A redox-responsive dihydroartemisinin dimeric nanoprodrug for enhanced antitumor exercise | Journal of Nanobiotechnology


Preparation and characterization of DHA dimeric NPs

Firstly, the DHA2-SS was synthesized via the esterification response of DHA with dicarboxylic acid (Further file 1: Fig. S1) [23]. After purification by silica gel column chromatography, DHA2-SS was obtained in excessive yields (> 90%) and the chemical building has been characterised through proton nuclear magnetic resonance (1H NMR) spectroscopy and a linear ion lure mass spectrometer (LTQ-MS). In 1H NMR spectra, the disappearance of proton of 10-hydroxyl group from DHA at round 4.7 ppm and the looks of latest peaks close to 3.66 ppm validated the success of esterification response, and the response web site was on the 10-hydroxyl group of DHA (Further file 1: Fig. S2). The height worth equivalent to DHA2-SS at round 737 in mass spectrometry was in step with the theoretical calculated worth (Further file 1: Fig. S3), additional confirming the construction of DHA dimer. With the intention to evaluate the redox responsiveness of DHA2-SS, we additionally synthesized one other DHA dimer with the identical size of carbon chain (DHA2-C6) as management (Further file 1: Figs. S1, S2 and S4).

It’s reported that the natural dimers may self-assemble into NPs in aqueous answer [25,26,27,28,29,30]. As anticipated, each sorts of ensuing dimers shaped spherical nanoparticles (abbreviated as SS NPs and C6 NPs, respectively) via nanoprecipitation methodology as noticed through transmission electron microscopy (TEM) (Fig. 1A, B). And SS NPs had a median hydrodynamic diameter of roughly 167.2 nm as decided by dynamic mild scattering (DLS), which was just like these of C6 NPs (181.4 nm) (Fig. 1C). These two sorts of NPs had been discovered to be unfavourable, and the zeta potential values had been round − 20 mV (Fig. 1D). The drug content material of SS and C6 NPs was 90.6% and 91.7%, respectively. As well as, SS and C6 NPs each possessed strong stability with negligible modifications in measurement and measurement distribution in a single week (Fig. 1E), and in addition stored secure in PBS (pH 7.4) containing 10% FBS after 24 h (Fig. 1F), wherein a slight measurement enhance within the first two hours was ascribed to the protein absorption on the floor of NPs. Moreover, these two sorts of NPs additionally had good structural stability after totally different therapies (Further file 1: Figs. S5, S6 and S7).

Fig. 1

Primary characterization of DHA dimeric NPs. TEM photos of (A) SS NPs and (B) C6 NPs. C Dimension distribution and (D) zeta potential of ready SS and C6 NPs. Modifications of hydrodynamic diameter and PDI of two sorts of NPs (E) in water and (F) in PBS with FBS (10%) over totally different instances measured by DLS. Information are expressed as imply ± SD (n = 3)

DTT and GSH triggered launch of DHA

As talked about above, disulfide bond possess distinct redox response functionality [47,48,49]. Subsequently, we investigated the responsiveness of DHA2-C6 and DHA2-SS through the use of dithiothreitol (DTT) and H2O2 as decreasing and oxidizing brokers, respectively. As proven in Fig. 2A, after incubation with 10 mM DTT, the HPLC peak of DHA2-SS at 10.6 min declined considerably and fully disappeared in 24 h, whereas the brand new peak for DHA at 6.4 min emerged and enhanced steadily (Further file 1: Fig. S8), indicating the dissociation of disulfide linkage and the discharge of DHA. Compared, drug launch from DHA2-C6 dimer was fairly gradual below the identical situation, and solely roughly 10% of DHA launched even after 48 h therapy (Fig. 2B). As well as, we additional investigated the oxidation responsiveness of those dimers. Just like discount responsiveness, DHA2-SS additionally exhibited delicate oxidation responsiveness. As offered in Fig. 2D, DHA2-SS may launch about 60% DHA after 48 h of H2O2 therapy (10 mM), whereas negligible degradation was detected for DHA2-C6 on the similar time (Fig. 2E). The discharge curves of DHA over time within the presence of DTT and H2O2 are revealed in Fig. 2C, F, respectively. These outcomes validate the redox responsiveness of disulfide bond linker and the managed launch of DHA. The proposed degradation mechanism of DHA2-SS dimers initiated by DTT or H2O2 are illustrated in Further file 1: Fig. S9 [23, 24, 26,27,28]. For DTT triggered drug launch, due to − SH of DTT assaults, a sulfhydryl − disulfide bond alternate response happens, and the generated thiol teams may facilitate the hydrolysis of the adjoining ester bond and the discharge of DHA from prodrugs. For H2O2 triggered drug launch, the disulfide bond of DHA2-SS could possibly be oxidized to hydrophilic sulfoxide or sulphone upon publicity to H2O2, resulting in hydrolysis of the adjoining ester bond and subsequent launch of DHA.

