CO) values were determined.Each evaluation was conducted for an average of 10 consecutive cardiac cycles, and all experiments were performed three times in a blinded manner.
Histological HE and masson staining-The heart tissue was fixed and embedded in paraffin after dehydration, followed by paraffinization and dewaxing.For HE staining, the sections were initially stained with hematoxylin solution for 3-5 min, followed by treatment with hematoxylin differentiation solution, hematoxylin Scott Tap Bluing, 85% ethanol for 5 min, and 95% ethanol for 5 min.Finally, the sections were stained with eosin dye for 5 min, dehydrated, and sealed with neutral gum.For Masson staining, the slices were soaked in Masson's dye solution overnight.Masson B and Masson C were mixed at a ratio of 1:1 to prepare Masson's solution.Tissues were stained with Masson's solution for 1 min and differentiated using 1% hydrochloric acid alcohol.The slices were immersed in Masson D for 6 min, Masson E for 1 min, and Masson F for 2-30 s.Next, the slices were rinsed in 1% glacial acetic acid, followed by dehydration with two cups of anhydrous ethanol, treatment with 100% ethanol for 5 min, and xylene for 5 min.Finally, sections were sealed with neutral gum and subjected to microscopic examination, image acquisition, and analysis.
Enzyme-linked immunosorbent assay-Myocardial damage, heart failure, and inflammatory cytokines in the myocardium and blood from the five groups of rats were measured using commercial enzyme-linked immunosorbent assay (ELISA) kits for MDA, SOD, GSH, NLRP-3, IL-1b, IL-18, and TNF-a (Biotech, MN, USA).All spectrophotometric readings were performed using a microplate reader (Multiskan MK3, Thermo Fisher Scientific, USA) according to the manufacturer's instructions.
Immunofluorescence staining of ROS-Frozen slides were incubated at room temperature and the objective tissues were marked with a liquid blocker pen.Spontaneous fluorescence quenching reagent was added for a 5 min incubation period.The marked area was incubated with ROS staining solution at 37 °C for 30 min in the dark, followed by DAPI solution at room temperature for 10 min in the dark.After removal of the liquid, the slides were cover-slipped with anti-fade mounting medium.The slides were examined, and images were obtained via fluorescence microscopy.DAPI displayed blue fluorescence under UV excitation at 330-380 nm Energy Environ.Mater.2024, 7, e12555 and emission at 420 nm.FITC displays green fluorescence at excitation and emission wavelengths of 465-495 nm and 515-555 nm, respectively.CY3 displayed red fluorescence under excitation at 510-560 nm and emission at 590 nm.
TUNEL analysis-The sections were then deparaffinized and rehydrated.Following liquid removal, the target tissue was marked with a liquid blocker pen.Tissues were covered with proteinase K working solution and incubated at 37°C for 25 min.After removal of the excess liquid, the permeabilization solution was added and incubated at room temperature for 20 min.Sections were slightly dried, and buffer was added to the tissues within the marked circle at room temperature for 10 min.Appropriate amounts of TDT enzyme, dUTP, and buffer from the TUNEL kit were used according to the number of slices and tissue size, mixed at a 1:5:50 ratio, and incubated at 37°C for 2 h.Next, Sections were incubated with DAPI at room temperature for 10 min in the dark and washed three times with PBS.The liquid was removed, and the sections were cover-slipped with anti-fade mounting medium.All sections were observed under a fluorescence microscope, and images were obtained for analysis.
Immunohistochemical analysis-The tissues were fixed with 10% formaldehyde, dehydrated over a low-to-high concentration alcohol gradient, rendered transparent with xylene, embedded in paraffin, and sectioned.Tissue sections were placed in a repair box filled with citric acid (pH 6.0) antigen retrieval buffer in a microwave oven and heated at medium power for 8 min until boiling.The microwave oven was turned off, and the sections were kept warm for 8 min and further subjected to medium-low power heating for 7 min.Next, the sections were placed in 3% hydrogen peroxide and incubated at room temperature in the dark for 25 min.After removal from the solution, the liquid was removed and the objective tissue areas were marked with a liquid blocker pen.BSA (3%) was added to the marked circle to evenly cover the tissue, which was then sealed for 30 min at room temperature.The sealing solution was gently removed, primary antibody prepared with PBS (PH 7.4) added, and sections placed on a flat surface in a wet box for overnight incubation at 4 °C.Next, the sections were dried, and tissues were incubated with secondary antibody (HRP-labeled) from the corresponding species of primary antibody at room temperature for 50 min.A newly prepared DAB color developing solution was added after drying the sections.The color developing time was controlled under the microscope, with brownishyellow color classified as positive.Sections were rinsed with tap water to terminate the reaction and counterstained with hematoxylin solution for about 3 min, followed by dehydration and mounting.Staining of tissue was visualized under a microscope and images were acquired for analysis.Upon immunohistochemical staining of paraffin sections, nuclei were stained blue with hematoxylin and sections with positive expression of DAB stained brownish-yellow.
Immunofluorescence analysis-Slides were deparaffinized and rehydrated, immersed in EDTA antigen retrieval buffer (pH 8.0) and maintained initially at sub-boiling temperature for 8 min followed by room temperature standing for 8 min and further incubation at sub-boiling temperature for 7 min.After removal of liquid, the objective tissue was marked with a liquid blocker pen.Marked tissue sections were incubated with 3% BSA to block non-specific binding for 30 min.The tissue area was immersed in 10% donkey serum or 3% BSA.Slides were incubated with primary antibody overnight at 4 °C, followed by secondary antibody at room temperature for 50 min in dark.Next, slides were treated with DAPI solution at room temperature for 10 min in the dark.After incubation with spontaneous fluorescence quenching reagent for 5 min, slides were washed in running tap water for 10 min.The liquid was subsequently removed and slides coverslipped with anti-fade mounting medium for analysis via fluorescence microscopy.DAPI displays blue fluorescence at UV excitation wavelengths of 330-380 nm and emission wavelength of 420 nm.FITC displays green fluorescence at excitation wavelengths of 465-495 nm and emission wavelengths of 515-555 nm.CY3 shows red fluorescence at excitation wavelengths of 510-560 nm and emission wavelengths of 590 nm.
Immunoblot analysis-Homogenates of LV myocardium samples were evaluated using immunoblot analysis.Briefly, following homogenization of frozen samples in ice-cold lysis buffer, proteins (40 L g) per lane were separated on 8%-15% SDS-polyacrylamide gels and transferred to PVDF membranes.Protein lysates were probed with primary antibodies diluted in 5% BSA overnight at 4 °C.Next, blots were rinsed, incubated with horseradish peroxidase-conjugated secondary antibodies in 5% BSA for 1 h, and developed using a chemiluminescent substrate.Densitometric quantifications were conducted using Image J software (version 1.44, Bethesda, Maryland, USA).
Statistical analysis-Experimental data (expressed as mean AE SEM) were assessed using one-factor analysis of variance (SPSS software, version 22.0, SPSS Inc.).Differences among various experimental and sham groups were analyzed using Student's t-test or ANOVA.The differences were considered statistically significant at *p < 0.05, **p < 0.01, and ***p < 0.001.

