Tumor-Targeting Peptide for Redox-Responsive Pt Prodrug and Gene

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Tumor-Targeting Peptide for Redox-Responsive Pt Prodrug and Gene Co-delivery and Synergistic Cancer Chemotherapy Yaxuan Bai, Zeyu Li, Liping Liu, Tiedong Sun, Xiaocheng Fan, Ting Wang, Zhenzhen Gou, and Shengnan Tan ACS Appl. Bio Mater., Just Accepted Manuscript • DOI: 10.1021/acsabm.9b00065 • Publication Date (Web): 13 Mar 2019 Downloaded from http://pubs.acs.org on March 15, 2019

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Tumor-Targeting Peptide for Redox-Responsive Pt Prodrug and Gene Co-delivery and Synergistic Cancer Chemotherapy Yaxuan BaiA‡, Zeyu LiA‡, Liping LiuB, Tiedong Sun*A, Xiaocheng FanA, Ting Wang*A, Zhenzhen GouA, Shengnan TanC ‡

Zeyu Li and Yaxuan Bai contribute equally to this article

A

Department of Chemistry, College of Science, Northeast Forestry University, 26 Hexing Road,

Harbin 150040, China B

Harbin First Specialist Hospital, 217 Hongwei Road, Harbin 150056, China

C

Testing& Analysis Center, Northeast Forestry University, 26 Hexing Road, Harbin 150040,

China KEYWORDS: gene delivery, tumor-targeting peptide, Pt prodrug, multidrug resistance

ABSTRACT: A new co-delivery system combining prodrug strategy, siRNA/BAplatin @CRGDK NPs, to overcome cisplatin (CDDP) resistance in human breast cancer was designed and researched. Negatively charged siRNA was deposited onto the surface of tumor-targeting


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peptide-functionalized BAplatin@CRGDK NPs. SiRNA/BAplatin@CRGDK NPs could facilitate cellular uptake of BAplatin and increase the cell proliferation suppression effect of Pt against MDA-MB-231/DDP cells. The tumor-to-kidney uptake ratio of the siRNA/BAplatin@CRGDK NPs in MB-231/DDP tumors is 2.4-fold higher than cisplatin in MB-231/DDP tumors, thus giving rise to more significant antitumor efficacy. It demonstrated that the siRNA/BAplatin@CRGDK NPs is a potential, safe and efficient therapeutic agent for cancer therapy.

Cisplatin is one of the widely known and used antitumor drugs against a large variety of solid tumors.1 However, the availability is limited by the drug resistance and side-effects. Different cellular adaptations can be raised from the drug resistance, including increased drug deactivation, reduced cellular drug uptake, increased DNA damage tolerance and/or DNA repair.2 Compared with Pt(II) drugs, Pt(IV) complexes are so inert that exhibit less toxicity, which is an alternative with great promise to avoid the side effects of Pt(II) drugs.3-5 After entering the cell, the Pt(IV) prodrugs are reduced by intracellular reducing molecules glutathione (GSH) to generate Pt(II) drugs, so that regain their cytotoxicity.6,7 Asplatin (a fusion of aspirin and cisplatin), exhibits significant cytotoxicity in tumor cells and almost fully overcomes the drug resistance of cisplatin resistant cells.8,9 Betulinic acid (BA) is potent antitumor drugs. Lei reported that the use of BA might elicit synergistic effects and prevent the emergence of drug resistance. 10 In this work, to generate a Asplatin-like prodrug, BAplatin (Figure 1), we designed a BA ligated Pt(IV) complex. We hypothesized and found that the generated Pt(II) drug was able to kill cancer cells and induce apoptosis more effectively because the BA ligand could regulate the cellular response to the Pt(IV) drug. Moreover, the cellular uptake pathways of both BA and platinum was changed by the ligation of BA to cisplatin. Therefore, BAplatin is an effective and potential therapeutic agent.


