Specially-Made Lipid-Based Assemblies for Improving

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Specially-Made Lipid-based Assemblies for Improving Transmembrane Gene Delivery: Comparison of Basic Amino Acid Residues-Rich Periphery Qian Jiang, Dong Yue, Yu Nie, Xianghui Xu, Yiyan He, Shiyong Zhang, Ernst Wagner, and Zhongwei Gu Mol. Pharmaceutics, Just Accepted Manuscript • DOI: 10.1021/acs.molpharmaceut.5b00967 • Publication Date (Web): 20 Apr 2016 Downloaded from http://pubs.acs.org on April 22, 2016

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Molecular Pharmaceutics

The peripheral properties of the assemblies are vital for complexation and interaction with physical barriers. Here, we report three cationic twin head lipids, and each of them contains a dioleoyl-glutamate hydrophobic tail and a twin polar head of lysine, arginine, or histidine. Such lipids were proven to self-assemble in aqueous solution with well-defined nanostructures and residual amino, guanidine or imidazole-rich periphery, showing strong buffering capacity and good liquidity. The assemblies with arginine- (RL) or lysine- (KL) periphery exhibited complete condensation of pDNA into nano-sized complexes. While assemblies composed of histidine-rich lipids (HL) showed relatively low cationic electric potential and poor DNA binding ability. The designed RL assemblies with guanidine-rich periphery enhanced the in vitro gene transfection up to 190-fold as compared with the golden standard PEI25k in the presence of serum. Meanwhile, interaction with cell and endo/lysosome membrane also revealed the superiority of RL complexes, that the guanidine-rich surface efficiently promoted transmembrane process in cellular internalization and endosomal disruption. More importantly, RL complexes also succeeded beyond others in vivo with significantly (~ 7-fold) enhanced expression in HepG2 tumor xenografts in mice, as well as stronger green fluorescence protein imaging in isolated tumors and tumor frozen sections. 47x26mm (300 x 300 DPI)

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Specially-Made Lipid-based Assemblies for Improving Transmembrane Gene Delivery: Comparison of Basic Amino Acid Residues-Rich Periphery Qian Jiang1, Dong Yue1, Yu Nie1,*, Xianghui Xu1, Yiyan He1, Shiyong Zhang1, Ernst Wagner2,†, Zhongwei Gu1,*

1 National Engineering Research Center for Biomaterials, Sichuan University, Chengdu 610064, Sichuan, P. R. China

2 Center for Drug Research, Department of Pharmacy, Pharmaceutical Biology-Biotechnology, and Center for NanoScience (CeNS), Ludwig-Maximilians-Universitat, Butenandtstr. 5-13, D-81377, Munich, Germany

KEYWORDS: Lipid Assemblies, Gene delivery, Basic amino acid residues-rich periphery, in vivo ABSTRACT: Cationic lipid based assemblies provide a promising platform for effective gene condensation into nano-sized particles, and the peripheral properties of the assemblies are vital for complexation and interaction with physical barriers. Here, we report three cationic twin head lipids, and each of them contains a dioleoyl-glutamate hydrophobic tail and a twin polar head of lysine, arginine, or histidine. Such lipids were proven to self-assemble in aqueous solution with


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well-defined nanostructures and residual amino, guanidine or imidazole-rich periphery, showing strong buffering capacity and good liquidity. The assemblies with arginine- (RL) or lysine- (KL) periphery exhibited positive charges (~ +35mV) and complete condensation of pDNA into nano-sized complexes (~ 120 nm). In contrast, assemblies composed of histidine-rich lipids (HL) showed relatively low cationic electric potential (~ +10 mV) and poor DNA binding ability. As expected, the designed RL assemblies with guanidine-rich periphery enhanced the in vitro gene transfection up to 190-fold as compared with the golden standard PEI25k and Lipofectamine 2000, especially in the presence of serum. Meanwhile, interaction with cell and endo/lysosome membrane also revealed the superiority of RL complexes, that the guanidine-rich surface efficiently promoted transmembrane process in cellular internalization and endosomal disruption. More importantly, RL complexes also succeeded beyond others in vivo with significantly (~ 7-fold) enhanced expression in HepG2 tumor xenografts in mice, as well as stronger green fluorescence protein imaging in isolated tumors and tumor frozen sections.

1. INTRODUCTION Gene therapy offers great promise as a powerful therapeutic tool for curing genetic disorders and cancer. Successful therapy requires transport of sufficient nucleic acids into cells in an efficient way. As a result, the delivery vehicle is vital, but also acts as a bottleneck for the successful development of gene therapy. Recombinant viruses are recognized as effective transfection vectors, however, issues of immunogenicity,



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carcinogenicity, and inflammation raise serious concerns for clinical applications. Thus development of safe and efficient nonviral vectors with well-defined nanostructure is keenly awaited. Nucleic acids are anionic biomacromolecules that cannot be efficiently translocated through the cell membrane. As a result, positively charged synthetic vectors are often used to condense nucleic acid via electrostatic interaction. More and more cationic nonviral vehicles based on polymers, dendrimers and liposomes emerge for efficient gene delivery in vitro by electrostatically bind to DNA. However, most synthetic systems are less efficient than viral ones, due to several challenges during the delivery such as stability in physiological conditions and membrane penetration. Therefore, the rational design of polycations should benefit effective complexation with DNA thus overcoming barriers including uptake through cellular membrane, escape from endosome by disturbing/destroying endo/lysosomal membrane, and final import of the DNA into the nucleus.1 Cationic periphery of the carrier directly interact with negatively charged genes during condensation and physical barriers in delivery, which dominate the gene transfection efficiency.2 Unfortunately, the benchmark cationic polymer polyethyleneimine and the commercially available poly(amido amine) dendrimer are non-degradable and display significant cytotoxicity. Moreover, many cationic lipids containing quaternary ammoniums and tertiary amines as positive head groups could inhibit critical enzyme activity such as protein kinase C.3 In fact, nature provides insight into DNA packaging where DNA wraps around histone octamers, which features a large proportion of basic residues for forming salt


