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Reversibly Stabilized Polycation Nanoparticles for Combination Treatment of Early- and LateStage Metastatic Breast Cancer Gang Chen,† Yixin Wang,† Pengkai Wu,† Yiwen Zhou,† Fei Yu,‡ Chenfei Zhu,† Zhaoting Li,† Yu Hang,‡ Kaikai Wang,† Jing Li,‡ Minjie Sun,*,† and David Oupicky*,†,‡ Downloaded via UNIV OF CAMBRIDGE on July 9, 2018 at 21:37:13 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

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State Key Laboratory of Natural Medicines, Department of Pharmaceutics, China Pharmaceutical University, Nanjing 210009, China ‡ Center for Drug Delivery and Nanomedicine, Department of Pharmaceutical Sciences, University of Nebraska Medical Center, Omaha, Nebraska 68198, United States S Supporting Information *

ABSTRACT: Metastatic breast cancer is a major cause of cancer-related female mortality worldwide. The signal transducer and activator of transcription 3 (STAT3) and the chemokine receptor CXCR4 are involved in the metastatic spread of breast cancer. The goal of this study was to develop nanomedicine treatment based on combined inhibition of STAT3 and CXCR4. We synthesized a library of CXCR4-inhibiting polymers with a combination of beneficial features that included PEGylation, fluorination, and bioreducibility to achieve systemic delivery of siRNA to silence STAT3 expression in the tumors. An in vivo structure−activity relationship study in an experimental lung metastasis model revealed superior antimetastatic activity of bioreducible fluorinated polyplexes when compared with nonreducible controls despite similar CXCR4 antagonism and the ability to inhibit in vitro cancer cell invasion. When compared with nonreducible and nonfluorinated polyplexes, improved siRNA delivery was observed with the bioreducible fluorinated polyplexes. The improvement was ascribed to a combination of enhanced physical stability, decreased serum destabilization, and improved intracellular trafficking. Pharmacokinetic analysis showed that fluorination decreased the rate of renal clearance of the polyplexes and contributed to enhanced accumulation in the tumors. Therapeutic efficacy of the polyplexes with STAT3 siRNA was assessed in early stage breast cancer and late-stage metastatic breast cancer with primary tumor resection. Strong inhibition of the primary tumor growth and pronounced antimetastatic effects were observed in both models of metastatic breast cancer. Mechanistic studies revealed multifaceted mechanism of action of the combined STAT3 and CXCR4 inhibition by the developed polyplexes relying both on local and systemic effects. KEYWORDS: siRNA delivery, fluorination, CXCR4, STAT3, metastasis, breast cancer

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Enhanced stability and improved pharmacokinetics are only the first necessary step in tumor delivery of siRNA, with additional challenges related to tissue penetration, cancer cell uptake, and endosomal escape requiring attention.8 Globally improved delivery strategies are needed to make polyplexes clinical reality.9−11 Modification of polycations with perfluorocarbons is a recent potential strategy for improving stability, membrane transport, and transfection efficacy of polyplexes. Perfluorocarbons display both hydrophobic and lipophobic properties and thus exhibit a high tendency toward stable

anoparticles (polyplexes) formed by self-assembly of polycations and nucleic acids have gained attention as delivery vectors of various anticancer therapeutic nucleic acids, including small interfering RNA (siRNA).1 Unfortunately, multiple challenges related to their dynamic nature and positive charge severely restricted their successful clinical translation.2,3 In particular, systemic administration of siRNA and other short-nucleic-acid polyplexes often suffers from destabilization and nonspecific interactions with blood components, such as electrolytes, proteins, and red blood cells, leading to rapid plasma clearance.1,4,5 siRNA polyplexes can be disassembled by heparan sulfate present in the glomerular basement membrane (GBM), which typically leads to fast renal clearance of siRNA decomplexed from the polycations.6,7 © XXXX American Chemical Society

Received: February 25, 2018 Accepted: July 3, 2018

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DOI: 10.1021/acsnano.8b01482 ACS Nano XXXX, XXX, XXX−XXX

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Figure 1. (A) Synthesis of CXCR4-inhibiting polycations and their siRNA polyplexes. (B) Proposed mechanism of action of the polyplexes with combined CXCR4 and STAT3 inhibition. (I) Increased plasma stability of the polyplexes due to stabilizing fluorous interactions. (II) Decreased susceptibility to disassembly in the kidney glomerular basement membrane. (III) Increased CXCR4-mediated tumor accumulation and CXCR4 inhibition. (IV) Enhanced cellular uptake and (V) endosomal escape induced due to fluorination. (VI) Cytoplasmic GSH-mediated degradation of the polycations to promote polyplex disassembly and release of siRNA. (VII) Target STAT3 silencing by the delivered siRNA. (VIII) Inhibition of lung metastasis.

phase separation in biological environment.12,13 In previous work, we have utilized the fluorous interactions to improve the siRNA delivery and stability of poly(amido amine) polyplexes in vivo.14−16 Breast cancer affects about 12% women but is responsible for 14% of all cancer-related deaths worldwide.17 Metastatic breast cancer (MBC) is the most deadly form of the disease that originates from the spread of cells from primary tumors to the organs and tissues in rest of the body.18 Even if metastases are not present at the initial diagnosis, 10−15% patients will develop metastasis within 3 years of the diagnosis. Despite improved breast cancer treatments, five-year survival of MBC patients remains low at only 5%.19−21 The process of metastasis is the consequence of multiple successive steps. First, cancer cells detach from the primary tumor and spread to the nearby tissue by penetrating the basement membrane. Then, the cells enter the circulatory or lymphatic system, evade immune attack, and survive as circulating tumor cells, only to extravasate at distant premetastatic niches,22 where they can proliferate and form a secondary tumor.23,24 Multiple steps in cell trafficking during the metastatic spread are orchestrated by an intricate system of chemokines and chemokine receptors.25 The metastatic pattern is not random or determined by only the features of the cancer cells but also

