PD-L1-targeted microbubbles loaded with docetaxel produce a synergistic effect for the treatment of lung cancer under ultrasound irradiation

: Immunotherapy is gradually becoming as important as traditional therapy in the treatment of cancer, but adverse drug reactions limit patient benefits from PD1/PD-L1 checkpoint inhibitor drugs in the treatment of non-small cell lung cancer (NSCLC). As a chemotherapeutic drug for NSCLC, docetaxel (DTX) can synergize with PD1/PD-L1 checkpoint inhibitors but increase haematoxicity and neurotoxicity. Herein, anti-PD-L1 monoclonal antibody (mAb)-conjugated and docetaxel-loaded multifunctional lipid-shelled microbubbles (PDMs), which were designed with biological safe phospholipid to produce synergistic antitumour effects, reduced the incidence of side effects and promoted therapeutic effects under ultrasound (US) irradiation. The PDMs were prepared by the acoustic-vibration method and then conjugated with an anti-PD-L1 mAb. The material features of the microbubbles, cytotoxic effects, cellular apoptosis and cell cycle inhibition were studied. A subcutaneous tumour model was established to test the drug concentration-dependent and antitumour effects of the PDMs combined with US irradiation, and an orthotopic lung tumour model simultaneously verified the antitumour effect of this synergistic treatment. The PDMs achieved higher cellular uptake than free DTX, especially when combined with US irradiation. The PDMs combined with US irradiation also induced an increased rate of cellular apoptosis and an elevated G2-M arrest rate in cancer cells, which was positively correlated with PD-L1 expression. An in vivo study showed that synergistic treatment had relatively strong effects on tumour growth inhibition, increased survival time and decreased adverse effect rates. Our study possibly provides a well-controlled design for immunotherapy 3 and chemotherapy and has promising potential as a clinical application for NSCLC treatment.


