Hepatic Carcinoma Selective Nucleic Acid Nanovector Assembled by

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A Hepatic Carcinoma Selective Nucleic Acid Nanovector Assembled by Endogenous Molecules Based on Modular Strategy Fang Xie, Luchen Zhang, Jinliang Peng, Chong Li, Jun Pu, Yuhong Xu, and Zixiu Du Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.6b00709 • Publication Date (Web): 30 Dec 2016 Downloaded from http://pubs.acs.org on January 1, 2017

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Molecular Pharmaceutics

A Hepatic Carcinoma Selective Nucleic Acid Nanovector Assembled by Endogenous Molecules Based on Modular Strategy Fang Xie a, Luchen Zhang a, Jinliang Peng a, Chong Li a, Jun Pu b, Yuhong Xu a & Zixiu Du a * a

School of Pharmacy, Shanghai Jiao Tong University, 800 Dongchuan Road, Shanghai 200240,

China b

School of Medicine, Shanghai Jiao Tong University, 280 Chongqing South Road, Shanghai,

200127, China * Correspondence to Zixiu Du ([email protected])

Abstract

We rationally formulated a nucleic acid nano-vector platform utilizing endogenous molecules in the following steps: nucleic acids are initially packed by a multifunctional peptide and a cationic liposome to form positively charged ternary complexes through electrostatic interaction; then the ternary complexes were coated with hyaluronic acid (HA) to form negatively charged quaternary nanocomplexes (Q-complexes). Among the components of Q-complexes, the multifunctional peptide was composed of a polysixteen-arginine (R16) and a hepatic tumor-targeted cell penetrating peptide (KRPTMRFRYTWNPMK); the cationic lipid component included DOTAP and fusogenic lipid DOPE; the HA component shielded the cationic ternary complexes and actively targeted the CD44 overexpressed on the surface of tumor cells. Q-complexes have showed a relatively high stability in the medium, and HA component partially separated from the nanocomplexes after the Q-complexes bound to the cancer cells. The Q-complexes showed significantly enhanced nucleic acid delivery activity than the corresponding quaternary complexes containing R16 and non-visible cytotoxicity in SCMM-7721 cells. In vivo, a selected Q-complex HLP1R specifically targeted and entered tumor cells without affecting normal tissues. Furthermore, HLP1R wrapped survivin siRNA efficiently and silenced the targeting gene in the liver orthotropic transplantation tumor models and showed nontoxic in vivo. This study reveals that Q-complexes are reasonable and feasible gene therapeutic carriers.

Keywords: Hepatic carcinoma selective delivery system; Modular design; Multifunctional peptide; Cationic lipid component; Hyaluronic acid.

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1. Introduction Studies on nucleic acid therapy have focused on synthetic delivery because of their large genetic payload, lower immunogenicity and easier synthesis compared with viral vectors1, 2. To achieve therapeutic effect, synthetic vectors must accomplish a series of tasks, including wrapping nucleic acids tightly, avoiding renal clearance from the blood, preventing non-specific interactions, reaching targeted cells through the enhanced permeability and retention (EPR) effect3 and active targeting property of vector, and mediating cell entry and endosomal escape to release cargoes in a time-dependent manner.4 However, most synthetic vectors have not achieved the ideal systemic delivery of nucleic acids because of highly chemical toxicity and low transfection efficiency.5, 6 Cationic polymers and liposomes are two primary synthetic vectors that generally wrap nucleic acids through electrostatic interactions, circulate in the blood through PEGylation, and bind to targeted cells through conjugation with pre-designed binding moieties.7-10. However, chemical conjugation of these molecules with target moieties leads to uncertain toxicity and metabolites, considering the limitations of PEGylation of pharmaceutics in clinical practice and animal models; such limitations include antibody production,11 hypersensitivity,12 and accelerated blood clearance phenomenon13. In addition, the highly chemical toxicity and low biocompatibility of packing vectors, such as PEI, PLL, and PAMAM, also hamper the clinical applications of synthetic delivery systems. Hyaluronic acid (HA), a biocompatible and biodegradable natural polysaccharide, is an alternative material for stealth behavior and tumor-targeted gene delivery; HA is anchored on the vector to shield the positive charge on the surface of nanoparticles and target CD44 overexpressed on the surface of tumor cells.14 For example, Lee, et al., used the stabilized calcium phosphate nanoparticles containing dopa-hyaluronic acid to achive target-specific delivery of siRNA15, Yamada, et al., investigated the sepecific target of HA-coating nanocarriers to CD44 positive tumor cells, and found HA could selectively lead nanocomplexes to enter cancer cells through CD44-mediated pathway.16 Since unmodified HA significantly decreases transfection efficiency duo to its anionic property.17, 18 HA is usually used in a modified manner and can be conjugated to cationic polymers by using chemical linkers.19, 20 However, the introduced linkers may increase the toxicity of the delivery systems; this effect nullifies the advantages of using HA


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as natural nontoxic molecules. Nonselective cell-penetrating peptides (CPPs) are used to improve the transfection efficiency of the vector, whereas their wide cell-penetrating property limits their application in target delivery. Recently, a series of tumor lineage-homing cell-penetrating peptides with specific tumor cell selectivity and dynamin-dependent endocytic pathway-mediated penetration have been obtained using mRNA display technology.21 These peptides had exhibit potential for cancer therapy and have been integrated into multifunctional drug vectors to achieve target delivery22, 23, their use for nucleic acid delivery has not been evaluated yet. Considering that the degradation products of peptides are endogenous amine acid residues and the cationic phospholipid liposomes composed of DOTAP and DOPE24 exhibits potential to be developed clinically,4 both of them have been used to package nucleic acids through electrostatic interactions for reducing toxicity and enhancing the biocompatibility of the packing vectors;4 however, the low transgene activity of these molecules limit their clinical application as therapeutic vectors. The lipopolyplexes, namely, cationic Receptor-Targeted Nanocomplexes (RTNs), are composed of cationic liposome, such as DOTAP/DOPE and DOTMA/DOPE, and integrin-targeted cationic peptide, such as K16GACRRETAWACG. These nanocomplexes were designed based on modular strategy and targeted the specific cells by the targeted acid amine sequences of the peptide, such as GACRRETAWACG, which exhibited higher gene transfection efficiency and improved specific targeting compared with binary polyplexes and lipoplexes.25-27 Our previous study confirmed the fusogenic lipid DOPE in RTNs could promote the endosomal escape of nanocomplexes after lipid bilayer fusion.28 In this study, we initially constructed positively ternary complexes based on similar selfassembly principles of RTNs28 by using DNA or siRNA, cationic liposomes, and multifunctional peptides fused by hepatic tumor cell-penetrating peptide KRPTMRFRYTWNPMK and polyarginine (R16). HA was then coated on the surface of the nanocomplexes to form negatively charged quaternary nanocomplexes (Q-complexes). The biophysical characteristics of Qcomplexes were examined by DLS, Zetasizer Nano and TEM analyses. The in vitro delivery efficiency of Q-complexes was investigated through DNA and siRNA transfections. The effects of peptide and HA on the delivery efficiency of Q-complexes were explored through DNA cellular uptake and intracellular transfection. The in vivo tissue distributions and siRNA target delivery efficiency of Q-complexes were assessed using subcutaneous xenograft and liver



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orthotropic transplanted tumors, respectively.