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bridges with the phosphate backbone of DNA.4 The high infection capacity of virus is partly due to their cell-penetrating peptides with positively charged amino acid clusters in the virus envelop or capsids.5 Thus, basic amino acids or oligopeptides have drawn intensive attention on the gene carrier design with excellent performances and biocompatibility.6, 7 Our group synthesized a series of amino acid derivatives, including

high

generation

lysine

dendrimer,

amino

acid-functionalized

polysaccharides, and arginine-rich nanohybrids, which were proven to significantly enhance delivery efficiency of genes and drugs.8-11 From our previous studies, different amino acid residues showed diverse characterizations. For instance, arginine derivatives present characteristics similar to cell penetrating peptides applied in cell translocation, providing driving force to permeate cellular membrane by hydration reaction between guanidine groups and phospholipid in the membrane.12 Lysine-rich segment could efficiently condense DNA with the protonated amine group,13 and histidine headgroup could prevent endo/lysosomal degradation by creating buffering capacity in endosomes through protonation of the imidazole groups.14 However, the previous work is not systemic, where the delivery system was assembled by different driving forces with diversified conformation, and amino acids located either inside or outside of the complexes. Condensation and delivery of gene require sufficient cationic molecules. Dendrimers are regarded as inherent and versatile nanocarriers, owing to their highly branched three-dimensional architecture and ampliative surface.15 However, the difficulty in manufacture of high generation dendrimers and high cost hinder their



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extensive biomedical applications and some systemic mechanism studies. Recently, self-assembled dendritic systems have drawn significant attentions in constructing sophisticated nanostructures with excellent performance.16 Using this structure as a starting point we found assemblies of low generation peptide dendrimers rich in amino groups in the external shell showed suitable flexibility, moderate toxicity and protein-like properties.17, 18 Thus, with our continuing interests in the amino acid-based gene carriers, we constructed DNA packaging agents using varying lipid-based assemblies containing three natural basic residues (lysine, arginine and histidine) with amino, guanidine or imidazole-rich periphery (Scheme 1). This design was expected to present following advantages: (1) facile manufacture, low cost and a well-defined nanostructure; (2) mild cytotoxicity due to the presence biodegradable amino acids, fatty chain and amide linkages; (3) favorable membrane fluidity and invasion, fast endosomal disruption capacity, as well as high delivery efficiency. Each step of the cationic lipid syntheses was characterized by 1H NMR spectroscopy and mass spectrum. Next, DNA complexes assembled by these lipid systems were studied systemically (Scheme 1), including DNA condensation, cellular internalization and intracellular tracking of the complexes. The in vitro transfection of the complexes was carried out in HepG2 and HEK293T cell lines and the in vivo transfection efficiency was further evaluated in a HepG2 hepatoma xenograft model.


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Scheme 1. Schematic Illustration of Lipid-based Assemblies with Basic Amino Acid Residues-rich Periphery for Gene Delivery, through penetration of cellular, endo/lysosomal and nuclear membranes.

2. MATERIALS AND METHODS 2.1

Materials. Oleyl amine was purchased from Aladdin (China) and

Boc-Lys(Boc)-OH,

H-Lys-OMe·2HCl,

Boc-Arg(pbf)-OH,


Boc-His(Boc)-OH,


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NH2-Glu-OH,

N1-(ethylimino)methylene)-N3,N3-

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dimethylpropane-1,3-diamine

(EDC), 1-hydroxybenzotriazole hydrate (HOBT) were from GL Biochem (Shanghai), N,N,N’,N’-tetramethyl-(1H-benzotriazol-1-yl)uronium hexafluorophosphate (HBTU), N,N-diisopropylethylamine (DIPEA), trifluoroacetic acid (TFA) were obtained from Astatech (China). Branched polyethylenimine 25 kDa (PEI25k) was obtained from Sigma-Aldrich (China). Lipofectamine™ 2000 reagent, Dulbecco’s modified Eagle’s medium with high glucose (DMEM-HG) and fetal bovine serum (FBS) were purchased from Life Technologies Corporation (Gibco®, USA). Nucleic acid labelling kit Label IT® Cy5™ was obtained from Mirus Bio Corporation (USA). The human hepatoma cell line (HepG2), and human embryonic kidney cell line (HEK293T) were obtained from Shanghai Institutes for Biological Sciences (China). Cell lysate and the luciferase reporter gene assay kit were purchased from Promega (USA). A BCA protein assay kit was purchased from Pierce (USA). Hoechst 33342 was purchased from Beyotime (China). LysoTracker® Green DND-22 was obtained from Life Technologies Corporation (USA). Plasmid pCMV-Luc was constructed by cloning the luciferase gene from pGL3 promoter vector into pcDNA3.1 and purified with EndoFree Plasmid Kit from Qiagen. All buffers were prepared in Milli-Q ultrapure water and filtered (0.22 µm) prior to use, and all the other chemicals were purchased from Sigma-Aldrich and used as received.

2.2

Chemical Experiments.

