Facile Hydrophobization of siRNA with Anticancer Drug for Non

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Facile Hydrophobization of siRNA with Anticancer Drug for Non-Cationic Nanocarrier-Mediated Systemic Delivery Congfei Xu, Dongdong Li, Zhiting Cao, Menghua Xiong, Xianzhu Yang, and Jun Wang Nano Lett., Just Accepted Manuscript • DOI: 10.1021/acs.nanolett.9b00657 • Publication Date (Web): 07 Mar 2019 Downloaded from http://pubs.acs.org on March 7, 2019

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Facile Hydrophobization of siRNA with Anticancer Drug

for

Non-Cationic

Nanocarrier-Mediated

Systemic Delivery Congfei Xu,†,§ Dongdong Li,‡,§ Zhiting Cao, Menghua Xiong,, Xianzhu Yang*,† and Jun Wang*, †

Guangzhou First People’s Hospital, School of Medicine, South China University of

Technology, Guangzhou, Guangdong 510006, China ‡

National Engineering Research Center for Tissue Restoration and Reconstruction, South China

University of Technology, Guangzhou, Guangdong 510006, P. R. China 

Key Laboratory of Biomedical Engineering of Guangdong Province, South China University

of Technology, Guangzhou 510006, P. R. China 

Key Laboratory of Biomedical Materials and Engineering of the Ministry of Education, South

China University of Technology, Guangzhou 510006, P. R. China 

Guangzhou Regenerative Medicine and Health Guangdong Laboratory, 510005 Guangzhou,

China §

C. Xu and D. Li contributed equally to this work.

* Address correspondence to: [email protected] (X. Yang), and [email protected] (J. Wang)

ACS Paragon Plus Environment

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ABSTRACT. The inherent features of small interfering RNA (siRNA), including a relatively high molecular weight, negative charge and hydrophilic nature, lead to the widespread use of cationic polymers and lipid-based nanocarriers, which might induce potential cytotoxicity, thus limiting their clinical application. Here, we report a facile strategy for changing the inherent features of siRNA molecules by achieving hydrophobization. We found that the simple mixing of siRNA and doxorubicin hydrochloride (DOX·HCl) could form a hydrophobic complex, which was readily encapsulated into non-cationic PEG-b-PLA micelles for systemic delivery. In addition to delivering DOX·HCl, this strategy could be extended to deliver other hydrochloride forms of anticancer drugs with large hydrophobic domains. This facile strategy efficiently avoids the use of cationic nanocarriers, providing a new avenue for siRNA delivery.

KEYWORDS: Hydrophobization of siRNA, Non-Cationic Nanocarrier of siRNA, siRNA Delivery, Codelivery, Cancer Therapy


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Small interfering RNAs (siRNAs) that selectively downregulate targeted gene expression are becoming promising therapeutics for treating numerous diseases.1,2 However, the therapeutic efficacy of siRNAs is impeded by difficulties in delivery.3-5 As such, a robust delivery system plays critical roles in the clinical translation of siRNA-based therapeutics.6-9 Despite the tremendous amount of effort spent on siRNA delivery thus far, cationic polymers and lipids remain the main materials for its delivery.10,11 Nevertheless, the cationic charge of these carriers may induce potential cytotoxicity, thus limiting their clinical application.12,13 Furthermore, cationic nanocomplexes enhance the adsorption of serum proteins, resulting in rapid clearance by the reticuloendothelial system.14 Therefore, there is an urgent desire to explore new strategies to design siRNA delivery systems.15-17 Small interfering RNAs (siRNAs) that selectively downregulate targeted gene expression are becoming a promising therapeutics for treating numerous diseases.1,2 However, the therapeutic efficacy of siRNAs is impeded by difficulties in delivery.3-5 As such, a robust delivery system plays critical roles in the clinical translation of siRNA-based therapeutics.6-9 Despite the tremendous amount of effort spent on siRNA delivery so far, the cationic polymers and lipids remain the main materials for its delivery.10,11 Nevertheless, the cationic charge of these carriers may induce potential cytotoxicity, thus limiting their clinical application.12,13 Furthermore, cationic nanocomplexes enhance the adsorption of serum proteins, resulting in the rapid clearance by the reticuloendothelial system.14 Therefore, there is an urgent desire to explore new strategies to design siRNA delivery systems.15-17 Cationic materials are widely used because of the inherent features of siRNA, a relatively large, negatively charged, and hydrophilic molecule.18,19 Changing these inherent features could be a possible avenue to develop other systems for efficient siRNA delivery. For instance, the


