We produced PCM-E2/PFPs nanoprobes through PCM conjugation, E2-loaded, and PFP encapsulation using a typical two-step emulsion process. A low-temperature process had to be adopted due to the relatively low boiling point of PFP (29 °C). As shown in Fig. 1, PCM-E2/PFPs nanodroplets had a milky white appearance (Fig. 1c) and presented an almost perfectly spherical morphology (Fig. 1a, b). It had an average diameter of 418 ± 11 nm with homogeneous distribution, as well as an average surface zeta potential of − 20 ± 1 mV (Fig. 1d, e). In addition, size distributions of different nanodroplets were compared to evaluate the effects of conjugation and encapsulation. We found no significant differences (p > 0.05) in size distributions between non-targeting E2 nanodroplets encapsulated with saline (E2/H2Os) and targeting PCM-conjugated E2 nanodroplets encapsulated with saline (PCM-E2/H2Os). After the addition of PFP to the targeting E2 nanodroplets (PCM-E2/PFPs), an apparent increase in average size was observed, although homogeneity and in vivo-favorable nanosize distribution were retained. This suggests that PFP can affect the size distribution of the droplets (Fig. 1f). Hyun et al. showed similar changes in size when PFP was encapsulated in echogenic glycol chitosan nanoparticles [32]. Additionally, the size of PCM-E2/PFPs showed no remarkable variation after 5 days of storage at 4 °C (Fig. 1g). The excellent stability of PCM-E2/PFPs during storage ensured their applicability during future experiments. The amount of E2 encapsulated in the PCM-E2/PFPs was determined using HPLC, with the encapsulation efficiency reaching 84.3 ± 2.8%.
Given that E2/PFPs nanoparticles do not have the ability to target cardiomyocytes on their own, they can accumulate at the cardiac site only through enhanced permeability. To improve their cardiomyocyte-targeting abilities, a 20-mer peptide with high binding affinity to cardiomyocytes was conjugated to the surface of the E2/PFPs. The extent of PCM conjugation was determined by detecting the connection between the FITC-labeled PCM and DiI-labeled nanodroplets. The merged orange images show a perfect connection between red E2/PFPs nanoprobes and green PCM peptides (Fig. 2b). The PCM conjugation efficiency was 97.33 ± 2.08% and accounted for only droplet-coupled PCM, given that all free conjugated nanodroplets were washed off (Fig. 2c).
Temperature-dependent phase transition process of PCM-E2/PFPs
To evaluate the phase transition behavior of PCM-E2/PFPs, size variations at different temperatures were visualized using an inverted fluorescence microscope equipped with a temperature-controlled stage. The PCM-E2/PFPs expanded gradually with an increase in external temperature (Fig. 2a). At lower temperatures (25 and 37 °C), no noticeable microbubbles appeared in the images due to insufficient thermal energy needed to vaporize the nanodroplets, demonstrating that PCM-E2/PFPs had a robust structure. Nevertheless, the boiling point of PFP is 29 °C, theoretically rendering it liquid at room temperature but gaseous at body temperature. Interestingly, PCM-E2/PFPs still remained liquid at 37 °C without undergoing phase transition. This phenomenon can be attributed to the effects of Laplace pressure at the boundary of the nanodroplets, which retarded the gas release and allowed the nanodroplets to retain their initial state at 37 °C [33, 34]. Continuously increasing temperature to 48 °C resulted in an increase in PCM-E2/PFPs size, and the initiation of microbubble formation. Almost all particles gradually expanded, while a large number of bubbles were generated when the temperature was further elevated to 60 °C, indicating that external temperature is a critical factor in the phase transition process of PCM-E2/PFPs. It is worth mentioned that during the process of microbubble formation, adjacent bubbles tended to coalesce with each other and form larger ones, similar to that found in previous research [35]. The strong hydrophobic interaction among PFP gases in the core of the generated microbubbles can be a good explanation for this phenomenon, which promotes adherence among bubbles. Two appealing advantages can be suggested from the temperature-dependent behavior of PCM E2/PFPs. One is that the relative stability of the nanodroplets at 37 °C ensures prolonged circulation time in vivo, while another is that the robust polymer shell of the nanodroplets retards gas release, which is important for enhanced US imaging after LIFU irradiation.