2.2.1 Synthesis of the hydrophobic moiety of cationic lipids (Compound 2). The hydrophobic part was synthesized as shown in Fig. 1A. Boc-glutamic acid (2.47 g, 10


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mmol), HOBT (4.86 g, 36 mmol), and EDC (6.90 g, 36 mmol) were weighted precisely, dissolved in anhydrous dichloromethane (DCM, 15 mL) and stirred at 0°C under N2 atmosphere for 30 min. Oleyl amine (10.14 g, 40 mmol) dissolved in anhydrous dichloromethane solution (15 mL) and DIPEA (16.52 mL, 100 mmol) were then added dropwise into the reaction system. After continuously stirring for 48 h at room temperature, the reaction mixture was washed with saturated sodium bicarbonate, diluted hydrochloric acid, saturated brine and dried with magnesium sulfate (MgSO4) overnight. Organic solvents were removed by a rotary evaporator and the resultant residue (Compound 1) was purified by chromatography on silica gel (ethyl acetate/petroleum ether = 1:1) yielding the product (83% yield) as an off-white solid. In order to react with the dendritic basic amino acid part in the next step,

Compound 1 (3 g, 4 mmol) was deprotected with TFA (3 mL, 40 mmol) in dichloromethane (3 mL) for 4 h. After removal of DCM and TFA, Compound 2 was obtained by ether precipitation. 2.2.2 Synthesis of dendritic arginine and lysine modified lipid (RL and KL). The synthesis of dendritic arginine-modified cationic of cationic lipid (RL) was shown in Fig. 1B. H-Lys-OMe·2HCl (4.66 g, 20 mmol), Pbf-Arg(Boc)-OH (16.63 g, 48 mmol), HBTU (6.48 g, 48 mmol), and HOBT (25.30 g, 48 mmol) were dissolved in anhydrous N,N-dimethylformamide (DMF, 30 mL). DIPEA (26 mL, 160 mmol) was added dropwise into the reaction system at 0°C and stirred under N2 atmosphere for 30 min. After reaction at room temperature for 48 h, the solvent was removed and the



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residue was washed with saturated sodium bicarbonate, diluted hydrochloric acid and saturated brine solution for several times. The solution was dried with magnesium sulfate overnight, and recrystalized from ethyl acetate (EtOAc, 250 mL) at 4oC to obtain a white powder (Compound 3) with a yield of 78%.

Compound 3 (5.9 g, 5 mmol) was treated with NaOH (1.6 g, 40 mmol) in methanol (MeOH) for 6 h. The mixture was evaporated, dissolved in H2O (250 mL) and adjusted to neutral pH value. Compound 4 was extracted by DCM and dried with MgSO4. Under nitrogen atmosphere, Compound 2 (0.64 g, 1 mmol), Compound 4 (1.40 g, 1.2 mmol), HBTU (0.76 g, 2 mmol) and HOBT (0.27 g, 2 mmol) were weighted and dissolved in anhydrous DMF (25 mL). After addition of DIPEA (0.66 mL, 4 mmol), the solution was stirred in ice bath for 30 min and at 25°C for 48 h. Organic solvents were removed by a rotary evaporator. The residue was redissolved in EtOAc, washed with saturated sodium bicarbonate, diluted hydrochloric acid, and saturated brine solution. The collected organic compound (Compound 5) was dried with MgSO4 and recrystallized from concentrated EtOAc. N-tert-butoxy arbonyl group in Compound

5 was removed by the same method as described in the section “2.2.1 synthesis of the hydrophobic moiety of cationic lipids” to obtain RL as off-white solid in 52% yield. As described in Fig. 1C, the synthesis of KL was similar to RL with an off-white powder in 43% yield. 2.2.3 Synthesis of dendritic histidine modified cationic lipid (HL, Fig. 1D).

Compound 2 (0.64 g, 1 mmol), Boc-Lys(Boc)-OH (0.42 g, 1.2 mmol), EDC (0.19 g,


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1.4 mmol), HOBT (0.28 g, 1.4 mmol) and DIPEA (1 mL, 5 mmol) were dissolved in anhydrous DCM (15 mL) at 0°C for 30 min and stirred at room temperature for 48 h. The reaction mixture was washed with saturated sodium bicarbonate, diluted hydrochloric acid, saturated brine solution. After dried with MgSO4 overnight,

Compound

9

was

obtained

by

column

chromatography

(silica

gel,

DCM/EtOAC/MeOH = 6:2:0.3) as white solid in 67% yield.

Compound 9 (0.9 g, 1 mmol) was deprotected by the same procedure as described above to obtain Compound 10, which was then mixed with (Boc)-His(Boc)-COOH (0.78 g, 2.4 mmol), EDC (0.43 g, 2.4 mmol), HOBT (0.34 g, 2.4 mmol) and DIPEA (1.2 mL, 8 mmol) in anhydrous DCM (15 mL). The solution was stirred under N2 atmosphere at 0°C for 30 min, followed by reaction at room temperature for 48 h. After washing with saturated sodium bicarbonate, diluted hydrochloric acid, saturated brine, the collected organic layer was dried with MgSO4 overnight, and concentrated under reduced pressure. Compound 11 was obtained by column chromatography (silica gel, DCM/EtOAC/MeOH = 6:2:1) as white powder. HL was obtained by removal of Boc groups in 75% yield.

A



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B

C


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D

Figure 1. Synthesis of dendritic amino acid-modified cationic lipid. 2.3

Assembly and Characterization of the Lipid-based Assemblies with

Basic Amino Acid Residues-rich Periphery. 2.3.1 Preparation, size, zeta potential and morphology of cationic lipid assemblies. Lipid assemblies were prepared by the methanol injection method as reported previously19 with some modifications. In brief, appropriate lipids (RL, KL or HL) were dissolved in about 10 µL of methanol, and injected into 1 mL of a fast string HBG solution (20 mM HEPES pH 7.4, 5% glucose). Cationic assemblies were formed spontaneously, followed by removal of residual methanol. The cationic lipid assemblies were diluted with HBG to a final concentration of 1 mg/mL for particle size and zeta potential measurement by a Zetasizer Nano-ZS (Malvern Instruments, UK) at 25°C. Moreover, appropriate amount of the sample dispersion was dropped on a 100 mesh copper grid, dyed using 2% phosphotungstic



