Enhanced antitumor efficacy of folate modified amphiphilic nanoparticles through co-delivery of chemotherapeutic drugs and genes.

Biomaterials xxx (2014) 1e10

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Enhanced antitumor efficacy of folate modified amphiphilic nanoparticles through co-delivery of chemotherapeutic drugs and genes Bojie Yu, Cui Tang, Chunhua Yin* State Key Laboratory of Genetic Engineering, Department of Pharmaceutical Sciences, School of Life Sciences, Fudan University, Shanghai 200433, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 April 2014 Accepted 21 April 2014 Available online xxx

Folate (FA) modified amphiphilic linoleic acid (LA) and poly (b-malic acid) (PMLA) double grafted chitosan (LMC) nanoparticles (NPs) with optimum grafting degrees of hydrophobic LA and hydrophilic PMLA were developed for the co-delivery of paclitaxel (PTX) and survivin shRNA-expressing plasmid (iSur-pDNA). The resultant NPs exhibited particle size of 161 nm and zeta potential of 43 mV. FA modification and the increasing grafting degrees of LA and PMLA were correlated with the suppressed protein adsorption, the inhibited release of PTX, and the accelerated dissociation of pDNA. PTX loading, cellular uptake, nuclear accumulation of pDNA, in vitro gene silencing efficiency, and cell growth inhibition were promoted by FA modification and higher grafting degree of LA, but impeded by increasing grafting degree of PMLA. In tumor-bearing mice, co-delivery of PTX and iSur-pDNA exhibited enhanced antitumor efficacy and prolonged survival period as compared with single delivery of PTX or iSur-pDNA. These results indicated that amphiphilic LMC NPs could serve as a promising platform for the co-delivery of antitumor drugs and genes, and highlighted the importance of adjusting the hydrophobic and hydrophilic grafting degrees. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Amphiphilic nanoparticles Co-delivery FA modification Hydrophobic modification Hydrophilic modification Antitumor efficacy

1. Introduction Due to the complexity of their signaling network, tumor cells may develop several pathways to escape from death induced by chemotherapeutics [1,2]. The curative effects of conventional chemotherapy hitting single target in tumor cells are therefore severely limited. The combination of chemotherapy and gene therapy provides a promising modality to improve the therapeutic index through simultaneous modulation of multiple signaling pathways in tumor cells [3]. Numerous delivery systems involving the co-delivery of antitumor drugs and genes have been developed including liposomes, dendrimers, and inorganic nanoparticles (NPs), intending to obtain a synergistic effect of the drug and gene in tumor therapy [4e6]. However, interferential influences between agents and toxicity induced by multi-component carriers are inevitable problems for these co-delivery systems [2,7e9]. Comparatively, a biodegradable amphiphilic copolymer can selfassemble coreeshell NPs with a hydrophobic core and a cationic

* Corresponding author. Tel.: þ86 21 6564 3797; fax: þ86 21 5552 2771. E-mail address: [email protected] (C. Yin).

hydrophilic shell in an aqueous solution, which has the ability of simultaneously loading a hydrophobic antitumor drug and a poly anionic gene in a single-component carrier [10], thereby avoiding aforementioned issues. The hydrophobic and hydrophilic modification of biodegradable chitosan has many advantageous effects for the delivery of antitumor drugs or plasmid DNA (pDNA). Hydrophobic modification, such as polylactide modification [11], stearic acid modification [12], and alkylation [13], can promote the encapsulation and cellular uptake of hydrophobic antitumor drugs and improve the protection from nuclease degradation, cellular uptake, and endosomal escape of pDNA. Hydrophilic modification, including PEGylation [14] and arginine modification [15], is able to facilitate the drug accumulation in tumor cells and intracellular release of pDNA. However, to our knowledge, few researches have concerned the application of amphiphilic chitosan derivatives as co-delivery systems. In our previous studies, amphiphilic linoleic acid (LA) and poly (b-malic acid) (PMLA) double grafted chitosan (LMC) NPs were developed as a safe and effective delivery system for paclitaxel (PTX) or pDNA [16,17]. Considering the fact that hydrophobic LA core and cationic PMLA shell of LMC NPs could be respectively evoke hydrophobic and electrostatic interactions, amphiphilic LMC

http://dx.doi.org/10.1016/j.biomaterials.2014.04.095 0142-9612/Ó 2014 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Yu B, et al., Enhanced antitumor efficacy of folate modified amphiphilic nanoparticles through co-delivery of chemotherapeutic drugs and genes, Biomaterials (2014), http://dx.doi.org/10.1016/j.biomaterials.2014.04.095

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B. Yu et al. / Biomaterials xxx (2014) 1e10

Abbreviations BSA DCC DMAP DMEM EB EDC$HCl EPR FA FITC GAPDH IC50 LA MTT NHS NPs PBS pDNA PMLA

bovine serum albumin dicyclohexylcarbodiimide 4-dimethylaminopyridine Dulbecco’s Modified Eagle’s Medium ethidium bromide N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride enhanced permeability and retention folate fluorescein isothiocyanate glyceraldehyde-3-phosphate dehydrogenase half maximal inhibitory concentration linoleic acid methyl tetrazolium N-hydroxysuccinimide nanoparticles phosphate buffered solution plasmid DNA poly (b-malic acid)