by the need for hospitable microenvironment in specific tissues. The chemokines expressed at the premetastatic sites provide chemoattractive signals that guide movement of cancer cellsa mechanism consistent with the seed-and-soil hypothesis of metastatic dissemination.26,27 Strong evidence supports a critical role of the chemokine receptor CXCR4 in the metastatic spread of breast cancer.28−30 For example, a clinical examination of invasive and preinvasive breast cancer samples showed that CXCR4 overexpression is associated with aggressive cancer and with negative survival prognosis.31 CXCR4 is a transmembrane G-protein coupled receptor whose activation with stromal cell-derived factor-1 (SDF-1) regulates proliferation, adhesion, and invasion of various cancer cells.32−36 The SDF-1/CXCR4 signaling also contributes to primary and metastatic tumor angiogenesis.37,38 Inhibiting CXCR4 leads to T-cell accumulation and synergistic interactions with checkpoint antagonists (α-PD-L1) to promote tumor regression.39 Current evidence strongly supports CXCR4 as one of the most promising chemokine receptors for therapeutic targeting in MBC. Signal transducer and activator of transcription 3 (STAT3) is a transcription factor,40 whose activation is involved in the accumulation and activation of immunosuppressive cells such as T-regulatory cells, T-helper-17 cells, and myeloid-derived B


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Figure 2. Polymer and polyplex characterization. (A) 1H NMR spectrum of PCA0 and PCA1 (a, aromatic protons of AMD3100; b, methylene protons of PEG). (B) siRNA condensation by the polymers. (C) Representative TEM images of PCA/siRNA polyplexes (scale bar 100 nm). (D) Hydrodynamic size and (E) ζ potential of the siRNA polyplexes.

suppressor cells.41−44 STAT3 is activated by phosphorylation and induced by multiple stimuli, including cytokines and growth factors.45 STAT3 activation results in dimerization, nuclear translocation, recognition by STAT3-specific DNA binding elements, and activation of target gene transcription including Bcl-2, MMP-9, VEGF, TNF, IFN-γ, and IL-6.46−49 Evidence shows that STAT3 functions as an oncogene and its constitutive activation in cancer cells is sufficient to induce tumor formation.50,51 Besides aberrant expression in tumor cells, STAT3 is also persistently activated in other cells, such as tumor-associated endothelial cells and a range of immune cells.52,53 STAT3 is frequently found in breast cancer patients with advanced disease, but not in normal breast epithelial cells.54 Constitutive activation of STAT3 induces cell proliferation and angiogenesis and contributes to tumor immune evasion. In the 4T1 breast tumor model, STAT3 inhibition by short hairpin RNA decreased tumor formation and occurrence of spontaneous metastases.55 Delivery of siRNA against STAT3 (siSTAT3) to 4T1 cells by a membrane-penetrating peptide inhibited 4T1 invasion and migration in vitro,56 while intratumoral administration of siSTAT3-expressing plasmid inhibited primary tumor growth and prevented formation of spontaneous lung metastases.57 Thus, STAT3 is an attractive target to inhibit primary breast tumor growth and spontaneous metastases.58 There is a vital need to develop therapies that focus on interventions aimed at preventing, delaying, and treating MBC. Despite extensive use of nanomedicine in anticancer drug delivery, the bulk of the research focuses on delivery to and treatment of primary tumors, with insufficient attention paid to the metastatic disease. In this study, we addressed the need by developing multifunctional polycationic drugs with the ability

to inhibit CXCR4 and silence STAT3 expression by delivering therapeutic siRNA in early- and late-stage models of MBC. The rationale was that combined targeting of two critical pathways involved in cancer cell metastasis and antitumor immunity will result in synergies that enhance the therapeutic outcomes in MBC (Figure 1). We synthesized a panel of polycationic CXCR4 antagonists and tested their ability to inhibit metastasis and deliver siSTAT3. We have used a combination of bioreducibility and fluorination as a way of reversibly enhancing systemic stability and transfection efficacy of the polyplexes. We investigated the mechanism of fluorination-induced enhancement of stability both in vitro and in vivo and assessed how combined fluorination and bioreducibility affects the cytotoxicity, cell internalization and intracellular trafficking, intracellular siRNA release, and gene silencing. Antitumor and antimetastatic efficacy and mechanism of action of the polyplexes were investigated in orthotopic syngeneic models of early-stage and late-stage breast cancer.

RESULTS AND DISCUSSION Two-Step Synthesis of Fluorinated and PEGylated Polycations That Inhibit CXCR4. We first synthesized a library of AMD3100-based polycations capable of inhibiting CXCR4. We synthesized both nonreducible (PHA: poly(HMBA-PEG/ABOL/AMD3100) and bioreducible copolymers (PCA: poly(CBA-PEG/ABOL/AMD3100) (Figure 1A and Figure S1). The copolymers were synthesized using a Michael-type polyaddition of a commercial CXCR4 inhibitor AMD3100 with a bis(acrylamide) (HMBA or CBA) and PEGNH2 to directly obtain PEGylated copolymers. This approach was used to avoid post-PEGylation which in our previous work C


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dynamic light scattering (DLS) (Figure 2D,E). In DLS analysis, all polyplexes had sizes ranging from 61 to 138 nm. Nonreducible PHA formed smaller polyplexes than the reducible PCA, and their size was less affected by the modifications. The relatively small size of the polyplexes is critical for effective internalization into cells.60 All the polyplexes also had a positive surface charge (ζ potential), which decreased with increasing PEG content and with decreasing w/w ratio. Fluorination had no significant effect on either the size or ζ potential. Polyplex Stability in Vitro. PEG shields the positive surface charge and increases colloidal stability of polyplexes.16 In order to determine how combining fluorination and PEGylation affect colloidal stability in simulated physiologic ionic conditions, we followed the effect of phosphate-buffered saline (PBS) on the hydrodynamic size of the polyplexes (Figure 3A and Figure S4). The results showed that polyplexes with no PEG (PHA0, PHA0F, PCA0, and PCA0F) began to aggregate immediately and the size grew to over 1 μm in 6 h. In contrast, all PEG-containing polyplexes exhibited significantly enhanced colloidal stability. However, our data showed that even though PHA1 and PCA1 polyplexes with low PEG content enhanced short-term (6 h) colloidal stability, they were not as effective as the high-PEG polyplexes in providing long-term stabilization (24 h). Interestingly, combining the favorable effects of PEG with fluorination appeared to provide additional colloidal stabilization as suggested by only 20 nm increase of size for PHA1F (vs 250 nm for PHA1) and a similar 30 nm increase for PCA1F (vs 140 nm for PCA1) in the span of 24 h. Both PHA2 and PCA2 stabilized the polyplex sizes effectively for the whole period of the experiment regardless of fluorination. The effect of supraphysiological ionic strength on the polyplex stability was evaluated by increasing NaCl concentration up to 0.25 M (Figure 3C). Similar stabilizing effect of the combined modification was observed with PEG and F-containing polyplexes showing negligible changes in mean sizes and size distributions. The fluorinated polyplexes were more stable in increasing NaCl, especially for PHA1F, PHA2F, PCA1F, and PCA2F polyplexes as indicated by negligible changes in their size. We then evaluated the stability of the polyplexes in fetal bovine serum (FBS) by assessing their integrity using fluorescence resonance energy transfer (FRET) (Figure 3B). The polyplexes were formed with a mixture of Cy3-siRNA and Cy5-siRNA as the donor/acceptor pair. We observed a significant (>50%) decrease in the FRET signal in case of PHA0 and PCA0 polyplexes with increasing FBS concentration suggesting at least partial disintegration of the polyplexes. PEG appeared to increase stability in FBS but it could not match the stabilizing effect of fluorination. Polyplexes that combined both modifications, regardless of their bioreducibility, showed the best FBS stability with 90%) based on the data in Figure 4B. First, we excluded polymer-mediated nonspecific effects by F


