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Acid-activated melittin for targeted and safe antitumor therapy Li Luo, Wei Wu, Da Sun, Hanbin Dai, Yi Wang, Yuan Zhong, Jinxuan Wang, Ali Maruf, Deti Nurhidayah, Xiaojuan Zhang, Ying Wang, and Guixue Wang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.8b00352 • Publication Date (Web): 27 Aug 2018 Downloaded from http://pubs.acs.org on August 28, 2018
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Acid-activated melittin for targeted and safe antitumor therapy Li Luo1,†, Wei Wu 1,†, Da Sun 2,†, Han-Bin Dai 1, Yi Wang 1, Yuan Zhong1, Jin-Xuan Wang 1, Ali Maruf1, Deti Nurhidayah1, Xiao-Juan Zhang1, Ying Wang3, Gui-Xue Wang 1,* 1
Key Laboratory for Biorheological Science and Technology of Ministry of Education, State and Local Joint Engi-
neering Laboratory for Vascular Implants, Bioengineering College of Chongqing University, Chongqing 400044, China 2
Institute of Life Sciences, Wenzhou University, Wenzhou 325000, China
3
Chongqing Key Laboratory of Translational Research for Cancer Metastasis and Individualized Treatment, Chongqing University Cancer Hospital, Chongqing, 400030, China
*Corresponding author. Email:
[email protected] KEYWORDS: acid-activated; melittin; tumor therapy; hemolysis; biocompatibility ABSTRACT: Melittin (MLT), as a natural active biomolecule, can penetrate the tumor cell membrane to play a role in cancer treatment, will attract more attention in the future development of anti-tumor drugs. The main component of natural bee venom MLT was modified by introducing pH-sensitive amide bond between the 2,3-dimethyl maleimide (DMMA) and the lysine (Lys) of MLT (MLT-DMMA). MLT and its corresponding modified peptide MLT-DMMA were used to anti-tumor and biocompatibility validation. The biomaterial characteristics were tested by MALDI-TOF MS, 1H NMR, IUPAC and HPLC, cell viability, hemolytic and animal experiment safety evaluation. Compared with the primary melittin, the modified peptide showed decreased surface charge and low cytotoxicity in physiological conditions. Moreover, cell assays confirmed the acid-activated conversion of amide bond resulting in adequately safe during its delivery and timely antitumor activity in tumor lesions. Thus, MLT-DMMA provided a feasible platform to improve the targeted and safe antitumor applications.
INTRODCTION Melittin (MLT), an antibacterial peptide from the main component of bee venom exerting biological activity, has well-known pharmacological effects1,2, including antibacterial, antitumor3, antiinflammatory4, analgesic5, radiosensitizer6, anti-platelet aggregation7, anti-HIV8 and other pharmacological effects9. Especially, for antitumor applications, MLT can not only attack tumor cells directly10, but also can crack the tumor cell membrane to increase the gap of the cell membrane11,12, exhibiting a broad ACS Paragon Plus Environment
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prospects in the future tumor therapy13–15. However, the biggest obstacle in the application of MLT comes from its notorious side effects of hemolysis and non-specific damage to the normal cells16,17. These unfavorable side effects greatly limit the promotion of MLT in clinical application. To this end, some strategies, typically the physical encapsulation by nanocarriers and the chemical modifications18,19, have been exploited to attenuate the side effects during circulation while enhance tumor cells apoptosis through subsequently accelerating the free MLT release in lesion location20. LaraArias
et al. formulated tetramericmelittin-carrying poly-D,L-lactic-co-glycolic acid nanoparticles
(PLGA-NPs) and deliver therapeutics effectively to breast cancer cells21. Xu et al. developed an environment-sensitive peptide delivery system, dual secured nano-sting (DSNS), and MLT loaded DSNS could kill almost all varieties cancer cells22. Despite all of these developed advantages in safety, the incomplete release of MLT may reduce its therapeutic efficacy, even lead to unexpected risk of cytotoxicity. Interestingly, to the solid tumor, the tumor microenvironment becomesslightly acidic, i.e. tumor tissue (pH ~6.5) versus normal tissue (pH ~7.4)23,24. This slight pH difference can then be utilized to realize tumor target and trigger drug release25. Therefore, a ultra pH-sensitive MLT modification, using tumoral acid labile imide conjugating on the active functional groups, may be “stealth” during blood circulation, and instantaneously recover “active” in slightly tumoral acidic environment26–30. Herein, in this study, 2,3-dimethylmaleic anhydride (DMMA) was proposed to facilely modify the active amino groups in MLT to obtain the ultra pH-sensitive MLT modification (MLT-DMMA)31. In this design, carboxyl groups of the DMMA’s modified imide structure convert the original positive charge of amino groups, which exhibits “stealth” to significantly attenuate hemolysis and clearance of reticuloendothelial system for its safety development32–34. Once arrived in tumor tissue, the pH labile imide bond can be readily cleaved at the slightly tumoral acid condition, then regenerate the primary “active” MLT for antitumor therapy (Scheme 1). Thus, this facile ultra pH-sensitive MLT modification exhibits sufficient safety during its blood circulation and targeted antitumor activity through completely and rapidly tumoral acid labile cleavage at tumor tissue35–38, which may provide a strong candidate to further improve its safety and efficacy in antitumor therapy39.