Materials and methods Materials The lipids 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-snglycero-3-phosphatidyl-ethanolamine (DOPE) were purchased from Sigma–Aldrich (Sigma, USA). Peptides R16KRPTMRFRYTWNPMK (P1), R16KMPNWTYRFRMTPRK (P2) and R16 (P3) were purchased from ChinaPeptides (Shanghai, China). Hyaluronic acid (Mw = 34 kDa) was obtained from Shandong Freda Biotechnology Co., Ltd., China. The pGL3 reporter plasmid (Promega, USA) was subcloned into the eukaryotic expression vector pCI (Qiagen, USA). Lipofectamine2000 (1 mg/mL) was purchased from Invitrogen (Life Technologies, USA), and PEI25kDa was purchased from Sigma-Aldrich (Sigma, USA). SMMC -7721 cells were provided by the Institute of Biochemistry and Cell Biology (SIBS, CAS, China). The siRNA sequences (Genepharma, China) used are as follows: negative siRNA labeled with or without Cy3 (sense: 5′-UUCUCCGAACGUGUCACGUTT-3; antisense: 5′-ACGUGACACGUUCGGAGAATT-3′); survivin

siRNA:

(sense:

AGCGCAACCGGACGAAUGCdtdt);

5′-GCAUUCGUCCGGUUGCGCUdtdt-3′; Luciferase

siRNA

(sense:

antisense: 5′-

CUUACGCUGAGUACUUCGATT-3′; antisense: 3′-TTGAAUGCGACUCAUGAAGCU-5′). All chemical reagents were purchased from Sigma–Aldrich (Sigma, USA). Preparation of Ternary complexes (LPD or LPR) and Q-complexes (HLPD or HLPR). The liposome DOTAP/DOPE was formulated at a 1:1 weight ratio of cationic and neutral lipids.28 Liposomes were prepared by briefly mixing individual lipids in chloroform, followed by rotary evaporation under a partial vacuum to produce a thin lipid film. Lipids were then rehydrated with distilled water while rotating the solution overnight. The solution was sonicated until clear to obtain a liposome solution of 2 mg/mL. Ternary complexes (LPD or LPR) were formed by adding the cationic liposome and peptide mixture to the nucleic acid solution at a weight ratio of 1:4:1 (i.e., charge ratio of 0.3:4.4:1 for P1 and P2; charge ratio of 0.3:8.4:1 for P3) in distilled water with DNA or siRNA at a final concentration of 1 µg per 100 µL. Afterwards, it was incubated for 30 mins at room temperature to allow for self-assembly. Next, hyaluronic acid to DNA or siRNA at weight ratios of 14:1 (i.e., 5.9:1 charge ratio) was added to the mixed solutions and mixed well. Afterwards, the solutions


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were incubated for an additional 15 mins at room temperature to allow for Q-complex formulation. The distilled water was replaced with Opti-MEM (Gibco, Life Technologies, USA) for cell transfection measurements, and Q-complexes formulated in the 5 % glucose for in vivo injection. Hydrodynamic size and zeta potential measurements. The hydrodynamic sizes and zeta potentials of Q-complexes in distilled water and 5 % glucose, were determined by dynamic light scattering (DLS) using a Malvern Nano-ZS90 (Malvern Instruments, Malvern, UK). Negative staining transmission electron microscopy (TEM). A 5 µL aliquot of the Qcomplexes was applied on a 230-mesh copper grid coated with a Formvar/carbon support film (Zhongjingkeyi Technology Co., Ltd., Beijing, China). Sample was then negatively stained with 1% phosphotungstic acid. Imaging was carried out with a Philips CM120 BioTwin Transmission Electron Microscope and operated at an accelerating voltage of 120 kV. Gel electrophoresis assay. Briefly, 1 µg of DNA and 10 µL of complex suspensions were used in the gel electrophoresis assay. All samples, except for the 1 kb DNA Ladder (Foregene, Chengdu, China), were added with 2 µL of 6× loading buffer (Thermo Scientific, USA) before loading the sample onto a 1% (for DNA) or 3% w/v (for siRNA) agarose gel in Tris-acetateEDTA (TAE) buffer, containing 3 µL of GelRed. The samples were then subjected to electrophoresis at 100 V for 30 min and visualized under UV light. Cell culture and flow cytometric measurements. SMMC-7721 and L02 cells were cultured at 37 °C in a humidified atmosphere with 5% CO2 in RPMI-1640 (Hyclone, USA) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco, Life Technologies, USA). Cells were seeded at a density of 5 × 104 cells/400 µL culture medium per well in 24-well plates. The culture medium was replaced by a complex solution (1 µg DNA in 400 µL) and incubated for 4 h. The fluorescent-labeled HLPD were prepared with liposome containing Cy5.5-DSPE at 1% of total lipid and HLPR were prepared with siRNA containig 50% of Cy3-siRNA. The cells were then digested using trypsin, and suspended using 500 µL of PBS. Flow cytometric measurements were performed using a BD FACSAria instrument. Confocal laser scanning microscopy (CLSM). 7.5 × 104 SMMC-7721 cells were seeded onto glass coverslips in 6-well plates and incubated for 24 h. The fluorescent-labeled HLP1R containing Cy5.5-DSPE and Cy3-siRNA (i.e., 30 nM siRNA, 1.6 mL/coverslip), was incubated with the cells for 0.5 h to track the entry of Q-complexes in the SMMC-7721 cells. Cells were