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acid solution (pH 7.4) and then put in a desiccator for morphology observation by transmission electron microscopy (TEM, JM-1011, JEOL). 2.3.2 Buffering capacity of various lipid assemblies. Acid-base titration was applied for the evaluation of the buffering capacities of the cationic assemblies. Before titration, each lipid assemblies was diluted in 1 mL Milli-Q® water to a final lipid concentration of 1 mg/mL. The solution of cationic assemblies was adjusted to pH 11 by 0.1 M NaOH solution, titrated by aliquots HCl solution (5 µL) sequentially in a small flask, and stirred well at a constant room temperature. The pH values were recorded with an Orion Star Series Meter (Thermo, USA). The buffering capacity was calculated according to the following equation: Buffering capacity = -∆CA/∆pH Where -∆CA is the amount of strong acid added per liter of solution, and ∆pH is the pH change induced by addition of acid. 2.3.3 Calorimetric analysis of the cationic assemblies. A gel-to-lipid crystalline phase transition temperature (Tc) of the lipid assemblies was estimated using differential scanning calorimeter (DSC), as a probe of the fusogenic potential. The cationic lipid dispersion (pure Milli-Q water with pH 6.5) containing 100 µg lipids was added to an aluminum pan, sealed and placed in a DSC cell compartment (Differential Scanning Colorimeter Q2000; TA Instruments, Newcastle, DE). Measurement was started from 0°C, and the temperature was raised at a rate of 1°C/min up to 80°C. 20 µL of distilled water was mounted in a reference pan as control.


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2.4

Preparation and Characterization of Lipid-based Assemblies/DNA

Complexes. DNA complexes were prepared by mixing pDNA (100 ng/µL) and cationic lipid assemblies (1 µg/µL) solution gently at different N/P ratios (in the range of 20 - 60) in HBG buffer and incubated at room temperature for 30 min before use. Size and zeta potential were measured at a final DNA concentration of 3 µg/mL using a Zetasizer Nano ZS (Malvern Instruments, Worcestershire, UK). For the measurement in transfection conditions, gene complexes were mixed with an equal volume of DMEM with or without 20% serum for 4 h, and diluted with water to a final volume of 1 mL.20 Gel retardation assay was used for DNA compaction ability evaluation of cationic assemblies with different N/P ratios (1 - 60). In order to evaluate the stability of gene complexes in the presence of DNase, the indicated complexes were incubated with DNase (166 U/mL) for 15 min, followed by the addition of EDTA buffer to quench the enzyme activity. The resulted mixture was optionally incubated with heparin (4 mg/mL) for 2 hours prior to the agarose gel electrophoresis. All samples containing with 200 ng DNA were loaded onto 1% agarose gel for electrophoresis (70 V, 1 h). The gel was stained with ethidium bromide, and analyzed on the Molecular Imager ChemiDoc XRS+ (Bio-Rad, USA) to visualize the location of DNA bands.

2.5

In vitro Gene Transfection of the DNA Complexes. HepG2 and HEK293T

cells were preserved in DMEM-HG medium containing 10% (v/v) heat-inactivated fetal bovine serum, 100 µg/mL streptomycin and 100 IU/mL penicillin. All cell cultures were performed in an incubator with a humidified environment of 5% CO2



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and a constant temperature of 37°C. The medium was changed twice or thrice a week. Cells were harvested with 0.02% EDTA and 0.025% trypsin. In transfection study, cells were seeded in 96-well plate at a density of 1×104 cells per well in 100 µL of 10% FBS containing DMEM and grown to reach 70 - 80% cell confluence. Prior to transfection, the medium was replaced with 100 µL of fresh medium with or without 10% serum, to which various gene complexes with different N/P ratios of 20 - 80 were added (200 ng pEGFP or pGL3 per well). Both PEI25k and Lipofectamine 2000 (LA, Invitrogen, Carlsbad, CA) were used as controls. PEI25k/DNA complexes were prepared at the N/P ratio of 10 for 20 min before use, while Lipofectamine 2000 was used according to the manufacturer’s guidelines. After 4 h incubation, the transfection medium was refreshed with new medium containing 10% FBS. Cells were grown until they were processed for analysis of reporter gene expression. Qualitative evaluation of pEGFP transfection was observed 48 h later by an inverted fluorescence microscope (Leica, Germany), and the quantitative measurement was performed by flow cytometer. In addition, quantitative measurement using luciferase assay was performed 24 h post transfection according to manufacturer’s protocols. Relative light units (RLU) was measured by the luciferase reporter gene assay kit on a microplate reader (Bio-Rad, Model 550, USA) and protein content of the lysed cell was determined by BCA protein assay. Luciferase activity was expressed as the relative fluorescence intensity per mg protein (RLU/mg protein).


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2.6

Cell Viability of the Gene Complexes with Different Formulations. Cell

viability of the cationic lipid solution was determined by CCK-8 assay (Dojindo Molecular Technologies, Japan). Cells were seeded in 96-well plates at a density of 1×104 cells per well and cultured overnight. The medium was replaced by 100 µL of fresh medium (with or without 10% serum), to which the materials were added together with 200 ng pDNA per well. PEI25k and Lipofectamine 2000 were used as controls. 24 h later, the medium was again replaced by 100 µL of fresh DMEM containing 10% CCK-8 solution. Plates were incubated for 2 h at the same incubation conditions after which the absorbance was read at 450 nm by a microplate reader (Bio-Rad, Model 550, USA). The cell viability of the complexes treated cells was expressed as a relative to untreated cell controls taken as 100% metabolic activity.

2.7

Cellular Uptake and Intracellular Tracking Study of Complexes by Flow

Cytometry and Confocal Laser Scanning Microscopy. In cellular uptake study, HepG2 cells were seeded into 6-well culture plates at a density of 3×105 cells per well and grown for 24 h. The cells were then treated with fresh medium with various complexes containing 40% Cy5-labelled pGL3 at 37°C for 1, 2 and 4 h. Thereafter, cells were harvested by trypsin, resuspended in 0.5 mL PBS, and analyzed using a flow cytometer (BD biosciences) by counting 1×104 events. Following, the intracellular fate of these complexes was probed by confocal laser scanning microscope (CLSM, Leica TCS SP5, Germany). HepG2 cells were seeded at a density of 1×104 cells per well in 35 mm confocal dish (Ф =15 mm) overnight for attachment and continued to incubate with various complexes of 300 ng 40% Cy5-labelled pGL3.