NPs were therefore adopted here as a co-delivery system carrying PTX and survivin shRNA-expressing pDNA (iSur-pDNA) simultaneously. Although the optimum hydrophobicity and hydrophilicity of LMC NPs have been elucidated for pDNA delivery [17], they are unavailable for the co-delivery of antitumor drugs and pDNA. Therefore, in this study, LMC NPs with various grafting degrees of LA and PMLA were prepared to probe the optimized hydrophobicity and hydrophilicity for a co-delivery system. Folate (FA) was further conjugated to endow LMC NPs with the active-targeting capacity. PTX and iSur-pDNA were loaded into LMC NPs through hydrophobic and electrostatic interactions, respectively. Assays of pDNA binding affinity, protein adsorption, pDNA protection, in vitro release, cellular uptake, intracellular distribution, and in vitro cell growth inhibition were performed. Finally, in vivo antitumor efficacies of various NPs were evaluated in tumor bearing mice. 2. Materials and methods 2.1. Materials, cell lines, and animals Chitosan with molecular weight of 100 kDa and deacetylation degree of 85% was obtained from Golden-Shell Biochemical Co., Ltd. (Zhejiang, China). Fluorescein isothiocyanate (FITC), ethidium bromide (EB), and Hoechst 33258 were purchased from Sigma (St. Louis, MO, USA). FA, rhodamine B (RhB), 4-dimethylaminopyridine (DMAP), and dicyclohexylcarbodiimide (DCC) were supplied by Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China). N-Hydroxysuccinimide (NHS) and N-(3dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride (EDC$HCl) were obtained from Yuanju Bio-Tech Co., Ltd. (Shanghai, China). DNase I was purchased from Worthington (Lakewood, NJ, USA). PTX was purchased from Hisun Pharmaceutical Co., Ltd. (Zhejiang, China). The iSur-pDNA and pGL3-control vector were amplified in Escherichia coli and isolated with EndoFree Plasmid Mega Kit (Tiangen Biotech Co., Ltd., Beijing, China). All other chemical reagents were of analytic grade. Human liver cell line L02 and human hepatoma cell line QGY-7703 were purchased from Chinese Academy of Sciences (Shanghai, China). Both cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, NY, USA) containing 10% fetal bovine serum. Female Kunming mice (6 weeks, 20 2 g) were purchased from Slaccas Experimental Animal Co., Ltd. (Shanghai, China). All animal experiments were performed following the protocol approved by the Institutional Animal Care and Use Committee, Fudan University. 2.2. Preparation and characterization of LMC and FA-LMC LMC was prepared according to our previous report [16]. In brief, poly (b-benzyl malate) (PMLABz) was synthesized through polymerization of lactic acid and lactone (1:10, m/m). PMLABz and oxalyl chloride (1:1.5, v/v) were reacted in dichloromethane at 40 C under N2 for 12 h to obtain PMLABz acyl chloride. LA acyl chloride

PMLABz poly (b-benzyl malate) PTX paclitaxel RhB rhodamine B SD standard deviation SDS sodium dodecyl sulfate TIR tumor inhibition ratio LMC LA and PMLA double grafted chitosan LMC1 LMC grafted with 20.1% LA and 4.1% PMLA LMC2 LMC grafted with 31.1% LA and 4.2% PMLA LMC3 LMC grafted with 65.9% LA and 4.1% PMLA LMC4 LMC grafted with 67.0% LA and 8.4% PMLA LMC3/pDNA NPs iSur-pDNA loaded LMC3 NPs LMC3/PTX NPs PTX loaded LMC3 NPs LMC/PTX/pDNA NPs PTX and iSur-pDNA co-loaded LMC NPs FA-LMC3 FA modified LMC3 FA-LMC3/pDNA NPs iSur-pDNA loaded FA-LMC3 NPs FA-LMC3/PTX/pDNA NPs PTX and iSur-pDNA co-loaded FA-LMC3 NPs FA-LMC3/PTX/pGL NPs PTX and pGL3-control vector co-loaded FA-LMC3 NPs.

was prepared using the similar method. Chitosan (1 g) was dissolved in 20 mL of methanesulfonic acid, followed by an addition of LA acyl chloride. After agitation for 1 h, PMLABz acyl chloride was added and the mixture was stirred for another 4 h before 30 g of ice water mixture was added to terminate the acylation reaction. The detailed feed amounts of LA acyl chloride and PMLABz acyl chloride for LMC1-4 were listed in Table 1. The grafting degrees of LA and PMLA were determined by 1 H nuclear magnetic resonance (1H NMR, Bruker, Germany) [16]. FA modified LMC (FA-LMC) was synthesized via Schiff base reaction. Briefly, 40 mg of EDC$HCl, 40 mg of NHS, 10 mg of DMAP, and 89 mg of FA were added into 50 mL of water. After stirred for 4 h, the mixture was reacted with 80 mL of LMC (10 mg/mL) for another 12 h to obtain FA-LMC. 2.3. NPs formation and characterization To form LMC NPs, 15 mL of LMC (2 mg/mL) was sonicated at 200 W for 15 min in an ice bath. LMC NPs were incubated at 37 C for 30 min before use. PTX and pDNA were loaded into LMC NPs via sonication and electrostatic adsorption methods, respectively. As for PTX encapsulation, LMC NPs and FA-LMC3 NPs were further sonicated for 10 min following the addition of 50 mL of PTX solution in ethanol (60 mg/mL). The PTX loaded LMC NPs and FA-LMC3 NPs was purified by silica column as previously described [16]. As for pDNA encapsulation, iSur-pDNA (100 mg/ mL) were added into PTX loaded LMC NPs and FA-LMC3 NPs at a weight ratio of 12:1 and kept under vortex for 15 s to obtain LMC/PTX/pDNA NPs and FA-LMC3/PTX/ pDNA NPs. Particle size and zeta potential of LMC/PTX/pDNA NPs and FA-LMC3/PTX/pDNA NPs suspended in HCl solution (pH 5.5) were measured with Zetasizer Nano (Malvern, Worcestershire, UK). Loading capacity and encapsulation efficiency of PTX were measured as described by Zhao et al. [16]. The association of pDNA with the NPs was evaluated by the gel retardation assay on a 1% (w/v) agarose gel, and the electrophoresis was performed at 120 V for 40 min. After the NPs were centrifugated

Table 1 Grafting degrees, particle sizes, and zeta potentials of LMC/PTX/pDNA NPs and FALMC3/PTX/pDNA NPs. Indicated values were mean SD (n ¼ 3). Sample

Grafting Feed Particle size Zeta potential amount of degree (%)a (nm) (mV) acyl chloride (g)

LMC1/PTX/pDNA NPs LMC2/PTX/pDNA NPs LMC3/PTX/pDNA NPs LMC4/PTX/pDNA NPs FA-LMC3/PTX/pDNA NPs

1.3 1.3 1.3 2.6 1.3

PMLA LA PMLA LA

a

0.6 1.2 1.8 1.8 1.8

4.1 4.2 4.1 8.4 4.1

20.1 31.1 65.9 67.0 66.0

206.7 172.8 151.7 219.3 161.9

2.7 2.0 2.6 7.4 5.3

45.6 44.8 43.3 41.4 43.0

2.3 1.6 2.6 0.4 1.0

The grafting degrees were determined by 1H NMR.


