Subscriber access provided by SUNY DOWNSTATE
Article
Nanoscale Coordination Polymers Co-deliver Chemotherapeutics and siRNAs to Eradicate Tumors of Cisplatin-Resistant Ovarian Cancer Chunbai He, Christopher Poon, Christina Chan, S. Diane Yamada, and Wenbin Lin J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.6b02486 • Publication Date (Web): 18 Apr 2016 Downloaded from http://pubs.acs.org on April 18, 2016
Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.
Journal of the American Chemical Society is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
Page 1 of 11
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Journal of the American Chemical Society
Nanoscale Coordination Polymers Co-deliver Chemotherapeutics and siRNAs to Eradicate Tumors of Cisplatin-Resistant Ovarian Cancer Chunbai He1, Christopher Poon1, Christina Chan1, S. Diane Yamada2, and Wenbin Lin1,* 1
Department of Chemistry, The University of Chicago, Chicago, IL 60637, USA
2
Department of Obstetrics and Gynecology, Section of Gynecologic Oncology, The University of Chicago, Chicago, IL 60637, USA. KEYWORDS: Nanoscale coordination polymers, chemotherapeutics, siRNA, tumor eradication, cisplatin-resistant ovarian cancer
ABSTRACT: Drug resistance impedes the successful treatment of many types of cancers, especially ovarian cancer (OCa). To counter this problem, we developed novel long-circulating, self-assembled core-shell nanoscale coordination polymer (NCP) nanoparticles that efficiently deliver multiple therapeutics with different mechanisms of action to enhance synergistic therapeutic effects. These NCP particles contain high payloads of chemotherapeutics cisplatin or cisplatin plus gemcitabine in the core and pooled siRNAs that target multidrug resistant (MDR) genes in the shell. The NCP particles possess efficient endosomal escape via a novel carbon dioxide release mechanism without compromising the neutral surface charge required for long blood circulation and effectively downregulate MDR gene expression in vivo to enhance chemotherapeutic efficacy by several orders of magnitude. Even at low doses, intraperitoneal injections of nanoparticles led to effective and long-lasting tumor regression/eradication in subcutaneous and intraperitoneal xenograft mouse models of cisplatin-resistant OCa. By silencing MDR genes in tumors, selfassembled core-shell nanoparticles promise a more effective chemotherapeutic treatment for many challenging cancers.
Introduction Ovarian cancer (OCa) is the fifth leading cause of death by cancer for women in the United States, accounting for 5% of cancer mortalities in this population. 1 Most patients present with advanced disease and are treated with upfront surgery followed by platinum/taxane-based chemotherapy, but the majority have tumors that either do not respond to chemotherapy or will eventually recur with multidrug resistant (MDR) OCa. 2-10 Intrinsically resistant and recurring ovarian cancers are terminal diseases that cannot be cured with existing therapeutics. 3,6 Thus, there is clearly an urgent need to develop novel therapeutic strategies to overcome drug resistance in OCa. Tumors may possess intrinsic or acquired drug resistant mechanisms that differ from patient to patient. 2-5,11-13 Moreover, cancer cells within one tumor can exhibit significant genetic heterogeneity with respect to the signaling pathways that promote drug resistance. 14,15 Small interfering RNA (siRNA) has the ability to disrupt cellular MDR pathways by silencing the expression of relevant genes. 16-25
Simultaneously delivering pooled siRNAs targeting multiple distinct molecular signaling pathways would provide an effective approach to overcoming drug resistance in OCa. 22,23,26 We hypothesized that co-administration of chemotherapeutics and siRNAs targeting MDR genes can increase the efficacy of existing OCa treatments. Because of the instability of siRNAs in systemic circulation and their inability to cross cell membranes, siRNAs must be either synthetically modified or delivered with effective vehicles to elicit gene silencing in vivo. Although nanoparticulate delivery systems have been shown to improve anticancer efficacy of chemotherapeutics, 27-37 efficient delivery of siRNAs to cancer cells in vivo has remained an unsolved problem due to the contradictory surface charge requirements for efficient endosomal escape (cationic) and long blood circulation (neutral). Although cationic lipids have been combined with nanoscale coordination polymers for the codelivery of cisplatin and pooled siRNAs to regress tumor growth in a cisplatin-resistant OCa mouse model via intratumoral injection, 38 such delivery systems exhibited poor
