Chemotaxis-driven delivery of nano-pathogenoids for complete eradication of tumors post-phototherapy

Cancer treatment often involves inflammation, and neutrophils play a key role in homing to inflammation sites. However, synthetic nanoparticles (NPs) lack chemotactic features and face challenges in overcoming biological barriers like the mononuclear phagocyte system (MPS), vascular barriers, and interstitial pressure. To address these issues, neutrophils are utilized as drug carriers, with the *in situ* hitchhiking strategy being more clinically advantageous. This study proposes using pathogen-mimicking nano-pathogenoids (NPNs) to hitchhike neutrophils. NPNs, coated with bacteria-secreted outer membrane vesicles (OMVs), mimic pathogens and are recognized by neutrophils via pathogen-associated molecular patterns (PAMPs). This approach is explored in eliminating residual microtumors after PTT, as PTT's limited light penetration can lead to tumor recurrence. The study hypothesizes that NPNs will effectively target residual microtumors after PTT by using neutrophils as vehicles.

The efficacy of nano-mediated drug delivery is often limited by biological barriers. Previous studies have explored using neutrophils as drug carriers, either by *ex vivo* assembly of NPs with neutrophils or by *in situ* hitchhiking. *Ex vivo* methods present challenges with neutrophil viability, cargo degradation, and cost. *In situ* hitchhiking requires designing NPs with high affinity for neutrophils. While denatured bovine serum albumin NPs and anti-CD11b antibody-decorated NPs have shown promise, more efficient and generalizable methods are needed. The pathogen-mimicking concept leverages the natural ability of neutrophils to recognize and ingest pathogens through PAMPs. Cloaking NPs with OMVs creates a nano-mimic of bacteria, enhancing neutrophil interaction while mitigating potential toxicity issues associated with live bacteria. Photothermal therapy (PTT) is also frequently used in cancer treatment, but suffers from incomplete tumor eradication due to limited light penetration. Therefore, combinatorial approaches to supplement PTT are actively researched.

The study used EMT6 (murine breast carcinoma) and CT26 (colon carcinoma) tumor-bearing mice. A photothermal transducer, PBIBDF-BT, encapsulated in PEG-*b*-PLGA micelles (NPs@PBT), was used for PTT. The temperature rise during PTT was controlled by adjusting the 808 nm laser's duty cycle. Neutrophil infiltration after PTT was analyzed by flow cytometry, revealing increased neutrophil percentages in tumors within 48 hours, peaking at 4 hours. The effect of tumor size and PTT parameters (temperature and time) on neutrophil infiltration was also investigated. Adoptively transferred, DiD-labeled neutrophils were used to further study PTT-induced neutrophil recruitment, showing increased tumor targeting in PTT-treated mice. Intravital microscopy of a dorsal-skin-fold window chamber model enabled real-time observation of neutrophil migration, showing successful transmigration from blood vessels into the tumor interstitium. NPNs were prepared by coating PEG-*b*-PLGA NPs with OMVs from *E. coli*, characterized by DLS and TEM. The ability of NPNs to hitchhike circulating neutrophils was assessed using DiO-labeled NPNs, showing significantly higher uptake by neutrophils compared to NPs. The role of TLR2 and TLR4 in NPN recognition by neutrophils was investigated using knockout mice and blocking antibodies. The release of NPNs from neutrophils and their subsequent uptake by tumor cells under inflammatory conditions (simulated using PMA) were studied using *in vitro* co-culture and *in vivo* intratumoral injection. The anti-tumor effect of PTT combined with cisplatin-loaded NPNs (NPNs@Pt) was investigated in EMT6 tumor-bearing mice. Treatment groups included PBS, cisplatin, OMVs, NPs@Pt, NPNs@Pt, PTT alone, and various combinations. Tumor growth, weight, and histological analysis (H&E, Ki67, TUNEL) were used to assess therapeutic efficacy. Neutrophil depletion and NETosis inhibition experiments were conducted to confirm the role of neutrophils and NETs in the therapeutic process. The efficacy of two treatments of PTT plus NPNs@Pt was investigated to assess complete tumor eradication.

PTT treatment created an inflammatory tumor microenvironment that recruited neutrophils. The number of neutrophils recruited was dependent on the tumor size, PTT temperature, and duration of irradiation. Adoptively transferred neutrophils showed significantly enhanced tumor targeting after PTT. Intravital microscopy demonstrated the ability of neutrophils to extravasate and migrate into the tumor interstitium. Pathogen-mimicking NPNs effectively hitchhiked circulating neutrophils, primarily through TLR4 and TLR2 signaling. NPNs accumulated in PTT-treated tumors to a greater extent than NPs alone. NPNs released their cargo (cisplatin) from neutrophils under inflammatory conditions (NETosis), which was then taken up by tumor cells. A single treatment of PTT combined with NPNs@Pt significantly suppressed tumor growth, and two treatments resulted in complete tumor eradication in all treated mice. Depletion of neutrophils or inhibition of NETosis significantly impaired the therapeutic efficacy of the combined therapy, highlighting the crucial role of neutrophil-mediated drug delivery and NETosis in the treatment outcome.

This study demonstrated a novel approach to overcome the limitations of traditional nano-mediated drug delivery by utilizing neutrophils as efficient drug carriers. The use of pathogen-mimicking NPNs, combined with PTT, significantly enhanced tumor targeting and therapeutic efficacy. The complete eradication of tumors in all mice treated with two cycles of the combined therapy is a significant advance. The mechanism involves the creation of an inflammatory environment by PTT, the recruitment of neutrophils, their specific targeting of NPNs, the transport of NPNs to the tumor, and the subsequent release of the drug upon NETosis. The findings highlight the potential of this approach for improving cancer treatment outcomes. Further investigation is needed to explore this method's clinical translation, including the optimization of OMV coating, detailed investigation of potential side effects, and exploring different types of drugs and cancer models.

This study successfully developed a novel nano-pathogenoid (NPN) system for *in situ* hitchhiking of circulating neutrophils, improving the delivery of therapeutic agents to tumors. The combined therapy of photothermal therapy (PTT) and cisplatin-loaded NPNs led to complete tumor eradication in all treated mice. This approach overcomes several limitations of traditional nanoparticle drug delivery and offers a promising strategy for enhanced cancer treatment. Future research should focus on optimizing the system for clinical translation and exploring its applicability to other cancer types and treatment modalities.

The study primarily used murine models, and the results may not directly translate to human clinical settings. The use of *E. coli* OMVs may trigger an immune response, although this was mitigated to some extent. The long-term effects of the treatment and potential toxicity need further evaluation. The study focused on specific tumor types, and further research is necessary to confirm the effectiveness in other cancer models.