B. Yu et al. / Biomaterials xxx (2014) 1e10 at 13,300 rpm for 30 min, the supernatant was collected and quantified for the pDNA by Hoechst 33258 staining and fluorimetry. The encapsulation efficiency of pDNA was calculated accordingly. QGY-7703 and L02 cells were seeded on 96-well plates at 1 104 cells/well and cultured at 37 C for 24 h. Blank LMC NPs and FA-LMC3 NPs were added into each well at the final concentrations of 0.01, 0.05, 0.2, 1, and 2 mg/mL, and incubated at 37 C for 72 h, followed by methyl tetrazolium (MTT) assay. Cells without NPs treatment served as 100% cell viability. 2.4. DNase I protection LMC/PTX/pDNA NPs and FA-LMC3/PTX/pDNA NPs containing 20 mg of pDNA were incubated with 5 U of DNase I. The variation in absorbance was continuously measured at 260 nm within a 1-h period at 37 C [13]. 2.5. EB exclusion LMC/PTX/pDNA NPs and FA-LMC3/PTX/pDNA NPs containing 4 mg of pDNA were diluted in 200 mL of 0.2 M phosphate buffered solution (PBS, pH 7.4), and then incubated with 20 mL of EB (0.1 mg/mL) for 10 min at room temperature. Fluorescence intensity was measured by fluorimetry (lex ¼ 518 nm, lem ¼ 605 nm). Results were expressed as the relative fluorescence intensity where 0 and 100% represented the presence of EB only and EB associated with naked pDNA, respectively. 2.6. Bovine serum albumin (BSA) adsorption LMC/PTX/pDNA NPs and FA-LMC3/PTX/pDNA NPs (2 mg/mL) were incubated with the equal volume of BSA (1.6 mg/mL) for 2 h at 37 C under shaking. The mixture was then centrifugated at 13,300 rpm for 30 min and the supernatant was collected to measure the amount of unadsorbed BSA by Lowry method. Results were represented as the percentage of adsorption compared with the control where BSA was incubated in the absence of the NPs.

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in the 0.5% SDS (w/v, pH 8.0) to quantify the content of PTX and pDNA in the nuclei. Results were represented as the relative percentage of internalized amounts of PTX and pDNA. 2.10. In vitro analysis of survivin expression QGY-7703 cells were seeded on 6-well plates at 2 105 cells/well and cultured for 24 h. The culture media were replaced by serum-free DMEM containing LMC/ pDNA NPs and FA-LMC3/pDNA NPs (5 mg of iSur-pDNA equivalent). After 4-h incubation, the culture media were replaced with fresh serum-containing DMEM and cells were incubated for another 72 h. RNA was isolated with Trizol reagent (Invitrogen, USA), and cDNA was reverse transcribed from 500 ng of total RNA according to the PrimeScriptÒRT reagent kit protocol (Takara Biotechnology Co., Ltd, China). Real-Time PCR was conducted with ABI PRISM 7900HT Real-Time PCR System (Applied Biosystems, USA) and the b-actin mRNA was used as an internal control. To determine survivin expression level, the transfected cells were washed with 0.2 M PBS (pH 7.4) and lysed in RIPA buffer (5 mM EDTA, 50 mM TriseHCl, 150 mM NaCl, 0.1% (w/v) SDS, 0.5% (w/v) sodium deoxycholate, 1% (v/v) NP-40, pH 8.0) containing protease inhibitor (Merck Millipore, Germany) for 20 min in ice. The extracts were mixed with solubilizing buffer (62.5 mM TriseHCl buffer pH 6.8, 0.01% (w/v) bromophenol blue, 2% (w/v) SDS, 5% (w/v) 2-mercaptoethanol, 7% (v/v) glycerol) and boiled for 10 min. Proteins were separated via 12% SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes (Merck Millipore, Germany). These membranes were incubated with rabbit anti-human survivin polyclonal antibody (Proteintech Group, USA) at 4 C overnight. After washed and probed with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature, membranes then reacted with ECL Plus Detection Reagent (Merck Millipore, Germany). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as an internal control. The protein bands were exposed with G:BOX Chemi XR5 (Syngene, UK). 2.11. Cell apoptosis and viability assay

