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Nanoscale Melittin@Zeolitic Imidazolate Frameworks for Enhanced Anticancer Activity and Mechanism Analysis Yawei Li, Na Xu, Wenhe Zhu, Lei Wang, Bin Liu, Jianxu Zhang, Zhigang Xie, and Wensen Liu ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b06125 • Publication Date (Web): 19 Jun 2018 Downloaded from http://pubs.acs.org on June 22, 2018

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ACS Applied Materials & Interfaces

Nanoscale Melittin@Zeolitic Imidazolate Frameworks for Enhanced Anticancer Activity and Mechanism Analysis Yawei Li,†§ Na Xu,†§ Wenhe Zhu,†§ Lei Wang,‡ Bin Liu,† Jianxu Zhang,‡ Zhigang Xie,*‡ and Wensen Liu*†

† Key Laboratory of Jilin Province for Zoonosis Prevention and Control, Institute of Military Veterinary Medicine, Academy of Military Medical Sciences, Changchun, 130122, P. R. China ‡State Key Laboratory of Polymer Physics and Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, 130022, P. R. China §Jilin Medical University, Jilin, 132013, P. R. China

*E-mail: [email protected], [email protected]

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ABSTRACT: The cytolytic peptide melittin (MLT) is an important candidate of anticancer drug owing to its hemolytic properties. Nevertheless, its clinical applications are severely restricted as a result of its non-specific toxicities like hemolysis. In this work, we reported MLT-loaded zeolitic imidazolate framework-8 (MLT@ZIF-8) nanoparticles (NPs). The formed MLT@ZIF-8 NPs not only possess excellent stability but also efficiently inhibit the hemolysis bioactivity of MLT. Confocal scanning imaging and cytotoxicity experiments revealed that as-synthesized MLT@ZIF-8 NPs exhibit enhanced cellular uptake and cytotoxicity toward cancer cells compared to MLT. The mechanism is well investigated by a series of transcriptome analysis, which indicates that MLT@ZIF-8 NPs can regulate the expression of 3383 genes, and the PI3K/Akt-regulated p53 pathway is involved in MLT@ZIF-8 NPs induced A549 cells apoptosis. Finally, MLT@ZIF-8 NPs exhibit enhanced antitumor activity than free MLT in vivo, while no obvious systemic toxicity has been found. This work emphasizes the great potential of utilizing MOF as a simple and efficient nanoplatform for deliverying cytolytic peptides in cancer treatment, and also the investigation on the anti-tumor mechanism could provide theoretical support for clinical usage of MLT.

KEYWORDS: melittin, ZIF-8, nanocarrier, anticancer activity, p53 pathway


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INTRODUCTION

Cytolytic peptides possess great potential as anticancer candidates due to their lytic properties,1 and their therapeutic function could be fully exerted by using delivery vectors.2-5 For example, melittin (MLT) is a linear 26-amino acid amphiphilic cationic peptide from the venom of bee,6-8 which could interact with cellular membranes in the form of a monomer, followed by destruction membranes by forming transmembrane pores, and subsequently induce cell death.8-10 Alternatively, after intracellular delivery, MLT can act on the cytomembranes of the internal organelles in a similar manner, inducing biochemical changes or transcriptional regulation. MLT exerts various impacts on functions of cancer cells, including proliferation, apoptosis, metastasis, angiogenesis, and cell cycles, and multiple signal pathways, genes, and molecules are activated or modulated to adjust those processes, suggesting that it could be an outstanding candidate for cancer treatment. However, the non-specific toxicities like hemolysis have hampered the clinical application of MLT severely.11-13 In order to solve these problems, there are two strategies used in the past decades. The first is chemical modification, which usually change the sequence of MLT, or conjugate with proteins or antibody to increase the specificity.14-18 However, the bioconjugation still can’t prevent the hemolytic activity completely.19 Another one is physical encapsulation, in which MLT is loaded into various nanoparticles, such as liposomes,20-22 polymeric nanoparticles23-25 and inorganic materials.26-27 These nanoscale formulations could protect the MLT from hemolysis during the circulation in blood, and then deliver them into disease sites. The robust stability is imperative



and important for nanoparticle formations of melittin.28 Up to now, some polymeric and inorganic nanoparticles have been used to carry melittin.23-24, 26 Although great advances have been made in nanoparticle formulations of MLT, their practical application is still limited due to the suboptimal loading efficiency and uncontrolled release.21, 29 It is urgent to design a simple and robust nanoplatform for delivery of cytolytic peptides.

