Although primary brain tumors are a rare malignancy and account for only 2% of all cancer diagnoses, they are a leading cause of cancer-related mortality. Gliomas represent the most prevalent primary brain tumor subtype, accounting for 80% of all malignant central nervous system tumors [1], and exhibit notoriously poor clinical outcomes, characterized by a low overall survival rate, high risk of recurrence, and poor prognosis [2,3]. Standard-of-care treatment consists of surgical resection followed by adjuvant radiotherapy and chemotherapy, with surgical resection being the primary therapy. However, the success of glioma surgery is heavily dependent on tumor location, and the risk of complete tumor removal often involves significant complications such as intracranial hemorrhage, brain nerve damage, and functional area impairment. Moreover, complete tumor removal is often not possible, and repeat surgeries are limited in their efficacy at controlling glioma recurrence. Despite significant therapeutic advancements, the median overall survival period for glioma patients remains <15 months, with 5-year survival rates rarely exceeding 6.8% [4].

In recent years, immune-based therapies such as gene therapy, immune checkpoint inhibitors, CAR-T cells, and neoantigen-based vaccines have emerged as promising treatment options for glioma and other cancers [[5], [6], [7], [8], [9]]. However, several practical challenges hinder their successful clinical translation. First, due to the remarkable specialization of the central nervous system and the existence of the BBB, drug penetration into brain lesions presents a significant obstacle. Invasive administration methods are technically challenging and associated with high risks, while oral or intravenous administration routes fail to deliver adequate drug concentrations to brain tumors. Developing delivery systems that can efficiently cross the BBB remains a vital research frontier [10,11]. Second, glioma harbors a unique tumor immune microenvironment due to the distinctive immune architecture of the central nervous system [12]. The immune ecosystem within the central nervous system presents a distinct contrast to the immune system in the periphery, resulting in a unique tumor microenvironment in glioma that differs significantly from other cancers. Consequently, immune therapies that exhibit efficacy in other cancers may face obstacles in glioma. Hence, optimizing treatment outcomes and suppressing glioma recurrence requires achieving three critical objectives: 1) efficient drug transit across the BBB; 2) potent delivery of drugs across the BBTB to the lesion; and 3) enhancing the repressive glioma immune microenvironment and activating effective antitumor immune responses.

In this study, we implemented a cholesterol-DP7-ACP-T7 (C-DP7-ACP-T7)-modified DOTAP liposome (DAT-LNP)/siRNA delivery system as a solution to three critical issues in glioma treatment: improving intravenous drug delivery across the BBB and BBTB, achieving targeted delivery of small nucleic acid drugs to brain tumors, and transforming the immune microenvironment of suppressive glioma to an activated state. Our liposome is equipped with cholesterol-DP7-ACP-T7, in which DP7 (VQWRIRVAVIRK) is a cell-penetrating peptide that serves as both a delivery carrier and an immune activator, as previously identified in our studies [[13], [14], [15], [16]]. In brief, as a delivery carrier, cholesterol-modified DP7 can efficiently deliver small nucleic acids and various antigen peptides into cells, and as an immune activator, it can promote the maturation of DC cells. In addition, another functional peptide on cholesterol-DP7-ACP-T7 is T7 (HAIYPRH), which has high affinity for the highly expressed transferrin receptor on gliomas and is commonly employed as a target head to penetrate the BBB and target brain tumors as a brain tumor-targeting peptide [17]. Cholesterol-T7 has also been shown to possess immune activation properties [18]. In the middle of the DP7 peptide and T7 peptide, we used ACP (6-aminocaproic acid) as a linker. ACP is a conformational flexible, FDA-approved, nontoxic, biodegradable material that is usually used as a linker to isolate two different materials [19]. Therefore, ACP was chosen for the isolation of two functional peptides. Through these modifications, our liposomes achieve efficient crossing of the BBB and precise targeting of brain tumors with a high affinity for the transferrin receptor (TfR) of brain tumor cells. Subsequently, DAT-LNP transports si slit2 into brain tumor cells via the caveolin and clathrin pathways, effectively inhibiting their migration and invasion by avoiding endosomal entrapment. Additionally, DAT-LNP/si slit2 (Slit2 is known to bolster glioma cell migration, invasion, and proliferation [18,20]) fosters a “hot” microenvironment in glioma by increasing the proportion of mature dendritic cells, M1 macrophages, and cytotoxic T cells. These combined measures foster a synergistic effect, improving the therapeutic efficacy of glioma. Therefore, this delivery system, designed to address three major scientific issues associated with glioma treatment, has the potential to become a universal strategy that leverages intravenous administration to target brain tumors across the BBB with nucleic acid drugs for immunotherapy of glioma, thus presenting a promising avenue for application.