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P-glycoproteins (P-gp) modulator siRNA could reverse the multidrug resistance (MDR) effectively. However, free siRNAs would be broken up by enzymatic degradation under physiological conditions. It means the siRNA delivery system should form small nanoparticles, prevent siRNA from being degraded, cross the cell membrane to the target site effectively and release siRNA. For this purpose, a CendR peptide, Cys-Arg-Gly-Asp-Lys (CRGDK), owing to specific binding of the CendR motif of CRGDK to the neuropilin-1 receptors (Nrp-1) which are overexpressed by tumor vessels and a variety of malignant tumors like breast tumors 11-13. So, the CRGDK can act as targeting moiety to selectively deliver BAplatin to Nrp-1 receptoroverexpressing cancer cells. CendR peptide also possess amphipathy, because the cell membrane is amphiphilic. On the one hand, the hydrophilic side interacts with the hydrophilic heads of the lipid bilayer and siRNA through electrostatic interaction. On the other hand, the hydrophobic side and hydrophobic anticancer drugs BAplatin is the hydrophobic core of the drug loading system. That can trigger the endocytosis pathways or assisting the direct translocation of peptide-cargo to the cytosol. Anticancer drugs BAplatin and the siRNA which modulates the P-glycoproteins (Pgp) are co-delivered by these smart nanocarriers to reverse the multidrug resistance (MDR). That was systematically demonstrated both in vitro and in vivo. First, the CRGDK peptide, as a carrier for delivering siRNA and BAplatin, possesses its cancer-specific ability. BAplatin is reduced by intracellular reducing molecules glutathione (high level in cancer cells) to generate Pt(II) drugs and kill the cancer cells.

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Second, CRGDK, It is not only conducive to the uptake of

intracellular nano-transport system through the cell membrane and the escape of endosome through the "proton sponge" effect. but can also targeted active siRNA delivery through cancer cell specific vectors.19 Third, the siRNA encapsulated within the CRGDK peptide can avoid the enzymatic degradation before entering the cell.20-22 Last but not least, peptide nano-carriers have


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the advantages of both traditional organic and inorganic nano-systems, which make the nanosystems have better biocompatibility. As depicted in Figure 1a, BAplatin was prepared in dimethylsulfoxide (DMSO). The mixture was stirred at room temperature. 24 hours later, the solution was lyophilized. And use acetone and diethylether to wash the product, then dried in a vacuum. Yield: 68%. Anal. calcd.(%) for C30H54N2O4Cl2Pt: C, 46.63; H, 7.04; N, 3.62. Found (%): C, 46.24; H, 7.74; N, 3.41. The product was characterized by 1H-NMR and ESI-MS (Fig. S1).1HNMR (400 MHz, DMSO): δ=3.84 (s, 2H); 3.71 (s, 3H), 3.53 (s, 1H), 2.65 (s, 3H). ESI-MS: m/z = 772.91 (calcd 772.74). To further enhance anticancer efficacy of BAplatin in vitro and in vivo, we made the encapsulation of BAplatin prodrug and P-glycoproteins (P-gp) modulator siRNA with tumor targeted peptide CRGDK to form the nanoparticle (siRNA/BAplatin@CRGDK) Figure 1b, Transmission electron microscopy (TEM) image manifests that siRNA/BAplatin@CRGDK have rather uniform diameters of ~ 50 nm

with

good

dispersion

(Figure

1d).

The

average

hydrodynamic

diameter

of

siRNA/BAplatin@CRGDK in solution, which is obtained by dynamic light scattering (DLS) analysis, is 97.4 ± 17.6 nm (Figure 1e). The nanoparticles siRNA/BAplatin@CRGDK are accumulated and endocytosed into the drug-resistant cancer cells due to the enhanced permeability and retention effect and further. Due to the proton sponge effect, they can escape from endo/ lysosomes and then promote the release of the transporter siRNA through the high redox potential within the cell. Therefore, the expression of efflux transporter p-gp was down-regulated, which resulted in the increase of intracellular BAplatin concentration. And then BAplatin can be rapidly reduced to platinum (II) by glutathione (Figure 1c), our previous study disclosed that the Pt(IV) prodrugs were pharmacologically inactive and must undergo reductive elimination by endogenous


