*3.8. Biodistribution of M-MTX/Cy5-siRNA in Tumor-Bearing Mice*

The efficient delivery of siRNA and MTX to the targeting site was a prerequisite for a successful treatment. Therefore, we investigated the biodistribution of M-MTX/siRNA in tumor-bearing mice. Mice injected with M-MTX/Cy5-siRNA showed a higher accumulation (about 6-folds higher radiant efficiency) in solid tumors than the M/Cy5-siRNA-treated group without MTX conjugated (Figure 5). At the sixth hour, the red fluorescence signal in the tumor injected with Cy5-siRNA and M/ Cy5-siRNA was barely visible. The same results were also reflected in the anatomical organs (Heart, Liver, Spleen, and Lung). The fluorescence signal of the free Cy5-siRNA injection group was barely detectable in major organs and tumors other than the kidney. This indicated a fast clearance of free siRNA in vivo. However, M-MTX/Cy5-siRNA exhibited the lowest fluorescence intensity in the kidney. This suggested that the Cy5-siRNA loaded in M-MTX had a long circulation time. Compared to M-MTX/Cy5-siRNA, M/Cy5-siRNA had a higher fluorescence signal in the spleen, liver, lung, and kidney but had a lower tumor fluorescence signal. This may be due to the larger size, higher positive charge, and non-targeting ability of the M/Cy5-siRNA which was easily captured by the reticuloendothelial system. The high and specific accumulation of M-MTX/Cy5-siRNA in tumor tissues further indicated a potential and promising imaging application of the carrier in the clinic.

**Figure 5.** The biodistribution of M-MTX/Cy5-siRNA in tumor-bearing mice. (**Left**) The real-time fluorescence imaging of tumor-bearing mice injected with naked Cy5-siRNA, M/Cy5-siRNA, and M-MTX/Cy5-siRNA via tail vein were obtained at the second, fourth, and sixth hours after administration (Cy5-siRNA = 1 nmol). (**Right**) The ex vivo imaging of the tumors and organs excised from tumor-bearing mice at the sixth hour after injection. The images were obtained by IVIS®In Vivo Imaging System with an optimized parameter (excitation, 640 nm; emission, 680 nm).

#### *3.9. Antitumor Efficacy of M-MTX/siRNA In Vivo*

The synergistic antitumor efficacy of MTX and survivin siRNA was further assessed in tumor-bearing mice. Consistent with the in vitro results, tumor growth inhibition and disease remission were significantly achieved in the M-MTX/siRNA treated group with a low MTX and siRNA dose (\*\* *p* < 0.01, *n* = 5) (Figure 6A–C). The M-MTX/siRNA-treated group had a large tumor volume and weight reduction (about 75% and 63%, respectively) compared to other groups. The HeLa cell lines are known for their high tumor growth rate. The Saline, MTX, and M-MTX/siRNA negative control groups showed a rapid increase in tumor size and weight (Figure 6A–C). Compared to MTX, M-MTX/siRNA negative control group had a lower tumor weight (Figure 6C). This suggested that the M-MTX/siRNA negative control had a more enhanced antitumor effect than free MTX, probably due to its extended circulation time (Figure 5) and an enhanced cellular uptake mediated by FR (Figure 2). The body weight of the mice was measured to evaluate the toxicity of the formulations. As shown in Figure 6D, the body weight of the different groups increased steadily, indicating that the formulations had no significant toxicity.

**Figure 6.** The therapeutic efficacy of M-MTX/siRNA in vivo. The tumor volume (**A**), photographs of the solid tumor (**B**), the tumor weight (**C**) of the animals treated with various drug formulations were obtained on day 24. (*n* = 5, \*\* *p* < 0.01, M-MTX/siRNA compared to saline). The body weight of the tumor-bearing mice treated with various drug formulations was also measured to assess the system toxicity (**D**). The values are the mean ± SEM, *n* = 5.

