Glioma is the most prevalent primary intracranial malignant tumor with high morbidity and mortality [1]. The median survival time of patients is only approximately 15 months, and fewer than 5% of the diagnosed patients can survive for about 5 years [2,3]. The standard treatment for glioma is surgical resection followed by postoperative radiotherapy and chemotherapy with temozolomide (TMZ), but no chemotherapeutics are available after patients display resistance to TMZ [4]. The existence of multiple biological barriers in intracranial tumors, especially the blood-brain barrier (BBB) and blood-brain tumor barrier (BBTB), greatly hinders the effective delivery of chemotherapeutics to the infiltrative glioma area. The BBB is composed of brain capillary endothelial cells surrounded by pericytes and astrocytes, responsible for maintaining homeostasis and protecting the brain from toxic substances in the blood, which means that only a few drugs can cross the BBB [5]. The BBB surrounding tumor cells in the brain parenchyma evolves into the BBTB during the development of primary intracranial tumors. The BBTB is heterogeneously permeable to many drugs, resulting in poor therapeutic efficacy [[6], [7], [8]]. Thus, it is of great need to develop a multifunctional brain-targeted drug delivery strategy that can help drugs traverse the BBB, BBTB, and then target the glioma cells to improve the anti-glioma efficacy of chemotherapeutic agents [8].

Two strategies have been applied in active targeted drug delivery [9]: one is to deliver drugs to the target site with the help of carriers (e.g., liposomes, micelles, nanoparticles, etc.), but there are no relevant active targeted drug delivery systems approved and marketed at present [10]; The other strategy involves connecting the drug with targeted molecules (e.g., antibodies, peptides, aptamers, polymers, etc.) through linkers to form conjugate drugs [11]. This strategy possesses the advantages of increased biological safety, reduced drug release in non-target areas, and fewer non-pharmacological components [12]. Among them, the most remarkable ones are antibody-drug conjugate (ADC) and peptide-drug conjugate (PDC), which are obtained by covalently coupling active targeted antibodies or peptides with highly cytotoxic drugs [13]. Compared with ADC, the advantages of PDC mainly include [14]: (1) Due to the small molecular size, PDC is easier to penetrate deep into the tumor tissue; (2) Peptides are biodegradable with low immunogenicity; (3) The modification site of the peptide is relatively confirmed, which enhances its productivity with simplified purification procedure. However, natural peptides are easily degraded by enzymes in the blood, which severely limits the development of PDC [15]. Up to now, only two PDCs have been approved worldwide, named Lutathera [16] and Pluvicto [17], both of which were developed by Novartis based on radiotherapy. Meanwhile, several PDC candidates have been authorized in clinical trials [18], which indicates that PDCs have a broad clinical application prospect in tumor-targeted drug delivery.

The selection of targeting peptides is crucial in the design of PDCs, as their structure and function can directly impact the stability, targeting ability, and therapeutic efficacy of the conjugates [19]. Moreover, the receptor of these targeting peptides should be overexpressed on tumor cells specifically to minimize latent damage from cytotoxic payload against normal tissues [20]. Glucose-regulated protein 78 (GRP78) is a key member of the heat shock protein family, exclusively expressed on tumor cells and neovasculature, and almost absent on the surface of normal cells [21,22]. It is thus expected to become a promising receptor for tumor-targeted therapy. DVAP (DPDADVDRDTDNDS), a stable peptide consisting of seven D-configuration amino acids with high affinity to GRP78, is a multifunctional peptide ligand for targeted drug delivery in glioma (e.g. the BBTB- and neovasculature-penetrating and glioma cell-targeting ability), but cannot penetrate the BBB [23]. Dopamine receptors belong to the G-protein coupled receptor family and play a crucial role in regulating various biological activities [24]. It has been reported that benzamide analogues have a high affinity to dopamine receptors in the central nervous system, and the small molecule ligand p-hydroxybenzoic acid (pHA) was capable of crossing the BBB [25]. Our previous study demonstrated that the conjugation of two peptides into a “Y”-shaped structure using 6-aminohexanoic acid (AHX) and modifying it on the same material molecule could increase the cmodification density of the targeting peptide on nanoparticles without affecting the targeting ability [26]. However, recent research has revealed that in addition to carbon chain length, the hydrophilicity of the linker also plays a role in determining receptor affinity for the “Y”-shaped peptide and solubility of the constructed PDC.

In this study, inspired by the structure of polyethylene glycol, we substituted the central carbon atom in AHX with oxygen to create a hydrophilic linker containing one ethylene glycol repeat unit, which was named AOHX. Subsequently, the chemotherapeutic agent doxorubicin (DOX) was conjugated with the “Y”-shaped targeting peptide (pHA-AHX-VAP or pHA-AOHX-VAP), and their targeting capability, solubility, and cytotoxicity in vitro were examined. The optimized pHA-AOHX-VAP-DOX was then selected for in vivo safety and anti-glioma efficacy evaluations. pHA-AOHX-VAP-DOX was expected to achieve remarkable anti-glioma efficacy because pHA can cross the BBB through dopamine receptors, pHA-AOHX-VAP can target the BBTB and glioma cells through GRP78 receptors and dopamine receptors, and DOX can effectively kill glioma cells (Fig. 1).