Recently, nanoscale metal-organic frameworks (NMOFs) attracted much attention in drug loading and delivery because of their porous structure, simple preparation and multifunctional feature.30 Several NMOFs with good biocompatibility and tunable pose size have been used for loading therapeutic or imaging agent, including UiO (UiO for University of Oslo),31 MIL (MIL for material from Institut Lavoisier)32-33 and ZIF (Zeotlitic imidazolate framework).34-35 Among these materials, zeotlitic imidazolate framework-8 (ZIF-8),36-38 formed by zinc ions and 2-methylimidazole, possess pH-sensitive behavior and tailoring pore size for loading various cargos, such as chemotherapy,34,

39-41

nucleic acid42-43 and proteins.44-45 The green synthesis in

aqueous media of ZIF-8 is a particular advantage for encapsulating water-soluble macromolecules.46 Several enzymes47-48 and vaccines49-50 have been loaded for catalysis and immune treatment. Relatively, peptides haven’t been explored yet.

In this work, the cytolytic peptide MLT was encapsulated into porous ZIF-8 in simple in situ loading in aqueous media as shown in Scheme 1. The formed MLT@ZIF-8 possesses nanoscale size and robust stability, and could not hemolyze


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red blood cells. More importantly, the MLT@ZIF-8 possesses enhanced cytotoxicity compared to MLT, and the mechanism has been investigated by a series of transcriptome analysis, which indicates that the expression of 3383 genes are adjusted by MLT@ZIF-8, and the PI3K/Akt-regulated p53 pathway is involved in MLT@ZIF-8 induced A549 cells apoptosis. This is the first attempt to carry out transcriptome analysis to clarify the anticancer mechanism of MLT@ZIF-8.

Scheme 1. Preparation of MLT@ZIF-8 NPs and their application in anticancer therapy.

RESULTS AND DISCUSSION

MLT@ZIF-8 particles with well-defined morphology and particles size were prepared based on earlier studies.34, 46 In a typical experiment, the zinc nitrate aqueous solution



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was mixed with another aqueous solution of 2-methylimidazole and MLT under vigorous stirring. After 3 min, MLT@ZIF-8 nanoparticles (NPs) were formed and purified by centrifugation and washing. Meanwhile, pure ZIF-8 samples were obtained using the same method as control. Detailed experimental procedure was depicted in the Methods section.

The sizes and morphologies of MLT@ZIF-8 NPs and pure ZIF-8 have been investigated via scanning electron microscopy (SEM) and transmission electron microscopy (TEM). MLT@ZIF-8 NPs and its ZIF-8 NPs control both show uniform rhombic dodecahedral shapes and monodisperse particles size with the mean diameter of approximately 100 nm (Figure 1A and 1B, Figure S1). And the hydrodynamic diameters of MLT@ZIF-8 NPs display a narrow size distribution (116.6 ± 2.7 nm) via Dynamic light scattering (DLS) analysis (Figure 1C), which is a little bigger than ZIF-8 control (100.7 ± 5.2 nm, Figure S2). The powder X-ray diffraction (PXRD) of the MLT@ZIF-8 NPs and ZIF-8 NPs (Figure 1D) are identical to the simulated one, indicating the successful formation of crystal structures.

To demonstrate the existence of MLT in MLT@ZIF-8 NPs, several experiments were carried out. A new absorption bond at 1667 cm-1 belonging to the carbonyls groups of MLT has been observed in the Fourier transform infrared (FTIR) of MLT@ZIF-8

NPs

(Figure

S3),

confirming

the

presence

of

MLT.