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mixed with 4% paraformaldehyde, and incubated for 5 min at room temperature with DAPI (0.2 mg/mL; Sigma-Aldrich, USA). Then, cells were washed five times in PBS for clearing away the substance outside of cell surface and sealed in a mounting media (Invitrogen, USA). Cytotoxicity assay and cell transfection. Cells were seeded into 96-well plates at 2.5 × 104 cells per well. All transfections were conducted with HLP1-3D and LP1-3D. Lipofectamine2000 (L2K) and PEI25kDa were used as the positive controls at 3:1 weight ratio to DNA; they were formulated in 200 µL Opti-MEM solution containing 250 ng of plasmid DNA, and in replicates of four. Culture medium was removed before adding the complex solution for a serum-free transfection. The complex solution was diluted to 200 µL per well using serum-free medium for a serum-free transfection or medium containing 10 % FBS (v/v) for a serum transfection. For the cytotoxicity assay, 10 µL of CCK8 /well (Signalway Antibody Co., Ltd, USA) was added and incubated for 1 h after an initial incubation of complexes and SCMM-7721 cells for 4 h with 10 % FBS or serum free medium. Viable cells were determined by measuring sample absorbance at 570 nm using an xMarkTM microplate absorbance spectrophotometer reader (BioRAD, USA). The percentages of viable cells were obtained by comparing the absorbance values of the samples without adding the complexes. SCMM-7721 and L02 cells were incubated for luciferase transfections. For serum free transfection, the cells were first incubated with complexes for 4 h at 37 °C then the culture medium was replaced with a complete growth medium, and the cells were incubated for an additional of 20 h. For transfection with serum, the cells were incubated with complexes in the medium containing 10 % FBS for 24 h. The cells were then lysed with the Reporter Lysis Buffer and a chemiluminescence assay was performed to measure transfected luciferase activity (Promega, USA). Protein concentrations in the lysate were determined using a Micro BCATM Protein Assay Kit (Thermo, USA). In vitro assay of luciferase and survivin gene silencing. The detail methods of luciferase and survivin gene silencing experiment was described previously.29 For luciferase siRNA delivery, SMMC-7721 cells stably expressing luciferase gene (Luc-SMMC-7721) was from Shanghai Medical Biotechnology CD., (Shanghai, China). Luc-SMMC-7721 cells were seeded in 96-well plate at 2.5 × 104 cells per well and cultured for 24 h. Then the complexes wrapping luciferase siRNA were added to the cells in four replicates, and the concentration of siRNA was 100nM. The untreated group was incubated in Opti-MEM. Lipofectamine2000 and PEI 25 kDa was used as the positive group. After the cells were incubated for 4 h at 37 °C, the complex


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solution was replaced with culture medium, then continued to incubate for 20 h again, Luciferase activity was determined by luminary intensity recorded (as RLU/mg) with a Sirius Luminometer (Berthold, Germany). For survivin siRNA delivery, SMMC-7721 cells were treated in the same way as described above. Real-time PCR was performed using a SYBR green method and universal reagents (catalog no. GMRS-001, GenePharma, Shanghai). hGAPDH was utilized as an internal standard to determine the relative expression levels for each gene. The RT-PCR assay was performed on MX3000P Real-time PCR Instrument (Stratagen, U. S. A.) Inhibition assay of HA and peptide. Cells were seeded into 48-well plates at 2 × 104 per well, for 24 h prior to transfection. After rinsing once with 1× PBS, the cells were incubated for 1 h at 37 °C with 80 µM dynasore (0.2 % DMSO in DMEM/F-12 as the control), or with antiCD44 antibody [rabbit monoclonal (EPR1013Y) to CD44] (1/250) for 1 h at 37 °C to inhibit CD44-mediated uptake. The inhibitor was removed, and the plates were first incubated with complexes for 4 h at 37 °C for flow cytometric measurements. FITC-labeled HA (FITC-HA) synthesized as reported30 and Cy5.5 labeled lipid were used to formulate Q-complexes for determining the internalization mechanism of Q-complexes. The culture medium was replaced with a complete growth medium, and the cells were incubated for an additional of 20 h for luciferase transfection. Animal experiments. All protocols involving the animals were approved by the Laboratory Animal Centre of Shanghai Jiao Tong University and were performed according to the guideline of the National Institutes of Health for the care and use of laboratory animals (NIH publication No 80-23). All efforts were made to reduce animal numbers used to the minimum required for valid statistical analysis. For Small animal imaging in vivo, male BALB/c nude mice (n = 6), aged 4 to 6 weeks, were subcutaneously injected with 1 ×106 SMMC -7721 cells. Once the volume of the tumor was approximated at 80 mm3, the mice were injected with HLP1D containing a 37.5-µg siRNA (50 % of siRNA labeled with Cy3) via the bilateral tail vein. In the untreated group (n = 3, control group), the mice were not injected with any complex. The animals were imaged post-injection at 0, 30, 45, 90, and 120 minute time points using the IVIS Lumina II small-animal imaging system (Caliper Life Sciences, Inc.). A Cy3 filter set was used to acquire Cy3-siRNA fluorescence values in vivo. Images were acquired and analyzed using the Living Image 4.3.1 software (Caliper, Alameda, CA, USA). The mice in the experiments were euthanized at 120 mins. major



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tissues (heart, liver, spleen, lung, kidney) and tumor were dissected, collected, and fixed in 10% formalin for 24 h and sectioned at 14 µm. The TdT-mediated dUTP Nick-End Labeling (TUNEL) staining was accomplished as directed by the reagent supplier's protocol (Beyotime, Inc., Shanghai, China). The DAPI mounting medium (Vector Laboratories, Inc., Burlingame, CA, USA) was added for nuclear staining. Tissue sections were imaged using a confocal fluorescence laser microscope (Leica TCS SP8, Germany). For survivin siRNA silencing in liver orthotropic transplanted tumors, the SMMC-7721 xenograft tumor in BALB\c nude mice was cut into about 1 mm3 fragments in the DMEM medium and embedded in the livers of BALB\c nude mice, aged 7−9 weeks. At the beginning of experiment, the mice were anaesthetized using Carbrital (10 mg/mL) dosed at 200 µL/20 g. After the tumors reached a size of 6 mm to 10 mm which were evaluated by random euthanizing three tumor-bearing mice, the mice were randomly divided into three groups. 200 µL of HLP1D wrapping survivin siRNA nanocomplexes in the 5 % glucose at 0.67mg/kg of survivin siRNA was injected via the tail vein (“n” represents the number of mice per group, n = 6) once every other day, and each mouse was intravenously injected 8 times. The body weights of mice were evaluated during the antitumor time. The mice were euthanized by cervical dislocation 48 h after the last injection, and the tumors were resected for RT-PCR assay. The untreated mice were used as controls (n = 6). The sample of naked survivin siRNA and HLP1R wrapping negative siRNA was used as the negative control group (n = 6). Statistical analyses. All experiments were repeated three times. Data were presented as mean ± S.D and compared using Student’s t-tests. An alpha value of “*” p < 0.05 was considered signiﬁcant. Analyses were completed using GraphPad Instat software (GraphPad, San Diego, CA, USA). The data of flow cytometry were analyzed using FlowJo software 7.6 (Flexera Software, Maryland, USA).