LIFU-triggered and temperature-dependent drug-release profile
To evaluate the temperature change of PCM-E2/PFPs triggered by the LIFU, the temperature were detected. As the trigger time of LIFU extended, the temperature gradually increased. When triggered for 10 min, the temperature reached to about 45 °C. When triggered for more than 10 min with LIFU, the temperature increase to 50 °C or even higher (Fig. 2d), which may result in the skin damage.
Given that PCM-E2/PFPs function as vessels for drug delivery, their drug-release profiles with and without LIFU exposure (2.4 W/cm2, 10 min) or heated (45 °C, 10 min) were verified. As expected, substantially higher E2 release rates were observed with LIFU-treated PCM-E2/PFPs (approximately 89% of E2) and with heated (approximately 82% of E2) than without any treatment (< 50% of E2) after 96 h (Fig. 2e). This indicated that external LIFU irradiation or heat treatment greatly enhanced the release of E2 from the nanodroplets. Meanwhile the E2 release rates was higher in LIFU-treated PCM-E2/PFPs than heat-treated PCM-E2/PFPs, this may due to the integral effect on all the nanodroplets, while LIFU, a focused ultrasound, may only function in small area. This is benefit for targeting drug release.
The high LIFU-triggered drug-release behaviors can maximize therapeutic efficacy through the expansion or rupture of the polymer shell. Considering the non-targeting nature of traditional non-focused ultrasonic devices and thermal damage from high-intensity focused ultrasound [36, 37], a LIFU-triggered drug delivery system could be an alternative method for promoting nanodroplet phase transition and drug release within the desired site. Similar to diagnostic US, LIFU can also generate acoustic waves outside the body and promote nanoprobe delivery to a specific organ.
US imaging of PCM-E2/PFPs in vitro and in vivo
To better understand phase transition in PCM-E2/PFPs, the effect of frequency, a crucial factor for inducing phase transition in PFP-encapsulated nanodroplets, should be investigated comprehensively.
Evaluation of the effect of LIFU frequency on US contrast imaging revealed that the images gradually brightened as frequency increased from 1.2 to 2.4 W/cm2. However, as LIFU frequency continuously elevated to 3.2 W/cm2, darkened images were observed. This probably indicated that the generated microbubbles had collapsed owing to the high frequency, which resulted in a remarkable decrease in the number of microbubbles (Fig. 3a). Furthermore, echo intensity analysis validated that the captured photographs were superior at a frequency of 2.4 W/cm2, which displayed the highest gray scale intensity (Fig. 3b). This result confirmed that LIFU frequency played an important role in improving phase transition by decreasing the droplet-to-bubble threshold. Therefore, 2.4 W/cm2 was the frequency selected for subsequent research, given that it was more suitable for PCM-E2/PFPs ultrasonography and prevented thermal injury to the skin. Moreover, after LIFU irradiation at 2.4 W/cm2, PCM-E2/PFPs were stable for more than 120 min in vitro (Fig. 3c), unlike the gas-filled sonovue solution, which was stable for only several minutes. These results demonstrated that PCM-E2/PFPs has great potential as an effective contrast agent for ultrasonic diagnosis.
