*2.8. The Viability of Cell Cultures Exposed to MTX-bPEI-LA and M-MTX*

The carrier cytotoxicity of the MTX-bPEI-LA and M-MTX was studied. The HeLa 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, 20 μL of the MTT solution (5 mg/mL) was added and incubated for 4 h to form formazan crystals. The medium was removed, and the formazan crystals were dissolved by adding 150 μL DMSO and were incubated for 15 min at 37 ◦C. Absorbance values at 490 nm were measured on Bio Tek SYNERGY4 (Winooski, VT, USA). Relative cell viability was determined and was presented as a viability percentage of the untreated cells.

#### *2.9. Cellular Uptake of the M-MTX/Cy3-Labeled siRNA Complexes*

Flow cytometry was first used to investigate the cellular uptake of M-MTX/siRNA complexes. The HeLa cells were seeded in 12-well cell culture plates at a concentration of 1 × 105 per well and cultured for 24 h to be attached to the plates. In order to study the FR targeting ability of the M-MTX/Cy3-labeled siRNA (Cy3-siRNA) complexes, the HeLa cells were first preincubated with free FA (0.1 mM and 1 mM) for 1 h to competitively bind to FR. Mixed micelles without an MTX conjugation but loaded with Cy3-siRNA (M/Cy3-siRNA) were set as a non-FR targeting control. Naked Cy3-siRNA, M/Cy3-siRNA, and M-MTX/Cy3-siRNA with an equivalent siRNA concentration of 100 nM were then added to the plates. After 4 h of incubation, the HeLa cells treated with different formulations were trypsinized, harvested by centrifugation, washed with cold PBS, and resuspended with 4% formaldehyde solution (*w/v*) (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China). The fluorescence intensity of the cells was measured on a Beckman Coulter EPICS XL flow cytometer (Brea, CA, USA). The cellular uptake of M-MTX/Cy3-siRNA was further visualized on a confocal laser scanning microscopy (CLSM). The HeLa cells were collected, counted, and then seeded at the bottom of glass flasks for 12 h. The medium was replaced with fresh opti-MEM (Thermo scientific, Rockford, IL, USA) and preincubated with free folic acid (1 mM) for 1 h. The cells were then treated with naked siRNA, M/Cy3-siRNA, and M-MTX/Cy3-siRNA complexes with an equivalent siRNA concentration of 100 nM at 37 ◦C. After 4 h of incubation, the medium was removed and the cells were washed gently three times with PBS (0.01 M, pH 7.4). Then, the cells were fixed with 4% (*w/v*) formaldehyde for 15

min at room temperature and washed repeatedly with PBS three times to remove the formaldehyde. Subsequently, the nuclei were stained with DAPI for 10 min, and the cells were collected after washing with PBS to remove the residual dye. The uptake of the M-MTX/Cy3-siRNA complexes in the HeLa cells was observed using an LSM710 microscope from Carl Zeiss (Oberkochen, Germany).

## *2.10. Internalization and Endosome Escape of M-MTX/FAM-siRNA Complexes in HeLa Cells*

The HeLa cells were seeded at the bottom of glass flasks at a cell concentration of 1 × 105 and cultured for 24 h. The medium was replaced with fresh opti-MEM and incubated with M-MTX/ FAM-siRNA complexes (100 nM) for 1 h, 2 h, and 4 h, respectively. After washing with PBS, the cells were then incubated with Lyso Tracker™ Red DND-99 for 30 min. Then, the supernatant was removed, and the cells were gently washed. The cells were then sequentially fixed with 4% (*w/v*) formaldehyde and stained with DAPI. After washing away the residual dye, the internalization and endosome escape of M-MTX/ FAM-siRNA complexes was observed on CLSM.

