Ultrasound Combined With PH-sensitive Dual-targeted Doxorubicin-loaded Prodrug-microbubble Complex And Evaluation Of Antitumor Activity

Background and objective:Doxorubicin hydrochloride (DOX), an anticancer agent belonging to the anthracycline class, is a leading clinical cytotoxic drug for breast cancer. However, the therapeutic efficacy of free DOX is compromised by various side-effects, including severe cardiotoxicity, nephrotoxicity and myelosuppression. Development of novel strategies to improve therapeutic efficacy and reduce side-effects is therefore crucial for the successful treatment of breast cancer.The pH value of normal tissue is around 7.4, while that of tumor tissue is as low as 6.0, due to hypoxia and high lactate metabolism of the tumor microenvironment. pH-responsive drug carrier can improve antitumor efficacy and reduce side effect. Different types of linking groups, specially stimuli-sensitive linkages including acid labile cisaconityl, acyl hydrazone groups, ester groups and disulfide groups have been explored to increase the in vivo drug delivery efficacy. For example, it has been reported that a pH-sensitive drug carrier conjugated with DOX showed lower toxicity to the normal cells, increased inhibition rate to the C6 glioma cells and enhanced BBB transport ratio. Here, we prepared a pH-sensitive prodrug composed of DOX and dual-targeted ligands, cRGD and folate, using heparin as the backbone. cRGD was decorated with polyethyleneglycol (PEG), with the aim of extending circulating time and escaping the reticuloendothelial system (RES) in vivo. Simultaneously, DOX was conjugated to heparin via a pH-sensitive hydrazone linkage to facilitate its release in the tumor microenvironment. Although the prodrug with high drug-loading capability exerts a greater anti-cancer effect than free DOX in vitro, it is inclined to aggregate and enlarge in size, which is unfavorable for drug dispersion and presents a significant obstacle for penetration into solid tumors.Ultrasound-targeted microbubble destruction (UTMD) is an adjuvant modality for drug delivery to localize intratumoral drug release and enhance intracellular drug accumulation. The inertial acoustic cavitation of microbubbles (MBs), including bubble implosion, microstreaming, shock waves and microjets, causes sonoporation (pore forming), which greatly improves intracellular uptake of drugs at the target site. Drug-loaded MBs with micron sizes ranging from 2-10 um have been widely investigated as carriers, and the well-characterized drug-loaded liposomes incorporated in MBs are uniformly nanosized. Aggregation is a common issue during preparation of the nanoparticle drug delivery system. In view of earlier findings that nanoparticles can be fragmented into smaller pieces under laser irradiation to promote drug release, we combined aggregated prodrug and MB and explored whether these large-sized drug-loaded particles could be disrupted to facilitate intracellular uptake into tumor cells, assisted by US as an external force.In the current study, aggregated dual-targeted pH-sensitive DOX prodrug was conjugated with MB via an avidin-biotin bridge to generate a DOX prodrug-MB complex (DPMC). We examined the morphological changes of DPMC before and after US destruction and focused on validating its tumor targeting specificity using in vivo fluorescence and ultrasound molecular imaging analyses. In particular, the anti-tumor efficacy of the complex with and without US was evaluated, both in vitro and in vivo. For prodrugs with significant cytotoxicity but relatively larger sizes, the newly generated complex assisted by US represents a promising approach to decrease size and extend their applications in vivo, providing an alternative therapeutic anti-tumor strategy.Materials and methods:1. Preparation of pH-sensitive dual-targeted DOX prodrug.To prepare pH-sensitive dual-targeted DOX prodrug, a proportion of succinylated heparin, Folate-NH2, Biotin-NH2, PEG-cRGD, EDC and NHS were added in Dimethylsufoxide(DMSO) and reacted at room temperature for 24 h. The mixture was dialyzed using a dialysis membrane for 48 h. DOX was conjugated to heparin through hydrazone linkage.2. Characterization of the DOX prodrugParticle size and zeta potential of pH-sensitive and pH-insensitive dual-targeted DOX prodrug were assessed using Dynamic laser light scattering (DLS) and their Morphology were further assessed via transmission electronic microscopy (TEM). Drug loading content of the prodrug was analyzed using UV-vis spectrophotometry.3. Preparation of pH-sensitive dual-targeted DOX prodrug-microbubble complexThe complex was prepared with the aid of avidin-biotin staining. A given amount of avidin was added to biotinylated MBs and incubated on ice for 30 min. MBs were washed