Results Characterization

of

ternary

complexes

and

Q-complexes. The sequences of

multifunctional peptides were designed as follows: the hepatic tumor-lineage homing cellpenetrating peptide conjugated with R16 (P1), as well as its sequence reversed conjugated with R16 (P2).

21

And R16 (P3) was used as the control peptide without specific “penetrating” domain

KRPTMRFRYTWNPMK. First, we prepared the formulation of the ternary complexes (LPD or


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LPR) then the ternary complexes were coated with HA to formulate Q-complexes. The formulation data of the ternary and Q-complexes correspond to the weight ratio of each component. “H” represents hyaluronic acid; “L” represents cationic liposome formulated by DOTAP/DOPE at a 1:1 weight ratio; and “D” and “R” represent “DNA” and “siRNA”, respectively. For example, “HLP1D” refers to hyaluronic acid, cationic liposome, P1, and DNA at 14:1:4:1 weight ratios. Moreover, HLPD and HLPR represent the Q-complexes wrapping DNA and siRNA, respectively. The sizes of most Q-complexes with the three peptides, except HLP3R at the size of 307 nm, were maintained at around 100 nm in diameter and were comparable with that of the corresponding ternary complexes. The zeta potential of the three ternary complexes was from +30 to +57 mV, and the zeta potential of Q-complexes ranged from −30 mV to −40 mV (Table S1, 2). Gel electrophoresis assay showed that both DNA and siRNA can be completely wrapped in the ternary complexes and Q-complexes (Figure S1). We tested the long term stability of HLP1D and found the size of HLP1D was unchangeable with time in the physiological saline of 5mg/ml BSA incubated for different time internal from 4 h to 24 h at 4 oC and 37 oC, respectively. At the same time, we also tested the size of Q-complexes in the medium containing 10% FBS, and found the size of Q-complexes still kept unchangeable with time. The size of HLP1D in the above solutions kept about 100 nm in diameter which is close to that in water (data not shown). The TEM results demonstrated that HLP1D and HLP2D formed spherical nanoparticles (Figure 1A, B). By contrast, the morphologies of HLP3D were alike spherical, the shape and size of which are differential from one another (Figure 1C). Furthermore, the morphologies of HLP12R

were similar to that of HLP1-2D (Figure 1D, E). Conversely, HLP3R formed larger spherical

nanoparticles with a tendency to agglomerate (Figure 1F).

Figure 1

Cellular uptake. Firstly, flow cytometry was used to examine the cellular uptake of Qcomplexes wrapping DNA (Cy5.5-DSPE labeled lipid as tracer) or siRNA (Cy3-siRNA as tracer). All Q-complexes can be effectively endocytosed by SMMC-7721 cells, whether they are packaging DNA (Figure S2A) or siRNA (Figure S2B), whereas the endocytosis efficiency of Q-



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complexes containing P1 and P2 was higher than that of Q-complexes containing P3. Then HLP1R and HLP3R as the representative samples were selected to observe their intracellular endocytosis through CLSM. Cy3-siRNA (green) and Cy5.5-DSPE (red) were used to monitor the trafficking of Q-complexes. As shown in Figure 2, the yellow dots represent the overlapping fluorescence of Cy3-siRNA and Cy5.5-DSPE from HLP1R and HLP3R. The observation of confocal microscopy indicates that more dots of HLP1R accumulated in the cells within 1.5 h than those of HLP3R, which was consistent with the result of flow cytometry. The above results demonstrated the specific “penetrating” domain KRPTMRFRYTWNPMK of the multifunctional peptide enhanced the cell uptake of Q-complexes.

Figure 2

In vitro cytotoxicity and nucleic acid transfection. Cytotoxicity results demonstrated that Q-complexes presented no evident toxicity against cell lines (Figure S3, 4). We further examined the transgenic activity of the Q-complexes. Results showed that Q-complexes displayed significantly higher luciferase transfection efficiencies than lipofectamine2000 (L2K) and PEI25kDa (Figure 3). We observed that the LPD displayed higher luciferase transfection activity than the corresponding HLPD in the serum-free medium (Figure 3A). However, the transfection efficiency of Q-complexes remained high in the medium containing 10 % serum as well and the transfection efficiency of the HLP1-2D was higher than that of LP1-2D (Figure 3B), which demonstrated that the HA coated on the surface of nanocomplexes can maintain the stability of Q-complexes and promote nanocomplexes to actively target the cell surface. Under both of these conditions, HL1PD and HL2PD showed higher transfection efficiency than HL3PD. We also carried out a study of luciferase transfection efficiency of the ternary complexes and Qcomplexes on the normal cells L02, and found these complexes showed extraordinary low transfection efficiency compared to that on SMMC-7721 cells (Figure S5). In the luciferase siRNA intracellular delivery experiment, HLP1R and HLP2R significantly silenced about 70% of luciferase expression in the SMMC-7721-luc cell line; it posed an equivalent delivery activity in siRNA with Lipofectamine2000 and PEI25kDa. In contrast, HLP3R showed moderate siRNA delivery capability (Figure 4A). The above results collectively indicated that Q-complexes containing P1 and P2 possess higher nucleic acid delivery capabilities


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than Q-complexes containing P3. HLP1R was selected to deliver survivin siRNA into SMMC7721 cells to silence survivin gene in mRNA level (Figure 4B). It indicated that HLP1R could silence over 85% expression of survivin gene which is higher than the silencing efficiency of PEI 25 kDa.