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Figure 5. Reduction-triggered disassembly of polyplexes. (A) Release of siRNA from polyplexes treated with GSH. (B) Effect of GSH on polyplex size by DLS and (C) particle morphology by TEM (scale bar 500 nm). (D) FRET analysis of intracellular disassembly of polyplexes prepared with a mixture of Cy3-siRNA and Cy5-siRNA. Scale bar: 20 μm.

Antimetastatic Activity of siScr Polyplexes in Experimental Lung Metastasis Model. We have previously reported that PHA and PCA function as effective CXCR4 antagonists.67 We first confirmed that neither PEG nor fluorination adversely affected the antagonism of the polymers at their anticipated dose range (Figure S10) and that both the polymers and their siScr polyplexes can inhibit CXCR4facilitated cancer cell invasion in a standard transwell assay (Figure S11).67 The strong anti-invasive effect in vitro inspired us to conduct antimetastatic SAR study of the polyplexes in an experimental lung metastatic mouse model established by tailvein injection of 4T1 cells in immunocompetent Balb/c mice.68−70 Clinically, for cancer with the risk of metastasis, administration of drugs to prevent metastasis is a potential strategy after surgery.71,72 In the present study, we used the experimental lung metastasis model to simulate the metastatic tumor cells in the circulation from the solid tumor. The mice were treated i.v. with AMD3100 and polyplexes to assess the CXCR4-related antimetastatic effect. The mice were sacrificed on day 11, and the total number of surface lung metastases was counted (Figure 8A and Figure S12). AMD3100 was used as a positive control. Despite nM activity of AMD3100 in vitro, the

drug showed only a limited antimetastatic activity in vivo, most likely due to rapid clearance after single bolus injection. Overall, we found less lung metastases in the mice treated with bioreducible PCA polyplexes than the nonreducible PHA polyplexes. The antimetastatic activity of PCA/siScr polyplexes was further improved by the presence of PEG (PCA0 vs PCA1) but fluorination appeared to have a slightly detrimental effect. Further increasing the PEG content, however, did not lead to additional improvement of the antimetastatic activity (PCA2 vs PCA1), a finding consistent with decreased in vitro CXCR4 antagonism of polymers with higher PEG. The gross observation of surface lung metastases was validated using H&E staining of the lungs (Figure 8B). A positive correlation was found between the number of metastases found in the H&E sections and the number of surface lesions. Compared to untreated animals, treatment with polyplexes not only decreased the number of lung metastases but also reduced their size. The reasons for the differences between bioreducible and nonreducible polyplexes remain unknown but we hypothesize that pharmacokinetic differences and the effect of polymer degradation and the role of small-molecule degradation products from PCA may play a role. We have G


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Figure 6. Reporter gene silencing. (A) Luc silencing efficacy of siLuc polyplexes in 4T1.Luc cells (red diamonds = Luc expression in cells treated with siScr polyplexes). (B) Effect of 30% FBS on transfection activity of the siLuc polyplexes in 4T1.Luc cells (PHA/siRNA w/w = 2, PCA/siRNA w/w = 8). *P < 0.05, **P < 0.01, ***P < 0.005, vs PBS. (C) Bioluminescence images of mice with 4T1.Luc tumors before and 2 days after i.v. injection of the polyplexes. (D) Ex vivo analysis of the Luc silencing in the isolated tumor tissue lysates. Data are shown as mean ± SD (3 mice in each group (n = 3)). **P < 0.01 vs saline.

without the pretreatment. As suggested by the result, GSH pretreatment increased the anti-invasive activity of the polyplexes providing an initial support for the hypothesis (Figure S13). We conclude that bioreducible PCA1 and PCA1F polyplexes exhibited strong CXCR4 antagonism and antiinvasive effects in vitro and the best antimetastatic activity in vivo. Polyplex in Vivo Stability and Biodistribution. Having shown strong antimetastatic activity of the bioreducible PCA1F/siScr polyplexes, we set to determine if the polyplexes can also deliver functional siRNA to primary tumors. Renal clearance due to disassembly by heparan sulfate in the kidney GBM has been recently recognized as a major reason for plasma clearance of siRNA polyplexes.4 Other significant contributors to the serum instability of siRNA polyplexes include salt and serum protein-mediated disassembly and clearance by the mononuclear phagocytic system. All these factors are further confounded by potential serum degradation of the disulfides in PCA. To predict how fluorination may affect the systemic stability of bioreducible polyplexes, we first challenged the FRET-active polyplexes with heparin (2 and 20 U/mL) and followed changes of the FRET signal for 2 h (Figure 9A). The PCA1F polyplex exhibited greater stability than PCA1 polyplex, suggesting that the fluorination did indeed decrease susceptibility to heparin-mediated disassembly. Tissue biodistribution of Cy5-labeled siRNA polyplexes was then examined in 4T1 tumor-bearing mice. Renal distribution was prominent within 15 min of i.v. injection for both PCA1 and PCA1F polyplexes (Figure 9B, C). At 3 h postinjection, PCA1F polyplexes exhibited higher concentration in the kidneys relative to PCA1 polyplex (Figure 9B,C), and the integrated fluorescence of all isolated organs was nearly 2-fold