#### *3.10. Protein Expression, Immunohistochemistry, and Histopathological Analysis*

Survivin expression levels in tumors were also analyzed by a western blot and an immunohistochemical analysis (Figure 7A–C). The western blot results showed a 40% reduction in survivin expression in M-MTX/siRNA-treated tumors compared to the saline group (\* *p* < 0.05, *n* = 3) (Figure 7A,B). The same results were also confirmed by the immunohistochemical analysis. The cells stained with brown and black nuclei were shown to be positive for survivin expression as indicated by the black arrow (Figure 7C). Furthermore, HE staining of the tumor tissues treated with M-MTX/siRNA showed a large amount of necrosis of the tumor cells as indicated by the black arrow (Figure 7D). The results of Figures 6 and 7 showed that M-MTX could efficiently deliver siRNA to the cells in tumor tissue, suppressing the related expression of proteins and finally leading to the death of a large number of tumor cells in tumor tissues. There was no significant toxicity in the major organs of mice treated with MTX, M-MTX/siRNA negative control, or M-MTX/siRNA compared to the saline group due to the reduced dose of MTX and siRNA (Figure 8). M-MTX exhibited good biocompatibility and low toxicity as a non-viral delivery for siRNA delivery.

**Figure 7.** The protein expression, immunohistochemistry, and histopathological analysis of tumor tissues. Survivin expression in tumor tissue was determined by western blot assay (**A**) and then quantified by image J (**B**) (*n* = 3, \* *p* < 0.05, M-MTX/siRNA compared to saline). The immunohistochemical analysis of protein expression (**C**) and the hematoxylin-eosin staining for tumor tissue necrosis (**D**) was also investigated.

**Figure 8.** The organ toxicity analysis of different formulations: Major organs (Heart, Lung, Liver, Kidney, and Spleen) were dissected and stained with hematoxylin-eosin to assess the systemic toxicity of the drug formulations.

#### **4. Discussion**

MTX, a FA analog, has long been used for cancer therapy but is often associated with severe systemic toxicity, bone marrow suppression, and drug resistance [6,7,43]. In addition, MTX monotherapy has limited effectiveness. Therefore, the new combination strategy of MTX and siRNA as well as a novel drug delivery system that reduces MTX toxicity and improves cancer efficacy is desirable [8,9]. As one of the strongest tumor apoptosis inhibitors, survivin not only promotes tumor cell proliferation but also is closely related to the development of tumor resistance [14,44,45]. Thus, the combination therapy of MTX and survivin-siRNA demonstrated a new possibility for enhanced tumor efficacy. In order to efficiently deliver MTX and survivin-siRNA into the cells, modified cationic polymer-based mixed micelles having an MTX targeting ability were designed due to their simplicity in synthesis and preparation.

LA is a polyunsaturated fatty acid that is essential for the body and is nontoxic. Moreover, recent studies reported that LA also inhibited tumor cell growth and metastasis [46–48]. Therefore, LA was selected to be separately conjugated to the b-PEI and mPEG to form two amphiphilic polymers of bPEI-LA and mPEG-LA in this work. The hydrophobic molecule of LA conjugated to bPEI was designed to reduce the high toxicity of bPEI [38]. MTX was then conjugated to the bPEI-LA by an esterase-stable amide linkage (Figure S1). MTX was conjugated to bPEI-LA by an amide linker to prevent and avoid MTX release in the blood and to enhance the targeting ability of MTX to the FA receptor. Then, M-MTX self-assembled by MTX-bPEI-LA and mPEG-LA was prepared and applied to efficiently co-deliver MTX and siRNA. M-MTX was aimed to target the tumor cells overexpressing FR and successfully release loadings into the cytoplasm as shown in Scheme 1. Actually, some researchers had revealed that MTX conjugated to the dendrimers showed a potential FR targeting ability through the tight-binding of MTX to the folate binding protein [49]. From the reversed cellular uptake (Figure 2) and biological activities (Figure 4) of M-MTX/Cy5-siRNA-treated groups preincubated with free FA and the high cellular uptake (Figure 2), biological activities (Figure 4) in vitro, and accumulation of M-MTX/Cy5-siRNA in the solid tumor (Figure 5), we point out that M-MTX nanocarrier exhibits an FR targeting ability and a much higher internalization efficiency via FA receptor-mediated endocytosis than M/siRNA complexes [50]. In Figure 4C, when M-MTX and MTX were incubated with cells for 4 h, the M-MTX-treated group exhibited a lower protein expression compared to free MTX. This also indicated that M-MTX entered more into the cells and had a higher internalization efficiency compared to free methotrexate. The siRNA and MTX-bPEI-LA conjugates with stable chemical linkage achieved endosome escape (Figure 3) and were released probably by the proton sponge effect of cationic polymer of bPEI [33,34]. As expected, M-MTX/survivin siRNA achieved tumor growth inhibition and disease remission with a low MTX and siRNA dose in tumor-bearing mice. There was no significant difference in the antitumor effect among saline, MTX, and the M-MTX/siRNA negative control with a low dose of MTX, which was not sufficient to achieve disease remission. However, the M-MTX/siRNA complexes exhibited an unexpectedly potent effect compared to MTX and the M-MTX/siRNA negative control. This may largely depend on the specific targeting and efficient delivery of siRNA by the bifunctional vector of M-MTX. Moreover, previous studies have shown that survivin siRNA may have a positive effect on chemotherapeutic drug sensitivity and may reduce drug resistance [14]. Thus, we propose that the effect of mutual promotion and the sensitivity of the two drugs at a low dose also contribute to the desired efficacy. The M-MTX carrier exhibited good biocompatibility and low toxicity as a non-viral carrier for siRNA co-delivery in vivo. However, the specific and detailed mechanisms and synergy of MTX and siRNA warrant further investigation.