Figure 3, 4

Functions of HA and KRPTMRFRYTWNPMK domain on internalization of Qcomplexes. We further investigated the mechanisms of Q-complexes internalization. Previous literature reported that the cellular entry of the hepatic tumor-targeted cell-penetrating peptide KRPTMRFRYTWNPMK was mediated by the dynamin-dependent endocytic pathway21 and HA were endocytosed through CD44-mediated endocytic pathways16. Thus, we first pretreated SMMC-7721 cells with the dynamin inhibitor (DI; dynasore)21, 31 and the CD44 blocker (antiCD44 antibody)16 before adding the Q-complexes, to investigate the functions of HA and KRPTMRFRYTWNPMK domain of the peptide in the delivery process, respectively. We used FITC-HA and Cy5.5-DSPE to track the HA and lipid components in Q-complexes, respectively. The flow cytometric analysis showed that dynasore significantly reduced the fluorescence intensity from the Cy5.5-labeled lipid component (Figure 5A) and exhibited no effect on the FITC-HA component (Figure 5B). Furthermore, anti-CD44 antibody reduced the fluorescence intensity of both FITC-HA and Cy5.5-lipid components from the samples (Figure 5C, D). These results indicated that the cellular uptake of HA component in Q-complexes depended on the CD44-mediated pathway, whereas the cellular uptake of lipid component in Q-complexes depended on both CD44-mediated and dynamin-dependent pathways. In addition, the cellular uptake of FITC-HA in Q-complexes was significantly higher than that of free FITC-HA at a similar concentration (Figure 5B, D), which indicated that the HA component failed to dissociate from the nanocomplexes before the Q-complexes attached to the surface of the target cells. In addition, the results of luciferase expression (Figure S6) indicated that dynasore significantly decreased the transfection efficiency of Q-complexes, whereas the anti-CD44 antibody had no effect on the transfection efficiency of Q-complexes. It demonstrated that most Q-complexes adhered to the surface of cancer cells through the interaction of HA and CD44, but HA had no effect on the transfection efficiency of nucleic acids, and KRPTMRFRYTWNPMK promoted the



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intracellular delivery of nucleic acids.

Figure 5

In vivo small animal image and tissue distributions of Q-complexes. A selected sample of HLP1R was administered to nude mice carrying subcutaneous xenograft tumor models through tail vein injection to investigate in vivo tissue distribution of Q-complexes. Cy3-siRNA was utilized as the tracer, followed by small animal in vivo imaging method. It demonstrated that the fluorescence intensity of Cy3 accumulated in the tumor (Figure S7A) and the fluorescent intensity in the local vicinity of the tumors rapidly developed over time (Figure S7B). Given sufficient time for the nanoparticles injected through the tail vein to overcome tumor blood vessel to reach the tumor location within 2 h, the mice were sacrificed. The major tissues, such as heart, liver, spleen, lung, kidney, and tumor were cryo-spliced at 120 min point after injection. Then, the nuclei were counter-stained with DAPI. Figure 6 shows the confocal microscopy exhibiting that the fluorescent signals can hardly be observed in the heart, liver and lung in the HLP1R group, which is similar to that of the untreated group. However, a few fluorescent signals were found in the kidney and spleen compared to the untreated group, which indicated that HLP1R can circulate in the blood and pass through the tumor vessel to reach the tumor sites. Moreover, the fluorescent signals of Cy3 in the HLP1R formulation was mainly observed inside the tumor cells, which was consistent with the observation in small animals. The absence of HLP1R in the main tissues showed that this multifunctional delivery system can efficiently achieved target transport of nucleic acid to cancer cells through EPR effect and active targeting. Moreover it also displayed no visible interference in the normal primary tissues.

Figure 6

Survivin siRNA silencing in liver orthotropic transplantation tumor models. In order to imitate the in vivo tumor environment, we established a liver orthotropic transplantation tumor model with SMMC-7721 cells in nude BALB/c mice, and 0.65 mg/kg survivin siRNA wrapped by HLP1R was injected every 48 h through a tail vein, for 8 times, we also monitored the body weight of the mice to evaluate the systemic toxicity during antitumor treatment. Body weights of


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mice were not great changed among experimental groups (Figure 7A), which showed HLP1R was no toxicity in vivo. Then the mice were euthanized 48 h after the final injection, the silencing efficiency of survivin siRNA was examined following injection. mRNA expression of the HLP1R group decreased to 77% compared with the untreated group; however, both naked survivin siRNA and negative-HLP1R groups reported no significant changes compared with that of the untreated group (Figure 7B).

Figure 7

Discussion In this study, our hypothesis was design of a safe and high nucleic acid delivery system using endogenous molecules based on the modular strategy. Firstly, the ternary complexes were formulated based on electrostatic interactions between cationic peptide and cationic liposomes with negatively charged nucleic acids. the assemble structure of ternary complexes described here before being coated with hyaluronic acid are similar to that of the reported cationic receptor-targeted nanocomplexes (RTNs)26 the structure of RTNs has been proved was a tightly condensed inner peptide–nucleic acid core surrounded by a disordered lipid layer, from which the neutral amine acid sequences of the peptide partially protrudes. Similarly, we proposed that the nearly neutral integrin-penetrating sequence of the peptide partially protrudes in the ternary complexes containing P1 or P2 (Figure 8A). This condition endowed the system with an additional tumor cell penetrating function. Furthermore, we shielded the positive charge of the ternary complexes with HA to achieve intravenous administration, as well as the enhanced tumor cell targeting via CD44 overexpressed on the surface of cancer cells.16 Compared with the corresponding ternary complexes, the coated HA showed no effect on increasing particle sizes (Table S1, 2) and Q-complexes presented efficient capabilities in completely wrapping DNA and siRNA (Figure S1). On the other hand, the ternary complexes containing P3, as a kind of cationic complexes without any neutral amine-acid sequence protrudes to the outside of surface, assembled with HA mainly through electrostatic interaction and completely coated on the surface of the ternary complexes which maybe slightly reduced the interaction between HA and the ternary complexes and increased the size of the accordingly Q-complexes. The highly negative zeta potential of Q-complexes indicated that HA could shield the