2.7. In vitro release LMC/PTX/pDNA NPs and FA-LMC3/PTX/pDNA NPs (0.5 mL) were sealed in dialysis bags (molecular weight cut-off of 14 kDa) and dialyzed in 100 mL of 0.2 M PBS (pH 7.4) containing 0.1% (w/v) Tween 80 under agitation at 37 C. At each predetermined time, 1 mL of the release medium was taken out and centrifugated at 13,300 rpm for 15 min, and the supernatant was determined for the amount of PTX by high performance liquid chromatography assay as previously described [16]. The accumulative release amount of PTX was calculated accordingly. LMC/PTX/pDNA NPs and FA-LMC3/PTX/pDNA NPs were suspended in 0.2 M PBS (pH 7.4) and incubated at 37 C under agitation. At predetermined time intervals, the NPs were centrifugated at 13,300 rpm for 15 min. Aliquots of the supernatants were collected and the equal volume of fresh PBS was added. The pDNA content in the supernatant was determined after staining with Hoechst 33258. The accumulative release amount of pDNA was calculated accordingly. 2.8. Cellular uptake PTX and iSur-pDNA were covalently labeled with RhB and FITC, respectively. PTX (100 mg) was reacted with RhB (1 mg) in 10 mL of dichloromethane in the presence of DCC (14.5 mM) and DMAP (10.3 mM). The mixture was kept stirring for 24 h in darkness, followed by vacuum distillation. Excess RhB was eliminated via washing with water and RhB labeled PTX was obtained after vacuum drying. FITC labeled pDNA was prepared as previously described [18]. Fluorescence labeled PTX and pDNA were loaded into LMC NPs and FA-LMC3 NPs as described in Section 2.3. QGY7703 and L02 cells were seeded on 24-well plates at 1 105 cells/well and cultured for 24 h. After treatment with the NPs for 4 h, cells were washed with 0.2 M PBS (pH 7.4) and lysed with 0.5% sodium dodecyl sulfate (SDS, w/v, pH 8.0). The cell lysate was quantified for PTX and pDNA with fluorimetry and protein content with Lowry method. To elucidate the mechanisms involved in the uptake of FA-LMC3/PTX/pDNA NPs, QGY-7703 cells were incubated at 4 C for 4 h. Additionally, cells were treated with sodium azide (10 mM), chlorpromazine (10 mg/mL), methyl-b-cyclodextrin (5 mM), genistein (200 mg/mL), colchicine (100 mg/mL), and wortmannin (50 nM) at 37 C for 30 min prior to the NPs application and throughout the 4-h uptake. Results were represented as the relative uptake percentage compared to cells incubated with FALMC3/PTX/pDNA NPs at 37 C for 4 h in the absence of inhibitors. 2.9. Intracellular distribution Intracellular distribution of PTX and iSur-pDNA was evaluated via nuclear isolation [19]. Briefly, QGY-7703 cells were collected through centrifugation at 1500 rpm for 5 min following 4-h incubation with LMC/PTX/pDNA NPs and FALMC3/PTX/pDNA NPs. The cell pellet was washed with 0.2 M PBS (pH 7.4) and resuspended in TM-2 buffer (0.5 mM PMSF, 2 mM MgCl2, 10 mM TriseHCl). The cell suspensions were incubated for 1 min at room temperature and for another 5 min in ice. After lysed with 1% Triton X-100 (v/v) for 5 min in ice, cells were centrifugated at 4 C and 800 rpm for 10 min. The supernatant was collected to measure the PTX and pDNA content in the cytoplasm while the pellet was further resuspended and lysed

QGY-7703 cells were seeded on 6-well plates at 2 105 cells/well and cultured for 24 h. After treatment with LMC/PTX/pDNA NPs and FA-LMC3/PTX/pDNA NPs for 72 h, cells were trypsinized, collected, resuspended in 1 mL of binding buffer, and stained with Annexin V-FITC and propidium iodide using Annexin V-FITC apoptosis analysis kit (Tianjin Sungene Biotech Co., Ltd, China) following the manufacturer’s instructions. Cells were monitored for apoptosis analysis on a FACSCalibur flow cytometer (BD, USA). QGY-7703 cells were incubated with various concentrations of LMC/PTX/pDNA NPs and FA-LMC3/PTX/pDNA NPs for 72 h. The cytotoxicity was evaluated by MTT assay and the half maximal inhibitory concentration (IC50) values were calculated using GraphPad Software. Cells without NPs treatment served as 100% cell viability. 2.12. In vivo antitumor efficacy Hepatoma bearing mice were established via subcutaneous inoculation of 0.2 mL of H-22 mouse ascites at the axillary region of mice. When the tumor volume had reached approximately 100 mm3, tumor bearing mice were randomly assigned to seven groups: saline (control); blank FA-LMC3 NPs; PTX injection; FA-LMC3/ pDNA NPs; FA-LMC3/PTX/pGL NPs; LMC3/PTX/pDNA NPs; FA-LMC3/PTX/pDNA NPs. PTX and iSur-pDNA dosages were set as 3 mg/kg and 2.5 mg/kg, respectively. Drug administration was conducted three times through tail vein injection with two days spaced between each administration, and the day of first administration was recorded as day 0. Tumor volumes and body weights were recorded every other day. Tumor volumes were calculated based on the equation: (L S2)/2, where L represents the long diameter and S represents the short diameter. Mice were sacrificed when the tumor volume of the control group exceeded 3000 mm3, and the excised tumors were weighed. The tumor inhibition ratio (TIR) was calculated using the following equation: TIR (%) ¼ (1 W T/WS) 100%, where W T and WS refer to the tumor weight of the test and saline groups, respectively. Another 42 tumor bearing mice were grouped and administered with various drug formulations according to the above method, and then carefully maintained for survival analysis. 2.13. Statistical analysis Data were expressed as the mean standard deviation (SD). Statistical analysis was performed using unpaired t test between two groups or single factor analysis of variance among three or more groups. Differences were judged to be significant at P < 0.05.

3. Results 3.1. Preparation and characterization of LMC and FA-LMC LMC was synthesized through double grafting of LA and PMLA onto chitosan. Different grafting degrees of LA and PMLA were


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achieved through adjusting their feed amounts as listed in Table 1. As depicted in Supplementary Information Fig. S1, the grafting degrees of LA and PMLA were determined by comparing peak areas of linoleic methyl protons of LA (d ¼ 0.8 ppm) and methylene protons of PMLA (d ¼ 4.7 ppm) with sugar protons of chitosan (d ¼ 2.9e3.7 ppm), respectively [16]. The 1H NMR spectrum of FALMC3 demonstrated the characteristic peaks for FA, which were assigned to the aromatic CeNH (6.7 ppm), 1-benzene CH (7.7 ppm), and 2-pyrazine CH (8.8 ppm), respectively, assuring that FA was conjugated to LMC. 3.2. NPs formation and characterization As shown in Table 1, particle sizes of LMC/PTX/pDNA NPs decreased with the increasing grafting degree of LA and the decreasing grafting degree of PMLA. LMC/PTX/pDNA NPs possessed similar positive zeta potential. Particle size and zeta potential of FALMC3/PTX/pDNA NPs were similar to those of LMC3/PTX/pDNA NPs, indicating the neglectable effect of FA on the compact nanostructure and positive surface charge of LMC/PTX/pDNA NPs. As listed in Supplementary Information Table S1, when pDNA was simultaneously loaded, PTX was effectively encapsulated into LMC NPs through sonication and the encapsulation efficiencies were above 80%. The loading capacity of PTX attenuated with the decreasing grafting degree of LA and the increasing grafting degree of PMLA. The encapsulation efficiency and loading capacity of PTX for LMC3/PTX/pDNA NPs was similar to those for LMC3/PTX NPs, meaning that pDNA exerted inappreciable influence on the PTX loading. Additionally, the loading capacity of PTX was significantly elevated after FA modification (P < 0.05). As illustrated in Supplementary Information Fig. S2, the migration of pDNA could be completely retarded at the LMC/pDNA weight ratio of 12, revealing the efficient entrapment of pDNA into LMC/PTX NPs. The strong binding affinity of LMC/PTX NPs for pDNA was further confirmed by the encapsulation efficiency, which exceeded 90% in all formulations (Supplementary Information Fig. S3). Blank LMC NPs and FA-LMC3 NPs exerted inappreciable cytotoxicity on QGY-7703 and L02 cells even at 2 mg/mL which was 10fold higher than the maximum dose applied in the other cellular studies (Supplementary Information Fig. S4), excluding their nonspecific cytotoxicity.