The

thermogravimetric analysis (TGA) was used to determine the content of MLT. A more distinct weight-loss of MLT@ZIF-8 NPs in the temperature range of 100 - 550 oC has


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been observed and given about 6 wt % of weight loss, which is ascribed to the removal of MLT from MLT@ZIF-8 NPs (Figure 1E). The protein loading and encapsulation efficiency of the MLT@ZIF-8 NPs were calculated to be 6.9 wt % and 25.5% using a standard BCA protein quantitative method (Figure S4), respectively, which is consistence with the above TGA analysis. The zeta potential value of MLT@ZIF-8 NPs and pure ZIF-8 are +14.4 mV and +13.8 mV, respectively (Figure S5), verifying that MLT was embedded into the framework of ZIF-8, rather than absorbed onto its external surface.

The stability of NPs carriers is one of the most important factors for drug delivery system, which can help NPs keep their activities before arriving at the target locations. As shown in Figure 1G, all tested MLT@ZIF-8 NPs could keep their hydrodynamic diameter and PDI for a week in aqueous solution. Moreover, no significant hydrodynamic diameter and PDI changes of MLT@ZIF-8 NPs (Figure 1H) were observed in neutral PBS with 10% FBS at 37 oC for 24 h, implying that these NPs also exhibit favorable stability in physiological environment. The slight increase of NPs size is mainly caused by the electrostatic interactions between the negatively charged proteins in serum and positively charged MLT@ZIF-8 NPs. As we known, ZIF-8 is constructed from organic linkers (2-methylimidazole) and metal ions (Zn2+). The coordination between them can dissociate in acidic environment, which results in the drug release. Therefore, in vitro peptide release experiments have been carried out in PBS at various pH values (Figure 1F). MLT@ZIF-8 NPs possess a very slow rate of release in pH 7.4 PBS, and the MLT released from MLT@ZIF-8 NP is only about



23.7% after 24 h. However, faster drug release rates can be observed in acidic PBS with pH 6.8 and 5.5, mimicking the acidity of tumor microenvironment and endosome/lysosome, and the cumulative release of MLT are about 77.3% and 80.6%, respectively. What’s more, TEM analysis also reveals the differences of these NPs in neutral and acidic PBS (Figure S6). Immersed in acidic PBS solution, MLT@ZIF-8 NPs become irregular and smaller, implying the dissociation of ZIF-8 NPs.

MLT can interact with lipid membranes and cause member broken, thus result in cell death, which can generate non-specific toxicities like hemolysis.7, 10, 51-52 In order to validate the subdued cytolytic effect of MLT@ZIF-8 NPs, an erythrocyte hemolysis assay was carried out in vitro utilizing red blood cells (RBC). As shown in Figure 1I, MLT@ZIF-8 NPs exhibit minimal hemolysis at all tested concentrations, even as high as 128 µg/mL, whereas free MLT lyses almost all the RBCs at a lower level (4 µg/mL). Those results imply that the encapsulation strategy is efficiently to inhibit the hemolysis of MLT, which is important for its further clinical application.


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Figure 1. Basic properties of MLT@ZIF-8 NPs. (A) SEM image and (B) TEM image of MLT@ZIF-8 NPs. (C) Particle size distribution of MLT@ZIF-8 NPs dispersed in water. (D) PXRD patterns of MLT@ZIF-8 NPs, ZIF-8 NPs and simulated ZIF-8. (E) TGA curves of ZIF-8 NPs and MLT@ZIF-8 NPs. (F) MLT release profiles from MLT@ZIF-8 NPs in PBS solution (pH = 5.5, 6.8 and 7.4). Changes of diameter and PDI of MLT@ZIF-8 NPs (G) in water and (H) in PBS with FBS (10 %) over different times measured by DLS. (I) Hemolytic assay for MLT@ZIF-8 NPs and free MLT samples at different concentrations in RBC. Insert: Photographs of MLT@ZIF-8 NPs samples at different concentrations in RBC, with Triton (1%) as a positive control (100% hemolysis, the first from left) and PBS (pH 7.4) as a negative control (the second from left). Data represent mean values ± standard deviation, n = 3. The cellular uptake of MLT@ZIF-8 NPs was performed by Confocal laser scanning microscopy (CLSM) against A549 cells. MLT were embedded together with Nile red (NR) into ZIF-8 NPs to facilitate fluorescence observation, and cellular nucleus were dyed in blue by Hoechst 33258. A strong and time-dependent red fluorescence signals ACS Paragon Plus Environment