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The intracellular behavior of gene complexes was monitored using CLSM at 2 and 4 h after addition of the fluorescent complexes. Thirty minutes prior to observation, the nucleus or lysosomes were stained by Hoechst 33342 or LysoTracker Green. Excited with a 543 nm laser, the emission signals of Cy5 was observed in Cy5 (dichroic mirror 655-755 nm) channels. The LysoTracker Green and Hoechst 33342 fluorophore were excited at 504 nm and 350 nm, the emission of them was detected with a 511 nm and 461 nm band pass filter, respectively. In the case of quenching extracellular fluorescence, Trypan blue was added at a final concentration of 0.4%.

2.8

In vivo Transfection Study. BABL/c nude mice (body weight 18 - 22 g)

were purchased from Jianyang Experimental Animal Centre (China), maintained in a germ-free environment in accordance NIH guidelines and allowed free access to food and water. After subcutaneous injection HepG2 cells (2×106 cells per mice) for three weeks, the tumor volume reached 170 - 200 mm3. Tumor size was measured using a vernier caliper across its longest (L) and shortest (W) diameters, and its volume was calculated using the formula of V [mm3] = 1/2 × LW2. Then, mice were randomly divided into four groups (n = 6) and intratumorally injected with a dosage of 20 µg pCMV-Luc or pEGFP plasmid DNA, respectively. Normal saline was used as negative control. At 48 h time point, mice were sacrificed. After washing with ice-cold PBS, tumors injected with pEGFP formulations were immediately froze in liquid nitrogen and cut into 10 µm thick cryosections. GFP fluorescence of the tissues was monitored under a confocal laser scanning microscope. For the treatment of


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pCMV-Luc, tumors and other tissues were taken out and homogenized using an IKA-Ultra-Turrax in 1 mL Promega cell lysis buffer. The supernatant was collected via centrifugation (12,000 g/min, 10 min) at 4°C for quantification of luciferase activity and protein content.

2.9

Statistical Analysis. Data were presented as means with standard deviations

(S.D.) of at least five independent samples and each measurement was performed in triplicates. Statistical analysis was determined by analysis of variance tests (ANOVA) using the software of Microsoft Excel 2007. Data sets were compared using two-tailed, unpaired t-tests. And a p value of < 0.05 was considered to be statistically significant.

3. RESULT AND DISCUSSION

3.1

Synthesis and Characterization of Cationic Lipid Assemblies. Lipid

carriers have already been demonstrated a promising delivery system for gene therapy in preclinical and clinical studies.21 However, the relatively high transfection expression level of current cationic lipid-based transfection reagents is usually associated with significant cytotoxicity;3 the search for optimum carriers has to continue. Based on our previous research of amino acid-modified gene carriers, in current work, novel lipids containing natural basic amino acid (arginines, lysines or histidines) with low dendritic generation were designed and synthesized as illustrated in Fig. 1 and Fig. S1. All of the linkages were convergently joined together by peptide bonds. After deprotection of primary amino and guanidine groups, the dendritic



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peptide-lipids were obtained with arginine- (Fig. 1B) or lysine-rich peptide (Fig. 1C) as hydrophilic head and dual-oleyl tails as hydrophobic segment. In terms of the histidine-modified lipid, the synthetic procedure had to be adjusted due to solubility problem of the twin histidine-containing head group during purification (Fig. 1D). The ESI-MS result and 1H-NMR spectrum confirmed the defined molecular structure of the targeting dendritic amino acid-modified cationic lipid (Fig. S1). All dendritic amino acid-based head groups were kept in the form of TFA salt. The research data from Takeoka’s group had demonstrated that the ionization states of the hydrophilic head group play a significant role in determining the properties of amino acid-based cationic assemblies, and result in the sequence of –NH3+TFA− > –NH3+Cl− > –NH2 in gene delivery efficiency.22

3.2

Characteristics of Cationic Lipid Assemblies with Arginine, Lysine or

Histidine Residues-rich Periphery. The size and zeta potential of cationic assemblies were shown in Table 1A, with diameters of all were almost around 100 nm. Zeta potentials of the arginine- and lysine-modified lipid assemblies were +32 ~ +38 mV, while histidine-type ones were much lower (~ +9.6 mV). The differences come from different protonation of residual amino, guanidine and imidazole groups in lysine, arginine and histidine-rich periphery,23 where pKa value of amino, guanidine and imidazole is 10.5, 12.5 and 6.0, respectively. Through the photos taken by transmission electron microscopy (Fig. 2A), it was shown that all these three assemblies were unilamellar vesicles. Assemblies from RL displayed uniform particles with spherical shape, while that of KL and HL showed


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irregular appearance due to big/small particles (diameter < 10 nm) aggregation. Although these three cationic assemblies had the same hydrophobic group, the different alkaline amino heads resulted in discrepancy in zeta potential and morphology of particles from different hydrophilicity and steric effect, which was in accordance with the report by Obata et. al.10, 24 In addition, unlike common cationic lipids,25, 26 all these three lipids could assemble relatively well without adding extra phospholipids or cholesterol, due to a proper balance between the hydrophilic and hydrophobic moieties.