3.3. DNase I protection As illustrated in Fig. 1A, naked pDNA was largely digested as indicated by the rapid increase in OD260 nm. In comparison, LMC/ PTX/pDNA NPs could effectively protect pDNA from enzymatic degradation as evidenced by the remarkable reduction in OD260 nm variation, in which LMC3/PTX/pDNA NPs exhibited the strongest protection capacity. The similar variation in OD260 nm between LMC3/pDNA NPs and LMC3/PTX/pDNA NPs indicated that the PTX loading did not affect the pDNA protection. However, variation in OD260 nm was elevated after FA modification, implying the decrease of protection capacity.

3.4. EB exclusion As shown in Fig. 1B, all the LMC/PTX/pDNA NPs could competitively occupy the binding sites on pDNA against EB as indicated by the notably decreased fluorescence intensity. The fluorescence intensity was negatively correlated with the binding affinity towards pDNA, which increased with the elevation of grafting degrees of LA and PMLA. There was no significant difference in fluorescence intensity between LMC3/pDNA NPs and LMC3/PTX/pDNA NPs, revealing that PTX entrapment did not affect the ability of LMC NPs to bind pDNA. Besides, the higher fluorescence intensity was observed in FA-LMC3/PTX/pDNA NPs as compared with LMC3/PTX/ pDNA NPs, demonstrating the decreased binding affinity towards pDNA after FA modification.

3.5. BSA adsorption The adsorption percentage of BSA to LMC/PTX/pDNA NPs and FA-LMC3/PTX/pDNA NPs was shown in Fig. 2. The less BSA was adsorbed to LMC/PTX/pDNA NPs with the higher grafting degree of LA and PMLA. Compared with LMC3/PTX NPs, the lower BSA adsorption to LMC3/PTX/pDNA NPs indicated that pDNA binding would enhance the suppression ability of protein adsorption owing to its negative charge. No significant difference in protein adsorption was detected between LMC3/pDNA NPs and LMC3/PTX/pDNA NPs, indicating that the PTX loading exerted minimal influence on the protein adsorption. In addition, the adsorption percentage of FA-LMC3/PTX/pDNA NPs was less than that of LMC3/PTX/pDNA,

Fig. 1. (A) DNase I protection assay determined by the alteration in OD260 nm of LMC/PTX/pDNA NPs and FA-LMC3/PTX/pDNA NPs following DNase I treatment for 1 h. (B) pDNA condensation efficiency of LMC/PTX/pDNA NPs and FA-LMC3/PTX/pDNA NPs determined by EB exclusion assay. Indicated values were mean SD (n ¼ 3). ***P < 0.001.



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LMC4/PTX/pDNA NPs were 36.5%, 39.9%, 46.4%, and 54.5%, respectively, indicating that higher grafting degrees of LA and PMLA promoted the pDNA disassociation. No significant difference in release profiles of pDNA between LMC3/pDNA NPs and LMC3/PTX/ pDNA NPs demonstrated that the PTX loading did not affect the pDNA release. After FA modification, the release rate of pDNA was further increased. 3.7. Cellular uptake

Fig. 2. The adsorption percentage of BSA to LMC/PTX/pDNA NPs and FA-LMC3/PTX/ pDNA NPs. Indicated values were mean SD (n ¼ 3). *P < 0.05, ***P < 0.001.

suggesting the superiority of FA modification in avoiding nonspecific protein adsorption. 3.6. In vitro release As demonstrated in Fig. 3A, PTX exhibited an initial burst stage followed by a slower and sustained release for LMC/PTX/pDNA NPs and FA-LMC3/PTX/pDNA NPs. The 72-h accumulative release percentages of PTX from LMC1/PTX/pDNA NPs, LMC2/PTX/pDNA NPs, LMC3/PTX/pDNA NPs, and LMC4/PTX/pDNA NPs were 95.8%, 91.0%, 87.0%, and 83.6%, respectively, indicating that higher grafting degree of LA and PMLA inhibited the PTX release. No differences in the release profiles of PTX between LMC3/PTX NPs and LMC3/PTX/ pDNA NPs demonstrated that the pDNA loading did not affect the PTX release. PTX release was further delayed by FA modification. Fig. 3B depicted the accumulative release of pDNA from LMC/ PTX/pDNA NPs and FA-LMC3/PTX/pDNA NPs within 72 h. Compared with PTX, the release rate of pDNA decreased in all NPs. At 72 h, the accumulative release percentages of pDNA from LMC1/ PTX/pDNA NPs, LMC2/PTX/pDNA NPs, LMC3/PTX/pDNA NPs, and