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transformation of siRNAs from hydrophilic to hydrophobic molecules would facilitate delivery by noncationic nanocarriers, such as clinically approved poly(ethylene glycol)-block-poly(D,Llactide) (PEG-b-PLA) micelles. Although we are well aware of this possibility, changing the features of siRNA is a major challenge. Herein, we report an unexpected phenomenon in which the simple mixing of siRNA and doxorubicin hydrochloride (DOX·HCl) can form a hydrophobic complex [siRNA&DOX], achieving the hydrophobization of siRNA molecules. After hydrophobization, the obtained hydrophobic siRNA complex was readily encapsulated into PEG-b-PLA micelles, resulting in a combinational anticancer effect of RNAi therapy and chemotherapy. Apart from DOX·HCl, this strategy could be extended to other hydrochloride forms of anticancer drugs with larger hydrophobic domains (e.g., epirubicin hydrochloride (EPI·HCl), daunorubicin hydrochloride (DAU·HCl), irinotecan hydrochloride (IR·HCl), and topotecan hydrochloride (TPT·HCl)), rendering a general and robust strategy to achieve the hydrophobization of siRNA. This approach completely subverts the traditional design of siRNA nanocarriers, providing a new avenue for the systemic delivery of siRNA with non-cationic nanoparticles. To achieve the hydrophobization of siRNA, an aqueous solution of siRNA and DOX·HCl was mixed at different molar ratios for 10 min, the mixture was centrifuged, and the precipitate and supernatant were collected for efficiency analyses. According to the siRNA concentration detected in the supernatant (Figure 1A and S1), approximately 74% of the siRNA was precipitated at a DOX:siRNA-nt (siRNA nucleotide units) molar ratio of 0.5:1, and close to 100% of siRNA was precipitated at a molar ratio of 1:1 or more. Interestingly, the molar ratio of DOX and siRNA-nt units in the collected precipitate reached approximately 1.25:1 at a molar ratio of 2:1 or 3:1.


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To demonstrate the mechanism underlying the hydrophobization of siRNA with DOX·HCl, we investigated this process using isothermal titration calorimetry (ITC). The addition of DOX·HCl to siRNA resulted in two peaks in the integrated enthalpy binding profile (Figure 1B). The initial interaction showed an endothermic reaction. As reported, a purely electrostatic interaction is entropy driven and endothermic.20 Thus, at this stage, with siRNA-nt in large excess, the interaction likely represents the electrostatic interaction of siRNA and DOX·HCl. The initial phase was followed by a transition stage when the molar ratios were close to 1.0, and the second phase was exothermic. The transition from endothermic to exothermic binding was likely due to the aggregation of the formed hydrophobic complex [siRNA&DOX]. At the same time, the hydrophobic domains of DOX·HCl could also interact with the formed hydrophobic [siRNA&DOX]. Thus, the interaction of DOX·HCl and siRNA could be divided into two stages (Figure 1C). To verify that the negative charge of siRNA plays a key role during hydrophobization, the change in DOX fluorescence of the [siRNA&DOX] formed at a molar ratio of 1:1 was detected. As a control, siRNA was first bound with polyethyleneimine (Mw = 2,500, PEI2.5k) to shield the negative charge of the former and then mixed with DOX·HCl (denoted as PEI2.5k/siRNA+DOX). Compared to that of free DOX, the fluorescence of the formed [siRNA&DOX] was significantly reduced (Figure S2) because of the aggregation-induced fluorescence quenching. In contrast, the decrease in the fluorescence intensity of PEI2.5k/siRNA+DOX was negligible, which implied that shielding of the negative charge of siRNA hindered its hydrophobization with DOX·HCl. Therefore, the negative charge of siRNA plays a key role in its hydrophobization, and electrostatic interactions could be the major driving force.


