**1. Introduction**

The combination of chemotherapy drugs and siRNA is recently emerging as a strategy for cancer therapy [1–3]. Rational design for the combination therapy strategy is essential to obtain maximum efficacy with minimum dosage, side effects, and drug tolerance. Methotrexate (MTX) is a dihydrofolate reductase (DHFR) inhibitor and has shown anticancer activity [4,5]. However, MTX monotherapy at high dosage is often associated with systemic toxicity, drug resistance, and low efficacy [6,7]. Therefore, MTX needs to be combined with other agents to reduce side effects and enhance tumor efficacy [8,9]. Small interfering RNA (siRNA) with a length of 20–25 base pairs has drawn much attention from researchers and is regarded as a potential therapeutic modality for cancer [10,11]. A siRNA could specifically silence the expression of the targeted gene through RNA interference [12]. Survivin has recently been found to be a crucial protein to tumor growth and metastasis and is a promising

therapeutic target for tumor [13]. Moreover, studies had indicated that the survivin could facilitate tumor drug resistance. The inhibition of survivin expression may boost the chemotherapeutic efficacy of cancer [14]. Thus, a co-delivery of siRNA targeting survivin expression and MTX may be a promising approach to overcome cancer drug resistance [1]. In addition, the distinct mechanisms of MTX and survivin siRNA suggest that their combination may produce synergy [15].

Nanocarriers such as liposomes, micelles, dendrimers, or supramolecular systems have been evaluated as vehicles for the co-delivery of chemotherapeutic drugs and gene agents [2,16]. Micelles for the co-delivery of chemotherapeutic drugs and nucleic acid have been shown to have good stability and to control drug release. They can increase the effectiveness of drug combination therapy and can reduce drug resistance [17–20]. Compared to the pristine micelles, mixed micelles self-assembled from two or more amphiphilic polymers provide greater flexibility. They have recently drawn much attention for use in combination cancer therapy [21,22]. The mixed micelles are easy to optimize, in terms of kinetic stability, drug loading capacity, size distribution, and the preparation of multifunctional carriers [21–24]. The mixed micelles also could be prepared with simplified procedures, could achieve a desirable antitumor efficacy, and could reduce variability when expanding to a large scale for clinical application [25,26]. However, the efficacy of the mixed micelles is still restricted due to rapid drug release especially for the co-delivery of two or more different types of therapeutic drugs in blood circulation. Polymer-drug conjugates have been studied as nanomedicine for the improvement of disease treatment efficacy recently [27]. Drug covalently bound to the polymers could avoid the drug dissociation and rapid clearance and could improve the drug stability during dilution or exposure to components in blood. Therefore, a system for the co-delivery of siRNA and MTX was developed based on mixed micelles consisted of functionalized polymer-drug conjugates [28].

MTX conjugated to dendrimers or polymers had previously been shown to retain good antitumor activity in vitro and in vivo [29,30]. Moreover, MTX has been suggested to act both as a targeting ligand and a therapeutic agent in recent studies [31]. When conjugated to dendrimers, MTX may directly target the folic acid (FA) receptor, be internalized into the cell, and then act on its target [32]. Polyethylenimine (PEI) has been used extensively for the delivery of nucleic acids such as siRNA, miRNA, and oligonucleotides because of its superior ability to electrostatically complex with nucleic acids and to facilitate the endosomal escape through its proton sponge effect. However, PEI has had limited clinical use due to its toxicity [33,34]. We previously demonstrated that fatty acid modified PEI showed reduced toxicity and enhanced the efficiency for oligonucleotides delivery [35,36]. Here, MTX was conjugated to linolenic acid modified branched PEI (MTX-bPEI-LA). The MTX-bPEI-LA has several advantages: (i) MTX conjugated to the polymers by amide bond is stable and the MTX conjugated micelles have a long circulation time; (ii) it reduced systemic toxicity compared to free MTX [37]; (iii) bPEI-LA with MTX conjugation may be less toxic than bPEI due to a reduction in charged amino groups [38], and (iv) polymer-MTX conjugates may act in dual roles of a targeting and a therapeutic agent [31,32]. In this study, we also synthesized linolenic acid (LA)-modified methoxy-polyethyleneglycol (mPEG-LA), which was combined with MTX-bPEI-LA to formed mixed micelles (M-MTX) (Scheme 1). In order to evaluate the co-delivery efficiency of the system, we studied their cellular uptake, cytotoxicity, siRNA target modulation, biodistribution and the therapeutic effect in vitro and in vivo.