Figure 7. Uptake and intracellular trafficking of polyplexes. (A) Cell uptake determined by flow cytometry at 4 h postincubation with the FAM-siRNA polyplexes in 4T1 cells. MFI = mean fluorescence intensity. Diamonds = FAM-positive cells (%) (mean ± SD of triplicate samples in each group (n = 3)). (B) Intracellular distribution of the FAM-siRNA polyplexes in 4T1 cells (lysosomes stained with Lysotracker Red, scale bar: 20 μm).

tested the hypothesis that small molecule degradation products of PCA may contribute to the observed enhancement of antimetastatic activity by conducting in vitro cell invasion assay using polyplexes that were either pretreated with GSH or used H


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taken up the polyplexes, we found that PCA1F polyplexes showed not only enhanced total tumor accumulation but also reached significantly higher percentage of tumor cells (75% vs 30%) than PCA1 polyplexes (Figure 9G). The results show the benefits of fluorination on systemic tumor accumulation and intratumoral distribution and cell uptake. Combined Antitumor and Antimetastatic Activity of siSTAT3 Polyplexes in Early Stage Breast Cancer. The high stability, enhanced tumor accumulation, effective gene silencing, and antimetastatic activity of the PCA1F polyplexes provided strong rationale to evaluate their combined antitumor and antimetastatic activities using siSTAT3 as the therapeutic siRNA. We used an early stage orthotopic 4T1 MBC model. The treatment was initiated when the tumors reached ∼80 mm3. At this early stage, there were no detectable lung metastases as verified by whole-body imaging (Figure S16). The animals were treated i.v. with PCA1F/siSTAT3 polyplexes. Control animals received i.v. PCA1/siSTAT3 to determine the effect of fluorination on the overall anticancer activity. Additional control groups received corresponding polyplexes prepared with siScr (i.e., CXCR4 inhibition alone), saline, and intratumoral (i.t.) PEI/siSTAT3 (i.e., STAT3 inhibition alone). This experimental design permitted us to determine the effect of combined CXCR4 and STAT3 inhibition. PEI/ siSTAT3 polyplexes were administered by i.t. injection and thus represent only the effect of local STAT3 silencing on the antitumor activity. In contrast, all other treatment groups were given by i.v. injection and thus capture both local and systemic effects of the combined CXCR4 and STAT3 inhibition. As shown in Figure 10A, both the i.v. treatment with PCA1F/ siSTAT3 and the i.t. treatment with PEI/siSTAT3 significantly inhibited primary tumor growth when compared with untreated (saline) group, indicating effective delivery of siSTAT3 and target gene silencing. Treatment with i.v. nonfluorinated polyplexes (PCA1/siSTAT3) only showed limited effect on tumor growth because of the relative poor delivery ability. CXCR4 inhibition alone with control PCA1/ siScr and PCA1F/siScr polyplexes also showed a small antitumor effect, indicating possible antiproliferative role of CXCR4 inhibition. After animal sacrifice, the tumors were excised and photographed (Figure 10B), and their terminal weights were recorded (Figure 10C). The average tumor weights in the i.v. PCA1F/siSTAT3 and i.t. PEI/siSTAT3 groups were similar and significantly lower than the other treatments. Similarly, H&E stained tumor tissues showed areas of tumor necrosis in these two groups (Figure 10D). To validate that the observed antitumor activity was caused by STAT3 silencing, the levels of activated p-STAT3 protein in tumors were analyzed by Western blot (Figure 10E). Both PCA1F/siSTAT3 and PEI/siSTAT3 significantly decreased STAT3 and p-STAT3 expression, but the saline and siScr controls showed no such effect. The orthotopic 4T1 model spontaneously forms lung metastases. The antimetastatic effect was evaluated as above by counting the number of surface lung metastases and following lung analysis by H&E staining (Figure 10F,G). Treatment with all of the PCA polyplexes significantly decreased the number of observed lung metastases when compared with the saline group. The PCA1F/siSTAT3 polyplexes showed the best activity due to the combined effect on CXCR4 and STAT3. As a result of its effect on the primary tumor growth, the i.t. injection of PEI/siSTAT3 polyplexes also showed reduction in the number of lung

Figure 8. Antimetastatic activity of siScr polyplexes in experimental lung metastasis model. (A) Total number of surface lung metastases. 4T1 cells were pretreated with AMD3100 or the polyplexes prepared with siScr and then injected i.v. in mice. The mice were then treated with repeated i.v. injections of AMD3100 or the polyplexes on day 3, 5, 7, and 9. Results shown as average of total number of surface lung metastases on day 11 ± SD of 5 mice in one group (n = 5). *P < 0.05, **P < 0.01, ***P < 0.005, vs nonreducible PHA polyplexes. Inset: representative images of the whole lungs with metastases. (B) H&E staining of the lung tissue sections (scale bar: 1 mm).

higher in case of PCA1F than PCA1 (Figure 9D). In agreement with these results, the calculated circulation half-life of PCA1F polyplex (1.27 h) was ∼4-fold greater than the half-life of PCA1 polyplex (0.3 h) (Figure S14A). These combined results suggest slower overall body clearance and improved overall biodistribution of the fluorinated PCA1F polyplexes. While the presence of PEG in the polymers provided clear stability benefits (Figure 3), PEG alone could not prevent the polyplex disassembly in the kidneys. The combined effects of PEG and fluorination achieved higher stability both in vitro and in vivo was needed to decrease susceptibility the renal clearance. Despite the clear benefits of fluorination, bioreducibility of PCA counteracted some of the positive effects. This was best demonstrated by decreased renal clearance of nonreducible PHA1F polyplexes when compared with bioreducible PCA1F polyplexes (Figure S14B,C). Further modifications of bioreducible polyplexes to decrease the renal clearance may be necessary to take full advantage of their apparent safety and CXCR4-inhibiting activity benefits. To evaluate if decreased renal clearance affected siRNA delivery to distant tumors, we used mice with 4T1 tumors. As shown in Figure 9E,F, the PCA1F polyplexes delivered 4.6times greater amount of siRNA to the tumors than PCA1 polyplexes. The tumor accumulation showed continuous increase during the 24 h after injection (Figure S15). When we analyzed the percent of cancer cells within the tumors that I