Figure 1 .
Figure 1.Schematic illustration of the preparation, drug release and targeting mechanisms of tanshinone IIA (TIIA)-encapsulating and reactive oxygen species-responsive MS loaded into the thermosensitive gel (Gel-MS/TIIA) system.

2. 2 .
Gel-MS/TIIA Preserves Cardiac Function after MI 2.2.1.Animal Model Preparation, Drug Injection, and Tissue Extraction The animal experiments are illustrated in Figure 3a.The rats were anesthetized and mechanically ventilated.Next, the heart was exposed

Figure 3 .
Figure 3. Intramyocardial injection of Gel-MS/TIIA and cardiac retention.a) Timeline of animal studies.b) Images obtained during injection (I.Exposure, II.Ligation, III.Injection, IV.Post-injection, and V. Open chest).c) Ex vivo IVIS imaging of heart following intramyocardial injection.d) Quantification of fluorescence intensities of Cy 7.5 in heart tissues on 1 min, days 7 and 14 after injection.e) Schematic diagram of the drug being "washed away," (n = 10), ***p < 0.001.

Figure 4 .
Figure 4. Therapeutic efficacy of injected Gel-MS/TIIA.a) Representative images of M-mode echocardiography; quantitative analysis of LV dimension and systolic function.LVEDD, LV end-diastolic diameter; LVESD, LV end-systolic diameter; LVEF, left ventricular ejection fraction; LVFS, left ventricular fractional shortening; CO, cardiac output.Representative images of b) HE staining and c) Masson staining of the whole heart and infarct and border zones.Quantitative analysis of d) left heart cavity area, e) ventricular wall thickness, f) percentage of collagen fiber area, and g) infarcted size (n = 10).*p < 0.05, **p < 0.01, ***p < 0.001.

Figure 5 .
Figure 5. Oxidative stress and apoptosis in cardiomyocytes.a) ROS staining to detect differences in ROS levels in post-MI heart tissue.b) Determination of levels of oxidative stress factors including MDA, SOD, and GSH, in post-MI heart tissue.c) Evaluation of apoptosis in post-MI heart tissue.(n = 10) *p < 0.05, **p < 0.01, ***p < 0.001.