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To further verify that electrostatic interactions were the major driving force during hydrophobization, we mixed DOX·HCl and Cy5-labeled siRNA (Cy5-siRNA) at a DOX:siRNAnt molar ratio of 1:1 and then added polyanionic heparin sulfate. During this process, the fluorescence of Cy5-siRNA was tracked. As shown in Figure S3, most of the Cy5 fluorescence was quenched without of addition of heparin. When heparin was added, the fluorescence recovery of Cy5-siRNA was clearly observed. Heparin has been widely used to disrupt the electrostatic interactions of siRNA and positively charged materials. Therefore, this result further confirmed that electrostatic interactions drive the hydrophobization of siRNA with DOX·HCl.

Figure 1. (A) The precipitation efficiency of siRNA at different molar ratios of DOX and siRNA-nt. (B) ITC analysis of the interaction of siRNA and DOX·HCl. Representative raw ITC data (upper) and integrated heat release (below). (C) Schematic illustration of the interaction of DOX·HCl and siRNA. (D) The precipitation efficiency of other hydrochloride forms of


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anticancer drugs after interaction with siRNA. (E) ITC analysis of the interaction of siRNA and GEM·HCl. Representative raw ITC data (upper) and integrated heat release (below). We next investigated whether the hydrophobization strategy can be extended to other hydrochloride forms of anticancer drugs. The representative anticancer drugs EPI·HCl, DAU·HCl, IR·HCl, TPT·HCl, cytarabine hydrochloride (CYT·HCl), and gemcitabine hydrochloride (GEM·HCl) were mixed with siRNA in aqueous solution at a drug:siRNA-nt molar ratio of 1:1 for 10 min. Then, the mixtures were centrifuged, and the supernatant was collected for analysis. As shown in Figure 1D, the analogs of DOX·HCl, EPI·HCl and DAU·HCl could efficiently form a hydrophobic precipitate with siRNA. In addition, most of the IR·HCl (83.4%) and TPT·HCl (79.0%) also induced the hydrophobization of siRNA but with slightly lower precipitation efficacies. In contrast, almost all siRNA was still in the supernatant after being mixed with CYT·HCl and GEM·HCl, indicating the failure of hydrophobization. To understand the reason, ITC was used to investigate the interaction of siRNA and GEM·HCl. At GEM:siRNA-nt molar ratios below 1.0, an endothermic peak that represented purely electrostatic interactions was detected (Figure 1E), similar to the peak for the mixture of siRNA and DOX·HCl. However, an exothermic peak was not observed even at molar ratios above 1.0, which means that there was no hydrophobic interaction. This phenomenon might be because GEM lacked large hydrophobic domains, indicating that the hydrophobic domain of an anticancer drug was a prerequisite for inducing the hydrophobization of siRNA. siRNA molecules that are hydrophobized with anticancer drugs might be readily encapsulated into the non-cationic nanoparticles. To demonstrate this possibility, we attempted to encapsulate hydrophobic [siRNA&DOX] obtained at a DOX:siRNA-nt molar ratio of 1:1 into PEG-b-PLA-based (PEG5k-b-PLA25k, where the subscript numbers represent the molecular


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weight of each block) nanoparticles using the nanoprecipitation method (Figure 2A). The PEGb-PLA nanoparticles efficiently loaded the hydrophobic [siRNA&DOX] with an encapsulation efficacy of 41.16±0.47%. The obtained nanoparticles, NP[siRNA&DOX], showed a spherical structure with a diameter of 53±5.7 nm (Figure 2B) and a zeta potential of approximately -13.2 mV (Figure S4). NP[siRNA&DOX] displayed excellent colloidal stability (Figure S5), and the encapsulated [DOX&siRNA] could be efficiently released (Figure S6). Furthermore, we evaluated the in vitro performance of NP[siRNA&DOX]. First, the internalization pathway of NP[siRNA&DOX] was examined. As shown in Figure S7, the internalization of NP[siRNA&DOX] was significantly inhibited by chlorpromazine and methyl-βcyclodextrin, which indicated that the internalization of NP[siRNA&DOX] depends on clathrin- and caveolae-mediated endocytosis. Then, we used the FAM-labeled siRNA to track the cellular uptake and intracellular distribution of NP[siRNA&DOX]. After incubation with MDA-MB-231 cells for 2 h, the uninternalized NP[FAM-siRNA&DOX] was removed. After further incubation for 0 h, 4 h or 8 h, the acidic organelles (including endosomes and lysosomes) and cell nuclei were counterstained, and then the distribution of FAM-siRNA and DOX within the tumor cells was detected by confocal laser scanning microscopy (CLSM). As shown in Figure 2C, slight FAMsiRNA (green) and DOX (red) fluorescence was observed at the beginning, and both fluorescence mostly overlapped with the purple fluorescence, indicating that the NP[FAMsiRNA&DOX]