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reductants to liberate active anticancer agent Pt(II) drugs to induce consequent apoptosis of cancer cells. 23 RNA interference (RNAi), for regulating MDR-related genes, is becoming one of the most effective means.24,25 It is the over-expression of P-gp that lead to the resistance of chemotherapeutic agents (MDR). SiRNA by CRGDK could efficiently inhibit the expression of P-gp and thus reverse the MDR of drug-resistant cancer cells. Figure S2a shows the RNA condensation capability for CRGDK with different mass ratios (MRs). At MR of 5, the band of siRNA disappears. It indicated that the high RNA-loading amount of CRGDK compared with those reported in the literature.26,27 We further verify that the siRNA could be protected by the structure of CRGDK from enzyme degradation (Figure S2b). The results showed that siRNA was difficult to be released from CRGDK (lane 2), while siRNA could occur under the action of negatively charged heparin (lane 3). And the bands in siRNA@CRGDK nanoparticles are stable and bright (lanes 4), indicating that siRNA can be protect by CRGDK from enzymatic degradation efficiently. Lanes 5 indicate that in the presence of RNase A, siRNA without nanoparticle’s protection cannot be detected on the gel. More importantly, CRGDK can efficiently release siRNA in the reductive microenvironment (Figure S2c). Obviously, siRNA was closely bound to CRGDK, and the bands of siRNA@CRGDK could not be detected. However, when 10mM GSH was added, bright bands could be clearly observed on the gel, indicating that the release rate of siRNA on the nanocarrier was very rapid. We first investigated the ability of siRNA/BAplatin@CRGDK NPs to generate BAplatin upon reduction. Our previous study disclosed that the PtIV-PW11 anions were pharmacologically inactive and endogenous reductants must undergo reductive elimination to release active anticancer agents, platinum (II)-substituted Keggin-type PtII-PW11 POM.11 20 μM siRNA/BAplatin@CRGDK NPs


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were incubated with 10 mM GSH for different time. And then the mixture was collected by freeze drying

and

analyzed

by

XPS.

After

24

h

incubation,

all

platinum

(IV)

in

siRNA/BAplatin@CRGDK NPs was reduced to platinum (II) (Figure S3), indicating that siRNA/BAplatin@CRGDK NPs could be rapidly reduced to platinum (II) by GSH. We further studied the drug release profiles of siRNA/BAplatin@CRGDK NPs in vitro by measuring its UV– vis absorption spectra. (Figure S4). after 16 h, 71.0 ± 4.1% of the encapsulated platinum (IV) is released at pH 7.0. In other words, the siRNA/BAplatin@CRGDK NPs exhibit a continuous release of platinum (IV) in 16 h and the release is almost at its maximum. The long release time helps the siRNA/BAplatin@CRGDK NPs circulate through the body. CRGDK peptide has the capability of binding to the transmembrane glycoprotein Nrp-1, thereby enabling the NPs to penetrate tumor cells and tissues. We further investigated the tumor cell internalization of siRNA/BAplatin@CRGDK NPs mediated CRGDK peptide. Fluorescein isothiocyanate (FITC) labeled siRNA/BAplatin@CRGDK NPs were incubated with two human breast tumor cell lines (MDA-MB-231 and MCF-7). After different incubation periods, the cells were washed and visualized using fluorescence confocal microscopy. As shown in Figure 2b, green fluorescence of FITC labeled siRNA/BAplatin@CRGDK NPs are rapidly internalized by MDA-MB-231 and MCF-7. Moreover, as expected, the siRNA/BAplatin@CRGDK NPs bind most abundantly to the MDA-MB-231 cells that possess the higher Nrp-1 expression level, whereas they bind least to the MCF-7 cells with lower Nrp-1 expression level (Figure 2a). To quantify the cellular uptake efficiency of siRNA/BAplatin@CRGDK NPs by MDA-MB-231 and MCF-7, the cells that had been incubated with the FITC labeled siRNA/BAplatin@CRGDK NPs were washed three times with PBS buffer solution to remove unbound NPs and then trypsinized for flow cytometric analysis. Flow cytometric analysis also reveals that higher uptake level of


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siRNA/BAplatin@CRGDK NPs is found in MDA-MB-231 cells with respect to MCF-7 cells (Figure

S5).