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Figure 9. (A) Stability of the polyplexes against disassembly with heparin. Polyplexes (Cy3-siRNA/Cy5-siRNA) were incubated in 2 and 20 U/mL of heparin, and their integrity was determined by FRET. Pharmacokinetics and biodistribution of polyplexes in 4T1 tumor-bearing mice. (B) Representative organ distribution of the Cy5-siRNA polyplexes 0.25 and 3 h after i.v. injection. (C) Biodistribution (Cy5-siRNA) in major organs determined from ex vivo fluorescence analysis. (D) Total body Cy5-siRNA retention 3 h after injection determined from the sum of fluorescence intensities of isolated major organs. (E) Whole-body Cy5-siRNA biodistribution in 4T1-bearing mice at different time after i.v. administration. Uptake of Cy5-siRNA polyplexes by the cancer cells in resected 4T1 tumors 24 h after injection: (F) mean fluorescence intensity/cell and (G) percentage of cells with uptaken Cy5-siRNA. Results shown as mean ± SD (3 mice in each group (n = 3)). *P < 0.05, **P < 0.01, ***P < 0.005.

suggesting no significant adverse effects related to CXCR4mediated mobilization of lymphocytes from bone marrow (Figure S17D).73 Overall, these findings support the safety of the PCA1F/siSTAT3 polyplexes. Antimetastatic Activity in Late-Stage MBC with Primary Tumor Resection. Upon diagnosis, majority of breast cancer patients opt for surgery to remove the primary tumor. To better simulate clinical reality, we next determined antimetastatic activity of the polyplexes following surgical excision of the primary tumors (Figure 11A). The 4T1 tumors were established as above and allowed to grow to ∼300−500 mm3. At this tumor size, microscopic metastases are present in the lungs as verified by imaging in Figure 11B. The primary tumors were excised and mice with established lung metastases were treated with i.v. polyplexes. As shown in Figure 11C,D, the treatment significantly decreased the number and size of lung metastatic lesions compared to saline group. As expected, PCA1F/siSTAT3 polyplexes which combine CXCR4 antagonism and STAT3 silencing showed the best activity. This was supported by the decreased levels of p-STAT3 in the lung metastases (Figure 11E). These in vivo results offer vital

metastatic lesions. These results showed that PCA1F/siSTAT3 polyplexes not only blocked primary tumor growth but also subdued metastatic spread in early stage MBC. This validates our overarching hypothesis that combining reversible stability, CXCR4 inhibition, and STAT3 silencing would result in improved antitumor and antimetastatic activity. Safety of PCA1F Polyplexes. The polycationic nature of PCA1F combined with systemic CXCR4 inhibition and STAT3 silencing may cause unexpected toxic side effects. The body weight was monitored and we observed a steady increase in the body weight of animals treated with the PCA1F/siSTAT3 polyplexes but the untreated group showed no increase in body weight (Figure S17A). Then, main organs were collected and histologically analyzed. Unlike other treatment groups, the animals treated with PCA1/siSTAT3 and PCA1F/siSTAT3 showed no signs of splenomegaly, indicative of a systemic effect of the polyplexes on the immune response. This finding was confirmed by an apparent increase in size of lymphoid follicles which were absent in the PCA1F/ siSTAT3 group (Figure S17B,C). No significant differences in blood cell profile were observed in the PCA1F/siSTAT3 group, J


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Figure 10. Anticancer and antimetastatic activity of siSTAT3 polyplexes in orthotopic 4T1 model of early stage breast cancer. (A) Primary tumor growth inhibition. (B) Primary tumor images on day 21 and (C) primary tumor weights on day 21. (D) H&E staining of tumors (scale bar: 400 μm). (E) Expression of STAT3 and p-STAT3 in tumor tissues determined by Western blot and semiquantitative analysis by ImageJ (three repeated experiments (n = 3)). (F) Representative photographs of the whole lungs and H&E staining of the lung tissue sections. Red circles and blue arrows denote metastases (scale bar: 1000 μm). (G) Average number of surface lung metastases. (mean ± SD of 5 mice in each group (n = 5)). *P < 0.05, **P < 0.01, ***P < 0.005, vs saline.

migration. These data indicate that PCA1F/siSTAT3 polyplexes increase apoptotic cells, decrease proliferative cells, and inhibit tumor cells invasion in MBC. The immunosuppressive tumor microenvironment compromises cancer therapy.75 STAT3 is crucial for tumor immune suppression. Increased STAT3 activity in tumors has a negative influence on the expression of immune-stimulating molecules, such as TNF, IFN-γ, and IL-6, leading to tumor immune suppression and evasion.76−78 We anticipated that PCA1F/ siSTAT3 polyplexes treatment will alter the immunosuppressive microenvironment and enhance antitumor immunity. We determined that the expression of IFN-γ, TNF-α, and IL-6 increased significantly in primary tumors and lung metastases from PCA1F/siSTAT3 treated mice (Figure 12B,C and Figure S18B). These cytokines enhanced antitumor activity and immune response by promoting infiltration of CD8+ cytotoxic T cells and subsequent IFN-γ production (Figure 12B,C and Figure S18B), which stimulated the tumor-killing activity. Immunocytokines are produced by both cancer cells and immune systems in vivo and our future studies will focus on more detailed analysis of the infiltrating immune cells and cytokines to antitumoral immune response.