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Scheme 1. Illustration of MLT-DMMA which can respond to the acidic pH-stimuli characteristic of tumoral microenvironments, promoting the targeted delivery of melittin while “stealth” its hemolysis and ultimately achieving the in vivo biocompatibility.
RESULTS AND DISCUSSION Design and synthesis of MLT-DMMA. Studies addressing the mechanisms of MLT are still subject to ongoing research40. The comprehensive studies have been presented for uptake of a MLT may occur by endocytosis and/or by direct membrane permeation. Verdine et al. showed that cell penetration is strongly related to the formal charge (due to electrostatic attraction of the peptide to the cell surface)41. MLT with positive charge is a prototypic membrane-active agent whose structural behavior and lipid interactions have been extensive investigated42. The results show that it has been effectively internalized into various types of cells without any significant non-specificity.43. The notorious side effects of hemolysis and unspecific cytotoxicity of MLT can be explained from the analysis of its chemical structure and positive charge. Hence, to avoid undesirable MLT-cell membrane interactions, the usual approach is to mask its positive charge31,44. In recent studies, regarding the modification of polypeptides, 4cyclohexene-1,2-dicarboxylic anhydride, 2,2,3,3-tetramethylsuccinic anhydride and other anhydrides have been used31. A major obstacle in the development of new therapeutic agents is the amides from those anhydrides hydrolyzed were very quickly. As our previous reports45–47, amidization using DMMA is ultra-sensitive to the slightly tumoral acid to regenerate the positively charged amino groups.
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3 Lys and 2 Arg in MLT are all positive charge, but the nonspecific interactions are mainly caused by the primary amines of Lys residues. In addition, compared with the cationic guanidino in Arg residues showing less susceptible to amidation45, Lys can be much easily amidated to accomplish the negatively charged modification. Thus, the amidized amines of the Lys residues of MLT may inhibit its nonspecific interactions and exhibit “stealth” properties during its blood circulation46. So MLT was modified using DMMA as protecting groups by 3 Lys amidation, to inhibit the MLT have interaction with normal cells before it arrives into tumor lesions. Compare to normal tissues, tumor tissues exhibited weak acidity (~pH 6.5). Therefore, once MLT-DMMA is delivered to the tumor lesions, the acid labile imide will hydrolyze into primary amines when exposed to the slight acid extracellular microenvironment in tumor, and restore the original structure of MLT then exert its transmembrane effect to destroy the tumor cells43. Characterization of MLT-DMMA. As determined by MALDI-TOF MS (Fig. 1A), the molecular weight difference between MLT and MLT-DMMA was 447, corresponding to three dimethylmaleate residues with three sodium ions, suggesting that the amidization of the three lysine residue amines to the dimethylmale amides, while the guanidino groups were not affected. Furthermore, 1H NMR was employed to confirm the amidization. The chemical shift of the methylene proton in CH2NH2 (marking “a*” in Fig. 1B) changed from 2.74 to 3.01 ppm after the amine was amidized, and the new chemical shift at 1.99 ppm of CH3 (marking “c*” in Fig. 1B) in DMMA, which indicated that the MLT-DMMA was successfully synthesized. According to the typical intensity change of the two peaks during the amidization of MLT-DMMA, the intensity of the CH2-amide (marking “b*” in Fig. 1B) peak increased while that of the CH2-amine (marking “a*” in Fig. 1B) decreased. The intensity ratio of the two peaks was used to calculate the purity of MLT-DMMA as 91.3%. In fact, because of the pH-sensitive property of MLT-DMMA, the slight hydrolysis would inevitably occur in the