#### *2.11. Cell Cytotoxicity of the M-MTX/Survivin-siRNA*

The cytotoxicity of the M-MTX/survivin-siRNA complexes was investigated. The HeLa cells were plated in 96-well microtiter plates (1 × 104 cells per well) and cultured overnight. The cells of the designed wells were pretreated with free FA (1 mM) for 1 h. MTX, M-MTX, M-MTX/survivin siRNA negative control, M, M/survivin-siRNA, and M-MTX/survivin-siRNA were added to the wells (survivin siRNA 50 nM, MTX 0.242 μg/mL). M-MTX loaded with a survivin siRNA negative control was defined as an M-MTX/siRNA negative control. After being cultured for 48 h, 20 μL of the MTT solution (5 mg/mL) was added and incubated for 4 h to form formazan crystals. The medium was removed, and the formazan crystals were dissolved by adding 150 μL dimethyl sulfoxide (DMSO, Sinopharm Chemical Reagent Co., Ltd., Shanghai, China). The absorbance values at 490 nm were measured on Bio Tek SYNERGY4 (Winooski, VT, USA). The relative cell viabilities were presented as a viability percentage of the cells treated with different formulations compared to the untreated cell samples which the viabilities were referred to be 100%.

#### *2.12. Western Blot Test*

The survivin expression was analyzed by western blot. The HeLa cells were seeded in 6-well cell culture plates at a concentration of 1.5 × <sup>10</sup><sup>5</sup> cells per well for 24 h at 37 ◦C in a 5% CO2 humidified atmosphere. Free FA (1 mM) was preincubated with the cells in the desired wells. MTX, M-MTX, M-MTX/siRNA, M-MTX/siRNA negative control, and M, M/siRNA with an equivalent siRNA concentration to 50 nM were then added to the wells. After 4 h of incubation, the medium was replaced with a fresh medium. After 48 h, the cells of different groups were collected, lysed in radio immunoprecipitation assay (RIPA) lysis buffer containing 1% protease inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA) and 2% phenylmethanesulfonyl fluoride (Sigma-Aldrich, St. Louis, MO, USA) and plated on ice for 15 min. Protein fractions were collected by centrifugation at 10,000 rpm at 4 ◦C for 10 min, quantified by a bicinchoninic acid (BCA) Protein Assay Kit (Thermo scientific, Rockford, IL, USA), subjected to polyacrylamide gel electrophoresis containing 10% sodium dodecyl sulfate (SDS-PAGE), and then transferred to polyvinylidene fluoride (PVDF) membranes (0.45 m, Merck Millipore, Billerica, MA). The membranes were blocked with a 5% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO, USA) solution (*w/v*) for 4 h at room temperature and incubated with survivin rabbit mAb (71G4B7E, Cell Signaling Technology Inc, Danvers, MA, USA) and GAPDH (ab181602, Abcam, cambridgeshire, UK) antibodies at 4 ◦C overnight, respectively. Horseradish peroxidase (HPR)-conjugated secondary antibody (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China) was added and incubated with the membranes at 4 ◦C for 4 h. The corresponding protein expression was measured by an electrochemiluminescence (ECL) detection kit (Merck Millipore, Billerica, MA, USA) and visualized by an imaging system (BioSpectrum 600, Analytik Jena US LLC., Upland, CA, USA).

#### *2.13. Dose-Dependent Inhibition Efficiency of MTX and M-MTX on Dihydrofolate Reductase (DHFR) Activity*

The inhibition efficiency of MTX and M-MTX on DHFR activity was carried out in accordance to the protocol of the Dihydrofolate Reductase Activity Kit (Colorimetric) from BioVision (S. Milpitas Blvd., Milpitas, CA, USA). Briefly, 40 μL of NADPH (500 μM), 60 μL of the DHFR substrate (15-fold dilution), and 50 μL of a series of concentrations of MTX or MTX-bPEI-LA (an equivalent MTX concentration 62.5 nM to 1000 nM) were added to each well of a 96-well clear plate. Finally, 50 μL of DHFR (250-fold dilution) was added to the wells to initiate the reaction with a total volume of 200 μL per well. The absorbance values at 340 nm were measured for 10 min at room temperature. The wells without MTX and MTX-bPEI-LA were set as the positive controls, and the wells without the DHFR enzyme were set as the negative controls. The Inhibition efficiency was defined as follows:

$$\text{Inhibition efficiency} \left(\%\right) = \frac{\left(\Delta OD \text{ positive control} - \Delta OD \text{ sample}\right)}{\left(\Delta OD \text{ positive control} - \Delta OD \text{ negative control}\right)}$$