As illustrated in Fig. 4AeB, the cellular uptake of pDNA and PTX in both QGY-7703 and L02 cells increased with the increase of grafting degree of LA, while declined with the increase of grafting degree of PMLA. Besides, the cellular uptake of PTX and pDNA in LMC3/PTX/pDNA NPs was similar to those in LMC3/PTX NPs and LMC3/pDNA NPs, respectively, implying the negligible mutual interference between PTX and pDNA in cellular uptake. FA modification dramatically enhanced the cellular uptake in both QGY7703 and L02 cells. Compared with L02 cells, QGY-7703 cells internalized higher amounts of PTX and pDNA. As demonstrated in Fig. 4C, low uptake temperature (4 C) or sodium azide pretreatment yielded remarkable depression in cellular uptake of FA-LMC3/PTX/pDNA NPs, indicating that an energy-dependent endocytic process was involved. Moreover, the internalization was inhibited by chlorpromazine and methyl-bcyclodextrin, implying the involvement of clathrin-mediated and cholesterol-dependent pathway. Genistein, colchicine, and wortmannin exerted negligible effect on the cellular internalization of FA-LMC3/PTX/pDNA NPs, excluding the involvement of caveolinmediated endocytosis, cytoskeleton reorganization or macropinocytosis. 3.8. Intracellular distribution Fig. 5 showed subcellular distribution of PTX and pDNA in QGY7703 cells after 4-h incubation with LMC/PTX/pDNA NPs and FALMC3/PTX/pDNA NPs. PTX mainly distributed in the cytoplasm with similar amounts among all formulations. Nuclear accumulation of pDNA was positively correlated with the grafting degree of LA, but negatively correlated with PMLA. The intracellular distribution of PTX and pDNA in LMC3/PTX/pDNA NPs was similar to those in LMC3/PTX NPs and LMC3/pDNA NPs, respectively, suggesting the negligible mutual interference between PTX and pDNA in intracellular distribution. After FA modification, the nuclear accumulation of pDNA was further elevated.

Fig. 3. In vitro release profiles of PTX (A) and iSur-pDNA (B) from LMC/PTX/pDNA NPs and FA-LMC3/PTX/pDNA NPs in 0.2 M PBS (pH 7.4) at 37 C. Indicated values were mean SD (n ¼ 3).


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Fig. 4. Cellular uptake of PTX (A) and iSur-pDNA (B) in QGY-7703 and L02 cells following 4-h incubation at 37 C. ***P < 0.001. (C) The effects of low temperature and chemical inhibitors on the internalization of FA-LMC3/PTX/pDNA NPs into QGY-7703 cells. Cells incubated at 37 C for 4 h without inhibitors pretreatment served as a control. Indicated values were mean SD (n ¼ 3). **P < 0.01, ***P < 0.001.

3.9. In vitro analysis of survivin expression As illustrated in Fig. 6A, the gene silencing efficiency of LMC1/ pDNA NPs, LMC2/pDNA NPs, LMC3/pDNA NPs and LMC4/pDNA NPs were 26.9%, 34.0%, 46.6%, and 39.4%, respectively, demonstrating the correlation of enhanced gene silencing efficiency with increasing grafting degree of LA and deceasing grafting degree of PMLA. Moreover, FA modification further improved the gene silencing efficiency to 71.8%. Western blot showed that FA-LMC3/ pDNA NPs induced the highest gene transfection in the protein level (Fig. 6B), which was well accorded with Real-Time PCR assay. 3.10. Cell apoptosis and viability assay As demonstrated in Fig. 7, after 72-h treatment, the percentages of apoptosis induced by LMC1/PTX/pDNA NPs, LMC2/PTX/pDNA NPs, LMC3/PTX/pDNA NPs, and LMC4/PTX/pDNA NPs were 60.23%, 83.13%, 95.06%, and 84.16%, respectively. These results indicated that an enhanced cell apoptosis was correlated with increasing grafting degree of LA and decreasing grafting degree of PMLA. Compared with LMC3/PTX/pDNA NPs, LMC3/PTX NPs and LMC3/ pDNA NPs decreased the percentages of apoptosis to 55.77% and 15.98%, respectively, revealing the synergistic effect arising from

the co-delivery of PTX and iSur-pDNA. Following treatment with FA-LMC3/PTX/pDNA NPs, cells exhibited the higher apoptosis rate (96.57%) as compared with LMC3/PTX/pDNA NPs. As depicted in Fig. 8, the IC50 values of LMC/PTX/pDNA NPs (0.09e0.28 mg/mL) were lower than that of free PTX (0.41 mg/mL). The IC50 values of LMC1/PTX/pDNA NPs, LMC2/PTX/pDNA NPs, LMC3/PTX/pDNA NPs, and LMC4/PTX/pDNA NPs were 0.28, 0.16, 0.09, and 0.14 mg/mL, respectively, indicating that more LA substitution and less PMLA substitution were favorable for stronger cytotoxicity. The IC50 value of LMC3/PTX NPs (0.32 mg/mL) was significantly higher than that of LMC3/PTX/pDNA NPs, also suggesting the synergistic effect from the co-delivery of PTX and iSurpDNA. Furthermore, FA-LMC3/PTX/pDNA NPs exhibited the highest cytotoxicity with the IC50 value of 0.06 mg/mL. 3.11. In vivo antitumor efficacy The increase in body weight among all groups throughout the experiment indicated the natural growth of mice (Supplementary Information Table S2). As depicted in Fig. 9AeB, blank FA-LMC3 NPs showed negligible antitumor efficacy. The antitumor efficacy of PTX injection was less effective compared with PTX encapsulated NPs. Significant tumor regressions were observed in FA-LMC3/

Fig. 5. Intracellular distribution of PTX (A) and iSur-pDNA (B) in QGY-7703 cells following treatment with LMC/PTX/pDNA NPs and FA-LMC3/PTX/pDNA NPs for 4 h. Indicated values were mean SD (n ¼ 3). *P < 0.05, **P < 0.01, ***P < 0.001.



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Fig. 8. In vitro viability of QGY-7703 cells following exposure to LMC/PTX/pDNA NPs and FA-LMC3/PTX/pDNA NPs at various PTX concentrations for 72 h. Indicated values were mean SD (n ¼ 6).