**Scheme 1.** The preparation of the methotrexate (MTX)-conjugated mixed micelles (M-MTX) and the co-delivery of MTX and siRNA to the cytoplasm. (**A**) The amphiphilic polymers of mPEG-LA (linolenic acid-modified methoxy-polyethyleneglycol) and MTX-bPEI-LA (MTX conjugated to linolenic acid-modified branch polyethylenimine) were first synthesized and then self-assembled in one step to form M-MTX and then incubated with siRNA to form the M-MTX/siRNA complexes (M-MTX/siRNA). (**B**) M-MTX/siRNA complexes could be efficiently taken up by the tumor cells through the folate receptor (FR)-mediated endocytosis, could successfully release the loadings to the cytoplasm and could produce a synergy between survivin siRNA and MTX-bPEI-LA with gene silencing and reduced enzyme activity.

#### **2. Materials and Methods**

#### *2.1. Materials*

Branched polyethylenimine (bPEI, 25 kDa) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Methotrexate (MTX) and 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) were purchased from Shanghai Yuanye Biological Technology (Shanghai, China). mPEG-NH2 (2000 Da) was purchased from Yarebio (Shanghai, China). Linolyl chloride (LC) was obtained from Tokyo Chemical Industry Co., Ltd. (Shanghai, China). Survivin siRNA: Sense (5 –3 ): mGCAGGUUCCUmUAUCUGUCAdTdT; Antisense (5 –3 ): UGAmCAGAmUAAGGAACCUGmCdTdT; Survivin siRNA negative control: Sense (5 –3 ): mUUCUCCGAACmGUGUCACGUdTdT; Antisense (5 –3 ): ACGmUGACmACGUUCGGAGAmAdTdT, and Cy3, 5 -FAM and Cy5-labeled survivin siRNA for cellular uptake and biodistribution studies were synthesized by Ribo Biochemistry (Guangzhou, China). HeLa cells were purchased from ATCC (Rockefeller, MD, USA). 4 ,6-Diamidino-2-phenylindole (DAPI) and Lyso Tracker™ Green DND-99 were purchased from Invitrogen Co. (Carlsbad, CA, USA). The Dihydrofolate Reductase Assay Kit was also purchased from BioVision (S. Milpitas Blvd., Milpitas, CA, USA). All chemical reagents used were of analytical grade.

#### *2.2. Synthesis and Characterization of the Amphiphilic Polymers*

LA was separately conjugated to the mPEG and b-PEI as shown in Figure S1 using a previously reported method [35,36]. Briefly, linolenic chloride (LC) dissolved in anhydrous dichloromethane (DCM) (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was added dropwise to the mPEG2000-NH2 and bPEI (25 kDa) anhydrous DCM solution, respectively. After 12 h, the reaction mixture was precipitated and washed three times by diethyl ether (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China). The products mPEG-LA and bPEI-LA were obtained by removing organic solvent in a rotary evaporator (Shanghai Yukang Scientific Instrument Co., Ltd., Shanghai, China) and then vacuumed for 2 h. MTX modified bPEI-LA(MTX-bPEI-LA) was prepared through the reaction of the amino groups of bPEI-LA and carboxy groups of MTX (Figure S1B). The activated reagents of 1-hydroxybenzotriazole (HOBT) (Xiya Chemical Industry Co., Ltd., Linshu, China), *O*-benzotriazole-*N*,*N*,*N* ,*N* -tetramethyl-uroniumhexafluorophosphate (HBTU) (Xiya Chemical Industry Co., Ltd., Linshu, China), and *N*,*N*-diisopropylethylamine (DIEA) (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) were first added to the MTX solution to activate the carboxyl groups of MTX for 2 h. Then, the bPEI-LA solution dissolved in anhydrous methanol (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was added dropwise to the MTX solution. The mixture was incubated at room temperature for 24 h under nitrogen atmosphere. The reaction mixture was placed in a dialysis bag with a molecular weight cutoff (MWCO) of 8000 to 14,000 Da and dialyzed against deionized water. The dialysate was changed every 4 h. After 48 h, MTX-bPEI-LA was freeze-dried on a Christ epsilon 2-6D LSC (Osterode, Germany). The structures of mPEG-LA, bPEI-LA, and MTX-bPEI-LA were confirmed by 1H NMR on a spectrometer from Bruker (Fällanden, Switzerland). mPEG-LA and bPEI-LA were dissolved in deuterated chloroform (CDCl3, Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA). MTX-bPEI-LA was dissolved in deuterated water (D2O, (Cambridge Isotope Laboratories, Inc., Tewksbury, MA, USA). The concentration of MTX in MTX-bPEI-LA was determined based on a calibration curve of MTX, and the drug reaction efficiency was calculated. The reaction efficiency of MTX was defined as the ratio of the weight of MTX which was conjugated to the bPEI-LA to the total weight of MTX added to the reaction. The drug loading efficiency was obtained by calculating the ratio of the weight of MTX conjugated to bPEI-LA to the total weight of MTX-bPEI-LA.