All

the

above

experimental

results

demonstrate

the

specificity

of

siRNA/BAplatin@CRGDK NPs towards Nrp-1 expressing MDA-MB-231 cells. CRGDK and siRNA@CRGDK possess high biosecurity (Figure S7). Then, the inductively coupled plasma mass spectrometry (ICP-MS) was used to detect the Pt concentration in cancer cells with free cisplatin, BAplatin@CRGDK, and siRNA/BAplatin@CRGDK were analyzed (Figure S6). The results show that Pt concentration from BAplatin@CRGDK nanocarriers is much higher than free cisplatin. Importantly, the Pt concentration can be further enhanced when siRNA is introduced. Under the joint mediation of CRGDK and siRNA (Figure S6), the cellular Pt uptake can be greatly increased, so that more cancer cells are killed (Figure S7). The assays of DNA damage and fragmentation were further to verify that the siRNA and prodrug BAplatin can efficiently improve the Pt concentration in MDA-MB-231/DDP cancer cells, which can be further verified by (Figure 2c). Antitumor effect was evaluated in mice bearing MDA-MB-231/DDP tumor xenograft to reveal the synergistic effect of siRNA and prodrug BAplatin. In Figure 3-a, the tumor-growth siRNA@CRGDK group is similar to the PBS group. The tumor volume of free cisplatin-treated group is 30.89% of the PBS group. Comparatively, The tumor growth inhibition of BAplatin@CRGDK group was further enhanced, the tumor volume was reduced by 74.59%. In the case of siRNA/BAplatin@CRGDK group, The antitumor activity was the highest. The tumor volume is only 1.73% of the PBS group, and is 39.37-, 17.87- and 14.70-folds lower than those of siRNA@CRGDK, free cisplatin, and BAplatin@CRGDK groups, respectively. Hematoxylin and eosin (H&E) staining analysis of tumor specimens collected on day 9, one can see that the MDAMB-231/DDP tumor cells after treated with BAplatin@CRGDK and siRNA/BAplatin@CRGDK


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NPs have mild necrosis, cytoplastic swelling, inflammatory and infiltrating. Notably, in contrast to cisplatin, which has considerable side effect, siRNA/BAplatin@CRGDK NPs treatment is not associated with appreciable toxicity, as measured by body weight change (Figure 3). The Jin's formula (q=EA+B/(EA+EB-EA*EB)) was used to analyses the effect of drug combination. E is the Tumor inhibitory rate. A and B are the different drugs. After the calculation, the q was calculated as 1.33 (>1.15). It means that the siRNA/BAplatin@CRGDK NPs has the synergistic effect of chemotherapy

and

gene

therapy.

These

in

vivo

models

highlight

that

the

siRNA/BAplatin@CRGDK NPs are a potential efficient anticancer drug. A detailed necropsy investigation was carried out to assess in vivo biodistribution of siRNA/BAplatin@CRGDK NPs. As seen from Figure 4a, cisplatin treatment results in significantly swell and granular degeneration in the kidney, as well as vacuoles degeneration in cytoplasm in the liver. On the contrary, in siRNA/BAplatin@CRGDK NPs-administered group, no necrosis is observed in any histological specimens except for tumors. These results indicate that the siRNA/BAplatin@CRGDK NPs has lower toxicity. Figure 4b outlines the comparative tissue distributions of siRNA/BAplatin@CRGDK NPs. After a intravenous dose of 2 mg/kg siRNA/BAplatin@CRGDK NPs, the tumors exhibit highest Pt concentration. Notably, in siRNA/BAplatin@CRGDK NPs group, the Pt level in the MDA-MB-231/DDP tumors is 2.4-fold of that in the cisplatin group, and 1.5-fold of that in the BAplatin@CRGDK group. The tumor targeting efficiency can reach up to approximately 50 %. The high therapeutic efficiency is due to the protection of CRGDK and stimuli-responsive with GSH so that the siRNA was delivered to the action site and released from the nanocarrier, then BAplatin can be reduced to cisplatin and BA, to kill the tumor cells after expression inhibition of MDR-related P-gp. Kidney is the main metabolic organ of cisplatin toxicity. Compared with cisplatin, the Pt level of


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siRNA/BAplatin@CRGDK NPs was lower. The lower Pt level in the kidney indicates that our system is less toxic to cisplatin (Figure 4b). In summary, this work first evaluated the tumor targeting peptide CRGDK as siRNA and Pt prodrug co-delivery carrier systematically. Further-more, this novel, efficient, and versatile carrier CRGDK peptide, could transport P-gp modulator siRNA and anticancer drugs Pt prodrug efficiently to reverse P-gp mediated MDR of cancer cells so that a better therapeutic effect can be achieved. In addition,, this carrier has the ability to protect siRNA from biodegradation, the function of RNAi to overexpress p-gp, and the anticancer effect of Pt precursor drugs, providing a new way for nanomedicine to treat cancer and reverse MDR.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website at DOI: Materials and characterizations; Experiments of the oxoplatin, BAplatin; Preparation of BAplatin@CRGDK NPs, siRNA/BAplatin@CRGDK NPs; Western blot analysis of Nrp-1 expression; Cell culture; Cellular uptake of siRNA/BAplatin@CRGDK NPs by MDA-MB-231 and MCF-7; MTT assay; Comet assay; Gel retardation assay; RNase a protection assay; Redoxresponsive BAplatin release; In vivo anticancer efficacy evaluation; Histopathological analysis; Biodistribution study. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. *E-mail: [email protected].