Fig. 2

Discount responsiveness of (A) DHA2-SS and (B) DHA2-C6 dimers degradation and (C) the speed of DHA launched from DHA dimers within the presence of 10 mM DTT at 37 °C. Oxidation responsiveness of (D) DHA2-SS and (E) DHA2-C6 dimers degradation and (F) the speed of DHA launched from DHA dimers within the presence of 10 mM H2O2 at 37 °C

Mobile uptake and in vitro cytotoxicity of DHA dimeric NPs

Human hepatoma HepG2 cells had been used to review the mobile internalization of those NPs through confocal laser scanning microscopy (CLSM). The fluorescent dye nile purple (NR) was utilized as a marker and encapsulated into the NPs by coassembly with DHA dimer, and the blue fluorescence of Hoechst 33258 was employed to localize the cell nucleus. As exhibited in Fig. 3A, the sturdy purple fluorescence alerts principally distributed within the cytoplasm, suggesting the environment friendly internalization of DHA dimeric NPs. And we may additionally observe that the fluorescence depth elevated clearly from 0.5 h to 4 h, implying that endocytosis continued in a time-dependent method (Further file 1: Fig. S10). Nevertheless, when the incubation time was prolonged to eight h, the fluorescence depth of SS NPs decreased considerably, indicating the dissociation of disulfide bond and the discharge of DHA, whereas C6 NPs nonetheless displayed sturdy fluorescence sign due to the insensitive responsiveness of C6 linker to the tumor redox microenvironment.

Fig. 3

(A) CLSM photos of HepG2 cells incubated with SS and C6 NPs at 37 °C for 4 h. Scale bars, 20 μm. In vitro cytotoxicity in opposition to (B) HepG2 cells and (C) HL-7702 cells of free DHA and SS NPs at totally different concentrations after incubation for 48 h. (D) Circulation cytometry information for apoptosis in HepG2 cells handled with totally different concentrations of SS NPs

Subsequent, the cytotoxicity of DHA dimeric NPs was evaluated in opposition to human HepG2 and HeLa cells via a typical MTT assay. As proven in Fig. 3B, each free DHA and SS NPs confirmed environment friendly suppression of HepG2 viability in focus dependent method for 48 h, and SS NPs exhibited larger cytotoxicity than free DHA. The cell viability of SS NPs was lower than 30%, whereas it was roughly 40% without cost DHA on the equal DHA focus. This outcome could also be attributed to the improved mobile uptake of NPs into tumor cells and the fast launch of DHA. And SS NPs may shortly launch lively DHA as soon as in dwelling cells due to the responsiveness of disulfide bond in redox surroundings. Nevertheless, C6 NPs exhibited obvious decrease cytotoxicity in contrast with SS NPs (Further file 1: Fig. S11). Equally, SS NPs nonetheless possessed the strongest mobile toxicity in direction of HeLa cells (Further file 1: Fig. S12). As well as, we evaluated the cytotoxicity of SS NPs towards regular hepatocytes (HL-7702), and in addition in contrast its toxicity on HepG2, HeLa and HL-7702 cells. As revealed in Fig. 3C, SS NPs exhibited no vital mobile toxicity in opposition to HL-7702 cells, and enhanced cytotoxicity in opposition to these two sorts of tumor cells (Further file 1: Fig. S13), indicating the selectivity of DHA2-SS prodrug in direction of tumor cells. This result’s ascribed to the totally different redox situations in regular cells and tumor cells.