#### *2.3. Preparation of MTX-Conjugated Mixed Micelles (M-MTX)*

M-MTX was prepared by the self-assembly of MTX-bPEI-LA and mPEG-LA. A specified volume of MTX-bPEI-LA and mPEG-LA solution (with a molar ratio of 1:200) dissolved in chloroform (Sinopharm Chemical Reagent Co., Ltd., Shanghai, China) was mixed together and sonicated for 3 min. Then, the mixture solution was evaporated by a rotary evaporator to remove the chloroform at 37 ◦C and further under vacuum for 2 h to remove the residual organic solvent and to obtain a film on the flask. To prepare M-MTX, diethyl pyrocarbonate (DEPC)-treated water (Coolaber, Beijing, China) was added to the flask and sonicated for 2 min. The particle size, zeta potential, and polydispersity index (PDI) of M-MTX were measured on a Zeta-sizer Nano ZS90 from Malvern Instruments (Malvern, UK) at 25 ◦C.

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

Gel retardation assays were performed to investigate the ability of M-MTX to complex siRNA using agarose gel electrophoresis. M-MTX with different concentrations and survivin siRNA solutions were first diluted to prepare the M-MTX/siRNA complexes with different N/P ratios. The desired amount of siRNA solution was then mixed with an equal volume of the M-MTX solution by gentle pipetting. The complexes were incubated for 10 min at room temperature before use. Then, 10 μL of the M-MTX/siRNA complexes with different N/P ratios were mixed with 2 μL of 6× loading dye and loaded into a 2% agarose gel. The voltage of electrophoresis (BIO-RAD Laboratories, Hercules, CA, USA) was set up at 100 V and run for 10 min in a Tris-acetate-EDTA (TAE) buffer (Beijing Dingguo Changsheng Biotechnology Co., Ltd., Beijing, China). After that, the gel was placed in a staining solution containing Molecular Probes SYBR®Gold nucleic acid (Invitrogen, Ltd., Willow Creek Road, Eugene, OR, USA) for 30 min. Free siRNA in the complexes could be detected as a band on the gel with a GelDoc-It Ts Imaging System (Analytik Jena US LLC., Upland, CA, USA). The particle size and the zeta potential of the M-MTX/siRNA complexes were measured by Nano ZS90 (Malvern, UK).

#### *2.5. In Vitro siRNA Release*

The in vitro siRNA release curve of FAM-siRNA loaded M-MTX (M-MTX/FAM-siRNA) in phosphate buffer saline (PBS) was studied. 1 mL M-MTX/FAM-siRNA complexes were transferred into a dialysis bag (MWCO 100 KDa, Shanghai Yuanye Biological Technology, Shanghai, China). The dialysis bag was immersed in 40 mL of PBS and stirred at 37 ◦C at a speed of 100 rpm. At fixed time intervals, 100 μL of the external solution was withdrawn and replaced with the same volume of fresh PBS. The fluorescence intensity of FAM-siRNA was measured by Bio Tek SYNERGY4 (Winooski, VT, USA) at λex = 485 nm and λem = 535 nm, and the concentrations of FAM-siRNA were measured based on a calibration curve of FAM-labeled siRNA with known concentrations.