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Author Contributions ‡

Zeyu Li and Yaxuan Bai contribute equally to this article

Notes The authors declare no competing financial interest ACKNOWLEDGMENTS This study is supported by the Fundamental Research Funds for the Central Universities of China (2572018BC11). REFERENCES [1] Abu-Surrah, A. S.; Kettunen, M. Platinum Group Antitumor Chemistry: Design and development of New Anticancer Drugs Complementary to Cisplatin. Curr. Med. Chem. 2006, 13, 1337-1357. [2] Kartalou, M.; Essigmann, J. M. Recognition of Cisplatin Adducts by Cellular Proteins. Mutat. Res. 2001, 478(1-2):1-21. [3] Hall, M. D.; Hambley, T. W. Platinum(IV) Antitumour Compounds: Their Bioinorganic Chemistry. Coord. Chem. Rev. 2002, 232, 49-67. [4] Ling, X.; Chen, X.; Riddell, I. A.; Tao, W.; Wang, J. Q.; Hollett, G.; Lippard, S. J.; Farokhzad, O. C.; Shi, J. J.; Wu, J. Glutathione-Scavenging Poly(disulfide amide) Nanoparticles for the Effective Delivery of Pt(IV) Prodrugs and Reversal of Cisplatin Resistance. Nano Lett. 2018, 18, 4618-4625; [5] Cong, Y. W.; Xiao, H. H.; Xiong, H. J.; Wang, Z. G.; Ding, J. X.; Li, C.; Chen, X. S.; Liang, X. J.; Zhou, D. F.; Huang, Y. B. Dual Drug Backboned Shattering Polymeric Theranostic


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Figure 1. Design and characterization of siRNA/BAplatin@CRGDK NPs. (a) Schematic illustration of preparation of BAplatin. (b) Schematic illustration of preparation of siRNA/BAplatin@CRGDK NPs. (c) Its effects on suppressing P-gp expression for the treatment of MDR. (d) TEM images of siRNA/BAplatin@CRGDK NPs. (e) DLS measurement of siRNA/BAplatin@CRGDK NPs in water solution.


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Figure 2. Cellular uptake of siRNA/BAplatin@CRGDK NPs by tumor cells. (a) Nrp-1 expression in MDA-MB-231 and MCF-7 cell lines. (b) Confocal images of MDA-MB-231 and MCF-7 cell lines treated with siRNA/BAplatin@CRGDK NPs at 20 µM for 3 h. (c) Comet assay analysis of DNA fragmentation of MDA-MB-231 and MCF-7 cells induced by siRNA/BAplatin@CRGDK NPs at concentrations of 20 μM, incubation time: 3 h. The figures represent a typical experiment of 3 independent repetitions.

Figure 3. Evaluation of in vivo anticancer efficacy of siRNA/BAplatin@CRGDK NPs. Mice with the right flank tumors derived from MDA-MB-231/DDP cells were intravenously administrated with siRNA/BAplatin@CRGDK NPs in PBS (cisplatin: 2 mg/kg), cisplatin in PBS (cisplatin: 2 mg/kg), BAplatin@CRGDK NPs in PBS (cisplatin: 2 mg/kg), siRNA@CRGDK or PBS (control) on the first day. (a, b) Tumor volume and body weight change with injection days. Data are presented as the mean standard deviation (n = 5). (c) H&E stained sections of tumor resected from mice sacrificed on day 9 after treatment.


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Figure 4. In vivo biodistribution and excretion of siRNA/BAplatin@CRGDK NPs. (a) H&E staining of liver and kidney slices from mice on day 9 after different treatment. (b) In vivo biodistribution profile of platinum in the liver, kidney and tumors. The mice were treated with either siRNA/BAplatin@CRGDK NPs (2 mg/kg), BAplatin@CRGDK NPs (2 mg/kg) or cisplatin (2 mg/kg). Error bars represent standard deviation (n = 5).


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Tumor-Targeting Peptide for Redox-Responsive Pt Prodrug and Gene

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