principally distribute in the cytoplasm, indicating efficient internalization of MLT@ZIF-8 NPs by A549 cells (Figure S7). Furthermore, the results of flow cytometry analysis (FCS) show that the endocytosis enhanced gradually with incubation time, confirming the time-dependent internalization (Figure 2A and 2B). Meanwhile, the fluorescence intensity also increases significantly with the increasing dosage of MLT, suggesting a dose-dependent manner (Figure S8). The colocalization with lysosome fluorescent dye was performed to discuss the endocytic pathways of MLT@ZIF-8 NPs (Figure S9). The red fluorescence of NR overlaps wonderfully with the green fluorescence of lysosome tracker, indicating that MLT@ZIF-8 NPs can be internalized via lysosomal-mediated pathway. The entry of NPs into cells is a complicate process, in most cases through one or more different endocytosis pathways. After endocytosis, MLT@ZIF-8 NPs possibly release the MLT in endosome or lysosome because the acid environment could destroy the ZIF-8 NPs. MLT could escape from endosome to the cytosol by destroying the membrane of endosome.

To evaluate the antitumor effects in vitro of MLT@ZIF-8 NPs, the cytotoxicity was performed using standard methyl-thiazolyl-tetrazolium (MTT) cell assay against A549 and HeLa cell lines. The cellular toxicity of ZIF-8 NPs was performed firstly (Figure 2C and S10). When A549 cells incubated with ZIF-8 NPs from 10 to 120 µg/mL for 24 h, more than 80% of cell survival rate has been detected. Even high up to 160 µg/mL, about 70% of cells are still alive, whereas MLT@ZIF-8 NPs with same concentration of ZIF-8 could produce more serious cytotoxicity, indicating the good biocompatibility of ZIF-8 carrier.53 Previous works have demonstrated that the cell


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death in the free MLT group is mainly due to the disintegration of cell membrane.8-10 Herein, the cellular toxicity of MLT@ZIF-8 NPs is stronger than free MLT in a concentration range (1.0 - 12.0 µg/mL) (Figure 2D). The half maximal inhibitory concentration (IC50) values of MLT@ZIF-8 NPs and free MLT are 6.7 and 8.2 µg/mL, respectively, implying the enhanced cytotoxicity of MLT@ZIF-8 NPs. The cytotoxicity assay toward HeLa cells also demonstrates the similar results that MLT@ZIF-8 NPs possess higher inhibition efficiency than MLT (Figure S11). The intensive cytotoxicity is largely due to the efficient internalization of MLT@ZIF-8 NPs by cancerous cells. Moreover, the change of cell morphologies observed by inverted microscope also confirms the cytotoxicity of MLT@ZIF-8 NPs (Figure S12). Most of cells with polygonal or spindle shape in the control group grow well, while the cellular survival rate obviously decrease and their morphologies become smaller and round after MLT@ZIF-8 NPs treatment with increased concentrations.