#### *2.14. Establishment of Tumor Model*

The animal experimental protocol was in compliance with the institutional guidelines and was approved by the Experimental Animal Ethics Committee of the School of Life Sciences, Jilin University. The number for the permit for the animal experiment was 201805003 from the Experimental Animal Ethics Committee of the School of Life Sciences, Jilin University. BALB/c nude mice (female, 6–8 weeks) were obtained from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Tumor-bearing mice were established through the subcutaneous injection of 5 × <sup>10</sup><sup>6</sup> HeLa cells into the right rear leg of nude mice after the mice were adapted to the new environment for a week.

#### *2.15. Accumulation of M-MTX/Cy5-Labeled siRNA (Cy5-siRNA) Complexes in Tumor Tissue*

Tumor-bearing mice were injected with Cy5-siRNA, M/Cy5-siRNA, and M-MTX/Cy5-siRNA via the tail vein with an equivalent amount of siRNA (1 nmol). The biodistribution of the complexes was visualized by an IVIS®spectrum system from Caliper Life Sciences (Hopkinton, MA, USA) at the second, fourth, and sixth hour after administration. The mice were anesthetized by administrating a 1% (*w/v*) pentobarbital sodium solution to the abdomen, and the optimized parameter (excitation, 640 nm; emission, 680 nm) was set up for image acquisition at various time points. At the sixth hour after administration, the internal organs (Heart, Liver, Spleen, Lung, and Kidney) and tumors were dissected and then visualized by the In Vivo Imaging System.

#### *2.16. In Vivo Antitumor Efficacy of M-MTX/Survivin siRNA Complexes*

When the average tumor volume of nude mice was grown to approximately 100–150 mm3 (Day 0), the animals were randomized into four groups, each group containing 5 nude mice. Tumor-bearing mice were then injected with saline, free MTX, the M-MTX/siRNA negative control, and M-MTX/siRNA via the tail vein, respectively. The formulations (MTX 500 μg/kg, siRNA 2 nmol) were administered every 3 days. Simultaneously, the volume of the tumor and body weight were measured every 4 days using a vernier caliper and scale. On day 24, the mice were anesthetized with sodium pentobarbital and sacrificed. The tumors were dissected, weighed, and fixed for hematoxylin and eosin (H&E) and immunohistochemistry staining. The expression of survivin in tumor tissue was also analyzed by a western blot assay. The pixel density of the survivin bands compared to the GAPDH bands was quantified for each sample using Image J software (National Institutes of Health, Bethesda, MD, USA).

#### *2.17. Histopathologic Analysis*

All mice were euthanized with a 1% pentobarbital sodium solution on day 24. Vital organs (Heart, liver, Spleen, Lung, and Kidney) and tumors were dissected in each group and fixed with 4% paraformaldehyde for histopathologic analysis. Tissue sections were cut into 5 microns thick and stained with H&E. For the immunohistochemistry analysis, the tumor tissue sections were first incubated with survivin rabbit mAb.

#### *2.18. Statistical Analysis*

Data were expressed as mean ± SEM and graphed by Origin 8.0 (OriginLab Corp., Northampton, MA, USA). The statistical analysis of two group differences and correlations was determined using Student's *t*-test. \* *p* < 0.05 was considered statistically significant. \*\* *p* < 0.01 and \*\*\* *p* < 0.001 were considered highly significant.