indication for antimetastatic activity of PCA1F/siSTAT3 polyplexes in an aggressive late-stage MBC model. We hypothesize that the polyplexes accumulate in the lung metastases to inhibit their growth and also inhibit further metastatic spread by interacting with circulating tumor cells in the bloodstream. Mechanism of the Combined Antitumor and Antimetastatic Activity. In the final set of studies, we focused on illuminating the mechanism of action of the combined CXCR4 and STAT3 inhibition by our polyplexes. Activated STAT3 is a critical mediator of tumor-cell proliferation, apoptosis, and invasion.74 We thus examined the effect of the PCA1F/ siSTAT3 polyplexes on selected biomarkers of these processes, including proliferating cell nuclear antigen (PCNA), cleaved caspase-3, and matrix metalloproteinase 9 (MMP-9) (Figure 12A and Figure S18A). Histopathological examination of the primary tumor sections showed extended areas with PCNApositive cells in the untreated group. PCA1F/siSTAT3 substantially lowered cell proliferation in the tumors. The extent of apoptosis was also clearly greater in the polyplex group when compared with saline. The polyplex treatment decreased MMP-9 expression in the tumors, which likely contributed to the inhibition of tumor cell invasion and K


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cally deliver siRNA to treat different stages of MBC. The combination of three features (bioreducibility, fluorination, PEG) resulted in excellent overall physical stability, decreased protein dissociation, and reduced susceptibility to dissociation in the kidney GBM. Fluorination and bioreducibility also significantly improved siRNA transfection efficacy of the polyplexes by promoting cell uptake, endosomal escape, and cytoplasmic siRNA release. The best performing PCA1F/ siSTAT3 polyplexes showed effective anticancer activity in both early- and late-stage MBC following systemic i.v. administration. Mechanistic studies revealed multifaceted mechanism of action of the polyplexes characterized by increased cancer cell apoptosis, inhibition of tumor cell invasion, induction of inflammatory cytokines, increased infiltration of CD8+ cytotoxic T cells, and inhibition of angiogenesis in both primary tumors and metastases. Overall, these results demonstrated the suitability of PCA1F/siSTAT3 as a therapeutic platform for the treatment of MBC in relevant preclinical cancer models. Although this study focused on MBC, the polyplexes have the potential of application to other metastatic cancer types (colorectal, liver, pancreatic, and lung) with known CXCR4 and STAT3 involvement.

MATERIALS AND METHODS Materials. AMD3100 was from Biochempartner (Shanghai, China). mPEG-NH2 (2 kDa) was from PengSheng Biological (Shanghai, China). HMBA, CBA, PEI (25 kDa), and heptafluorobutyric anhydride (HFBA) were obtained from Sigma-Aldrich (St. Louis, MO). 4-Amino-1-butanol (ABOL) was from Meryer Chemical Technology Co., Ltd. (Shanghai, China). Fluorescently labeled FAMsiRNA, Cy3-siRNA, Cy5-siRNA, and siScr (sense strand, 5′-UUC UCC GAA CGU GUC ACG UTT-3′), siLuc (sense strand, 5′-GGA CGA GGA CGA GCA CUU CUU-3′) and siSTAT3 (sense strand, 5′-GGU CAA AUU UCC UGA GUU GUU-3′) were from GenePharma (Shanghai, China). DAPI, LysoTracker Red, Luciferase reporter assay kits, and BCA protein assay kits were from the Beyotime Institute of Biotechnology (Haimen, China). STAT3 rabbit mAb, Phospho-STAT3 rabbit mAb and β-tubulin rabbit mAb, antirabbit IgG, and HRP-linked antibody were purchased from Cell Signaling Technology (Danvers, MA). Human SDF-1was from Shenandoah Biotechnology, Inc. (Warwick, PA). FBS, RPMI-1640 medium, trypsin, penicillin, and streptomycin were from Hyclone (Waltham, MA). All other materials were from Nanjing Wanqing Chemical Glassware Instruments. Synthesis of Polycationic CXCR4 Antagonists. PHA and PCA were prepared by Michael addition of HMBA or CBA, ABOL, mPEGNH2, and AMD3100 (Table 1). Briefly, HMBA (0.5 mmol) or CBA (0.5 mmol), mPEG-NH2 (0, 0.05, and 0.1 mmol) and ABOL (0.1, 0.05, and 0 mmol) dissolved in 4 mL methanol/water (4:1 v/v) were reacted at 45 °C under nitrogen. After 6 days, AMD3100 (0.4 mmol) was added and the reaction was let to proceed for another 3 days at 37 °C in nitrogen atmosphere. AMD3100 (0.04 mmol) was then added for another 6 h to consume remaining acrylamide groups. Ethanolic HCl was then added to adjust the pH to 4 and the mixture was extensively dialyzed (MWCO 3.5 kDa) and lyophilized. The polymers were analyzed by 1H NMR in D2O. The molecular weight of each sample was tested by gel permeation chromatography (GPC) using Agilent 1260 Infinity LC system with TSKgel G3000PWXL-CP (Part No. 0021873, Tosoh Bioscience LLC, King of Prussia, PA) column. The eluent system was 0.1 M sodium acetate buffer (pH 5.0) at a flow rate of 0.5 mL/min, 25 °C. The detection system was equipped with a miniDAWN TREOS multiangle light scattering (MALS) detector and a Optilab T-rEX refractive index detector from Wyatt Technology (Santa Barbara, CA). Astra 6.1 software (Wyatt Technology) was used for analyzing the results. The polymers were fluorinated by dissolving in methanol and adding methanolic HFBA (one-third molar equivalent of AMD3100 moieties in the copolymers) and

Figure 11. Antimetastatic activity in late-stage breast cancer following surgical excision of the primary tumor. (A) Metastatic model after surgical excision of the primary tumor. (B) Representative bioluminescence image of 4T1.Luc primary tumor and lung metastasis. (C) Representative photographs of the whole lungs from mice in different treatment groups and H&E staining of the lung tissue sections. Red circles and blue arrows denote metastases (scale bar: 1 mm). (D) Average number of surface lung metastases. (E) p-STAT3 expression in the lung metastases (scale bar: 25 μm). Semiquantitative analysis of the protein expression by ImageJ. (mean ± SD with 5 mice in each group (n = 5)). *P < 0.05, **P < 0.01, ***P < 0.005, vs saline.

Angiogenesis has long been identified as an essential factor in tumor growth, progression, and metastasis.79,80 A previous study demonstrated VEGF expression is up-regulated by STAT3, indicating that STAT3 is also a target for blocking angiogenesis in cancers.48 In this study, PCA1F/siSTAT3 treatment decreased the number of VEGF-positive cells significantly (Figure 12D and Figure S18C). The endothelial cell-specific surface marker CD31 was also stained to identify disorganized vascular endothelium and angiogenesis. The polyplex-treated group showed marked reduction in the CD31 expression. The combination results indicated significant inhibition of tumor angiogenesis by PCA1F/siSTAT3 polyplexes.