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positive charge of the ternary complexes. The approximately equivalent transgene efficiencies of Q-complexes containing P1 and P2 (Figure 3 and 4) identified similar functions of P1 and P2 in the nucleic acid delivery. Moreover, both of Q-complexes containing P1 and P2 have higher transfection efficiency than Q-complexes containing P3. These findings showed the promoted effect of specific “penetrating” peptide domain on the nucleic acid delivery activity whenever R16 was linked with the N-terminal region or C-terminal region of KRPTMRFRYTWNPMK. Given that the amine and the carboxylic acid groups on the peptide protrude on the surface of ternary complexes, they can form hydrogen bonds with the hydroxyl groups on the HA,32 and these hydrogen bonds add to the stabilizing effects on Q-complexes containing P1 and P2, in addition to the fore-mentioned electrostatic interactions with positively charged ternary complexes. HLPD containing P1 and P2 achieved a higher transfection efficiency than the corresponding LPD in the medium containing 10 % serum (Figure 3B), which indicated that Qcomplexes were relatively stable in the medium and the HA component did not separate from Qcomplexes before the nanoparticles bound to the cancer cell surface. This result can be accounted for by the far lower cellular uptake of free HA than that of Q-complexes containing P1 and P2 at the same concentration of HA (Figure 5B and D). In the intracellular pathway study of Q-complexes using Cy5.5-labbeled lipid and Cy3siRNA as the trackers, we found the lipid component and siRNA wrapped together in the cells (Figure 2). Interesting, the flow cytometry assay showed the separation endocytosis pathway of HA from other components of most Q-complexes (Figure 5) and had no effect on the DNA transfection efficiency (Figure S6). Since the high-molecular weight extracellular HA has been reportedly tethered to the cancer cell surface by the combined efforts of overexpressed CD44 and the GPI-anchored enzyme Hyal-2,33 in the process, hyal-2 cleaved high molecular weight HA to products of approximately 20 kDa, and then, the hyal-2-generated HA fragments are internalized and delivered to the endosomes by CD44, and ultimately to lysosomes.34 Combination with our experimental results, on one hand, we can infer that some HA of Q-complexes underwent both cleavage and CD44-mediated endocytosis, which resulted in the shedding of HA from the other components. On the other hand, Q-complexes failed to undergo complete HA separation during the binding process in the extracellular environment; consequently, CD44 mediated the endocytosis of a minority of Q-complexes (Figure 5C). A dynamin-dependent, CD44independent pathway for transgene expression associates with the higher transfection activities


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of HLP1D and HLP2D compared with that of HLP3D (Figure S6), which indicated that the “penetrating” peptide domain played an important promoted role in the intracellular transport of the nucleic acids. Conversely, as shown in Figure 8B, the intracellular route of the monitory Qcomplexes mediated by CD44 was possibly similar to the metabolic pathway of HA in which the lipid component hardly disrupted the endosomal membrane duo to the barrier of HA. However, the detail mechanism of internalization of Q-complexes would be further investigated in our future study, here we mainly explored the different effects of HA and peptide on the nucleic acid delivery efficiency of Q-complexes. Furthermore, the transfection of Q-complexes containing P1 and P2 was higher than the corresponding ternary complexes in the medium containing serum, which suggested that HA promoted cancer cell binding of Q-complexes containing P1 and P2.

Figure 8

Once the nanocomplexes entered the endosome through the dynamin-dependent pathway, the exposed lipid component containing DOPE can fuse with the endosomal membrane to release nucleic and peptide/nucleic acid complexes into the cytoplasm, which had been investigated and verified by Du Z and Munye M et al., through the DNA intracellular delivery of RTNs 27, 28 , the proposed mechanisms of Q-complexes trafficking was shown in Figure 8B. Following the release of cargoes in the cytoplasm, Nucleic acids could be continuously released from the peptide/nucleic acid complexes in the degradation process of the multifunctional peptide.25 From the in vivo tissue distributions of siRNA, it could be found that Q-complexes HL1PR can overcome the biological barriers to achieve specific targeting without interfering with normal tissues, which also, to some extent, indirectly reflected Q-complexes kept stable and nonpositively charged in the physiological flood and passed over the tumor vessel through EPR effect, At the same time, the result of survivin siRNA silencing efficiency using HL1PR in the liver orthotropic transplantation tumor models (Figure 6, 7) further confirmed the great nucleic acid therapeutic potential of Q-complexes with rationally-designed peptides. In terms of toxicity, the components we adopted were either endogenous or proven-as-safe. As a major component of extracellular matrix, HA has been widely used in pharmaceutical products and drug vectors because of its safety, biocompatibility, and biodegradability in vivo.35 Both DOTAP and DOPE are known to have the potential to serve as commercialized non-toxic



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lipids for the clinical application of gene therapy.4 Peptides can be biodegraded to the amino acid fragments beneficial to humans.21, 25 Thus, Q-complexes reported no evident cytotoxicity in the presence or absence of serum in vitro, as well as no obvious in vivo toxicity from the observation that no weight change in the nude mice occurred after continuous tail vein injection in the liver orthotropic transplantation tumor model.

Conclusion In summary, we rationally designed a novel platform using peptides and lipids and HA for nucleic acid delivery based on the modular design. In this text, we studied the effects of the peptide component and HA on the nucleic acid delivery efficiency of Q-complexes. The experimental results showed KRPTMRFRYTWNPMK could enhanced nucleic acid transfection efficiency, and HA could shield the positive charges of ternary complexes and help Q-complexes bind to the cancer cells but had no effect on the intracellular transfection. We also utilized the membrane fusogenic lipid DOPE in Q-complexes to disrupt the endosomal membranes for promoting in vivo delivery efficiency based on our previous study.28 The nucleic acid delivery system was initially found to be relatively stable in the culture medium in vitro and the cellular uptake assay showed HA component separated from the nanocomplexes after Q-complexes binding to the target cells, and the rest complexes were mainly endocytosized through the dynamin-dependent pathways. Q-complexes could reach the tumor sites without interfering with normal tissues. This design rationally utilized the structure, function, and synergistic effect of each component to construct a biologically responsive nucleic acid delivery system, gaining ideal properties such as being non-toxic, biocompatible, biodegradable and high effect. In addition, it offered a rational and optimized platform for the development of the in vivo delivery system in nucleic acid therapeutics. However, further investigation has to be carried out on Qcomplexes although which primary confirmed our hypothesis, for examples, the detail mechanism of internalization of Q-complexes is still unclear, Are the particles separating somehow during binding to cells or endocytosis? Additional studies will be investigated in our future study.

Supplementary data The supplementary data associated with this article can be found in the online version of the


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paper.

Author information

Corresponding author *E-mail: [email protected]

Competing financial interests

The authors declare no competing financial interests.

Acknowledgements

Zixiu Du was financially supported by the National Natural Science Foundation of China (Grant No. 30901881) and the Engineering and Medical Cooperation Projects of Shanghai Jiao Tong University (No. YG2013MS42 and YG2014QN04 and YG2016MS22). We also thank Wangxi Hai (Shanghai Jiao Tong University) for his help in drawing Figure 8. Additional information

Supplementary information accompanies this paper at http://www.nature.com/scientificreports.