#### **3. Results**

#### *3.1. Synthesis and Characterization of MTX-bPEI-LA and mPEG-LA*

The chemical structure characterization of mPEG-LA, bPEI-LA, and MTX-bPEI-LA was confirmed by 1H NMR (Figures S2–S4). The peak assignment and peak integration were marked with character. In Figure S2, the new characteristic peak of mPEG-LA was found at 3.37 ppm for 1α-NC=O–C (C). The peak assignment of the 1α-NC=O–C indicated that the LC was successfully conjugated to mPEG-NH2. Respectively, the characteristic peaks for 1-ethylene, methyl, and methylene of LA were seen at 5.34 ppm (A), 0.88 ppm (F), and 1.62 ppm (E). The characteristic peaks for mPEG were marked with B at 3.65 ppm for ethyl (–H–CH–CH–H). The characteristic peak for amide near 1α-C(=0)–N and 2α-C=C of mPEG-LA was at 2.04 ppm (D). The purity of the mPEG-LA was then calculated to be 91% based on the peak integration of C (1α-NC=O–C, δ = 3.37ppm, 1.01) and F (–H–CH2–CH–H, δ = 0.88 ppm, 1.67). As shown in Figure S3, the characteristic peak assignments and peak integrations of PEI and LA were both studied. The characteristic peaks of PEI were marked with C (about 2.3–3.00 ppm, 14.27), and the characteristic peak of LA was marked with A (δ = 5.34 ppm, 0.93) for 1-ethylene, E (1β-C–(C=O)–N, δ = 1.56 ppm, 0.40), and F (–H–CH2–CH–H, δ = 0.88 ppm, 0.81). A new characteristic peak was found in B (1α-N-(C)-C and 1-N–C, δ = 3.63 ppm, 1.14) indicating a successful synthesis of bPEI-LA (Figure S3). The purity of bPEI-LA was calculated to be 94.7% based on the peak integration of B and F (Figure S3). Then, the degree of functionalization of bPEI with LA moieties was calculated to 12. The 1H NMR spectra of MTX-bPEI-LA conjugates contained not only the characteristic peaks of bPEI (G, δ = 2.89 ppm) and LC (D, δ = 5.32 ppm; F, δ = 0.90 ppm) but also the characteristic signals for 1-benzene (B, δ = 7.71 ppm; C, δ = 7.34 ppm) and 2-pyrazine (A, δ = 8.47 ppm) of MTX (Figure S4). Furthermore, the new characteristic peak at 3.52 ppm (E) due to the generation of an amide bond between bPEI and MTX was also characterized. The results indicated that the MTX was successfully conjugated to the bPEI-LA-NH2. The purity of MTX-bPEI-LA was calculated to be 95.6% based on the characteristic peak integration of B (2.08), C (2.08), and E (4.50). The absorption curve peak of the MTX-bPEI-LA was measured at 303 nm. The MTX reaction efficiency and the drug loading efficiency were calculated to be 59.6% and 12.1% respectively through the standard curve of MTX with a known concentration measured by a UV spectrophotometer. The degree of functionalization of bPEI-LA with MTX moieties was also calculated to 15.

#### *3.2. Preparation and Characterization of M-MTX and M-MTX/siRNA Complexes*

M-MTX was prepared by self-assembly of MTX-bPEI-LA and mPEG-LA and had a narrow particle size of 141.8 ± 2.4 nm (PDI = 0.212 ± 0.012) (Figure 1A). SiRNA was then complexed by M-MTX with different N/P ratios (Figure 1B). The result showed that siRNA could be successfully condensed via electrostatic adsorption at an N/P ratio of 16/1 (Figure 1B). M-MTX/siRNA complexes also had a narrow particle size of 124.7 ± 9.1 nm (PDI = 0.231 ± 0.011) (Figure 1C). SiRNA adsorption to the cationic polymer of the carrier may contribute to a decrease in particle size and zeta potential (12.23 ± 0.47 mv decreased to 1.63 ± 0.14 mv). The MTX and the survivin siRNA drug loading efficiencies in the M-MTX/siRNA complexes were then calculated to be 0.17% and 2% (*w/w*) respectively with an N/P ratio of 16/1.

**Figure 1.** The characterization of the M-MTX and M-MTX /siRNA complexes. (**A**) The particle size of M-MTX was measured by Zeta-sizer Nano ZS90. (**B**) SiRNA was then condensed by M-MTX with a different N/P ratio through the electrostatic interaction. (**C**) The particle size of the M-MTX/siRNA complexes at an N/P ratio of 16/1 was further determined.