Considering its outstanding performance during in vitro US imaging, the cardiac-targeting US imaging ability of PCM-E2/PFPs was confirmed in vivo by intravenous injection of targeting PCM-E2/PFPs and non-targeting E2/PFPs in rats. However, at an acoustic intensity of 2.4 W/cm2, no US contrast enhancement was found in the cardiac region (data not shown), which indicated insufficient energy for inducing phase transition of PCM-E2/PFPs within cardiac tissues. At a fixed LIFU acoustic intensity of 3.2 W/cm2, US imaging enhancement was apparent after irradiation for 10 min (data not shown). Therefore, in vivo US imaging experiments were performed at 3.2 W/cm2 for 10 min. We investigated the post-injection imaging performance of groups with and without LIFU stimulus. The results showed no obvious differences in US imaging between the PCM-E2/PFPs and E2/PFPs + LIFU groups. Nevertheless, LIFU-triggered PCM-E2/PFPs showed markedly enhanced capability for US imaging. Moreover, the change in echo intensity from 48.01 ± 7.94 to 33.68 ± 10.3 within 60 min during cardiac US imaging (Fig. 3e, f) indicated that LIFU can enhance the US imaging capability of PCM-E2/PFPs and thereby improve its accuracy during cardiac diagnosis. We also found that quantitative echo intensity values were substantially higher in the PCM-E2/PFPs + LIFU group than in the E2/PFPs + LIFU group (Fig. 3e), indicating effective cardiac accumulation. Primers also showed that acoustic nanodroplets were able to detect abnormalities in myocardial perfusion. Nevertheless, further studies are needed to optimize these nanodroplets in order to lower their vaporization threshold in vivo. This would increase nanodroplets vaporization in targeted tissues given the relatively lower imaging enhancement observed in nanodroplets than in microbubbles despite injecting greater amounts thereof.
In vivo biodistribution of PCM-E2/PFPs in rats
The targeted transportation and distribution of PCM-E2/PFPs in vivo were determined using DiI-labeled nanodroplets. Prominent and wide-ranging red dots representing DiI-labeled PCM-E2/PFPs distribution were observed in the cardiac cryosections in the PCM-E2/PFPs + LIFU group than PCM-E2/PFPs and E2/PFPs + LIFU groups under CLSM 12 h after injection, suggesting excellent cardiac targeting. Moreover, cardiac nanodroplet accumulation in the PCM-E2/PFPs + LIFU group was more prominent (Fig. 4a) than other tissues (liver, kidney, lung, spleen) (Fig. 4b), given that LIFU can be focused and can penetrate nanodroplets deep within the target regions. In addition, fluorometric analysis of DiI signals showed a 50% reduction in DiI serum concentration within 30 min of injection in the PCM-E2/PFPs + LIFU group and that the DiI signal lasted for more than 24 h (Fig. 4c). These results suggest that the combination of PCM-E2/PFPs with LIFU could greatly improve the efficiency of drug delivery in terms of PCM-guided active targeting, LIFU-triggered passive targeted drug release, and cavitation-induced enhancement of vessel permeability.
Assessment of safety
Histopathological evaluation of major organs, including the lungs, liver, spleen, kidneys, and brain, after PCM-E2/PFPs + LIFU treatment was performed using HE staining. As shown in Additional file 1: Fig. S1a, no noticeable morphological abnormalities in tissue architecture were detected in the PCM-E2/PFPs + LIFU group. To further assess the biosafety of this synergistic strategy, blood biochemical tests, including those for liver and renal function, were carried out. No significant variance in biochemical indicators of liver and kidney function were found among any group (Additional file 1: Fig. S1b), indicating excellent biocompatibility of PCM-E2/PFPs in rats. This suggested that PCM-E2/PFPs may have the potential to effectively reduce the side effects of E2.
Prevention of LV dysfunction in rats with cardiac hypertrophy
During the study, no morality of the animals was observed. Compared to the sham group, the TAC rats exhibited a significant increase in HM/TL, LM/TL, LVPWd, and IVSD, but a decrease in LVDD, indicating the occurrence of cardiac hypertrophy (Fig. 5a–g). In general, all four E2-treated groups exhibited slightly better LVDD and significantly higher HM/TL, LW/TL, LVPWd, and IVSD compared to the untreated hypertrophic animals (p < 0.05). The results indicated that E2 treatment attenuates cardiomyopathy. Furthermore, the greatest differences in the five parameters above were observed in PCM-E2/PFPs + LIFU group. In comparison, LM/TL and LVPWd were much lower in the PCM-E2/PFPs + LIFU group than in other groups (p < 0.05). LVEF, as determined by echocardiography, was similar in all groups, indicating that LV function remained compensated in all groups with TAC surgery.