Table 1. Characteristics of Various Cationic Assemblies and Gene Complexes

A. Size, Zeta Potential and Transition Temperature of Arginine-, Lysine-, Histidine-rich Assemblies. RL

KL

HL

Z-average size (nm)

63.1 ± 0.6

116.6 ± 12.3

82.6 ± 6.2

PDI

0.15 ± 0.026

0.45 ± 0.035

0.19 ± 0.009

Zeta potential (mV)

32.7 ± 1.0

38.0 ± 0.9

9.6 ± 0.6

Transition temp (°C)

42.5

51.3

66.3

B. Size Distribution and Zeta Potential of Gene Complexes.

RL/DNA

N/P

Z-average size (nm)

PDI

Zeta potential (mV)

20

117.3 ± 2.0

0.28 ± 0.006

42.2 ± 2.1

40

116.5 ± 2.6

0.16 ± 0.006

39.0 ± 1.8

60

112.9 ± 1.5

0.18 ± 0.020

40.9 ± 1.1

20

115.6 ± 2.0

0.26 ± 0.007

38.4 ± 0.1

40

124.6 ± 0.6

0.26 ± 0.010

39.3 ± 0.9

KL/DNA



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60

3.3

125.8 ± 2.5

0.20 ± 0.005

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40.7 ± 1.6

Buffering Capacity of Basic Amino Acid Residues in Various Lipid

Assemblies Periphery.

It was reported that an efficient gene delivery system should

have suitable buffering capacity in the mild acidic environment for facilitating the escape from endosomes.27 Higher buffering capacity means more increase in endosomal osmotic pressure and cationization to disrupt endocytic vesicles to facilitate complex release in the cytoplasm.28 As shown in Fig. 2B, the optimal buffering capacities of three cationic assemblies were all in the range of pH 5.0 – 8.0. KL exhibited the highest buffering capacity of 4.6 × 10-3 at pH 7.0, followed by the RL with 4.5 × 10-3 buffering capacity value at pH 7.1, which was almost the same as KL. While the buffering capacity of HL was about 4.0 × 10-3 pH 7.1. In accordance with other research,8 the better buffering capacity was owing to more α-amine groups in RL and KL than HL, which might benefit pDNA release and high transfection efficiency.


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Figure 2. Physicochemical characterization of lipid assemblies with basic amino acid residues-rich periphery: (A) TEM microscopy of various gene complexes together with extra magnification. (B) The buffering capacity of cationic assemblies: arginine(■, red), lysine- (▲, blue) and histidine-modified (♦, black) assemblies (1 mg/mL) in 1 mL Milli-Q® water (pH 11, adjusted with 0.1 M NaOH) were titrated by HCl.

3.4

Calorimetric Analysis of the Cationic Assemblies. The transition

temperature (Tc) is related with the fluidity of the cationic assemblies, which could further influence the final gene expression efficiency.29 Prepared assemblies were in the relatively stable gel state at room temperature. They could change to liquid-crystalline phase at the transition temperatures (Tc), showing improving fusion ability with biological membrane. Hence, lower phase transition temperature might result in higher membrane fusion potential.30 In our study, the transition temperature of the amino acid-modified lipids KL, RL and HL were 42.5, 51.3, and 66.3°C, respectively (Table 1A). The distinction in Tc might result from different ionization



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states of the polar head of various lipids31-33 (not from alkyl chain length in hydrophobic moiety10). In the dispersion medium (pure Milli-Q water, pH 6.5), the residual amino and guanidine were at ionized state, while imidazole was at non-ionized state. Previous research31, 32 found that the more component of lipid is ionized, the lower Tc will be for the lipid assemblies. It might be some explanation for the lower transition temperature of RL (or KL) than HL.

3.5

DNA Binding Ability of Cationic Assemblies. We analyzed complex

formation by varying the lipid-to-pDNA ratio in agarose gel retardation assay (Fig. S2A). Both RL and KL could retard DNA migration completely as the lipid-pDNA ratio increased to 20 and 10, respectively. In contrast, HL was unable to bind DNA even at the high N/P ratio of 60. The phenomena were in accordance with the zeta potential data, due to lower pKa values of the imidazole in HL.34 The imidazole groups of histidine were insufficiently cationized at physiological pH and consequently turned to be ineffective in electrostatic interaction with pDNA. Thus, no further characterization of HL was performed in this study. It was obviously that after treatment with DNase and release by heparin, gene condensed by all cationic assemblies showed well integrity, while naked plasmid DNA was completely degraded (Fig. S3). The DNA bands corresponding to plasmid in cationic lipid complexes were of stronger intensity than that in PEI group after decompression by heparin (with DNase), suggesting stronger compassion capacity of RL and KL than PEI. And the brightest band originated from RL and KL complexes might contribute to better transfection efficiency.


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3.6

Size and Zeta Potential of Cationic Complexes in the Presence and

Absence of Serum. Data of the hydrodynamic particle size and zeta potential of various complexes were showed in Table 1B. The cationic assemblies composed of RL or KL were able to condense plasmid DNA efficiently (N/P = 20, 40, 60), with approximate diameter of 110 - 125 nm and a narrow size distribution. Compared with DNA-free assemblies (Table 1A), the diameter of nanoparticles increased from several dozen (60 - 80 nm) to more than one hundred (110-125 nm) with constant zeta potential of +40 mV. Interestingly, the size and zeta potential did not change so much with increasing N/P ratio, which were in accordance with the study by Huang et al.35 Particle size of both complexes formed with RL or KL in DMEM medium without serum were almost twice as big as formed in HBG (Fig. S2B), indicating that electrolytes in the solvent induce complexes aggregation.36 The presence of serum in DMEM also clearly affected the size of complexes depending on electrostatic interaction with the negatively charged serum components.37 This “protein corona” forming phenomenon was common for the positively charged gene complexes, together with surface charge reversal (Fig. S2C). The zeta potential of arginine- and lysine-containing complexes changed from strongly positive in serum-free medium (+34.8 mV and +35.6 mV) to negative with serum protein coating (-12.2 mV and -11.7mV).