Figure 2. Cellular uptake and cytotoxic effects against A549 cells. (A) FCS of cells incubated with MLT@ZIF-8 NPs at 37 oC for 0.5, 2, and 4 h. (B) Mean fluorescence intensity (MFI) quantification analyses against cellular uptake. In vitro cytotoxicities of (C) ZIF-8 NPs and MLT@ZIF-8 NPs, and (D) free MLT and MLT@ZIF-8 NPs against A549 cells at different concentrations after 24 h. Data represent mean values ± standard deviation, n = 3. Apoptosis is an evolutionary conserved form of cell death.54-58 To determine the role of MLT@ZIF-8 NPs on apoptosis, the cell apoptosis was tested using FCS with Annexin V-FITC/ PI dyeing. As revealed in Figure 3, the percentage of early apoptosis of Annexin-V are 8.91%, 20.36% and 32.54% with the increasing dosage of MLT (2, 4 and 8 µg/mL), respectively, and negligible apoptosis were found in control group of blank, suggesting the occurrence of cell apoptosis. These differences are significant in the quantitative analysis of cells incubated with various concentrations of MLT@ZIF-8 NPs (Figure S13). Furthermore, we also observed the changes of


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cytomorphology in concentration-induced apoptosis by CLSM analysis. The cell nuclei in control group showed diffused dyeing of chromatin, while the cells treated with different concentrations of MLT@ZIF-8 NPs exhibited representative morphological changes, for example chromatin condensation, margination and shrunken nucleus, indicating the occurrence of apoptosis (Figure S14). Above results indicate that MLT@ZIF-8 NPs can induce apoptosis of A549 cells, and apoptotic ratio increases with the increasing of concentration. However, the necrotic cells are increased dose-dependently after treatment with free MLT, the percentages of the necrotic cells of Annexin-V/PI are 11.7%, 20.62% and 25.34% (Figure S15), confirming that the effects of free MLT and MLT@ZIF-8 NPs on cancer cells are different.

Figure 3. The rate of apoptosis in A549 cells treated with different concentrations of



MLT@ZIF-8 NPs. A: Control group, the groups of different concentrations of MLT@ZIF-8 NPs: B: 2 µg/mL, C: 4 µg/mL, D: 8 µg/mL. To further explore the anticancer mechanism of MLT@ZIF-8 NPs, transcriptome analysis was used to study the gene expression changes of A549 cells before and after MLT@ZIF-8 NPs treatment by using RNA isolation, microarray analysis and the western blot. The total RNA extracted from A549 cells treated with MLT@ZIF-8 NPs was analyzed using an ultraviolet spectrophotometer, and untreated cells as control. The ratio of A260/A280 is 2.1 for the all samples, which validates the purity of the RNA. And the quality and integrality of extracted RNA were further evaluated by formaldehyde agarose gel electrophoresis. The electrophoretograms (Figure S16) show distinctive strips matching 28S and 18S ribosomal RNA, indicating the obtain RNA is complete, undegraded, and suitable for the following microarray analysis. Meanwhile, the method of principal component analysis (PCA) of samples has been carried out based on the complete data set.59 The first step in PCA is to subtract the mean from each of the data dimensions. Next, the covariance matrix and the eigenvectors and eigenvalues of the covariance matrix are calculated. Data compression and reduced dimensionality are performed when converting the data into components. The PCA results demonstrate that the samples were clearly separated into 2 clusters (Figure 4A), indicating an obvious directionality among diverse groups on the basis of similarities in gene expression.

Following, differentially expressed genes (DEGs) caused by MLT @ ZIF-8 NPs treatment were described by Volcano plots and Heatmap, using a false discovery rate


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threshold of 0.05 and a fold change of 1.5. 60 By comparing with the genechip results from the untreated control cells (C) and the MLT@ZIF-8 treatment cells (M), we identify 3383 DEGs, including 1945 up-regulated genes and 1438 down-regulated genes (Figure 4B and 4C). All these data suggest that MLT@ZIF-8 NPs have a vital function on transcriptional regulation of multiple genes in A549 cells.