Fig. 6. (A) Survivin mRNA expression in QGY-7703 cells following treatment with LMC/ pDNA NPs and FA-LMC3/pDNA NPs for 72 h. Silencing efficiency was expressed as percentage values of QGY-7703 cells without NPs treatment. Indicated values were mean SD (n ¼ 3). *P < 0.05, **P < 0.01, ***P < 0.001. (B) Western blot analyses on survivin expression of QGY-7703 cells treated with LMC/pDNA NPs and FA-LMC3/pDNA NPs. Untreated QGY-7703 cells served as a control.

pDNA NPs and FA-LMC3/PTX/pGL NPs with the TIR of 45.67% and 78.53%, respectively, while FA-LMC3/PTX/pDNA NPs exhibited the highest TIR of 94.75% (Supplementary Information Table S2), suggesting the synergistic in vivo antitumor effect of PTX and iSurpDNA. The active targeting ability of FA was evidenced by the stronger antitumor efficacy of FA-LMC3/PTX/pDNA NPs than that of LMC3/PTX/pDNA NPs. However, LMC3/PTX/pDNA NPs were more effective than FA-LMC3/PTX NPs in tumor regression, which proposed that the synergistic antitumor effect of PTX and iSur-pDNA resulting from co-delivery systems could outperform the antitumor efficacy of active targeting mediated by FA modification. Fig. 9C exhibited the survival rate of mice. Mice in the groups of saline and blank FA-LMC3 NPs suffered rapid death within 30 days. Compared with PTX injection treatment (died within 44 days) or single administration with FA-LMC3 NPs (43 days for iSur-pDNA and 53 days for PTX), the survival rate of LMC3/PTX/pDNA NPs and FA-LMC3/PTX/pDNA NPs groups remained 100% up to 80 days.

Fig. 7. Evaluation on the apoptosis of QGY-7703 cells treated with LMC/PTX/pDNA NPs and FA-LMC3/PTX/pDNA NPs for 72 h by flow cytometry.


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Fig. 9. (A) Tumor growth curves of H-22 tumor bearing mice following treatment with PTX injection and various NPs formulations. Indicated values were mean SD (n ¼ 6). (B) Photographs of tumor from each treatment group excised on day 14. (C) Survival curves of H-22 tumor bearing mice treated with PTX injection and various NPs formulations.

4. Discussion Numerous NPs-based delivery systems have been attempted to achieve high selectivity and efficiency in the treatment of tumor. However, due to the complex molecular mechanisms of tumor, their ultimate antitumor efficacy is actually unsatisfactory in therapies aiming at single target. Therefore, co-delivery strategy involving PTX and iSur-pDNA was tended to achieve enhanced antitumor efficacy through the synergistic effect. PTX as a hydrophobic chemotherapeutic drug can stabilize microtubule and retain cell cycle in the late G2/M phase, thereby mediating tumor cell death [20]. iSur-pDNA as a therapeutic gene can suppress the expression of oncogene survivin, thereby promoting apoptosis of tumor cells [21]. Owing to their different antitumor mechanisms, PTX and iSur-pDNA were selected here for the synergistic effect in the treatment of tumor. In this study, amphiphilic LMC NPs with LA chains as a hydrophobic inner core and PMLA chains as a cationic hydrophilic shell can simultaneously encapsulate PTX and pDNA. Moreover, various grafting degrees of LA and PMLA were achieved to elucidate the effects of hydrophobic and hydrophilic fragments on the co-delivery behaviors of LMC NPs. FA as an active targeting moiety to tumor cells was conjugated to further enhance the antitumor efficacy of LMC/PTX/pDNA NPs. Particle size and zeta potential were known to influence the pharmacokinetic behaviors of NPs, which ultimately affected their tumor accumulation [22,23]. The higher grafting degree of LA was correlated with more compact hydrophobic core and smaller particle size, whereas PMLA substitution increased the particle size owing to the extended hydrophilic shell. As a result, LMC3/PTX/ pDNA NPs with higher grafting degree of LA and lower grafting degree of PMLA exhibited the compact nanostructure with the

smallest particle size of about 150 nm (Table 1), which is more beneficial to avoid the capture by reticuloendothelial system [24]. The positive charges of LMC NPs at pH 5.5 allowed them to condense the negatively-charged pDNA. As revealed by the gel retardation assay (Supplementary Information Fig. S2) and the encapsulation efficiency determination (Supplementary Information Fig. S3), pDNA was effectively condensed into all the NPs. PTX loading capacity attenuated with the increase of PMLA moieties because of the thicker hydrophilic shell, whereas increased with the higher grafting degree of LA (Supplementary Information Table S1) owing to the hydrophobic interactions between LA moieties and PTX. The ability against enzymatic degradation was regarded as the prerequisite for efficient gene therapy. LMC/PTX/pDNA NPs with higher hydrophobicity possessed the stronger protection capacity of pDNA against the nuclease degradation (Fig. 1), consistent with a previous report [17]. This result might be attributed to the stronger shielding of the enzymatic action sites by stronger hydrophobic interaction and lower permeability of DNase I [13]. In addition, it was known that the adsorption of non-specific proteins in the blood stream would induce the aggregation and rapid clearance of NPs [25,26]. Since LA modification could compact the hydrophobic core, the surface area was decreased, which led to the reduced opportunity for BSA to bind onto LMC/PTX/pDNA NPs (Fig. 2). Interestingly, owing to the decrease of the zeta potential after loading negatively-charged pDNA, co-delivery systems showed lower adsorption of non-specific protein than LMC3/PTX NPs. Ideal delivery systems should exhibit minimal premature loss of cargos prior to releasing them efficiently within target cells. As for LMC/PTX/pDNA NPs, the rapid premature release of PTX and poor intracellular dissociation of pDNA represented the major rate-



