Liang et al. NPG Asia Materials (2019) 11:38 https://doi.org/10.1038/s41427-019-0138-6 NPG Asia Materials ARTICLE Open Access Menthol-modified BSA nanoparticles for glioma targeting therapy using an energy restriction strategy Jianming Liang1,2,3,YingZhu2,CaifangGao1,3, Chengli Ling4, Jing Qin1,QiWang5, Yongzhuo Huang6,WeigenLu3 and Jianxin Wang 1,5 Abstract Current chemotherapeutics for glioma are not sufficiently effective due to their low tolerance and poor blood–brain barrier (BBB) permeability. Energy restriction based on co-delivery of albendazole (Abz) and nano-silver has been proven to suppress tumor growth without adverse effects. Accordingly, menthol was conjugated to BSA (MeB) to overcome the BBB-permeability issue. In this study, Abz-loaded MeB-silver nanoparticles (MBS-Abz) were developed by self-assembly of MeB, albendazole and nano silver for glioma targeting therapy. The nanoparticle entered the brain across the BBB and specifically accumulated in the glioma region. MeB delivered the nanoparticles across the brain capillary endothelial cell monolayer by promoting internalization and BBB disruption. MBS-Abz hindered ATP generation via the inhibition of glycolytic and mitochondrial pathways both in vitro and in vivo. The anti-glioma mechanisms of energy restriction were related to the cytotoxicity, proliferation inhibition, cell cycle arrest, and apoptosis induced by ATP exhaustion, and the effects of MBS-Abz were significantly better than those induced by monotherapy nanoparticles or unmodified nanoparticles. These results demonstrated that by combining the energy 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; restriction effect of albendazole and nano silver, as well as the BBB penetration ability of menthol, MBS-Abz achieves superior anti-glioma efficacy and can be an effective strategy for glioma therapy. Introduction nitrosoureas and platinum-based drugs, can curb the Cancer has replaced cardiovascular disease as the growth of glioma cells, they also cause serious adverse leading cause of death in the United States1. Although effects, such as nephrotoxicity by cisplatin and pulmonary many antitumor drugs have been developed, the global toxicity by nitrosoureas3,4, which lead to poor tolerance burden of cancer is still high. Brain cancers account for and limited efficacy5. Therefore, the need to develop safe approximately 3% of the global cancer cases2, among and effective strategies for glioma treatment is pressing. which, gliomas are associated with high morbidity and Since tumor cells proliferate rapidly and infinitely6, they mortality. Chemotherapy is a major therapeutic modality consume substantial amounts of energy to maintain this for glioma. Although cytotoxic agents, such as abnormal growth process7. Abnormal energy metabolism is an important physiological characteristic of glioma8,9. Adenosine triphosphate (ATP) is the most direct source Correspondence: Yongzhuo Huang ([email protected])or of energy, and compared with normal cells, glioma cells Weigen Lu ([email protected]) or Jianxin Wang ([email protected]) 10 1 are more sensitive to ATP levels . Energy restriction was Department of Pharmaceutics, School of Pharmacy, Fudan University & Key 11,12 Laboratory of Smart Drug Delivery, Ministry of Education, 201203 Shanghai, PR confirmed to effectively inhibit glioma growth . Thus, China energy metabolism inhibition may be an effective 2 Institute of Tropical Medicine, Guangzhou University of Chinese Medicine, approach for glioma therapy. Glioma cells mainly gen- 510006 Guangzhou, PR China Full list of author information is available at the end of the article. erate ATP via the glycolytic pathway rather than oxidative These authors contributed equally: Jianming Liang, Ying Zhu © The Author(s) 2019 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a linktotheCreativeCommons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. Liang et al. NPG Asia Materials (2019) 11:38 Page 2 of 18 phosphorylation, which is known as the Warburg effect. systems, the most commonly used method, may enhance The inhibition of glycolytic enzyme activities can effec- brain penetration34, there are still some limitations. First, tively decrease ATP production and cell proliferation13. while it is very likely that the aromatic resuscitation When glycolysis is inhibited, tumor cells compensatively components reach the BBB, the nano-carriers may still increase