References 1. Fire, A.; Xu, S.; Montgomery, M. K.; Kostas, S. A.; Driver, S. E.; Mello, C. C. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998, 391, (6669), 806-11. 2. Fire, A. Z. Gene silencing by double-stranded RNA (Nobel Lecture). Angewandte Chemie 2007, 46, (37), 696684. 3. Yu, M.; Zheng, J. Clearance Pathways and Tumor Targeting of Imaging Nanoparticles. ACS nano 2015, 9, (7), 6655-74. 4. Yin, H.; Kanasty, R. L.; Eltoukhy, A. A.; Vegas, A. J.; Dorkin, J. R.; Anderson, D. G. Non-viral vectors for genebased therapy. Nature reviews. Genetics 2014, 15, (8), 541-55. 5. Keswani, R. K.; Lazebnik, M.; Pack, D. W. Intracellular trafficking of hybrid gene delivery vectors. Journal of controlled release : official journal of the Controlled Release Society 2015, 207, 120-30. 6. Pu, J.; Yuan, A.; Shan, P.; Gao, E.; Wang, X.; Wang, Y.; Lau, W. B.; Koch, W.; Ma, X. L.; He, B. Cardiomyocyteexpressed farnesoid-X-receptor is a novel apoptosis mediator and contributes to myocardial ischaemia/reperfusion



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 18 of 30

injury. European heart journal 2013, 34, (24), 1834-45. 7. Hu, Y.; Li, K.; Wang, L.; Yin, S.; Zhang, Z.; Zhang, Y. Pegylated immuno-lipopolyplexes: A novel non-viral gene delivery system for liver cancer therapy. Journal of controlled release : official journal of the Controlled Release Society 2010, 144, (1), 75-81. 8. Luo, X.; Pan, S.; Feng, M.; Wen, Y.; Zhang, W. Stability of poly(ethylene glycol)-graft-polyethylenimine copolymer/DNA complexes: influences of PEG molecular weight and PEGylation degree. Journal of materials science. Materials in medicine 2010, 21, (2), 597-607. 9. Nie, Y.; Gunther, M.; Gu, Z.; Wagner, E. Pyridylhydrazone-based PEGylation for pH-reversible lipopolyplex shielding. Biomaterials 2011, 32, (3), 858-69. 10. Venkataraman, S.; Ong, W. L.; Ong, Z. Y.; Joachim Loo, S. C.; Ee, P. L.; Yang, Y. Y. The role of PEG architecture and molecular weight in the gene transfection performance of PEGylated poly(dimethylaminoethyl methacrylate) based cationic polymers. Biomaterials 2011, 32, (9), 2369-78. 11. Dewachter, P.; Mouton-Faivre, C. Anaphylaxis to macrogol 4000 after a parenteral corticoid injection. Allergy 2005, 60, (5), 705-6. 12. Chanan-Khan, A.; Szebeni, J.; Savay, S.; Liebes, L.; Rafique, N. M.; Alving, C. R.; Muggia, F. M. Complement activation following first exposure to pegylated liposomal doxorubicin (Doxil): possible role in hypersensitivity reactions. Annals of oncology : official journal of the European Society for Medical Oncology / ESMO 2003, 14, (9), 1430-7. 13. Ishida, T.; Kashima, S.; Kiwada, H. The contribution of phagocytic activity of liver macrophages to the accelerated blood clearance (ABC) phenomenon of PEGylated liposomes in rats. Journal of controlled release 2008, 126, (2), 162-5. 14. Li, J.; Wang, Y.; Zhu, Y.; Oupicky, D. Recent advances in delivery of drug-nucleic acid combinations for cancer treatment. Journal of controlled release : official journal of the Controlled Release Society 2013, 172, (2), 589-600. 15. Lee, M. S.; Lee, J. E.; Byun, E.; Kim, N. W.; Lee, K.; Lee, H.; Sim, S. J.; Lee, D. S.; Jeong, J. H. Target-specific delivery of siRNA by stabilized calcium phosphate nanoparticles using dopa-hyaluronic acid conjugate. Journal of controlled release 2014, 192, 122-30. 16. Yamada, Y.; Hashida, M.; Harashima, H. Hyaluronic acid controls the uptake pathway and intracellular trafficking of an octaarginine-modified gene vector in CD44 positive- and CD44 negative-cells. Biomaterials 2015, 52, 189-98. 17. Chono, S.; Li, S. D.; Conwell, C. C.; Huang, L. An efficient and low immunostimulatory nanoparticle formulation for systemic siRNA delivery to the tumor. Journal of controlled release 2008, 131, (1), 64-9. 18. Ruponen, M.; Yla-Herttuala, S.; Urtti, A. Interactions of polymeric and liposomal gene delivery systems with extracellular glycosaminoglycans: physicochemical and transfection studies. Biochimica et biophysica acta 1999, 1415, (2), 331-41. 19. Urbiola, K.; Sanmartin, C.; Blanco-Fernandez, L.; Tros de Ilarduya, C. Efficient targeted gene delivery by a novel PAMAM/DNA dendriplex coated with hyaluronic acid. Nanomedicine 2014, 9, (18), 2787-801. 20. Fan, Y.; Sahdev, P.; Ochyl, L. J.; J, J. A.; Moon, J. J. Cationic liposome-hyaluronic acid hybrid nanoparticles for intranasal vaccination with subunit antigens. Journal of controlled release 2015, 208, 121-9. 21. Kondo, E.; Saito, K.; Tashiro, Y.; Kamide, K.; Uno, S.; Furuya, T.; Mashita, M.; Nakajima, K.; Tsumuraya, T.; Kobayashi, N.; Nishibori, M.; Tanimoto, M.; Matsushita, M. Tumour lineage-homing cell-penetrating peptides as anticancer molecular delivery systems. Nature communications 2012, 3, 951. 22. Zhang, D.; Wang, J.; Xu, D. Cell-penetrating peptides as noninvasive transmembrane vectors for the development of novel multifunctional drug-delivery systems. Journal of controlled release 2016, 229, 130-9. 23. Gao, H.; Zhang, Q.; Yang, Y.; Jiang, X.; He, Q. Tumor homing cell penetrating peptide decorated nanoparticles used for enhancing tumor targeting delivery and therapy. International journal of pharmaceutics 2015, 478, (1), 240-50. 24. Vitor, M. T.; Bergami-Santos, P. C.; Zompero, R. H.; Cruz, K. S.; Pinho, M. P.; Barbuto, J. A.; de la Torre, L. G. Cationic liposomes produced via ethanol injection method for dendritic cell therapy. Journal of liposome research 2016, 1-15. 25. Hart, S. L. Multifunctional nanocomplexes for gene transfer and gene therapy. Cell biology and toxicology 2010, 26, (1), 69-81. 26. Mustapa, M. F.; Bell, P. C.; Hurley, C. A.; Nicol, A.; Guenin, E.; Sarkar, S.; Writer, M. J.; Barker, S. E.; Wong, J. B.; Pilkington-Miksa, M. A.; Papahadjopoulos-Sternberg, B.; Shamlou, P. A.; Hailes, H. C.; Hart, S. L.; Zicha, D.; Tabor, A.