ACS Paragon Plus Environment
Journal of the American Chemical Society
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
pharmacokinetics and biodistribution upon systemic administration and are unsuitable for clinical translation. We herein report a robust self-assembled, core-shell nanoscale coordination polymer (NCP)-based nanomedicine platform for the co-delivery of chemotherapeutic agent(s) and siRNAs targeting MDR genes for the effective treatment of resistant OCa. The novel nanomedicine, NCP-1/siRNAs, carries cisplatin in the core and siRNAs in the lipid layer with built-in mechanisms for triggered release and endosomal escape and shows prolonged blood circulation, improved tumor uptake, and enhanced anticancer efficacy in subcutaneous and intraperitoneal xenograft mouse models of resistant OCa. By modifying the nanoparticle core to include gemcitabine, the resulting NCP-2/siRNAs particles retained all the above merits while simultaneously potentiating anticancer efficacy. Given the dramatically improved therapeutic window of NCP-1/siRNAs and NCP-2/siRNAs, we believe that these particles hold great promise for clinical translation for the treatment of resistant OCa. Results Synthesis and characterization of NCP-1/siRNAs 1,2-dioleoyl-sn-glycero-3-phosphate (DOPA)-capped NCP-1 particles containing a cisplatin prodrug, cis,cis,trans[Pt(NH3)2Cl2(OCONHP(O)(OH)2)2] (PtBp), were synthesized according to our previous report. 39 We developed a strategy to incorporate siRNA in the shell while being shielded by the PEG layer to prevent nuclease degradation in physiological environments. N-succinimidyl-3-(2-pyridyldithio)propionyl1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPESPDP) was synthesized from succinimidyl 3-(2pyridyldithio)propionate and 1,2-distearoyl-sn-glycero-3phosphoethanolamine, and further conjugated with thiol siRNA (IDT, USA) to afford the DSPE-siRNA conjugate. The disulfide linkage is placed on the 5’ end of sense strand of siRNA duplexes in order to avoid potential inhibition on the antisense strand. DOPA-NCP-1 particles were then coated with cholesterol, DOPC, DSPE-siRNA conjugates, and 20 mol% DSPE-PEG2k to afford a core-shell nanostructure with a solid core carrying chemotherapeutics and a lipid layer containing siRNAs (Figure 1A). Transmission electron microscopy (TEM) images indicated that NCP-1/siRNAs form spherical particles (Figure 1B). The Z-average size, PDI, and zeta potential of NCP-1/siRNAs were 105.3±6.2 nm, 0.112±0.004, and -4.8±1.3 mV, respectively, by DLS measurement. The cisplatin loading was determined to be 25 wt.% by inductively coupled plasma-mass spectrometry (ICPMS) and the siRNA loading was 6 wt.% by Quant-iT RiboGreen RNA kit. The siRNA release of NCP-1/siRNAs was evaluated in PBS supplemented with 4.5 µM glutathione (GSH, extracellular environment) or 10 mM GSH (intracellular environment). siRNA release was slow in PBS without GSH, but significantly enhanced in PBS containing 10 mM GSH (Figure 1C). Inside cells, the disulfide bond of DSPE-siRNA was rapidly cleaved by a reducing agent, which led to enhanced siRNA release. We evaluated the capability of NCP-1/siRNAs to protect siRNAs from nuclease degradation by incubating NCP-1/siRNAs with serum and observing siRNA integrity by gel electrophoresis analysis over time. As depicted in Figure 1D, the intensity of free siRNA bands decreased rapidly with time; ~46% of the siRNA maintained its integrity in the DSPE-siRNA conjugate after 24h, only one
Page 2 of 11
half of the ~91.9% of siRNA intact after loading onto NCP1/siRNAs (Figure S2), suggesting that NCP-1/siRNAs effectively protected siRNA from degradation by nuclease in the serum.