Histological analyses of HE and Masson’s staining were performed using paraffin-embedded cardiac tissues (Fig. 6a, c). Cardiomyocyte disorganization and hypertrophy were accompanied by an altered collagen network structure in the studied animals. Moreover, CSA and CVF were significantly higher in the TAC group than in sham and other treated groups. Compared to the TAC group (719.08 ± 93.19 μm2 and 13.58 ± 2.05%), CSA and CVF were significantly lower in the E2/PFPs (596.45 ± 79.87 μm2 and 9.9 ± 2.48%), E2/PFPs + LIUF (561.17 ± 88.57 μm2 and 7.45 ± 1.08%), and PCM-E2/PFPs (536.27 ± 85.07 μm2 and 6.7 ± 1.98%) groups. Furthermore, CSA and CVF were significantly lower in the PCM-E2/PFPs + LIFU group (462.31 ± 74.04 μm2 and 2.88 ± 0.67%) than in other treatment groups (Fig. 6b, d).
RT-PCR analysis clearly showed that cardiac tissue-targeted delivery of therapeutic payloads combined with LIFU irradiation significantly regressed the cardiac hypertrophy as evidenced by the reduced expression of hypertrophy markers (Fig. 6e) and the higher expression of β-MHC, Collagen 1, and Collagen 3 in TAC group compared to the sham group (p < 0.05). Remarkably, E2-treated groups showed significantly lower expression levels of β-MHC, Collagen 1, and Collagen 3 than the TAC group. Moreover, the PCM-E2/PFPs + LIFU group exhibited the lowest expression of β-MHC, Collagen 1, and Collagen 3 among the treatment groups (p < 0.05).
Therapeutic efficacy data showed that LIFU-irradiated PCM-E2/PFPs might have increased the local concentration of the released drug in cardiac tissues, maximizing its anti-hypertrophic efficacy. Theranostic approaches have attracted major attention, given that they allow simultaneous diagnosis and treatment. The current study is the first to report on the development of a theranostic E2-loaded droplet-to-bubble nanoprobe for cardiac-targeted imaging and treatment. Three main factors may explain the synergistic mechanism through which PCM-E2/PFPs + LIFU irradiation affects cardiac hypertrophy. First, the excellent targeting ability of PCM peptides and cavitation-induced enhancement of vessel permeability increased the accumulation of nanodroplets in cardiac tissues. Second, LIFU-triggered passive targeted drug release promotes E2 release from PCM-E2/PFPs, accelerating E2 accumulation in cardiac tissues while minimizing systemic toxicity. The third factor may be attributed to the effects of myocardial cavitation-enabled therapy (MCET). Myocardial contrast echocardiography has been shown to be capable of causing lethal injury to cardiomyocytes, resulting in scattered microlesions throughout the scanned region. Interestingly, these microlesions can innocuously heal within a few weeks with minimal scarring, leaving a marked reduction in tissue volume [38], This noninvasive and relatively gentle method of tissue reduction has been shown to be advantageous in the treatment of cardiac hypertrophy [39]. Therefore, we hypothesis that this “droplets-to-bubbles” nanodroplets will have the similar effects on hypertrophic heart, which may be one potencial mechanism of PCM-E2/PFPs with LIFU irradiation in preventing myocardial hypertrophy, the precise MCET of PCM-E2/PFPs need to further be testified.
Taken together, our study has provided extensive evidence to strongly suggest that PCM-E2/PFPs combining with LIFU technique have a great potential in facilitating targeted imaging and delivery of E2 for the prevention of cardiac hypertrophy thus minimizing adverse effects to other organs.