To additional perceive the contribution of SS NPs on apoptosis, we stained HepG2 cells with Annexin V-FITC and PI, and analyzed them by movement cytometry. As offered in Fig. 3D, the ratio of early apoptotic cells was 9.40%, 15.00%, and 24.56%, respectively, because the drug focus will increase (20, 40 and 60 μM), validating the cell apoptosis in a concentration-dependent method. Moreover, we additionally examined the nuclear morphological modifications by CLSM. The cell nucleus emerged as a homogeneous blue chromatin with an organized construction in regular cells, whereas the cells incubated with SS NPs displayed consultant morphological modifications (Further file 1: Fig. S14), together with intense fluorescent spots, nuclear pyknosis, and in depth blebbing, additional verifying the apoptosis of tumor cells.

Antitumor mechanism of SS NPs

To elucidate the mechanism of SS NPs in inhibiting tumor cell proliferation and inducing apoptosis, RNA sequencing (RNA-seq) know-how was utilized to gather the gene expression [50]. The full RNA extracted from SS NPs therapy group (SS) and the management group (C) have been analyzed for high quality and integrity by using formaldehyde agarose gel electrophoresis. The outcomes verified that the obtained RNA was intact, undegraded, and appropriate for RNA-seq evaluation. In the meantime, principal part evaluation (PCA) of samples was carried out on the entire dataset [51], which might show modifications of total gene expression. As proven within the PCA outcomes (Further file 1: Fig. S15), there have been two clusters, verifying a definite directionality between SS NPs therapy and the management teams based mostly on the similarity of gene expression. Subsequently, the differentially expressed genes (DEGs) induced by SS NPs have been recognized and described by volcano plots and heatmaps. After evaluating with the untreated management group (log2 fold-change ≥ 2.0 and adjusted P worth < 0.05), we distinguished 6546 DEGs, together with 3288 up-regulated and 3258 down-regulated expression genes (Fig. 4A, B). The above outcomes suggest that SS NPs play an necessary position on gene expression of the handled tumor cells.

Fig. 4

A Volcano plots to find out DEGs of SS NPs therapy group and management group. The x-axis represents the log 2.0-fold modifications (FCs) of genes and the y-axis represents the − log10 of the p-values for the varied situation pairs. Every dot represents a gene. The grey factors characterize a non-statistically vital distinction in gene expression. The purple area represents the upregulated genes and the blue area represents the downregulated genes. B Warmth map displaying the overview of the differentially expressed genes induced by SS NPs therapy

In accordance with the RNA-seq information, we carried out the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway evaluation for 6546 differentially expressed genes. Three domains had been concerned: mobile parts (CC), organic processes (BP) and molecular operate (MF). Within the BP area, a big proportion of genes had been related to mobile course of, metabolic course of, organic regulation (Fig. 5A). For the CC area, the enriched genes lined cell, cell half, organelle, membrane and so forth (Fig. 5B). Binding, catalytic exercise, transcription regulator exercise and molecular operate regulator had been primarily influenced for the MF area (Fig. 5C).

Fig. 5

GO and KEGG pathway purposeful enrichment evaluation of DEGs in SS NPs handled HepG2 cells. A Biochemical processes. B Mobile parts. C Molecular operate. D KEGG pathway purposeful enrichment of DEGs

On the idea of the DEGs outcomes, we additionally executed KEGG purposeful enrichment evaluation and pathway classification. The KEGG purposeful enrichment evaluation outcome (Fig. 5D) demonstrated that a number of pathways have been affected by DHA2-SS NPs, and the signaling pathways of carbon metabolism, biosynthesis of amino acids, fatty acid metabolism, HIF-1 signaling pathway and pathways in most cancers had been among the many prime 20 pathways. Alternatively, KEGG pathway classification outcomes (Further file 1: Fig. S16) displayed that there have been six branches for KEGG pathways, together with mobile processes, environmental info processing, genetic info processing, human illnesses, metabolism, and organismal programs. And the DEGs in HepG2 cells after SS NPs therapy had been primarily enriched in PI3K-Akt signaling pathway, MAPK signaling pathway and pathways in most cancers.