CONCLUSION We have previously reported that CXCR4 antagonist polymers can function as antimetastatic agents in vivo. However, when used as delivery systems of therapeutic siRNA, their use was restricted to local administration because of systemic instability and insufficient transfection efficacy. In the present study, we have successfully synthesized multifunctional polycations and demonstrated their ability to inhibit CXCR4 and to systemiL


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Figure 12. Mechanism of anticancer and antimetastatic activity of the polyplexes. (A) PCNA, cleaved caspase-3, and MMP-9 expression in primary tumors. (B) TNF-α, IFN-γ, and CD8 expression in primary tumors. (C) Quantitative RT-PCR analysis of IL-6 mRNA expression in primary tumors and lung metastases. The mRNA levels were normalized to the levels of GAPDH mRNA and then fold to saline group. (D) VEGF, and CD31 levels in primary tumors. The scale bar indicates 200 μm. Semiquantitative analysis of the protein expression by ImageJ. Data are shown as the means ± SD with 5 mice in each group (n = 5). *P < 0.05, **P < 0.01, ***P < 0.005, vs saline group. sulfate. Changes in the FRET signal were calculated as ICy5/(ICy5 + ICy3) × 100%, where ICy5 and ICy3 are fluorescence intensities of Cy5 at 670 nm and Cy3 at 570 nm, respectively. For protein-binding evaluation, polyplexes were incubated with mouse serum for 1 h at 37 °C. The mixture was then centrifuged at 14000g for 1 h at 4 °C and the pellet was washed with PBS. The pellet was resuspended in lysis buffer containing 2% SDS and 4% Triton X100 to dissociate the bound proteins. After another centrifugation, the supernatant protein concentration was analyzed by the BCA assay and individual proteins separated by SDS-PAGE. As a control, 200 μL serum was incubated with 1 mL PBS for 1 h at 37 °C and analyzed as above. Cytotoxicity. Cytotoxicity was evaluated by the MTT assay in 4T1, U2OS, and B16F10 cells. Ten thousand cells were seeded per well in 96-well plates, grown for 12−16 h, and incubated for 24 h with increasing concentrations of the polymers or polyplexes in 100 μL of fresh media. MTT reagent was added, followed by DMSO and absorbance was measured at 570 nm. Viability was calculated relative to untreated controls and showed as the mean ± SD, n = 3. Polyplex Disassembly by Disulfide Reduction. Polyplexes (w/ w 4) were incubated with or without 10 mM GSH at 37 °C for 1 h and analyzed by agarose gel electrophoresis at 100 V for 15 min. Intracellular polyplex disassembly was studied by FRET with polyplexes prepared with Cy3-siRNA and Cy5-siRNA as above. 4T1

triethylamine (1.2 mol equiv of HFBA). The reaction was kept under stirring at room temperature for 48 h. The isolated polymers were extensively dialyzed against pH 4 HCl (MWCO 3.5 kDa). The fluorinated polymers PHAF and PCAF were obtained after lyophilization. The fluorine content and conjugation ratio were determined from elemental analysis (F, Center of Modern Analysis, Nanjing University, China) and 19F NMR in D2O. Preparation and Physicochemical Characterization of Polyplexes. Polymer and siRNA solutions in 10 mM HEPES (pH 7.4) were mixed and vortexed for 30 s to achieve siRNA concentration of 0.020 mg/mL. Polyplexes were kept at room temperature for half hour prior to use. Complexation was evaluated by agarose gel electrophoresis at 100 V for 15 min with polyplexes prepared at w/w 0−2 and quantifying % of free unbound siRNA using ImageJ. Hydrodynamic size and zeta potential were determined by ZetaPlus (Brookhaven, Long Island, NY). Particle shape was observed by transmission electron microscopy (TEM, H-600, Hitachi, Japan). Stability of Polyplexes in Vitro. To evaluate colloidal stability, the polyplexes were incubated in PBS (pH 7.4), 0.1 M NaCl, and 0.25 M NaCl at 25 °C and hydrodynamic size changes were followed by DLS. Polyplex stability in the presence of FBS and heparan sulfate was evaluated by FRET using a 1:1 mixture of Cy3-siRNA and Cy5siRNA complexed with the polymers. Polyplexes (100 nM siRNA) were incubated with 10, 20, and 30% FBS or 2 and 20 U/mL heparan M