#### **5. Conclusions**

In this study, an M-MTX/siRNA co-delivery system was developed based on the mixed micelles composed of mPEG-LA and MTX-bPEI-LA. M-MTX/siRNA exhibited a good ability to target tumor cells overexpressing FR. M-MTX/siRNA was internalized by FR-mediated endocytosis and was able to release siRNA into the cytoplasm. Studies in tumor-bearing mice showed that M-MTX/siRNA could greatly target tumor tissue and inhibit tumor growth in vivo. All above, M-MTX could be applied as a promising and safe nanoplatform for siRNA or even other therapeutic agent deliveries and may find utility in anticancer therapy.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1999-4923/11/2/92/s1, Figure S1. The synthesis procedure of the amphiphilic polymers. mPEG-LA was synthesized by the reaction of oleic acid chloride and mPEG-NH2 (A). MTX-bPEI-LA was synthesized by two steps (B). BPEI-LA was first synthesized, and then, the MTX was conjugated to finally obtained MTX-bPEI-LA; Figure S2. The 1H NMR of mPEG-LA. mPEG-LA was dissolved in CDCl3 and measured in a nuclear magnetic resonance instrument (500 MHZ). The characteristic peaks of mPEG and LA were marked with the colour characters A, B, C, D, E, and F; Figure S3. The 1H NMR of bPEI-LA. bPEI-LA was dissolved in CDCl3 and measured in a nuclear magnetic resonance instrument (500 MHZ). The characteristic peaks of bPEI and LA were marked with the colour characters A, B, C, D, E, and F; Figure S4. The 1H NMR of MTX-bPEI-LA. MTX-bPEI-LA was dissolved in D2O and measured in a nuclear magnetic resonance instrument (500 MHZ). The characteristic peaks of bPEI, MTX, and LA were marked with the colour characters A, B, C, D, E, F, and G; Figure S5. The in vitro siRNA release profile of FAM-siRNA-loaded mixed micelles in PBS (pH = 7.4); Figure S6. The hemolytic analysis of M-MTX and MTX-bPEI-LA on murine erythrocytes (n = 3, mean ± SEM). 200 μL various concentrations of M-MTX and MTX-bPEI-LA were incubated with 1 mL erythrocytes in a 2% phosphate buffer saline for 3 h. The suspensions were centrifuged, and the absorbance of supernatant (100 μL) was measured at 450 nm; Figure S7. The cytotoxicity of M-MTX and MTX-bPEI-LA (*n* = 3, mean ± SEM). The cells were plated in 96-well microtiter plates (5000 cells per well) and cultured overnight. M-MTX and MTX-bPEI-LA at 3 concentrations (1, 5, and 20 μg/mL) were added. After another 24 h, the relative cell viability was determined and was presented as a percentage of the viability of untreated cells.

**Author Contributions:** Conceptualization, L.T., J.X. and F.H.; methodology, F.H., C.Y., L.Z. and Y.S.; software, S.D.; validation, Z.C., Q.M. and J.L.; formal analysis, F.H.; investigation, F.H.; resources, L.T.; data curation, F.H.; writing—original draft preparation, F.H.; writing—review and editing, R.J.L.; visualization, F.H.; supervision, L.T. and Q.M.; project administration, L.T., L.T. and J.L.

**Funding:** This research received no external funding.

**Acknowledgments:** The biodistribution and histopathologic analysis in this article were offered help with the IVIS®spectrum system and technical support from the Key Laboratory of Pathology and Biology Teaching of Ministry of Education and Basic Medical Experimental Teaching Center of Jilin University.

**Conflicts of Interest:** The authors declare no conflict of interest.