From GO and pathway evaluation outcomes, we found that the metabolic course of was concerned within the inhibition of tumor cells proliferation, thus we carried out community statistical evaluation on the protein–protein interplay (PPI) of those genes correlated with metabolism. As proven in Fig. 6, the metabolism of carbohydrates, amino acids and lipids have been remarkably regulated within the cells after SS NPs therapy, indicating that SS NPs may induce metabolic reprogramming in tumor cells. Subsequently, we targeting the metabolism reprogramming of tumor cells after being handled with SS NPs.

Fig. 6

PPI community of the DEGs correlation with (A) carbohydrates, (B) amino acids and (C) lipids metabolism

Apoptosis is a extremely regulated technique of cell loss of life, which is necessary to keep up the inherent stability of multicellular organisms, and includes a wide range of sign pathways [52,53,54]. The Bcl-2 protein household accommodates pro-apoptotic and anti-apoptotic regulators of programmed cell loss of life/apoptosis, and performs a dominant position in regulating cell apoptosis. On this household, Bax gene, a pro-apoptotic member, can kind heterodimers with Bcl-2 protein, and the ratio of Bax/Bcl-2 may decide the sensitivity of cells to apoptosis. The activation of Bax can launch cytochrome c (Cyt C), and Cyt C prompts caspase-9 and downstream caspase-3 via a cascade response to advertise cell apoptosis, whereas the anti-apoptotic Bcl-2 operates within the reverse approach. Subsequently, we detected expression of Bax, Bcl-2, cleaved caspase-3, cleaved caspase-9 and Cyt C by western blotting. As displayed in Fig. 7, the expression ranges of Bax, caspase-3, caspase-9, and Cyt C had been clearly elevated, and the expression ranges of Bcl-2 was decreased in comparison with the management group. The above outcomes display that mitochondrial apoptosis pathway is concerned within the apoptosis of HepG2 cells induced by SS NPs.

Fig. 7

Results of SS NPs on expression of mitochondrial apoptosis pathway relative proteins. A Protein expression was analyzed utilizing western blotting and (B) quantified in relation to β-actin. Densitometric values had been normalized by β-actin and expressed as imply ± SD, n = 3. Statistical significance: *p < 0.05, and **p < 0.01

As we recognized, tumor cells typically reveal aberrant metabolism because of metabolic reprogramming [55]. As a trademark of tumor cells, the Warburg impact signifies that tumor cells rely closely on glycolysis for power, relatively than oxygen [56,57,58]. To analyze whether or not SS NPs therapy may suppress the glycolysis of tumor cells, the glucose uptake, contents of lactic acid and ATP merchandise of HepG2 cells handled with SS NPs had been detected. As proven in Fig. 8, the glucose uptake was decreased, and the contents of intracellular ATP and extracellular lactic acid had been additionally declined, indicating that SS NPs could inhibit glycolysis of tumor cells.