ATP synthesis via the mitochondrial pathway to circulate in other parts of the body, making it difficult for meet the need for physiological functions14, which ren- the active substance to pass the BBB and reach the brain ders the curative effect of monotherapy (glycolysis inhi- tumor site. Second, the physical mixture of BPEs and drug bitor only) unsatisfactory15. Therefore, ideal energy delivery systems may open the BBB for too long, causing inhibition should block ATP synthase by simultaneously the entrance of harmful substances into the brain and inhibiting the glycolytic and mitochondrial pathways. In possible damage to the brain tissue. Third, because the our previous study, we developed a novel antitumor delivery efficiency of simple mixing is very low, a higher strategy based on energy restriction by utilizing albenda- dose of aromatic resuscitation components might be zole (Abz) and silver nanoparticles (SNP) as glycolysis required, which might cause severe damage to the blood inhibitors and mitochondrial inhibitors, respectively16. vessels and other organs31. Chemical coupling could be an Abz inhibits glycolytic enzyme activities and down- effective method35,36 to overcome these shortcomings by regulates hypoxia-inducible factor I expression, thus synchronizing delivery of aromatic resuscitation compo- inhibiting glycolysis17,18. Moreover, Abz nanoparticles nents and nanoparticles at the BBB, and potentially efficiently suppressed the growth of solid tumors19, and enhancing the water solubility of BPEs. Menthol, the main Abz decreased tumor markers, in addition to being well active component of corn mint, is one of the most widely tolerated in a phase I clinical trial20. SNP reduced mito- used aromatic resuscitation drugs in traditional Chinese chondrial function and inhibited ATP generation via the medicine. This molecule enhances the penetration of mitochondrial pathway21,22. Therefore, our strategy based many drugs into the brain by downregulating the level of on the combination of Abz and SNP was expected to TJ proteins37,38. effectively and safely restrict glioma growth, even though Penetration of the brain tissue across the BBB is not no cytotoxic antitumor agent was used during the sufficient, as the nanoparticles need to accumulate in treatment. satisfactory concentrations in the glioma region. Tumor The delivery of therapeutic agents is more difficult in cells need a substantial amount of nutrients, such as glioma than in other tumors because of the blood–brain albumin, for rapid proliferation39. Specifically, glioma cells barrier (BBB). The BBB is a continuous endothelial up-regulate the expression of albumin-binding protein monolayer coupled by tight junctions (TJs), with extre- and secreted protein acidic and rich in cysteine (SPARC) mely low permeability for the exchange of materials23. For to uptake albumin40,41. However, SPARC-albumin bind- brain targeting, drug-delivery strategies, such as receptor- ing has not attracted much attention. Despite the limited mediated transcytosis and BBB disruption, effectively studies reporting that the SPARC pathway can be applied enhance the distribution of drugs to the brain via trans- in glioma-targeting biomimetic delivery, SPARC was cellular and paracellular pathways, respectively24,25. shown to prominently improve treatment outcome42,43. However, receptor-mediated transcytosis is strongly lim- Moreover, albumin can be modified with functional ited by the amount of receptors and their recycling26.To ligands and encapsulate various antineoplastic drugs, thus maximize BBB penetrability, BBB disruption methods, constituting an ideal and natural glioma-targeting carrier. such as focused ultrasound, which delivers drugs to the For example, menthol-modified bovine serum albumin brain by directly opening TJs, have been frequently (MeB), prepared by conjugating 8-mercaptomenthone applied27,28. However, BBB disruption is commonly with bovine serum albumin (BSA), not only induces BBB associated with severe side effects29. Thus, it is important disruption, but also penetrates the BBB directly through to find a safe and effective way for BBB opening. the pineal body pathway44. Thus, MeB can be used to During the clinical practice of traditional Chinese deliver drugs to the brain safely and effectively and allow medicine for thousands of years, some aromatic resusci- the selective accumulation of these drugs in