3.7

In vitro Transfection Study. Transfection ability of the RL and KL carriers

was evaluated with pGL3 (Fig. 3A and B) and pEGFP (Fig. 3C and S4) plasmid at various N/P ratios (20 – 80) in the presence or absence of serum. The quantitative



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evaluation of luciferase gene expression showed that both RL and KL had comparable or better gene transfection efficiency than the positive controls in HepG2 (Fig. 3A) and HEK293T cells (Fig. 3B). Under serum-free conditions, cells incubation with KL gene complexes showed similar luciferase expression with controls, while that incubation with RL complexes at optimal N/P reached the highest gene transfection level. For example, transfection of RL at N/P ratio of 40 was up to 5- fold higher than that of Lipofectamine 2000 in HepG2 cells, while gene expression at optimal N/P ratio of 20 was up to 26-fold than that of PEI25k in HEK293T cells. In the presence serum, transfection efficiencies of controls (PEI25k and Lipofectamine 2000) decreased evidently, which is a known serious obstacle for the application of cationic carriers in vivo.22 However, luciferase expression of the RL group in serum increased gradually with increasing lipid-to-pDNA ratios and even reached the transfection level under serum-free conditions at N/P ratio of 80, which was up to 190-fold higher than that of PEI25k. It indicated that the improved transfection abilities induced by the arginine-rich periphery is not being inhibited by serum. Interestingly, the transfection efficiency of KL complexes was relatively moderate in free-serum over N/P ratio of 40, but increased at least 10-fold by incubating in serum-containing medium. This result might be related with the decreased cytotoxicity after “protein coating” (Fig. 4).


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Figure 3. Transfection efficiency of various complexes on HepG2 and HEK293T cells: Quantitative evaluation of luciferase gene expression on HepG2 (A) and HEK293T

cells

(B) for arginine

(RL) and

lysine

(KL)-type

cationic

assemblies/DNA complexes at N/P ratios of 20 - 80. *p < 0.05 vs RL40 group. (C) Transfection of cationic assemblies containing EGFP plasmid on two kinds of cells at N/P ratio of 40. Qualitative and quantitative study utilized a DNA encoding EGFP reporter gene gave similar results (Fig. 3C and S4). The boost in transfection efficiency for RL and KL complexes was contributed from well-dispersive morphology of lipid assemblies, efficient DNA binding ability to form nano-sized complexes, strong buffering capacity and good liquidity. More importantly, it was obviously that RL showed better efficiency in transfection than KL. According to the buffering capacity and calorimetric analysis, the superiority of arginine-rich periphery in RL complexes does



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not rely on the strong buffering capacity and good liquidity. We speculated that it may result from the guanidine groups in the carriers, which could link to the phosphate groups in the lipid membrane to form bidentate hydrogen bonds, and lead to the enhanced cell-penetrating ability.12 Thus further insight research on cytotoxicity, cellular uptake, endosomal disruption and nuclear import have to be performed.

3.8

Cytotoxicity of Gene Complexes. In order to condense negatively charged

genes, effective and stable carriers are usually positively charged,24 which often lead to serious cytotoxicity. From the data presented in Fig. 4, it was obviously that under serum-free medium PEI25k was the most toxic carrier for both HepG2 (~71% viability) and HEK239T (~20% viability). Importantly, complexes composed of lysine or arginine-based lipids (RL and KL) showed lower cytotoxicity than either PEI25k or Lipofectamine 2000. For instance, the cellular metabolic activity remained 90% after treatment with RL and KL at N/P ratio of 40 in HepG2 cells, whereas for the control it was only 70% (PEI25k) and 80% (Lipofectamine), respectively. In the conditions of serum-containing medium, all gene complexes were well tolerated, showing > 90% cell viability in both cell lines. PEI25k/DNA complexes became nontoxic at the expense of losing transfection efficiency, while RL and KL complexes showed a win-win scenario with a gain in metabolic cell activity (Fig. 4) and high transfection conservation (Fig. 3). All of the delivery systems were positively charged, the diversity in toxicity might also derive from different biodegradability of PEI25k, Lipofectamine 2000 and peptide based cationic lipids after internalization into cell and cargo release.3, 10 Considering of in vitro transfection efficiency and cytotoxicity


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of the designed RL and KL complexes, we chose N/P ratio of 40 for further study.

Figure 4. Cell viability of the complexes (RL and KL) in HepG2 (A) and HEK293T cells (B) in the absence (left) or presence (right) of serum. *p < 0.05, **p < 0.01.

3.9

Observation of Internalization and Intracellular Fate of the Complexes.

One of essential premise of successful transfection is sufficient cellular internalization of gene complexes. In order to get insight into the mechanism through which arginine-rich complexes induced a higher delivery of pDNA, the uptake of these complexes by HepG2 cells with serum-containing DMEM was investigated. After 4 h incubation with Cy5-labelled complexes, flow cytometry results indicated that the ranking of internalized complexes was RL > KL > LA > PEI, which was the same as the confocal laser scanning microscope images (red spots) (Fig.5A). The fluorescent intensity of the RL and KL groups were 9-fold and 7-fold higher than of PEI25k (Fig. S5), respectively, showing a similar trend as the in vitro transfection study. The difference of cellular uptake was evident from the first 1 h incubation (Fig. S6), which indicated that RL with guanidine-rich periphery contribute to the internalization.



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Figure 5. Cellular uptake study (A) and intracellular fate (B) of various gene complexes with serum in HepG2 cells: (A) The fluorescence intensity of Cy5-positive cells was indicated by qualitative histogram plots and confocal microscopy; (B) Image of endosomal disruption of gene complexes containing Cy5-labelled plasmid (Red) in cells with LysoTracker Green (Green) after 2 h and 4 h incubation: LysoTracker Green channel (1), Cy5 channel (2), bright field (3), overlay (4), and enlarged view (5).