Fig. 8

SS NPs suppress glycolysis degree in HepG2 cells. A After HepG2 cells had been handled with SS NPs, glucose content material within the tradition media was instantly examined, and glucose uptake was calculated. B Lactic acid product was detected. C ATP content material was detected via bioluminescence assay. Information arepresented as imply ± SD, n = 3. Statistical significance: *p < 0.05, and **p < 0.01

Most cancers metabolic reprogramming is regulated by a number of pathways, which incorporates the PI3K-AKT signaling pathway [59]. The activated PI3K-AKT can promote the transition to cardio glycolysis, and AKT may outcome within the phosphorylation of some necessary downstream targets, equivalent to Bcl-2 apoptosis-related household, and mammalian goal of rapamycin (mTOR), to guard cells from apoptosis. In the meantime, this pathway additionally may regulate HIF-1α via mTOR, and activated HIF-1α is expounded to the up-regulation of glucose transporters (Gluts) and glycolytic enzymes. To grasp whether or not the activation of PI3K/AKT and HIF-1α had been concerned in metabolic reprogramming of tumor cells handled by SS NPs, we detected the associated protein expression. As displayed in Fig. 9, SS NPs therapy may lower the expression of glycolytic enzymes, equivalent to PFKP, HK2, LDH, Glut1. The ratio of p-PI3K/PI3K, p-AKT/AKT, p-mTOR/mTOR and HIF-1α had been additionally decreased accordingly. Collectively, these findings verify that SS NPs may induce apoptosis and suppress glycolysis by regulating the PI3K/AKT/HIF-1α signaling pathway.

Fig. 9

Results of SS NPs on expression of PI3K/AKT/HIF-1α signaling pathway relative proteins. A Protein expression of glycolytic enzymes had been analyzed utilizing western blotting and (B) quantified in relation to β-actin. C Protein expression of PI3K/AKT pathway had been analyzed and (D) quantified in relation to β-actin. Densitometric values had been normalized by β-actin and expressed as imply ± SD, n = 3. Statistical significance: *p < 0.05, and **p < 0.01

In vivo antitumor efficacy of DHA dimeric NPs

We additional evaluated the anti-cancer exercise of SS NPs on H22 tumor-bearing Kunming mice. Mice bearing the tumors had been randomly divided into 4 teams with totally different therapies: PBS, free DHA, C6 NPs, and SS NPs, and injected intravenously at equal DHA doses each second day. As illustrated in Fig. 10A, DHA therapy exhibited a average inhibitory impact on tumor progress in contrast with the management group, which is especially owing to its intrinsic toxicity. Notably, SS NPs exhibited evident antitumor exercise, which is stronger than free DHA group. The improved therapeutic efficacy of SS NPs ought to be ascribed to the a number of benefits of nanoparticle formulations, together with enhanced tumor accumulation, efficient endocytosis, and fast drug launch in tumor websites. Unsurprisingly, C6 NPs group displayed the weakest tumor progress inhibition impact in these therapy teams on account of the insensitivity of C6 linker to redox microenvironment. What’s extra, the tumor weight (Fig. 10B) and the pictures of resected tumors (Fig. 10C) visually demonstrated the best tumor inhibition efficacy obtained by SS NPs, additional validating the improved antitumor impact of disulfide-bond bridged prodrug nanoparticles. As well as, all mice had no vital weight fluctuation throughout the entire therapy interval (Fig. 10D), and there was additionally no detectable histological injury noticed after SS NPs therapy from the hematoxylin and eosin (H&E) stained tissue sections of main organs (coronary heart, liver, spleen, lung, and kidney) (Further file 1: Fig. S17). The above outcomes validate SS NPs at present doses possess favorable biosafety and ignorable systemic toxicity. The H&E staining of tumor slices revealed that SS NPs group exhibited probably the most extreme mobile injury in contrast with the management group. The tumor cells after SS NPs therapy shrank largely and the tumor tissue considerably decreased. (Fig. 10E). The entire outcomes substantiate that DHA2-SS NPs could possibly be safely used for the in vivo therapy, and possess higher therapy results in distinction with free DHA.

Fig. 10

In vivo antitumor efficacy analysis. A Tumor progress curves and (B) tumor weights of H22 tumor-bearing mice after totally different therapies. C Images of excised tumors after the final therapy. D Physique weight curves of tumor-bearing mice in every group throughout therapies. E H&E-stained photos of tumor slices collected from mice after totally different therapies. Scale bars, 50 μm. Information are expressed as imply ± SD (n = 3). Statistical significance: **p < 0.01, and ***p < 0.001


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