later dialysis against water. Subsequently, the final obtained MLT-DMMA had a relatively high purity of 91.3% instead of 100%. As the measured free primary amine contents can be seen from Fig.1C, being different from the free primary amine groups of Lys in MLT showed an extremely high content (up to 93.0%), that in MLT-DMMA exhibited a relatively low amount (5.2%), which was coincided with the 1H NMR result, suggesting almost complete conversion of the free primary amine groups of Lys in MLT to carboxylate residuals through amidation reaction. The IUPAC (2,2-Dihydroxyindane-1,3-dione) assay revealed that a high purity (94.8%) of MLT-DMMA was successfully obtained. To study the pH-sensitive hydrolysis based reversibility of MLT-DMMA, the acid labile hydrolysis of the MLT-DMMA was evaluated by monitoring its time-dependent hydrolysis at pH 5.5, 6.5, 7.4 and 8.5 using HPLC (Fig.1D and Fig. S1). According to the quantified results measured by HPLC, under acidic pH conditions (≤ 6.5), MLT-DMMA exhibited ACS Paragon Plus Environment
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rapid hydrolysis, and more than 80% of the MLT-DMMA was converted to MLT in 12 h. However, under neutral and alkaline conditions, less than 10% of MLT-DMMA hydrolyzed. MLT-DMMA were sufficiently stable at pH 7.4 and pH 8.5 but underwent fast hydrolysis at pH ≤ 6.5, which suggested the safety and functionality of MLT can be guaranteed simultaneously30. In other word, the bio-toxicity of MLT after DMMA modification could be shield under normal physiological pH conditions, while timely acted therapeutic activity for killing tumor cells.
Figure 1. (A) Molecular weights of primary MLT and MLT-DMMA (after amidization) as determined by MALDITOF MS. The molecular weight difference corresponds to three -OCCH2CH2COONa groups. (B) The detection and analysis of the structures of MLT and MLT-DMMA by 1H NMR spectrum in DMSO-d6. (C) Quantification of the free primary amine contents of Lys in the MLT and MLT-DMMA by IUPAC. (D) The hydrolysis rate of MLT-DMMA after 37 °C of treatment in different pH (pH 5.5, 6.5, 7.4, and 8.5).
Hemolytic study of modified peptide. For intravenous injection, it is greatly important to confirm that MLT-DMMA could efficiently quench the hemolytic activity. A hemolytic assay was carried out by ACS Paragon Plus Environment
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using the different pH treated MLT-DMMA and primary MLT at different concentrations (10, 20, 50 and 100 µg/mL) (Fig. 2A). The visual hemolytic images showed no significant hemolysis of both MLTDMMA and MLT at any concentration below 20 µg/mL. While MLT-DMMA showed a differential hemolysis at the extremely high concentrations of 100 µg/mL, it is showed that significant hemolysis under acidic conditions (the hemolysis ratio: 99.54% at pH 6.5 and 101.02% at pH 5.5), but only slight hemolysis was observed under neutral and alkaline conditions (the hemolysis ratio: 22.72% at pH 7.4 and 17.51% at pH 8.5) (Fig. 2B). Above all there was significant differences in hemolytic performance at concentration of 50 µg/mL. Compared with the MLT treated sample, MLT-DMMA did not exhibit significant hemolysis under both neutral and alkaline treated conditions, but presented a hemolysis under the acid treated conditions. It is suggested that the amide bond was easy to cleave at acidic condition, and subsequently regenerated the original MLT. MLT was capable of forming a tetramer state that binds to the cell membrane more efficiently than the monomer36. Therefore, the hemolytic results also advised us the critical concentration of MLT-DMMA for safe intravenous injection because it could always cause lysis of red blood cells, and eventually lead to serious red-cell damage17.
Figure 2. (A) Images of the results after hemolytic assay. (B) The hemolytic activity of melittin, MLT-DMMA treatment at different pH and different concentrations (***p