Abstract:
Immunotherapy is gradually becoming as important as traditional therapy in the treatment of cancer, but adverse drug reactions limit patient benefits from PD1/PD-L1 checkpoint inhibitor drugs in the treatment of non-small cell lung cancer (NSCLC). As a chemotherapeutic drug for NSCLC, docetaxel (DTX) can synergize with PD1/PD-L1 checkpoint inhibitors but increase haematoxicity and neurotoxicity. Herein, anti-PD-L1 monoclonal antibody (mAb)-conjugated and docetaxel-loaded multifunctional lipid-shelled microbubbles (PDMs), which were designed with biological safe phospholipid to produce synergistic antitumour effects, reduced the incidence of side effects and promoted therapeutic effects under ultrasound (US) irradiation. The PDMs were prepared by the acoustic-vibration method and then conjugated with an anti-PD-L1 mAb. The material features of the microbubbles, cytotoxic effects, cellular apoptosis and cell cycle inhibition were studied. A subcutaneous tumour model was established to test the drug concentration-dependent and antitumour effects of the PDMs combined with US irradiation, and an orthotopic lung tumour model simultaneously verified the antitumour effect of this synergistic treatment. The PDMs achieved higher cellular uptake than free DTX, especially when combined with US irradiation. The PDMs combined with US irradiation also induced an increased rate of cellular apoptosis and an elevated G2-M arrest rate in cancer cells, which was positively correlated with PD-L1 expression. An in vivo study showed that synergistic treatment had relatively strong effects on tumour growth inhibition, increased survival time and decreased adverse effect rates. Our study possibly provides a well-controlled design for immunotherapy 4 for ultrasound (US)-targeted delivery 15 . Microbubbles' smaller size allows extravasation from blood vessels into surrounding tissues, improving stability and giving longer residence times in the systemic circulation 16 . Therefore, lipid-shelled microbubbles might be an appropriate drug delivery system for the combination of a PD1/PD-L1 checkpoint inhibitor and docetaxel (DTX).
As one of the most common noninvasive physical radiation sources, US plays an important role in clinical diagnosis and therapy. Lung US as an emerging theranostic modality exhibits non-ionizing properties, high local resolution, real-time imaging, and low cost 17 . Clinical trials have verified the efficacy and safety of pulmonary US irradiation treatment combined with corresponding drugs 18,19 . The cavitation and sonoporation effects are generally believed to contribute to the therapeutic effect of US irradiation, which can ensure specialized targeted delivery of proteins, genes, exosomes or traditional chemotherapeutic drugs [20][21][22] . Hence, we hypothesized that immunochemotherapeutic phospholipid microbubbles combined with US irradiation might enhance the efficacy and reduce the adverse effects of the combination of a PD1/PD-L1 inhibitor and DTX.
To verify our hypothesis, we designed a multifunctional microbubble system in which the membrane was DTX loaded and then anti-PD-L1 monoclonal antibody (mAb)-modified (PDMs). The anti-PD-L1 mAb could block the immunosuppressive PD1/PD-L1 pathway, while the PDMs directly kill tumour cells via DTX. Moreover, US irradiation was used to rupture the PDMs to further increase drug concentrations in the tumour. The cavitation and sonoporation effects improve the ability of drugs to enter 0.964 mV after coupling to an anti-PD-L1 mAb. These increases were concluded to be the result of successful synthesis of the antibody. The dispersions of the microbubbles are shown in Fig. 2B. The change in size could be a sign of successful binding of the antibody to the surface of the microbubbles. The encapsulation and loading efficiency of DTX in the DTX-loaded microbubble (DM) were 59.21 ± 1.97% and 4.75 ± 0.65%, respectively, and in the PDMs were 57.34 ± 2.61% and 4.45 ± 0.91%, respectively (Table S2).
The profiles of the release of DTX from the microbubbles with/without US irradiation were examined to assess the effects of sonication on DTX release. As shown in Fig. 2C, US irradiation significantly promoted drug release. CY5-labelled anti-PD-L1 mAb conjugation with FITC-labelled DTX microbubbles were observed by the laser scanning confocal microscopy (LSCM) (Fig. 2D). The microbubbles consistently maintained a round shape and were bound with a fluorescein-labelled antibody.
The BMs, DMs, and PDMs had similar contrast imaging capabilities in vitro and in vivo (Fig. 2E); the peak intensity and time to peak were not significantly different among the microbubbles ( Fig. 2F and Fig. 2G). In addition to the expression on a number of cancer cell lineages, PD-L1 was also expressed on non-parenchymal cells and non-haematopoietic lineages 5 . The distribution of PD-L1 in other organs might prevent the enhancing effect of the microbubbles from being obvious over a short period of time.
We also investigated whether BMs, DMs, and PDMs can induce haemolysis ( Fig.   S1A and B), the result showed none of the formulations was haemolytic. The toxicity study showed that no weight loss was observed, and serum biochemical parameters were within the corresponding reference ranges (Fig. S2A-E). HE staining showed that the free combo group resulted in thickened alveolar walls and lymphocyte infiltration in normal lung tissue. Our findings illustrate the immunochemotherapeutic microbubbles are biologically safe and lung protective.

Microbubble cytotoxicity in vitro
The PD-L1 expressions of one type of mouse cell (LLC) and three types of human cells (NCI-H460, NCI-H1299, and A549) were tested by flow cytometry. As shown in Fig.   3A and B, the expression of LLC cells was similar to that of NCI-H460 cells, while NCI-H1299 cells had the highest expression and A549 cells had the lowest. Free DTX and DMs had similar cytotoxicity in the four tumour cell lines. PDMs had stronger efficacy because of the conjugated anti-PD-L1 mAb, and this efficacy was strengthened by US irradiation (Fig. 3C-F). The results demonstrated that anti-PD-L1 mAb targeting and US irradiation could both enhance the cytotoxicity of DTX to the target tumour tissues.

In vitro enhancement of cellular drug uptake mediated by PD-L1
To further assess the relationship between the targeting efficiency of PDMs and affinity of DTX for LLC cells in vitro, the cellular uptake of DTX in different formulations was determined by fluorescence imaging and flow cytometry. C6 to indicate drug uptake was contained in microbubbles at a concentration equimolar to DTX. As shown in Fig.   3A and D, CLSM images indicated elevated intracellular drug uptake of C6-PDMs, especially with US irradiation. The flow cytometry results were also consistent with the 8 fluorescence imaging results (Fig. S4). Therefore, PD-L1-mediated internalization was more efficient than passive diffusion and nonspecific target. US irradiation could promote drug uptake, which might account for the increased cellular toxicity of PDMs when combined with US irradiation.