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ACS Nano cells were treated with polyplexes (100 nM siRNA) for 6 h, either with or without 0.5 h pretreatment with NEM (1 mM). FRET analysis was performed on Zeiss CLSM (Cy3 λexc/em 550/570 nm, Cy5 λexc/em 550/670 nm) confocal microscope with identical settings for all groups. Reporter Gene Silencing. 4T1.Luc cells (4 × 104 cells per well) were seeded and cultured for 12−16 h in 48-well plates. Polyplexes (100 nM siRNA) were prepared with siLuc or siScr (w/w 1, 2, 4, and 8) and added without or with 30% FBS. After 4 h, the medium was fresh medium with 10% FBS replaced the incubation medium and the cells were grown for 24 h. After that, the cells were washed with PBS, lysed, and the Luc activity determined according to the kit supplier’s protocol. Relative light units (RLU) per mg protein in the cell lysate measured by a BCA protein assay kit were calculated. Female Balb/c mice (5 weeks old) were used in compliance with the Institutional Animal Care and Use Committee of China Pharmaceutical University. One ×105 4T1.Luc cells were injected to the mammary fat pad of mice and allowed to grow to ∼100 mm3. Anesthetized animals were given intraperitoneal injection of 0.2 mL D-luciferin (15 mg/mL) and Luc bioluminescence imaged by Tanon 5200 Multi imaging system on day 0. The animals were divided into 5 groups with 3 mice/group and administered i.v. with polyplexes (siLuc, siScr) in 5% glucose buffered with 20 mM HEPES (1.2 mg/kg siRNA). The injections were also given on day 1 and bioluminescence measured on day 2. The mice were euthanized, and the tumors excised and homogenized in a lysis buffer followed by centrifugation at 12000g for 10 min. Luc expression was measured as above. Cellular Uptake and Intracellular Trafficking. Cell uptake was analyzed with polyplexes prepared with fluorescently labeled siRNA (FAM-siRNA) in 4T1 cells seeded in confocal dishes 12−16 h before 4-h transfection. Cells were detached by trypsin and uptake measured by flow cytometry. Endosomal escape was assessed by confocal microscopy in cells treated for 1−6 h with FAM-siRNA polyplexes and stained with LysoTracker Red. CXCR4 Redistribution Assay. U2OS cells expressing EGFPlabeled CXCR4 receptor were plated in 96-well black plates with clear glass bottom. Cells were washed twice with 100 μL assay buffer (DMEM supplemented with 2 mM L-glutamine, 1% FBS, 1% PenStrep, and 10 mM HEPES) and treated with AMD3100 (300 nM), all of the polymers (2.5 μg/mL polymer, excluding the PEG and fluorocarbon content), and their siScr polyplexes (w/w 2, 4, 8) for 30 min. SDF-1 (10 nM) was added to each well for 1 h, and the cells were washed four times with PBS, fixed with paraformaldehyde, and imaged by EVOS fluorescence microscope. Cancer Cell Invasion. Transwell inserts were coated with 40 μL ice-cold Matrigel diluted 1:3 (v/v) with serum-free medium. 4T1 cells (1 × 105) were treated with AMD3100 (300 nM), polycations (2.5 μg/mL excluding the PEG and fluorocarbon content) or siScr polyplexes (w/w 2, 4, 8) in serum-free medium. The treated cells were placed in the top chamber and SDF-1 was added as the chemoattractant to the lower transwell chamber. The noninvaded cells were removed after 24 h using a cotton swab and the invaded cells were fixed, stained with CrystalViolet (0.2%). Cells were counted and imaged by EVOS xl microscope and the data shown as mean number of invaded cells ± SD (n = 3). Antimetastatic Activity in Experimental Lung Metastasis Model. Five-week old female Balb/c mice were assigned into 14 groups (n = 5). Five ×105 4T1 cells were pretreated with AMD3100 (1 mM), and polyplexes (w/w 4) for 4 h before i.v. injection in 100 μL PBS (Day 1). The animals were then treated i.v. with AMD3100 (1.25 mg/kg) and polyplexes (1.2 mg/kg siScr) on day 3, 5, 7, and 9 and sacrificed on day 11. The isolated lungs were perfused with 30% sucrose, fixed in Bouin’s solution for 18 h, and stored in 70% ethanol before counting all surface metastases under dissecting microscope. The lungs were also sectioned and H&E stained. Polyplex Biodistribution. Cells (1 × 105 4T1.luc) were inoculated to the fat pad of 5 week old female Balb/c mice (3 mice in each group). When the tumor size reached ∼80 mm3, mice were treated with i.v. injection (100 μL) of the polyplexes (1.2 mg/kg Cy5siRNA, w/w 4, which corresponds to N/P 13). IVIS Lumina imaging

system (Xenogen Co., USA, excitation/emission, 640/680 nm) was used to detect the fluorescent signal in vivo. The fluorescence imaging in anesthetized animals and the main organs were carried out 0.25−48 h postinjection. IVIS Living imaging software was used for the analysis of the amount of Cy5 in tissues. In vivo uptake by 4T1 tumor cells was also analyzed by flow cytometry as follows.4 Tumors were isolated 24 h after injection, cut into small pieces and digested by an enzyme mix containing collagenase (0.5 mg/mL) and DNase (0.19 mg/mL) in DMEM for flow cytometry and analysis using FlowJo. Anticancer and Antimetastatic Activity in Early-Stage MBC. Orthotopic 4T1 breast tumor model was established as above. When the tumors reached ∼80 mm3, the mice were randomly divided into 6 groups (5 mice in each group (n = 5)) and administered i.v. with saline, and siScr or siSTAT3 polyplexes (1.2 mg/kg siRNA, w/w 4, which corresponds to N/P 13) every other day (10 injections). Tumor size was measured using digital calipers and the volume (mm3) was calculated as 0.5 × length × width2. Body weight was recorded every other day. On day 21, mice were sacrificed and tumors collected and weighed. Tumors were also fixed in 4% paraformaldehyde, embedded in paraffin and sections were prepared for H&E staining conducted by Wuhan Servicebio Technology. The lung tissues were also collected and analyzed as described above. Collected blood was analyzed by Wuhan Servicebio Technology. Major organs (liver, spleen, heart, and kidneys) were collected and stained with H&E. Anticancer Activity in Late-Stage MBC. The 4T1.Luc tumors were established as above and allowed to grow to ∼300−500 mm3. The primary tumor was then excised under anesthesia through a small skin incision. The mice were randomized to receive i.v. saline and polyplexes of siScr or siSTAT3 (1.2 mg/kg siRNA, w/w 4, which corresponds to N/P 13) every 3 days. After 5 weeks, mice were sacrificed, lungs were obtained, and the surface tumors were counted, sectioned, and stained with H&E as described above. Statistics. Statistical analysis was performed using two-sided Student’s t test for two groups. (P < 0.05 was considered statistically significant). *P < 0.05, **P < 0.01, ***P < 0.005 vs saline or the relevant group are illustrated in figure legends. Results are shown as mean ± SD.

ASSOCIATED CONTENT S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.8b01482. Detailed immunofluorescence protocol, polymer synthesis scheme, NMR of the polymers, colloidal stability of siRNA polyplexes in PBS, cytotoxicity, polyplex disassembly, gene silencing in vivo, Manders’ overlap coefficient to analyze intracellular distribution of polyplexes, CXCR4 antagonism and cell invasion inhibition, antimetastatic activity of siScr polyplexes in experimental lung metastasis, effect of disulfide reduction on cancer cell invasion inhibition by polyplexes, pharmacokinetics and tumor accumulation of polyplexes, bioluminescence imaging of 4T1.Luc tumors, in vivo safety of the polyplexes, in vivo mechanism of action of polyplexes (PDF)

AUTHOR INFORMATION Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Gang Chen: 0000-0002-1368-5681 Minjie Sun: 0000-0003-0582-6189 David Oupicky: 0000-0003-4710-861X N


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The authors declare no competing financial interest.

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