Figure 4. (A) Principal component analysis of A549 cells based on (C) untreated control and (M) MLT@ZIF-8 NPs treatment groups. (B) Volcano plots to determine DEGs of C vs. M. The x-axis represents the log 1.5-fold changes (FC) of genes and the y-axis represents the -log 10 of the p-values for the various condition pairs. Each dot represents a gene. The red colored field represents the up-regulated genes and the blue colored field represents the down-regulated genes which met the selection threshold of FC > 1.5 and p < 0.05. (C) Heatmap displaying the overview of the



differentially expressed genes induced by MLT@ZIF-8 NPs treatment. Based on the data of gene chip study, we also perform Gene Ontology (GO) and pathway analysis for 3383 DEGs caused by ZIF-8@MLT NPs. According to GO classification, three domains are covered, including cellular components (CC), biological processes (BP) and molecular function (MF).61 A high propertion of differentially expressed genes are related to growth cone or actin cytoskeleton (Figure 5A). In the BP domain, the enriched parts cover cell differentiation, cell migration, multicellular organism development, negative regulation of growth, regulation of cell proliferation transcription (Figure 5B). And among MF domain, transcription regulatory region DNA binding, transcriptional activator activity, RNA polymerase II core promoter proximal region sequence-specific binding and protein C-terminus binding are affected (Figure 5C). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis reveal that diverse pathways are influenced by MLT@ZIF-8 NPs treatment, the top 10 pathways contain transcriptional misregulation in cancer, mineral absorption, pathways in cancer, drug metabolism, metabolism of xenobiotics by cytochrome P450, complement and coagulation cascades, chemical carcinogenesis, systemic lupus erythematosus and bacterial invasion of epithelial cells. (Figure 5D).


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Figure 5. GO and pathway analysis of DEGs in MLT@ZIF-8 NPs treated A549 cells. (A) Cellular components. (B) Biochemical processes. (C) Molecular function. (D) Percentage of DEGs in top 10 (p ≤ 0.05 & Over lappings ≥ 5) enriched pathways.



The above pathway enrichment results reveal that MLT@ZIF-8 NPs treatment can influence diverse pathways, and the pathways in cancer possess the highest percentage (26.7%) of DEGs in the top 10 enriched pathways. In current study, we concern about the effect of MLT@ZIF-8 NPs on p53 signaling pathway (Figure S17). As we known, P53 can coordinate cells against different stress and antitumor agents as a principal mediator. And it can lead to the up-regulation of Bax and down-regulation of Bcl-2, which performs a significant function for the change of Bcl-2/Bax ratio.64 If this balance of Bcl-2/Bax ratio is broken, mitochondrial permeabilization and subsequent apoptosis activation would arise.65 Western blot analysis (Figure 6A and 6B) shows that treatment of A549 cells with MLT@ZIF-8 NPs could increase the protein expression levels of p53 and Bax, whereas markedly decrease the level of Bcl-2, which result in the enhancement of Bax/Bcl-2 ratio with the increasing concentrations of MLT@ZIF-8 NPs. Besides, more Cytochrome c (Cyt c) also can be delivered from mitochondria to cytosol in a concentration-dependent manner. All above data demonstrate that MLT@ZIF-8 NPs can cause apoptosis through the mitochondrial dependent pathway.

The PI3K/Akt pathway performs important functions in the process of cellular apoptosis, proliferation or survival in different types of cells.66 PI3K/Akt cellular kinases are crucial signal pathways of influencing the expression and effects of p53, which can restrain various tumor suppressor proteins, including Bad, FOXO transcription factors, the tuberin/hamartin complex and GSK3.67 In addition, AKT activation also can promote cellular survival through interacting with certain proteins


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participated in apoptosis, for example the pro-apoptotic Bcl-2 family representative member Bax.68 Therefore, interruption of this signal pathway can simultaneously prevent the tumor cells proliferation and trigger cellular apoptosis. In order to analyze whether inhibition of PI3K and phosphorylation of Akt could affect p53 expression for MLT@ZIF-8 NPs induced A549 cells apoptosis, we detected the expression of PI3K, total and phosphorylated Akt using western blotting method (Figure 6C). The results demonstrated that MLT@ZIF-8 NPs could inhibit the expression of phosphorylated Akt and PI3K in a dose dependent manner, which indicates that MLT@ZIF-8 NPs may induce apoptosis through inhibition of PI3K/Akt pathway.