Apoptosis induction and cell cycle inhibition in LLC cells in vitro
The cell apoptosis study assessing different formulations utilized the Annexin V-FITC/PI method to further explore tumour killing. LLC cells were treated with US irradiation, free DTX, DMs, PDMs, or PDMs + US. After 24 h of incubation, the total apoptosis rate was analysed by flow cytometry. As shown in Fig. 3B and E, PDMs + US induced the highest apoptosis rate, free DTX and DMs produced similar apoptosis rates, and US irradiation monotherapy had no influence on apoptosis.
For cell cycle analysis, the PI/RNase method was employed and the remaining steps were the similar. In agreement with the cytotoxicity, cellular uptake, and apoptosis studies, the cell cycle analysis showed that cell cycle inhibition increased in response to different formulations ( Fig. 3C and F). Hence, using the aforementioned data, it can be concluded that the PDMs combined with US irradiation increased drug uptake, inhibited the cell cycle, promoted apoptosis, and enhanced drug toxicity to the cells.
Tumour resistance to Paclitaxel family agent has been a problem troubling clinicians for a long time. Until studies on transcription and post-transcriptional mechanisms revealed that Paclitaxel family agent induces the expression of PD-L1 immunosuppressive molecules through the mitogen-activated protein kinase (MAPK) pathway 23 . The evidence suggests patients may benefits from a synergy of docetaxel interest analysis (Fig. 5C) showed that PDMs combined with US irradiation displayed the highest fluorescence efficiency, which might portend enhanced therapeutic effects.
As predicted, US irradiation promoted rapid drug release in tumours, and the active targeting by the anti-PD-L1 mAb on the surface of PDMs promoted drug accumulation.
Comparing the distributions of different drugs in major metabolic organs showed that the distribution of free drug in the liver was distinctly lower than that of the microbubbles. It is deduced that increasing the time between injections might reduce liver injury, and this was confirmed in the biotoxicity experiment.
Due to the side effects of immunochemotherapy, a variety of materials have been used as adjuncts [25][26][27][28] . The targeted drug delivery system of ultrasonic microbubbles, coupled with an antibody that targets a corresponding antigen overexpressed on tumour cells, provides a promising strategy for reducing the severe adverse effects associated with chemotherapeutic drugs 29,30 . The fabricated PDMs could effectively target the tumour tissue and thus reduce off-target toxicity. PD-L1 is highly expressed on the surface of various tumour cells and helps tumour cells evade antitumour immunity.
Previous studies have indicated that anti-PD-L1 mAb-coupled drug delivery systems can target tumour cells 31 and activate the immune system 32 , and this approach can synergize with chemotherapy. Therefore, it can be concluded that PDMs combined with US irradiation exhibited an increased drug concentration and extended drug duration in tumours and were therefore beneficial for therapeutic effects of increased strength and duration. days, the mice were weighed, the tumour volumes were measured, and blood was obtained to assess liver and kidney functions. After all the treatments, the mice were sacrificed for subsequent histopathological study of the tumours and vital organs to evaluate efficacy and safety.

Inhibited tumour growth in a subcutaneous tumour model after treatments with
As shown in Fig. 6B-D, the PDMs combined with US irradiation group had the highest survival rate through the termination of experiment, and this group showed the best inhibition of tumour growth. The PDMs alone had a better therapeutic effect than the free-drug combination and ranked second. In addition, the synergistic therapy had more obvious efficacy than chemotherapy. The DMs produced slightly improved therapeutic effects on the tumour volume inhibition and survival rate. The difference in body weight was not obvious differences among the groups.
Apoptosis and proliferation in tumours were analysed via the TUNEL assay and CD31 and Ki67 immunohistochemistry ( Fig.7 A-F). The data showed that the PDMs combined with US irradiation group had the highest apoptosis rate and lowest proliferation level. The trend in the data was in accordance with the tumour growth observations. The results for cleaved caspase-3, cleaved caspase-8, and cleaved caspase-9 in Western blot assays also revealed that the PDMs combined with US irradiation group had the highest apoptosis rate (Fig. 8A-D).
It has been verified that US irradiation can enhance checkpoint inhibitor therapy 33 . US irradiation combined with immunotherapeutic nanomaterials has been studied in colorectal cancer 33 , B-cell lymphoma 34 , and breast cancer 35 . US irradiation combined with microbubble therapy for local lesions owns many advantages. The cavitation effect caused by microbubble rupturing under US irradiation can achieve high drug enrichment 18,36 . The sonoporation effect promotes drug uptake and enhances the delivery of small and large molecules 37,38 . Moreover, studies of US irradiationenhanced microbubble tumour treatments indicated that this approach could induce rapid vascular damage and shut down blood flow 36 . Our results demonstrated that PDMs combined with US irradiation can produce a strong antitumour effect.

