nRGD modified lycobetaine and octreotide combination delivery system to overcome multiple barriers and enhance anti-glioma efficacy
Graphical abstract
Introduction
Brain malignant glioma has been one of the most common and deadly cancer [1]. Despite achievements in surgical resection and radiotherapy, the average median survival for patients with glioma was less than 18 month [2]. Chemotherapy as a common used cancer treatment strategy has played an important role in treating glioma. However, like other treatment options, chemotherapy also gained very limited treatment outcome. The undesired anti-glioma efficacy was mainly attributed to the multiple biological barriers in glioma greatly hindering the tumor-specific delivery and tumor accumulation of therapeutic agent. Briefly, biological barriers such as blood-brain barrier (BBB), blood-brain tumor barrier (BBTB), areas of hypoxia and tumor invasiveness have posed obstacles for delivering drugs to tumor sites [3], [4], [5]. In addition to the presence of biological barriers, glioma cells have good capacity to escape from anti-tumor therapy through several pathways. For example, vasculogenic mimicry (VM) channels are responsible for the invasion and refractoriness of brain cancer, exhibiting high resistance to the chemotherapy [6]. What’s more, tumor associated macrophages (TAMs) could contribute to angiogenesis, tumor progression and recurrence, which present additional challenges on effective chemotherapy against glioma [5], [7]. Thus, developing a novel drug delivery system which could overcome multi-barriers to efficiently deliver antitumor drugs to glioma area and inhibit the development of glioma by multi-therapeutic mechanisms is urgently demanded.
Recently, nanocarriers modified with targeting peptides in glioma treatment have received extensive attentions. First, nanocarriers can encapsulate both hydrophilic and hydrophobic drugs, and their composition, size and surface properties can be easily tailored to achieve site-specific drug delivery [8]. Next, peptide ligands can specifically interact with receptors/transporters overexpressed on BBB and efficiently mediate transcytosis across BBB. For example, iRGD, a tumor-penetrating cyclic peptides [9], has been widely used in glioma targeted drug delivery systems [10], [11]. However, some reports indicated that iRGD cannot eradicate the existing tumor cells completely [9], [12]. In our previous study, a multi-target peptide nRGD was developed by conjugating alanine-alanine-asparagine (AAN) with cyclic iRGD [13], and exhibited a stronger anti-tumor efficacy against breast cancer compared with iRGD. However, whether nRGD could achieve a strong anti-tumor effect in glioma model is unclear, considering the presence of multiple biological barriers we have discussed above. Thus, we hypothesize nRGD could transport to cross BBB and target the glioma cell and VM channels by iRGD module interacting with αv integrin receptors [14], and target TAMs through the AAN module [15]. To validate the hypothesis, nRGD was used as a targeting ligand in this study to investigate the anti-glioma efficacy.
In recent years, concurrent loaded nanotechnology has been an effective strategy to achieve anti-angiogenesis and anti-tumor effect, and combination therapies also have been proven to regress tumors and some are already in clinical testing [16], [17]. For example, Li et al. used paclitaxel and artemether (ARM) concurrently loaded liposomes to treat glioma, which showed that ARM could inhibit angiogenesis and VM channels formation. Those combination therapies demonstrated synergistic antitumor effect against glioma [18]. In this study, lycobetaine (LBT), a topoisomerase inhibitor [19], [20], exhibiting significant cytotoxicity against various tumor cells [21], was used to kill glioma cells and TAMs. However, the anti-angiogenesis effect of LBT is unknown. Thus, to enhance the anti-angiogenesis effect and inhibit VM channels, octreotide (OCT) was co-delivered. OCT has high affinity to somatostatin receptor (SSTR) subtypes 2 and 5, which are overexpressed in many tumors including glioma [22], [23]. In previous reports, OCT usually being used as a ligand to target SSTR, has played a key role in scintigraphic visualization of tumors and anti-angiogenesis [24], [25]. Therefore, LBT and OCT with different anti-tumor mechanisms were used to achieve a stronger anti-glioma effect.
To achieve a better anti-glioma effect, LBT and OCT were co-encapsulated in the liposomal carrier. Next, a multi-target peptide, nRGD, was further covalently conjugated to liposomes as a targeting ligand to facilitate glioma-specific drug accumulation and penetration. We systematically investigated the cellular uptake efficiency, brain tumor penetrating capability, and VM channels destructive capability in vitro. Also, the therapeutic efficacy of LBT and OCT loaded multi-functional liposomes was evaluated in a glioma-bearing mice model.
Section snippets
Materials
iRGD peptide (CRGDKGPDC) and nRGD peptide (AAN-iRGD, CRGDK(NAA)GPDC) with a C-terminal cysteine were synthesized by GL Biochem (Shanghai, China). Lycobetaine (LBT) acetate and octreotide acetate (OCT) were offered by Hongsheng Plant Raw Material Co. Ltd. (Shifang, China). Soybean lecithin (SPC) and oleic acid (OA) were purchased from Lipoid (Ludwigshafen, Germany). PEG2000-DSPE and Mal-PEG2000-DSPE were purchased from Advanced Vehicle Technology (Shanghai, China). Cholesterol and sodium oleate
Characterization of liposomes
To increase the lipophilicity and achieve a high entrapment efficiency of LBT and OCT, OA and sodium oleate were used to incorporate LBT and OCT to form complex, respectively [26], [27], [34]. With the increasing lipophilicity of LBT and OCT, drugs-loaded liposomes were prepared.
TEM photographs showed that LBT-OCT LPs, LBT-OCT LPs-iRGD and LBT-OCT LPs-nRGD remained spherical (Fig. 1C). The z-average size distributions of different liposomes were about 140 nm (PDI < 0.30) with negative zeta
Discussion
Despite the malignancy of brain glioma, significant breakthroughs in glioma therapy are still lacking [35]. Recently, attentions have been focused on the ligand modified delivery system for CNS-targeted drug delivery [36]. Besides, combination chemotherapy has long been considered effective in antitumor treatment in clinic especially in reversing multidrug resistance [18]. Thus, to overcome several barriers and enhance anti-glioma efficacy, we developed a multi-target peptide modified
Conclusion
In this study, a multi-target peptide nRGD modified, LBT and OCT co-loaed liposomes has been demonstrated as a comprehensive and safe antitumor drug delivery strategy to overcome multi-barriers in glioma and enhance the anti-glioma efficacy. The combination of LBT and OCT showed a synergistic anti-glioma effect, LBT and OCT co-loaded liposomes exhibited excellent anti-tumor and anti-angiogenesis activity both in vitro and in vivo. nRGD increased the tumor cell targeting, glioma penetrability
Acknowledgements
We are grateful for the financial support from the National Basic Research Program of China (973 Program, 2013CB932504) and the National Natural Science Foundation of China (No. 81673359).
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