Above all, our current results clearly reveal that PI3K/Akt-regulated p53 pathway is related to MLT@ZIF-8 induced A549 cells apoptosis and perfectly validates the microarray data. The MLT@ZIF-8 NPs could suppress the proliferation of A549 cells via down-regulating the expression of PI3K and p-AKT, up-regulating p53, activating pro-apoptotic proteins Cyt c, caspase-3, caspase-9 and Bax, and suppressing anti-apoptotic protein Bcl-2, which result in cell apoptosis. These findings demonstrate the possible molecular mechanisms of MLT@ZIF-8 NPs induced A549 cells death and suggest that MLT@ZIF-8 NPs may be a promising platform as an effective drug in cancer therapy.



Figure

6.

MLT@ZIF-8

NPs

induced

the

A549

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cells

apoptois

by

the

PI3K/Akt-regulated p53 pathway. A549 cells were treated with gradient concentrations of MLT@ZIF-8 NPs for 24 h. The whole cell lysates were analyzed by western blotting method. (A) The expression levels of p53 protein expression in A549 cells and the relative quantitative analysis. (B) Western blot analysis was conducted using anti-Bax, Bcl-2, Cleaved caspase-3, Cleaved caspase-9, Cyt c antibodies and the relative quantitative analysis. (C) The expression level of PI3k, p-AKT, AKT and the relative quantitative analysis. Data represent mean values ± standard deviation, n = 3. Subsequently, we proceeded to study in vivo antitumor activity on U14 tumor-bearing Kunming mice. After tumor grew to a size of 150 mm3, we divide these mice into four groups (n = 4 per group) at random. Mice were administered ZIF-8 NPs (at a dose equivalent to MLT@ZIF-8 NPs), free MLT (1 mg/kg), MLT@ZIF-8 NPs (14.5 mg/kg, containing 1 mg/kg of MLT) and PBS as control by intravenous


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method daily for three days, respectively. Tumor volume and mice weight were measured every other day in 10 days. As described in Figure 7A, PBS and ZIF-8 NPs groups both showed rapid tumor growth during the whole treatment period, implying ZIF-8 NPs alone had a slight impact on tumor growth. In contrast, the MLT@ZIF-8 NPs treated mice exhibited obvious tumor growth inhibition, which was more remarkable than free MLT group. The changes of body weight over time can be utilized to illustrate the general toxicity. As shown in Figure 7B, free MLT treatment displayed evident body weight reduction during injection as a result of nonspecific toxicities and by-effects of MLT, like hemolysis, while the body weight of the mice in MLT@ZIF-8 NPs treated group increased gradually, and also had no significant difference compared with the control group, suggesting that MLT@ZIF-8 NPs could reduce the side effects of MLT to some extent. After 10 days of observation and measurement, the mice of four groups were sacrificed and tumors were weighed. Photographs of mice in each group and the excised tumors are exhibited in Figure 7C and S18. The average tumor weight of MLT@ZIF-8 NPs treated group was smaller than the other three groups (Figure7D), which was agree with the result of the tumor volume. And the quantitative tumor inhibitory rate is calculated to be about 71%, which is higher than MLT treated group (49%), indicating MLT@ZIF-8 NPs possessed better antitumor effect.

Furthermore, we also evaluate the potential systemic toxicity of MLT@ZIF-8 NPs in vivo. After the healthy Kunming mice were injected intravenously MLT@ZIF-8 NPs (14.5 mg/kg, containing 1 mg/kg of MLT) daily for three days, the blood was



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collected from the eye socket on the third and seventh days after administration. The serum biochemistry and a whole blood panel were tested. As shown in Figure S19, the red blood cells (RBC), white blood cells (WBC), hemoglobin (HGB), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelet (PLT) and the liver and

kidney

function

parameters

(alanine

transaminase

(ALT),

aspartate

aminotransferase (AST), uric acid (UA), and creatinine (CREA)) of treatment group at different time points all showed non-significant changes compared with PBS group, indicating negligible systemic toxicity of MLT@ZIF-8 NPs. These results above all clearly reveal that MLT@ZIF-8 NPs can be safely and effectively used for antitumor therapy in vivo, which possess excellent anticancer effect and reduced systemic toxicity compared with free MLT.