Figure 1. Preparation and characterization of NCP1/siRNAs. (A) Schematic representation of NCP-1/siRNAs carrying cisplatin in the solid core and siRNAs in the lipid shell. (B) TEM image of NCP-1/siRNAs showing the spherical and mono-dispersed nanostructure. Bar = 100 nm. (C) siRNA release from NCP-1/siRNAs in the presence or absence of reducing agents. (D) Improved siRNA stability in serum of NCP-1/siRNAs compared to free siRNA and DSPEsiRNA conjugate as evaluated by electrophoresis (2% agarose gel). “M” stands for untreated siRNA marker. Efficient endosomal escape NCP-1/siRNAs have a novel built-in endosomal escape mechanism that is not reliant on cationic excipients. Two carbon dioxide molecules are generated as byproducts of the intracellular release of cisplatin (Figure 2A), which we hypothesize can change the osmotic pressure to disrupt the endosomal membrane, facilitating the escape of siRNAs from endosomal entrapment and triggering the formation of RNAinduced silencing complexes (RISCs) in the cytoplasm to mediate gene silencing (Figure 2B). The efficient generation of CO2 was first confirmed in a solution, where we observed efficient CO2 generation from PtBp in reducing environments. Vigorous gas bubbling was observed when 5 mM cysteine (Cys) was added to a solution of PtBp in PBS, but no gas generation was noted before the addition of Cys. The identity of the gas was confirmed to be CO2 by gas chromatography, which displayed a peak with a retention time identical to that of authentic CO2 (Figure S3). We used confocal laser scanning microscopy (CLSM) to evaluate endosomal escape efficiency of Alexa Fluor 647 labeled NCP-1/siRNAs (NCP-1/Alexa-siRNAs) in A2780/CDDP cells. Human OCa cells A2780/CDDP were
ACS Paragon Plus Environment
incubated with NCP-1/Alexa-siRNAs for different time periods, fixed, stained with Lysotracker Green and DAPI, and observed by CLSM. The co-localization of green fluorescence (Lysotracker Green-stained endosome) and red fluorescence (Alexa Fluor 647-labeled siRNA) was calculated by Image J. NCP-1/Alexa-siRNAs was rapidly internalized by cells, evidenced by the co-localization of siRNA and endosome/lysosome. A linear decrease in co-localization occurred over 30 min, indicating that siRNAs can successfully escape endosomal entrapment and form RISCs in the cytoplasm (Figure 2C-2D).
groups, suggesting that pooled siRNAs may work synergistically to silence multiple drug-resistant genes. At a total siRNA dose of 6 nM, NCP-1/siRNAs efficiently downregulated both survivin and Bcl-2 gene expression in SKOV-3 and A2780/CDDP cells by >70%, as determined by Realtime-PCR and enzyme-linked immunosorbent assays (ELISA). For SKOV-3 cells, NCP-1/siRNAs outperformed the industry standard Lipofectamine RNAiMAX at 3 nM in gene silencing (33.9±2.88% vs. 52.8±3.6%). ELISA quantification indicated that NCP-1/sisurvivin silenced the gene (~75% silencing effect) in SKOV-3 cells for up to 4 days (Figure S4). mRNA expression %
A 120
control NCP-1
B
1.5 1.2
NCP-1/siRNAs
90 60 30
Bcl-2
120
Figure 2. Novel endosomal escape mechanism. (A) Scheme showing the CO2 generation of PtBp in the reducing environment. (B) Schematic showing the endosomal escape of NCP-1/siRNAs. (C) Time-dependent endosomal escape of NCP-1/siRNAs (Alexa Fluor647-labeled, red fluorescence) in SKOV-3 cells. Endosomes and nuclei were stained with Lysotracker Green (green fluorescence) and DAPI (blue fluorescence), respectively. Bar = 20 µm. (D) Percent colocalization of siRNA and endosome quantified by Image J based on (C). In vitro transfection efficiency and cytotoxicity NCP-1/siRNAs successfully overcame several key barriers to gene transfection, including siRNA encapsulation, protection, release, and endosomal escape. As a result, NCP1/siRNAs evoked potent gene silencing, significantly reducing mRNA expression (Figure 3A&3C) and protein production (Figure 3B&3D) in two cisplatin-resistant ovarian cancer cell lines: SKOV-3 and A2780/CDDP. We compared the gene silencing efficiency of NCP-1/siRNAs containing two siRNAs to those containing only an individual siRNA to determine if there was synergy in the siRNA downregulation of relevant proteins responsible for drug resistance. Pooled siRNAs in NCP-1/siRNAs contain the same total siRNA dose as that of NCP-1/individual siRNA, with each individual siRNA contributing equally. Unappreciable differences were noticed for the specific gene silencing efficiency between treatment