Page 19 of 30

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B. Biophysical characterization of an integrin-targeted lipopolyplex gene delivery vector. Biochemistry 2007, 46, (45), 12930-44. 27. Munye, M. M.; Ravi, J.; Tagalakis, A. D.; McCarthy, D.; Ryadnov, M. G.; Hart, S. L. Role of liposome and peptide in the synergistic enhancement of transfection with a lipopolyplex vector. Scientific reports 2015, 5, 9292. 28. Du, Z.; Munye, M. M.; Tagalakis, A. D.; Manunta, M. D.; Hart, S. L. The role of the helper lipid on the DNA transfection efficiency of lipopolyplex formulations. Scientific reports 2014, 4, 7107. 29. Du, Z.; Xiang, S.; Zang, Y.; Zhou, Y.; Wang, C.; Tang, H.; Jin, T.; Zhang, X. Polyspermine imine, a pH responsive polycationic siRNA carrier degradable to endogenous metabolites. Molecular pharmaceutics 2014, 11, (10), 3300-6. 30. Hua, Q.; Knudson, C. B.; Knudson, W. Internalization of hyaluronan by chondrocytes occurs via receptormediated endocytosis. Journal of cell science 1993, 106 ( Pt 1), 365-75. 31. Macia, E.; Ehrlich, M.; Massol, R.; Boucrot, E.; Brunner, C.; Kirchhausen, T. Dynasore, a cell-permeable inhibitor of dynamin. Developmental cell 2006, 10, (6), 839-50. 32. Suh, K. Y.; Yang, J. M.; Khademhosseini, A.; Berry, D.; Tran, T. N.; Park, H.; Langer, R. Characterization of chemisorbed hyaluronic acid directly immobilized on solid substrates. Journal of biomedical materials research. Part B, Applied biomaterials 2005, 72, (2), 292-8. 33. Stern, R. Hyaluronan catabolism: a new metabolic pathway. European journal of cell biology 2004, 83, (7), 317-25. 34. Stern, R. Hyaluronan metabolism: a major paradox in cancer biology. Pathologie-biologie 2005, 53, (7), 37282. 35. Zhang, L.; Gao, S.; Zhang, F.; Yang, K.; Ma, Q.; Zhu, L. Activatable hyaluronic acid nanoparticle as a theranostic agent for optical/photoacoustic image-guided photothermal therapy. ACS nano 2014, 8, (12), 12250-8.



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Figure legends Figure 1 Transmission electron microscopy of Q-complexes: (A) HLP1D, (B) HLP2D, (C) HLP3D, (D) HLP1R, (E) HLP2R, and (C) HLP3R. Figure 2 Confocal microscopic images of the intracellular localization of Q-complexes identified by Cy3-siRNA (red) and Cy5.5-labelled lipid (green). Representative images of HLP1R (A1-A5) and HLP3R (B1-B5) are shown after being incubated for 1.5 h with SMMC-7721 cells. The nucleus is identified using DAPI (blue). Panels A1-B1 indicated DAPI labeled nucleus (blue); Panels A3-B3 showed Cy3-siRNA lipid only (green); Panels A4-B4 identified the localization of cells (bright field); A5-B5 represent the merged images showing Cy3-siRNA (green) and Cy5.5labeled lipid (red) taken up by SMMC-7721 cells. Scale bar = 25 µm for all images.

Figure 3 Luciferase transfection of Q-complexes in SMMC-7721 cells incubated for 24 h: (A) in serum-free medium; (B) in medium containing 10 % FBS. Values are the means of four replicates ± standard deviation. “*” represents p < 0.05 and “***” represents p < 0.001.

Figure 4 (A) Relative luciferase expressions transfected with luciferase siRNA in the presence or absence carriers in SCMM-7721 cells stably expressing luciferase including the untreated (incubated in Opti-MEM) and the naked siRNA (transfected with free luciferase siRNA); (B) the silencing effect of survivin gene of LP1R and HLP1R. Lipofectionamine (L2K) and PEI25 kDa were used as the positive controlled groups. “ns” represents p > 0.05.

Figure 5 Cellular uptake of Q-complexes in SMMC-7721 cells incubated for 4 h in the absence of any inhibitor (Normal) and in the presence of dynamin inhibitor I (DI; Dynasore) or CD44antibody. Values of the FITC-HA and Cy5.5 labeled lipid components are the means of four replicates ± standard deviation. “**” represents p < 0.01 and “***” represents p < 0.001.

Figure 6 The distribution of Cy3-siRNA (red) in heart, liver, spleen, lung, kidney, and tumor after an IV injection of HLP1R for 120 mins. The nucleus is identified by DAPI (blue). Scale bar = 75 µm for all images.


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Figure 7 (A) Body weight during the period of treatment. (B) Relative expressions of survivin genes in the tumor tissue in the liver orthotropic transplantation tumor model with SMMC-7721 cells in the nude BALB/C mice tested by real time-PCR assay. “Untreated” represents mice without any injections, “Negative-HLP1R” represents mice injected with Q-complexes of HLP1R wrapping negative siRNA, and “HLP1R” represents mice injected with Q-complex of HLP1R wrapping survivin siRNA. “*” represents p < 0.05 and “**” represents p < 0.01.

Figure 8 (A) Schematic diagram of the model construction of Q-complexes. (B) Two main routes for the intracellular delivery of nucleic acids using Q-complexes after Q-complexes attached to the cancer cell surface. Route 1: (1) most HA component interacting with CD44 and hyaluronidase (Lyal-2) easily separated from the rest of the nanocomplexes, (2) endocytosed through dynamin-dependent pathways introduced by the tumor lineage-homing cell-penetrating peptide sequence and the positive nanocomplexes charges; (3) exposed positive lipid components fused with negative endosomal membranes, and the nucleic or peptide/nucleic acid particles dissociate from the nanocomplexes and are released into the cytoplasm (peptide/nucleic-acid particles may easily enter the nucleus through penetrating the nuclear membrane fit for the DNA transfection). Route 2: (I) minority of Q-complexes are still coated by HA are endocytosed through CD44-mediated pathways; (II) the Q-complexes entered the lysosome and the hydrolytic enzymes in which the contents of the late endosomes are digested.



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Figure 1


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Figure 3


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Figure 5


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Figure 7


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Schematic diagram of the model construction of Q-complexes Figure 8A 89x20mm (150 x 150 DPI)


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Hepatic Carcinoma Selective Nucleic Acid Nanovector Assembled by

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