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Figure 7. In vivo antitumor study of MLT@ZIF-8 NPs. (A) The relative tumor volume growth rates and (B) body weight curves of U14 tumor-bearing mice after different treatments. (C) Photographs of the excised tumors. (D) Tumor weight in each group. The level of significance was set at probabilities of **p < 0.01, and ***p < 0.001. Data represent mean values ± standard deviation, n = 4.



CONCLUSIONS

To summarize, an effective and mild method to synthesis cytolytic peptide melittin-loaded ZIF-8 (MLT@ZIF-8) NPs has been established. The formed MLT@ZIF-8 NPs possess nanoscale size and robust stability, and effectively protect MLF from hemolyzing. The anticancer experiments demonstrate that MLT@ZIF-8 NPs possess enhanced cytotoxicity compared to MLT, which hold great potential for delivery of cytolytic peptides. More importantly, we also perform transcriptome analysis to detect differentially expressed genes (DEGs) caused by MLT@ZIF-8 NPs, and further carry out the GO and pathway analysis of the DEGs, which can help us clarify the function of MLT@ZIF-8 NPs on A549 cells. Meanwhile, the western blot analysis has been used to validate that PI3K/Akt-regulated p53 signal pathway is related to MLT@ZIF-8 induced A549 cells apoptosis. In this work, we explore the anticancer mechanism of MLT@ZIF-8 NPs through transcriptome analysis for the first time, which could provide significant understanding for clinical application and development of MLT@ZIF-8 NPs. Therefore, this work not only emphasizes the potential of using MOF as a desired platform for delivering cytolytic peptides, but also provides new insights for understanding how MLT@ZIF-8 NPs exert their effects against lung cancer.


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METHODS

Synthesis of MLT@ZIF-8 NPs. MLT@ZIF-8 particles with well-defined morphology and particles size were prepared based on earlier studies.34, 46 In a typical experiment, the zinc nitrate aqueous solution was mixed with another aqueous solution of 2-methylimidazole and MLT under vigorous stirring. After 3 min, MLT@ZIF-8 NPs were formed and purified by centrifugation (14000 rpm, 10 min) and washing with deionized water three times. Finally, the products were freeze-dried and stored at -20 oC until further use. Yield is calculated to be about 80% based on the amount of zinc. Meanwhile, pure ZIF-8 samples were obtained using the same method as control. NR-MLT@ZIF-8 NPs were prepared by embedding MLT with Nile red (NR) together into ZIF-8 NPs to promote fluorescence observation of cellular internalization, free NR was removed by centrifugation.



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ASSOCIATED CONTENT.

Supporting Information.

The Supporting Information is available free of charge on the ACS Publications website.

Materials, general measurements, details of the experiments, supporting data including SEM, TEM and size distributions of ZIF-8 NPs, FTIR spectra and Zeta potential of ZIF-8 and MLT@ZIF-8 NPs, standard curve of peptide, CLSM images

of

A549

cells

incubated

with

different

concentrations

of

NR-MLT@ZIF-8 NPs for different time at 37 oC, in vitro biocompatibility of free MLT and cytotoxicity of MLT@ZIF-8 NPs against HeLa cells at different concentrations, morphological apoptosis in A549 cells and the quantitative analysis of the rate of apoptosis, RNA electrophoretogram analysis of A549 cells in different groups, the gene expression in the p53 signaling pathway was regulated by MLT@ZIF-8 NPs, blood biochemical analysis and in vivo experiment results (PDF)

AUTHOR INFORMATION Corresponding Authors * E-mail: [email protected], [email protected].


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Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT Financial support was kindly provided by China Postdoctoral Science Foundation of China (No. 2017M613412). Author 1, Author 2 and Author 3 contributed equally to this work.



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TOC Figure


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