control NCP-1
0.6
0.0
survivin
survivin
D NCP-1/siRNAs
90 60
Bcl-2 control NCP-1 Zncontrol/siRNAs NCP-1/siRNAs NCP-1/Bcl-2 siRNA NCP-1/survivin siRNA
4 3 2 1
30 0
0.9
Protein (ng/mL)
C
control NCP-1 Zncontrol/siRNAs NCP-1/siRNAs NCP-1/Bcl-2 siRNA NCP-1/survivin siRNA
0.3
0
mRNA expression %
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Journal of the American Chemical Society
Protein (ng/mL)
Page 3 of 11
Bcl-2
survivin
0
survivin
Bcl-2
Figure 3. Efficient in vitro gene silencing. mRNA expression and protein production of Bcl-2 and survivin in cisplatinresistant OCa cells (A&B for SKOV-3 and C&D for A2780/CDDP) transfected with NCP-1/siRNAs at an siRNA concentration of 6 nM (n=3). Efficient gene silencing dramatically enhanced the efficacy of NCP-1/siRNAs compared to free cisplatin with free siRNAs, NCP-1, and Zn Control/siRNAs in the two cisplatin-resistant ovarian cancer cell lines SKOV-3 and A2780/CDDP. By co-delivering cisplatin and two siRNAs, NCP-1/siRNAs resensitized the cells to platinum drug treatment, drastically decreasing the dose of a drug required for 50% inhibition (IC50) of cisplatin relative to that of free cisplatin with free siRNAs or NCP-1. As shown in Table 1 and Figure S5-S7, the cisplatin IC50 of NCP-1/siRNAs decreased by two orders of magnitude, specifically a 368- and 113-fold decrease compared to free cisplatin with siRNAs in SKOV-3 and A2780/CDDP cells, respectively. In comparison, NCP-1/individual siRNA was still more potent than NCP-1 but less powerful than NCP-1/pooled siRNAs, indicating that simultaneously silencing two MDR genes was more effective than targeting a single gene in overcoming drug resistance. NCP-1 alone did not significantly differ from free cisplatin with siRNAs in cytotoxicity. In contrast, in the cisplatinsensitive ovarian cancer cell line A2780, we observed similar cytotoxicity in all five treatment groups. This result further confirmed our hypothesis: the efficient knockdown of MDR genes by NCP-1/siRNAs is vital to enhancing chemotherapeutic cytotoxicity in drug-resistant cells. Table 1 Cisplatin IC50 values of free cisplatin+siRNAs, NCP1, and NCP-1/siRNAs in SKOV-3, A2780, and A2780/CDDP cells after 72 h incubation.
ACS Paragon Plus Environment
SKOV-3 (µM)
A2780 (µM)
A2780/CDDP (µM)
Journal of the American Chemical Society 2.25±0.15
15.79±2.99
2.10±0.09 2.02±0.15
12.72±0.68 0.30±0.20
NCP-1/sisurvivin NCP-1/siRNAs
3.62±0.15 0.14±0.06
2.11±0.17 1.89±0.02
0.52±0.09 0.14±0.09
A 40
B
5 min 1h 3h 8h 24 h 48 h
30 20
60 siRNA (ID%)
51.63±10.76 45.77±12.42 1.17±0.18
Pt (ID%/g)
Free cis+siRNAs NCP-1 NCP-1/siBcl-2
10 0
In vivo pharmacokinetic and biodistribution The pharmacokinetics and biodistribution of NCP1/siRNAs was carried out on CT26 tumor-bearing Balb/c mice by intravenous or intraperitoneal injection (Figure 4). The platinum distribution was quantified by ICP-MS and the serum siRNA amount was quantified by fluorimetry (excitation: 650 nm; emission: 670 nm). By i.v. injection, both the cisplatin and siRNA concentrations in blood were best fit by a onecompartment model with nonlinear elimination, with blood circulation half-lives of 10.2±0.6 and 10.7±1.2 h, respectively. The ratio of cisplatin to siRNA remained consistent in the blood for up to 48 h post injection, suggesting that NCP1/siRNAs remained structurally intact during both abdominal absorption and systemic circulation. In addition to the prolonged blood circulation time, tissue distribution profiles of NCP-1/siRNAs showed a resistance to uptake by the mononuclear phagocyte system (MPS), as evidenced by the low % ID/g (percent injected dose/gram tissue) in the liver (