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After entering cells, efficient endosomal escape and nuclear import of cargo DNA are vital for the successful gene transfection.19 The intracellular fate of gene complexes was thus visualized by confocal microscope (Fig. 5B and Fig. S7). As shown in the image of the initial 2 h, yellow florescent spots only appeared in the RL and KL groups, as a result of an overlay of Cy5-labelled pGL3 (red) and LysoTracker labelled endo/lysosomes (green). Compared with the controls (Fig. S7), RL showed the highest strength and amount of florescence, in accordance with the results of cellular uptake study (Fig. 5A and S6). In all experimental groups, some red spots were also detected at the inner side of cell membrane, which might be complexes delivered into early endosomes. Following another two hours incubation, the yellow fluorescence was observed in the PEI25k and Lipofectamine 2000 groups (Fig. S7), reflecting increasing cellular uptake and a colocalization of complexes in the lysosomal compartment. In contrast, additional fluorescent feature were detected in cells treated with RL and KL complexes. Especially, in the RL group some pattern turned orange or red and accumulated at nuclear periphery (Fig. 5B), resulting from getting closer to nucleus by endosomes migration and successful endosomal disruption. The semi-quantificated R (Mander’s overlap coefficient) value inflected the colocalization of DNA and endosomes more directly (Fig. S8). At 2 h point, the Mander’s coefficient of RL was the highest among all groups, implied that the most degree of Cy5-labelled DNA encapsulated. Compared with the increasing R of other three groups, the opposite trend of R in RL group (decreased from 0.37 to 0.15) suggested that separation of endo/lysosome and RL complexes instead of endocytosis.



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The green fluorescence in RL group attenuated weak at 4 h time point, suggesting a weaker acid condition in endo/lysosome due to arginine penetration property.16, 38 Changes of the endo/lysosomal acidic environment could prevent or delay the degradation of DNA, and benefit for gene transport.39 Importantly, some red dots seemed to localize in the position of the nucleus (Fig. 5B). For better distinction, the nucleus was dyed by Hoechst 33342 (blue fluorescence in Fig. 6). It was obvious that fraction of dim purple stains appeared in the nuclear of cells treated with RL and KL complexes, suggesting import of cargo pDNA into nuclear environment. The Cy5-labelled DNA delivered by PEI25k or Lipofectamine 2000 appeared around but not inside the nucleus. The difference between DNA-loaded arginine- and lysine-rich assemblies in cell uptake and intracellular process is not unexpected, for high density of arginine could work like well-known arginine-containing cell-penetrating peptides to facilitate membrane disruption or fusion.40, 41


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Figure 6. CLSM images for nuclear delivery of complexes containing 300 ng Cy5-lablled pGL3 after 4 h: The images were Cy5-labelled DNA fluorescence in cells (1), cell nuclei stained by Hoechst 33342 (2), and overlay images (3).

3.10

In vivo Transfection Study. To validate the feasibility of the in vivo

application, gene transfer of pCMV-Luc encoding luciferase by RL and KL complexes was carried out in BALB/c nude mice with HepG2 hepatoma xenografts. After intratumoral administration, gene expression in the tumor, heart, liver, spleen, lung and kidney was assayed. Different levels of protein expression in the tumor were found for the three experimental groups (Fig. 7). RL and KL complexes induced a high level of luciferase activity in the tumor, which was about 7- and 2.4- fold higher than PEI25k/DNA complexes, respectively (p < 0.01). These results were in accordance with the transfection in vitro (Fig. 3), which implies potent in vivo gene delivery for RL lipoplexes. Besides these quantitative studies, a DNA encoding EGFP reporter gene gave the same tendency as the luciferase transfection in HepG2 tumors (Fig. 7A). Gene expression of RL lipoplexes was also the strongest as shown from the highest brightness of isolate tumors and the area of green fluorescent regions in tumor frozen sections (Fig. 7B).



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Figure 7. Gene transfer efficiency after intratumoral injection of various complexes formulations in vivo: (A) In vivo imaging of isolated tumors after injection of pEGFP formulations for 48 h; (B) Representative fluorescence micrographs of HepG2 tumor frozen sections after injecting pEGFP formulations into tumors for 2 days. (Scale bar = 50 µm); (C) Luciferase assay in HepG2 tumors and tissues after intratumoral treatment of DNA complexes. (n = 6, dose: 15 µg DNA per mouse; *p < 0.05, **p < 0.01 vs PEI group).

4. CONCLUSION In summary, we synthetized three dendritic amino acid (arginine, lysine and histidine)-modified cationic lipids with amino, guanidine or imidazole-rich periphery, which self-assembled into nano-sized carriers with defined molecular structure. Among them, lipid assemblies with histidine-rich surface showed poor binding and


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condensation capacities with pDNA. The other two lipids displayed greater DNA binding ability and strong buffering capacity. The arginine-rich lipid assemblies displayed a high level of gene transfer efficiency with minimum

cytotoxicity. Its

transfection activity was up to 190-fold higher than that of commercial PEI25k in the presence of serum. The superiority of RL assemblies does not result from the strong buffering capacity and good liquidity but may due to more efficient transmembrane capacity in cellular internalization, endosomal disruption and nuclear import. In vivo studies showed that complexes formed from these lipid-based assemblies with basic amino acid residues-rich periphery are promising candidates for further practical application.

ASSOCIATED CONTENT

Supporting Information 1

H NMR spectra and mass spectra; gel retardation assay; DLS data; EGFP

transfection studies; CLSM images

AUTHOR INFORMATION

Corresponding Author *E-mail: [email protected]. Tel.: +86-28-85415928. * E-mail: [email protected]. Tel.: +86-28-85410336. Fax: +86-28-85410653.

Notes



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Page 36 of 38

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENT This study was supported by National Natural Science Foundation of China (NSFC, No. 51133004, 81571794, 31271020), Fund from Sino-German Center for Research Promotion (GZ 756), Sichuan Province Science and Technology Support Program (2015SZ0122 and 2015SZ0189).

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