with double-distilled water three times to remove unreacted avidin, and the corresponding amounts of prodrugs added and incubated for a further 30 min. Complexes were washed 3-4 more times to remove free prodrugs.4. Ultrasound exposureUltrasound exposure was achieved with 20 mm US probe of a therapeutic US system (CZ906A, Chongqing Medical University, Chongqing, China). Irradiation parameters were set as follows:1 MHz,50% duty cycle, duration of 1 min and US intensity of 1 W/cm2 for in vitro and 2 W/cm2 for in vivo experiments.5. Characterization of pH-sensitive dual-targeted DOX prodrug-microbubble complexParticle size and zeta potential of the complex before and after ultrasound were assessed using DLS and their Morphology were further assessed via TEM. To characterize the morphologic differences between the complex with and without US exposure, the suspension was mounted on a slide with a coverslip and visualized using confocal microscopy. The size distribution of the complex was further estimated using a Coulter Multisizer.6. Evaluation of antitumor activity for the complex in vitro and in vivo6.1 In vitro experiments6.1.1 In vitro release of doxorubicinEqualvalent content of DOX, pH-sensitive dual-targeted DOX prodrug and the complex were dialysised in PBS at different pHs (5.0 and 7.4), the complex was exposed to ultrasound according to the above parameters in advance. At predetermined time intervals,2 mL of release medium was removed for drug concentration measurement by a UV/Vis spectrophotometer.6.1.2 Cellular uptakeFor quantitative evaluation of the cellular uptake of pH-sensitive dual-targeted DOX prodrug and the complex with US was evaluated using flow cytometry compared to free DOX on MCF-7 cells.6.1.3 In vitro cytotoxicity assaysThe cytotoxicity of pH-sensitive dual-targeted DOX prodrug and the complex with US was evaluated using MTT assay compared to free DOX on MCF-7 cells.6.2 In vivo experiments6.2.1 Ultrasound Molecular ImagingTumor-bearing mice were anesthetized and visualized by a Philips iU22 ultrasound system. After a single bolus injection of 0.2 mL blank MBs or complexes (1×109/mL) through the tail vein, the process was continuously monitored via ultrasonography. After 4 min, targeted MBs generally adhered firmly to the tumor site. Blank MBs or complexes were destroyed using the Flash mode of the ultrasound system. Datasets from all mice were analyzed offline in a random order with MCE software.6.2.2 In Vivo Fluorescence Imaging AnalysisFree Cy-5.5, Cy-5.5-loaded complex and Cy-5.5-loaded complex with US (similar absorption intensity as Cy5.5,200 μL) were intravenously injected into tumor xenograft mice. The complex with US exposure group was irradiated with US after the injection as described earlier. At 0.5,12,24,48 and 72 h post-injection, biodistribution in MCF-7 tumor-bearing mice was visualized using an in vivo fluorescence imaging system.6.2.3 In vivo antitumor efficacyTumor-bearing mice were randomized into four groups (5 mice/group):Control, free DOX DOX, complex without US and complex with US. Each mouse was intravenously injected with the corresponding formulations at 2.5 mg DOX equivalent per kg within a final volume of 200μL through the tail vein five times every 4 days. For the complex with US group, tumors were irradiated with US after every injection. At the end of the experimental period, all mice were sacrificed, and tumors harvested and weighed. Throughout the experiment, body weights of mice and tumor volumes were measured every other day.6.2.4 Histological and immunohistochemical analysesAt the end of the experiment, different organs, including heart, liver, spleen, lung, kidney and tumor, were collected and analyzed by eosin (H&E) staining, immunohistochemical analyses of Capase-3, Ki67 and CD34. Tissue slices were visualized under an optical microscope and subsequently analyzed with Image J2x software.7. Statistical analysisStatistical analysis was conducted with SPSS software (version 19.0). Data are presented as means±SD. Significant differences between groups were determined using Student’s t-test, one-way ANOVA and Repeated Measures ANOVA. Data were considered statistically significant at p<0.05. All experiments were repeated at least three times.Results:1. Charaterization of pH-sensitive dual-targeted DOX prodrugTEM images revealed approximately spherical, small-sized pH-sensitive prodrug, along with aggregated morphology. Corresponding DLS revealed its inhomogeneous size distribution (average diameter:149.6±29.8 nm and 1036.2±38.8 nm) and zeta potential of -19.8±4.5 mV. The DOX loading content detected via UV-vis analysis was 18.9%.2. Charaterization of pH-sensitive dual-targeted DOX prodrug-MB complexCompared with aggregated prodrug, application of external US induced