limiting processes for their functions. Delicate regulation of drug release rate through optimizing grafting degrees of LA and PMLA would therefore be required. Increased conjugation of LA and PMLA endowed LMC NPs with stronger hydrophobic binding with PTX and denser hydrophilic layer, respectively, thus reducing the premature release of PTX. In addition, hydrophobic LA and negativelycharged PMLA would attenuate the electrostatic interaction between pDNA and amino groups of chitosan, resulting in accelerated release of pDNA. From this point of view, LMC3/PTX/pDNA NPs and LMC4/PTX/pDNA NPs were favorable for the extracellular encapsulation and intracellular unpacking of both PTX and pDNA. Upon arrival at tumor cells, cellular uptake and the relevant intracellular fate turned out to be the dominant parameters for therapeutic effect of NPs. Enhanced hydrophobicity caused by more LA substitution would be favorable for cellular uptake of both PTX and pDNA due to their hydrophobic interaction with lipid membrane (Fig. 4AeB). By contrast, the higher PMLA modification led to lower cellular uptake, which might be attributed to corresponding larger particle size and thicker hydrophilic shell. Uptake mechanism was closely related to the intracellular fate of NPs [27]. FALMC3/PTX/pDNA NPs were internalized via clathrin-mediated pathway (Fig. 4C), thus lysosomal entrapment would be the prevailing limitation. According to our previous study, LMC NPs were capable of escaping the endo-lysosomes via proton-sponge effect [16]. Following the internalization, the majority of PTX was distributed into the cytoplasm (Fig. 5A), leading to mitotic arrest. By contrast, LMC3/PTX/pDNA NPs induced higher nuclear accumulation of pDNA (Fig. 5B), which might be resulted from their superiority in cellular uptake (Fig. 4B) and dissociation of pDNA (Fig. 1B). Such result might be advantageous for pDNA transfection. The silencing efficiency of iSur-pDNA against survivin was a comprehensive reflection of the delivery efficacy of LMC NPs. As for LMC3/pDNA NPs, owing to their supreme protection ability, highest cellular uptake, as well as the preferable nuclear accumulation, the most robust inhibition of survivin was achieved (Fig. 6). Together with the highest internalization of PTX and the negligible influences of PTX loading on the protection, binding affinity, protein adsorption, in vitro release, cellular uptake, and intracellular accumulation of pDNA, the strongest cell growth inhibition was observed in LMC3/PTX/pDNA NPs (Fig. 8), in agreement with their highest apoptotic ratio (Fig. 7). In spite of high colloidal stability against negatively charged proteins and fast pDNA dissociation, LMC4/PTX/pDNA NPs presented poor cellular uptake (Fig. 4AeB) and insufficient protection against DNase I (Fig. 1A), which might collaboratively decrease the intracellular concentration of pDNA, leading to poorer inhibition of survivin, lower apoptotic ratio, and weaker cell growth inhibition compared to LMC3/PTX/pDNA NPs. To further enhance the antitumor efficacy of LMC/PTX/pDNA NPs, the conjugation of active-targeting ligand that could bind to specific receptor in tumor cells was needed. Based on the highest in vitro antitumor efficiency of LMC3/PTX/pDNA NPs, we prepared FA-LMC3/PTX/pDNA NPs to evaluate the effects on tumor therapy exerted by the active targeting. After FA modification, the loading capacity of PTX increased (Supplementary Information Table S1), which might be ascribed to the enhanced hydrophobic interaction of PTX with FA. Moreover, FA modification reduced non-specific protein adsorption because of the shielding of the binding sites. After the arrival at the tumor tissues via enhanced permeability and retention (EPR) effect, FA ligand could actively interact with the overexpressed FA receptor on tumor cell surfaces, resulting in the increased cellular uptake in QGY-7703 cells (Fig. 4A). FA modification exerted the negative effect on the pDNA binding affinity of LMC NPs as evidenced by EB exclusion assay (Fig. 1B) and induced rapid pDNA release (Fig. 3B). The accelerated intracellular dissociation of pDNA facilitated the entry of free pDNA via the nuclear pore,

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resulting in high pDNA accumulation in the nuclei. Collectively, the decreased protein adsorption, improved cellular uptake, and enhanced nuclear accumulation were responsible for the superiority of FA-LMC3/PTX/pDNA NPs in gene silencing, cell apoptosis, and cell growth inhibition. Considering the outstanding performance of FA-LMC3/PTX/ pDNA NPs in the in vitro investigations, they were selected for evaluating the in vivo synergistic effect of PTX and iSur-pDNA. As compared with single delivery of PTX or pDNA, FA-LMC3/PTX/ pDNA NPs showed the higher antitumor efficiency and longer survival time of tumor-bearing mice (Fig. 9), in accordance with the results of in vitro cell apoptosis (Fig. 7) and cytotoxicity assays (Fig. 8). The in vivo active-targeting ability of FA-LMC3/PTX/pDNA NPs was also confirmed by their superior antitumor efficacy compared with LMC3/PTX/pDNA NPs. In addition, it was worth noting that the TIR and survival ability of mice treated with LMC3/ PTX/pDNA NPs was superior to those treated with FA-LMC3/PTX/ pGL NPs, suggesting that the synergistic antitumor effect of PTX and iSur-pDNA induced by targeting multiple mechanisms could be more effective than improvement in the monotherapy mediated by ligand modification. This finding might provide an evidence for the superiority of co-delivery systems for antitumor drugs and genes. 5. Conclusions FA modified amphiphilic LMC NPs were synthesized for the codelivery of PTX and iSur-pDNA. Through combining the therapeutic effects of PTX and pDNA and avoiding the disadvantageous interactions between PTX and pDNA in the entire delivery processes, this co-delivery system showed a significantly enhanced in vitro and in vivo antitumor efficiency compared with single administration with PTX or pDNA. Notably, the hydrophobicity and hydrophilicity of LMC/PTX/pDNA NPs were demonstrated to exert significant effects on the entire delivery processes. Hydrophobic LA modification could improve PTX loading, resistance of protein adsorption, and cellular uptake, while hydrophilic PMLA modification contributed to the reduced premature release of PTX and the accelerated intracellular disassociation of pDNA. Moreover, the antitumor efficacy of LMC/PTX/pDNA NPs with an elaborate balance between hydrophobicity and hydrophilicity was further improved through the active targeting induced by the ligand modification. The idea of simultaneously optimizing the hydrophobic, hydrophilic, and targeting ligand modification to promote the antitumor efficacy could provide critical insights into the design of amphiphilic nanocarriers as co-delivery systems for tumor therapy. Acknowledgments The authors are thankful for the financial support from the National Natural Science Foundation of China (No. 30873204) and the Science and Technology Commission of Shanghai Municipality of China (No. 14XD1400800 and 1052nm03500). Appendix A. Supplementary data Supplementary data related to this article can be found online at http://dx.doi.org/10.1016/j.biomaterials.2014.04.095. References [1] Bock C, Lengauer T. Managing drug resistance in cancer: lessons from HIV therapy. Nat Rev Cancer 2012;12:494e501. [2] Greco F, Vicent M. Combination therapy: opportunities and challenges for polymer-drug conjugates as anticancer nanomedicines. Adv Drug Deliv Rev 2009;61:1203e13.


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