#### *3.3. In Vitro FAM-siRNA Release*

The in vitro siRNA release curve of FAM-siRNA loaded in M-MTX (M-MTX/FAM-siRNA) was shown in Figure S5. The cumulative release percentage of FAM-siRNA loaded in the M-MTX/siRNA complexes was less than 30% at 48 h. A strong interaction of siRNA with the cationic carrier may play an important role in the slow release of FAM-siRNA loaded in M-MTX. The results showed a controlled release of siRNA and high stability of the M-MTX/FAM-siRNA complexes in PBS (pH = 7.4). The release curve of free MTX in M-MTX in a dialysis bag with an MWCO of 100 KDa was also investigated in PBS (pH = 7.4). However, no free MTX was detected within 72 h. Since MTX was conjugated to the polymers (MTX-bPEI-LA) with a stable amide bond, MTX was hardly released in the release medium at pH = 7.4. The results were consistent with those of the previous study [39].

#### *3.4. Carrier Toxicity of M-MTX and MTX-bPEI-LA*

PEG has widely been applied to reduce the cytotoxicity, to prolong the circulation time in the blood, and to avoid uptake by the mononuclear phagocyte system of the cationic carriers due to its well-known good biocompatibility [23,24,40]. In order to investigate whether mPEG-LA had a good ability to reduce the toxicity of the mixed micelles, we performed a hemolytic activity assay and cytotoxicity analysis of M-MTX and MTX-bPEI-LA, as shown in Figures S6 and S7. As shown in Figure S6, MTX-bPEI-LA had a much stronger hemolysis effect when the concentrations of MTX-bPEI-LA were greater than 50 μg/mL compared with M-MTX at the same MTX-bPEI-LA concentration. In Figure S7, MTX-bPEI-LA and M-MTX had the same cytotoxicity when the concentration of MTX-bPEI-LA was low (1 μg/mL). When the concentration of MTX-bPEI-LA was 5 μg/mL, MTX-bPEI-LA showed significant carrier toxicity while M-MTX also had low cytotoxicity. When the concentration was 20 μg/mL, both MTX-bPEI-LA and M-MTX showed higher carrier toxicity. These results indicated that the mPEG-LA could reduce the toxicity of the cationic polymer (MTX-bPEI-LA) within a moderate concentration range which may interact with the cell membrane as the previous study suggested [23,24,40]. Thus, the mixed micelles were expected to have a greater potential in the clinical application compared with MTX-bPEI-LA.

#### *3.5. Cellular Uptake of M-MTX/Cy3-siRNA in FR-Overexpressing HeLa Cells*

The cellular uptake of M-MTX/Cy3-siRNA was investigated in the FR-overexpressing HeLa cells. Flow cytometry results showed that the M-MTX/Cy3-siRNA-treated groups had the highest mean fluorescence intensity and had significant improvement compared with the Cy3-siRNA- and M/Cy3-siRNA-treated groups (\*\*\* *p* < 0.001) (Figure 2A). Moreover, the fluorescence intensity of the M-MTX/Cy3-siRNA-treated group could be greatly reduced by adding a free FA (0.1 mM and 1 mM) to cells in advance. The similar results were further visualized by CLSM. The red signal in the cytoplasm treated with M-MTX/Cy3-siRNA was markedly stronger than that of the naked Cy3-siRNA and M/Cy3-siRNA and was also greatly reduced due to the free FA (1 mM) preincubated with HeLa cells (Figure 2B).

**Figure 2.** The cell uptake of M-MTX/Cy3-siRNA in the FR-expressing HeLa cells. (**A**) Flow cytometry was used to quantify the cellular uptake of different formulations. The HeLa cells were incubated with M-MTX/Cy3-siRNA, M/Cy3-siRNA, and Cy3-siRNA for 4 h at 37 ◦C. Two concentrations of free folic acid (FA) (0.1 mM and 1 mM) was added 1 h before the M-MTX/Cy3-siRNA (100 nM) was incubated. \*\*\* *p* < 0.001. (**B**) The confocal micrographs of HeLa cells were also obtained after incubation with M-MTX/Cy3-siRNA, M/Cy3-siRNA, and Cy3-siRNA for 4 h at 37 ◦C. Free FA (1 mM) was also added 1 h before the M-MTX/Cy3-siRNA (100 nM) addition. The green bar in the image was 20 μm. The values were the mean ± SEM (n = 3).