were mainly trapped in acidic endosomes/lysosomes after internalization. After the

removal of uninternalized NP[FAM-siRNA&DOX] and further incubation for 4 h, the intracellular FAM-siRNA and DOX fluorescence was enhanced, and slight DOX fluorescence was observed in the cell nuclei. Considering that the FAM-siRNA and DOX fluorescence was partially quenched after the formation of hydrophobic [FAM-siRNA&DOX], the enhanced FAM-siRNA


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and DOX fluorescence should be attributed to the dissociation of FAM-siRNA and DOX within the tumor cells. When the incubation time was further extended to 8 h, most of the DOX fluorescence was localized in the cell nuclei, while FAM-siRNA fluorescence was still retained in the cytoplasm. In addition, FAM-siRNA and DOX gradually escaped from the endosomes/lysosomes with the extension of incubation times. The intracellular FAM-siRNA and DOX fluorescence was also determined by flow cytometric analysis after receiving the same treatment. As shown in Figure S8, the intracellular FAM-siRNA and DOX fluorescence significantly increased after further incubation for 4 h and 8 h, which is consistent with the CLSM analyses. Thereafter, polo-like kinase 1 (Plk1), as the oncogenic target gene,21 was selected to evaluate the silencing efficacy of the NP[siRNA&DOX]. MDA-MB-231 cells were incubated with NP[siPlk1&DOX] at different siRNA concentrations, and the expression of Plk1 at the mRNA and protein levels was analyzed. As shown in Figure 2D, the negative control siRNA (siN.C.)-loaded NP[siN.C.&DOX] did not affect Plk1 expression. In contrast, NP[siPlk1&DOX] efficiently downregulated Plk1 gene expression in a dose-dependent manner. For instance, with the NP[siPlk1&DOX] treatment, the Plk1 mRNA levels were significantly decreased to approximately 87.9%, 78.9%, 51.7%, 30.3%, and 18.1% when the siPlk1 concentration was 20 nM, 40 nM, 60 nM, 80 nM and 100 nM, respectively. Treatment with NP[siPlk1&DOX] also induced a dose-dependent gene silencing phenomenon in Plk1 protein expression (Figure 2E).


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Figure 2. (A) Schematic illustration of the encapsulation of the hydrophobic complex [siRNA&DOX] into PEG-b-PLA nanoparticles. (B) Particle size distribution of NP[siRNA&DOX].


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The inserted image represents a transmission electron micrograph of NP[siRNA&DOX]. The scale bar is 50 nm. (C) CLSM images of the intracellular distribution of NP[siRNA&DOX]. After incubation with NP[FAM-siRNA&DOX] for 2 h and removing the uninternalized nanoparticles, MDA-MB-231 cells were further incubated for 0 h, 4 h and 8 h for CLSM analyses. The scale bar is 10 μm. FAM-labeled siRNA (green) was used; acidic endosomes/lysosomes and cell nuclei were counterstained with LysoTrackerTM Deep Red (purple) and 4′,6-diamidino-2-phenylindole (DAPI; blue). (D, E) Expression of Plk1 mRNA (D) and protein (E) in MDA-MB-231 cells following incubation with NP[siPlk1&DOX] or NP[siN.C.&DOX] (n = 3). (F) Induction of apoptosis in MDA-MB-231 cells following incubation with different formulations. Downregulation of Plk1 expression induces cancer cell apoptosis.22 Thus, after treatment with the above formulations for 48 h, the MDA-MB-231 cells were stained with annexin V-FITC and propidium iodide (PI) to evaluate cell apoptosis. As shown in Figure 2F, transfection with NP[siN.C.&DOX] at siN.C. doses of 60 nM and 100 nM induced 20.4% and 32.1% cell apoptosis, respectively. In contrast, cell apoptosis was significantly elevated to 34.0% and 46.5% when the MDA-MB-231 cells were treated with NP[siPlk1&DOX] at siPlk1 doses of 60 nM and 100 nM, respectively. Encouraged by the combinational antitumor effect of NP[siPlk1&DOX] in vitro, we then performed in vivo animal experiments. Mice with MDA-MB-231 xenografts were treated with various formulations via intravenous injection. As shown in Figure 3A, treatment with free Plk1 exhibited negligible anticancer efficacy, whereas tumor growth was mildly suppressed with free DOX and NP[siN.C.&DOX], which was due to the therapeutic effect of DOX. In contrast, the NP[siPlk1&DOX] group exhibited high anticancer efficacy, which confirmed that NP[siPlk1&DOX] was capable of realizing a combination of siRNA therapy and chemotherapy and inducing a