Immune activation and cytokine production alleviation by microbubbles
To study the infiltration of immune cells into the tumour site after treatment, tumorinfiltrating lymphocytes (TILs) were harvested from tumours and analysed by immunofluorescence and flow cytometry on day 15 of the experimental process. The flow cytometry results also showed that CD8 + and CD4 + cell infiltration was approximately 4-fold greater in the PDMs combined with US irradiation group than in the control group ( Fig. 8E and F). Immunofluorescence staining revealed that the tumours from the PDMs combined with US irradiation group were remarkably infiltrated by both CD8 + and CD4 + T cells, while untreated tumours exhibited limited infiltration (Fig. 8G).
We also observed that the level of TNF-α, which induces tumour cell apoptosis, of the mice.  ℃ with/without US irradiation (2.0 W/cm 2 , 1 MHz, duty cycle 50% for 5 minutes).
After 1 mL of dialysate water was removed from the container at predetermined intervals and stored at 4 ℃ for analysis, the same volume of PBS was used to replenish the sampled mixture. The release amount was measured by the same HPLC method described above. Three independent samples from each group were tested and analysed.

Toxicities of different microbubble formulations
All animal procedures were performed in accordance with the Guidelines for Care and atmosphere, the supernatant was removed, and the cells were rinsed carefully with PBS twice, followed by the addition of RPMI 1640 medium (100 μL) and CCK-8 (10 μL) for 1 h. The OD was detected at 450 nm using a microplate reader. Relative cell viability (RCV) (%) was calculated as RCV (%) = OD test /OD control × 100%.

Evaluation of specific DTX cellular uptake mediated by PD-L1
C6 was added to microbubbles to analyse cellular drug uptake. LLC cells were seeded in six-well plates with the corresponding incubation medium at a density of 5 × The antitumour efficacies of the microbubbles in the orthotopic tumour models were evaluated by CT scan, body weight, and survival rate statistics.

Statistical analysis
All statistical analyses were performed using SPSS 21.0 software and GraphPad Prism 8 software. Data are expressed as mean ± standard deviation unless otherwise noted.
An unpaired two-tailed t-test was used to compare between two groups. When comparing multiple groups, one-way ANOVA with the Newman-Keuls post hoc test was performed. Kaplan-Meier survival curves were analysed using the log-rank test with the Tukey post hoc test. Differences were considered statistically significant when P < 0.05. Statistical significance was noted as follows: # P < 0.05 and ## P < 0.01 compared with the control group, and * P < 0.05 and ** P < 0.01 between groups.

Conclusion
Here, we developed anti-PD-L1 mAb-conjugated and DTX-loaded multifunctional lipid-shelled microbubbles and verified that the anti-PD-L1 mAb and US irradiation could promote DTX uptake. We validated that a checkpoint inhibitor could be integrated into a therapeutic microbubble to produce a combination with synergistic effects. We also proved that US irradiation-mediated immunochemotherapeutic microbubble therapy could be used to treat lung cancer. This immunochemotherapeutic microbubble approach illustrates a successful treatment strategy that can be extended to other combinations based on clinically approved antibodies (e,g, anti-CTLA-4, anti-41-BB, or anti-TIM-3 antibodies) or traditional chemotherapeutic drugs (e.g. † These authors contributed equally to this work.

Conflicts of interest
The authors have declared that no competing interest exists.  biologically independent mice, analysed using one-way ANOVA with the Newman-Keuls post hoc test. *P < 0.05 and **P < 0.01 among groups. the control group (normal saline); *P < 0.05 and **P < 0.01 between groups compared using a paired two-way Student's t-test. The log-rank test followed by Tukey's post hoc test was performed to determine statistical significance in D; n = 6 biologically independent animals. #P < 0.05 and ##P < 0.01 compared with the control group, *P < 0.05 and **P < 0.01 compared with the control group (normal saline).  View Article Online saline); *P < 0.05 and **P < 0.01 among groups. biologically independent mice. #P < 0.05 and ##P < 0.01 compared with the control group (normal saline); *P < 0.05 and **P < 0.01 between groups using a paired twoway Student's t-test. The log-rank test was performed followed by Tukey's post hoc test to determine statistical significance in E; n = 6 biologically independent mice. #P < 0.05 and ##P < 0.01 compared with the control group (normal saline); *P < 0.05 and