fragmentation of complex into uniformly smaller particles (average diameter:128.6± 42.3 nm, PDI:0.21, zeta potential:-20.6±3.4 mV), as measured with DLS, corresponding to TEM findings. The morphology of the complex was additionally confirmed via confocal microscopy. The surface of MB was clearly surrounded by the prodrug with red fluorescence before US dispersion. After US radiation, complexes were disrupted into small fragments displaying red fluorescence. In addition, the DOX loading content of the prodrug detected via UV-vis analysis was 18.9%. The average diameter of the complex was 5.87±0.08μm with PDI of 0.09, as estimated using a Coulter counter.3. Evaluation of antitumor activity in vivo and in vitro for the complex3.1 In vitro experiments3.1.1 In vitro release of doxorubicinCompared with 30% obtained at pH 7.4, cumulative release of DOX from both prodrug and complex with US at pH 5.0 was estimated as～90% after 8 h, indicating comparative stability in neutral conditions. The data clearly demonstrate that the introduction of MB into the prodrug does not hinder its pH responsiveness, but rather, promotes DOX release due to US cavitation.3.1.2 Cellular uptakeThe fluorescence intensity of the prodrug was higher than that of free DOX, indicating that the dual ligand-targeting effect and pH-sensitive properties of the prodrug contribute to enhanced cellular internalization. Moreover, complex with US exhibited the greatest internalization ability among the three agents.3.1.3 In vitro cytotoxicityThe in vitro cytotoxicity of the prodrug and complex with US, compared with free DOX, was estimated in MCF-7 cells using the MTT assay. After 48 h incubation, cytotoxicity was increased in the order of free DOX< prodrug< complex with US (IC50:310.35 ng/mL,217.43 ng/mL,120.23 ng/mL, respectively).3.2 In vivo experiments3.2.1 Ultrasound Molecular ImagingThe targeting ability of complex was investigated compared with that of blank MBs. Videos were recorded for the whole process and analyzed offline in a random order using MCE. The color intensity localized in the tumor region from mice treated with complex was obviously higher than that from mice treated with blank MBs, indicating that the proportion of complex adhering to the tumor site is significantly higher than that of blank MBs.3.2.2 In vivo fluorescent imagingMice treated with Cy5.5-labeled complex combined with US presented higher accumulation of the complex at the tumor site, compared with other groups, with the strongest fluorescence intensity detected within the initial 0.5 h after intravenous injection.3.2.3 In vivo antitumor efficacyThe control group showed a progressive increase in tumor volume while tumors in all drug-treated groups displayed growth retardation. DOX-treated group displayed greater antitumor efficiency than mice treated with complex without US exposure. Notably, treatment with complex assisted by US led to a higher tumor inhibition rate than DOX alone. The DOX group showed an obvious weight decrease, compared to the control, suggesting a certain extent of systemic toxicity, while the group exposed to complex plus US presented better drug tolerability.3.2.4 Histological and immunohistochemical analysesH&E staining revealed that organs and tumors showed no visible necrosis or acute inflammatory cell infiltration associated with animal toxicity in all groups, including mice treated with free DOX.For immunohistochemical analysis, compared to the control group, tumor tissues of mice exposed to complex combined with US showed relatively higher levels of capase-3-positive and lower levels of Ki-67-positive cells. Significant differences were observed relative to the other groups. Complex with US exerted the greatest antiangiogenic effect, and the corresponding MVD per field was significantly lower than that of other treated groups. It suggest that various mechanisms contribute to the antitumor effects of complex assisted by US, including enhancement of cell apoptosis, inhibition of cell proliferation and antagonism of angiogenesis.Conclusion:1. The dual-targeted pH-responsive doxorubicin prodrug-microbubble complex with excellent molecular imaging ability and antitumor ability has been successfully prepared.2. With the assistance of localized US, aggregated prodrug loaded in complex could be fragmented into smaller nanoparticles, facilitating intracellular accumulation and antitumor activity in vivo.2. Ultrasound combined with dual-targeted pH-responsive doxorubicin prodrug-microbubble complex could increase the tumor targeting ability and improve release of DOX thereby enhancing its antitumor efficacy.4. The combined strategy exerted significant antitumor efficacy through inducing apoptosis, inhibiting cell proliferation and antagonizing angiogenesis.