The tumor microenvironment (TME) plays a pivotal role in cancer progression, particularly through the accumulation of hyaluronan (HA), which contributes to increased interstitial pressure and reduced drug perfusion. High molecular-weight HA (HMW-HA) further promotes immunosuppression by suppressing M1 macrophage polarization while enhancing M2 polarization. While hyaluronidase-based therapies aim to degrade HA and improve drug delivery, they inadvertently accelerate tumor metastasis—a major cause of mortality. To address this challenge, we developed a novel exosome-based drug delivery system named Exos-PH20-FA, engineered to express human hyaluronidase PH20 and modified with folic acid (FA) via self-assembly. Our results demonstrate that Exos-PH20-FA effectively degrades HMW-HA into low molecular-weight HA (LMW-HA), which reprograms macrophages toward an anti-tumorigenic M1 phenotype and reduces immunosuppressive cell populations, thereby shifting the immune landscape from immunosuppressive to immunosupportive. Furthermore, FA modification confers tumor-targeting capability, significantly enhancing chemotherapy delivery. Notably, Exos-PH20-FA directly counteracts the pro-metastatic effects induced by hyaluronidase activity, thus mitigating a key side effect of conventional HA-degrading treatments. These findings highlight Exos-PH20-FA as a promising all-in-one strategy for HA-rich tumors, offering improved therapeutic efficacy and safety by simultaneously targeting the extracellular matrix, modulating immunity, and preventing metastasis.RNPEP Antibody medchemexpress
Exos-PH20-FA was generated by genetically engineering HEK 293T cells to express membrane-bound PH20, followed by isolation of exosomes via ultracentrifugation.147127-20-6 SMILES Folic acid was then incorporated using DSPE-PEG-FA through non-covalent self-assembly, preserving exosome integrity and functionality. Transmission electron microscopy and dynamic light scattering confirmed spherical morphology with a diameter of approximately 100 nm. Western blot analysis verified the presence of PH20 and exosomal markers CD63 and CD9. FTIR spectroscopy confirmed successful FA conjugation, with characteristic absorption peaks at 1413.2 and 1607.5 cm⁻¹ corresponding to benzoic and aromatic ring vibrations. Enzymatic assays revealed high hyaluronidase activity—1971 U/mg for Exos-PH20 and 2004 U/mg for Exos-PH20-FA—indicating that FA conjugation did not impair enzymatic function. Mass spectrometry confirmed degradation of HMW-HA into disaccharide and tetrasaccharide fragments, confirming effective LMW-HA generation.PMID:34676498
In vitro studies demonstrated that Exos-PH20-FA efficiently degraded HA in PC3 and 4T1 tumor cells, with minimal effect observed in HK-2 normal cells. Fluorescent labeling revealed enhanced internalization of Exos-PH20-FA in tumor cells due to overexpression of folate receptors, whereas uptake was limited in HK-2 cells. In vivo biodistribution studies using DiR-labeled exosomes showed significantly higher fluorescence intensity at tumor sites in the Exos-PH20-FA group compared to controls, confirming superior tumor targeting. Scanning electron microscopy and immunofluorescence analysis revealed substantial remodeling of the extracellular matrix, with reduced HA deposition and loosened ECM structure in tumors treated with Exos-PH20-FA.
Functional analysis showed that LMW-HA derived from PH20-mediated degradation promoted M1 macrophage polarization, as evidenced by downregulation of M2 markers (IL-4, IL-10) and upregulation of M1 markers (TNF-α, IL-6). This shift was dependent on TLR4 signaling, as blockade abolished the effect. In orthotopic 4T1 breast cancer models, Exos-PH20-FA treatment led to increased M1-like TAMs, decreased M2-like TAMs, elevated IL-12, reduced IL-10, and diminished regulatory T cell infiltration. Concurrently, tumor-infiltrating CD8+ T cells increased significantly, indicating restoration of anti-tumor immunity.
Critically, Exos-PH20-FA inhibited hyaluronidase-induced tumor cell migration both in vitro and in vivo. Transwell assays showed that Exos-PH20 alone enhanced migration of PC3 and 4T1 cells, while Exos-PH20-FA significantly suppressed this effect. Scratch assays confirmed reduced horizontal migration in PC3 cells. In a lung metastasis model, mice treated with Exos-PH20 exhibited widespread pulmonary metastases after 14 days, whereas those receiving Exos-PH20-FA showed markedly fewer nodules. H&E staining confirmed extensive metastatic lesions in the Exos-PH20 group but minimal spread in the Exos-PH20-FA group, demonstrating that FA modification effectively counteracts the pro-metastatic side effect of HA degradation.
Finally, Dox@Exos-PH20-FA exhibited potent antitumor activity in vivo. In BALB/c mice bearing 4T1 tumors, intravenous administration of Dox@Exos-PH20-FA resulted in significant tumor growth inhibition, reduced tumor weight, and increased apoptosis as confirmed by TUNEL staining. Enhanced Dox accumulation within tumors was visualized via fluorescence imaging. Serum levels of IFN-γ and TNF-α were elevated, and flow cytometry revealed increased CD3+CD8+ and CD3+CD4+ T cells in tumor tissues. Immunofluorescence staining confirmed greater infiltration of CD8+ T cells and reprogramming of TAMs toward the M1 phenotype. Importantly, no lung metastases were detected in the Dox@Exos-PH20-FA group, underscoring its dual benefit in therapy and metastasis prevention.
These findings establish Exos-PH20-FA as a multifunctional nanotherapeutic platform capable of overcoming the limitations of current hyaluronidase-based strategies. By integrating HA degradation, immune modulation, tumor targeting, and metastasis suppression, it offers a comprehensive solution for treating HA-rich solid tumors with enhanced efficacy and safety.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com