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combinational antitumor effect. We also recorded the weights of the tumor tissues (Figure S9) and photographed the tumor mass (Figure S10), which visually confirmed that the NP[siPlk1&DOX] exhibited a high anticancer effect. These treatments did not significantly affect the body weights of the mice (Figure S11). Finally, cell apoptosis and proliferation in the tumor tissues were analyzed by immunohistochemical staining after treatment. As shown in Figure 3B, treatment with NP[siPlk1&DOX] effectively increased the number of terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL)-positive tumor cells and reduced the percentage of proliferating cell nuclear antigen (PCNA)-positive tumor cells.

Figure 3. The combinational tumor suppression effect of NP[siPlk1&DOX] in an MDA-MB-231 xenograft murine model. (A) MDA-MB-231 tumor growth curves of various groups after


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intravenous administration (n = 5). (B) mRNA and protein expression of Plk1 in the tumors analyzed at the end of the treatment. MDA-MB-231 xenograft tumor-bearing mice received one intravenous injection every two days. (C) PCNA and TUNEL analyses of tumor tissues after the indicated treatment. The scale bar is 40 µm. **p < 0.01. Subsequently, the Plk1 mRNA and protein expression levels in the tumor tissues were further analyzed. As shown in Figure 3C, the Plk1 mRNA levels showed a 63.4% reduction for the NP[siPlk1&DOX] group (p < 0.01), while the other groups did not show reductions in the Plk1 mRNA levels in the tumor after treatment. Western blot analyses also revealed that only the Plk1 protein levels of the NP[siPlk1&DOX] groups were significantly reduced. These results demonstrated that administration of NP[siPlk1&DOX] significantly reduced Plk1 expression, resulting in a combinational anticancer effect in the MDA-MB-231 xenograft murine model. In summary, we report for the first time a facile strategy to achieve the hydrophobization of siRNA molecules with the assistance of DOX·HCl. This strategy could be extended to other hydrochloride forms of anticancer drugs with large hydrophobic domains. The obtained hydrophobized siRNA and drug were readily encapsulated into noncationic mPEG-b-PLA nanoparticles, realizing simultaneous systemic delivery and combinational RNAi-chemotherapy. This siRNA hydrophobization strategy addresses the dependence of drug delivery systems on cationic nanocarriers. Considering the similarity of nucleic acid drugs, this strategy should also be applicable to protein-expressing plasmid DNA, messenger RNA, antisense oligonucleotides, microRNA, and so on, providing a simple and robust strategy for gene delivery and combination therapy.


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ASSOCIATED CONTENT Supporting Information. Experimental methods and supporting figures. This material is available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author Address correspondence to: [email protected] (X. Yang), and [email protected] (J. Wang)

Author Contributions C. Xu and D. Li contributed equally to this work. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS. This work was supported by the National Key R&D Program of China (2017YFA0205601), National Natural Science Foundation of China (51822302, 51773067), the Natural Science Foundation for Distinguished Young Scholars of Guangdong Province (2017B030306002), outstanding Scholar Program of Guangzhou Regenerative Medicine and Health Guangdong


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Facile Hydrophobization of siRNA with Anticancer Drug for Non

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