Cytoplasmic Reactive Cationic Amphiphiles for Efficient ... - NSFC

Article pubs.acs.org/Macromolecules

Cytoplasmic Reactive Cationic Amphiphiles for Efficient Intracellular Delivery and Self-Reporting Smart Release Jinming Hu,†,§ Xiao Wang,‡,§ Yinfeng Qian,‡ Yongqiang Yu,*,‡ Yanyan Jiang,† Guoying Zhang,† and Shiyong Liu*,† †

Downloaded by UNIV OF NEBRASKA-LINCOLN on August 31, 2015 | http://pubs.acs.org Publication Date (Web): August 4, 2015 | doi: 10.1021/acs.macromol.5b01110

CAS Key Laboratory of Soft Matter Chemistry, Hefei National Laboratory for Physical Sciences at the Microscale, iChem (Collaborative Innovation Center of Chemistry for Energy Materials), Department of Polymer Science and Engineering, University of Science and Technology of China, Hefei 230026, China ‡ Department of Radiology, The First Affiliated Hospital of Anhui Medical University, Hefei 230022, China S Supporting Information *

ABSTRACT: Nonviral gene delivery vectors need to overcome both extracellular and intracellular obstacles before releasing plasmid DNA in the transcriptionally active form. However, serum and transport stability desired for cationic polymer/pDNA polyplexes contradicts with the eventual plasmid release requirement; chain lengths of cationic polymer vectors render additional compromise between cytotoxicity and transfection efficiency. Although the introduction of stimuli-triggered degradable cationic polymers can partially solve these issues, the quest for novel design criteria and elucidation of elementary cellular transport pathways are highly desirable. Herein we report a supramolecular approach to construct fluorogenic gene delivery vectors via self-assembly of intracellular milieu-reactive cationic amphiphiles. This new type of micellar nanocarriers can effectively bind pDNA to form polyplexes due to multivalent cationic segments at micellar coronas. Upon cellular uptake and endosomal escape, hydrophobic micellar cores are subjected to fluorogenic Michael addition reactions with highly hydrophilic cytoplasmic thiols, leading to micellar disintegration, pDNA release, and emission turn-on for image-guided delivery.

■

INTRODUCTION Although nonviral gene delivery vectors based on lipids,1 polymers,2−9 and functionalized nanoparticles10−12 have been developed to alleviate safety issues of recombinant viral vectors, their delivery efficiency is relatively low.13−17 Specifically, polyplexes of cationic polymer vectors and plasmid DNA (pDNA) need to overcome both extracellular and intracellular obstacles before releasing pDNA for exogenous gene transcription in the cell nucleus. Serum and trafficking stability of polyplexes is necessary for cationic vectors to prevent pDNA from unwanted enzymatic degradation. However, this prerequisite contradicts with the final crucial step of polyplexes disintegration and pDNA release. Another dilemma between transfection efficiency and cytotoxicity also exists for cationic vectors. Although cationic polymer vectors such as poly(Llysine) (PLys), poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA), and polyethylenimine (PEI) typically exhibit enhanced transfection efficiency at higher molecular weights (MWs), their cytotoxicities are also considerably higher.18−24 These two compromises remarkably limit the design of efficient nonviral cationic polymer vectors. In order to solve these major issues, intracellular biodegradable polycations have been designed to promote polyplex unpackaging and decrease cytotoxicity.25−36 However, polyplex formation retards polycation degradation kinetics, which might lead to incomplete plasmid release on the transfection time scale.32−34 This limitation was getting worse when cytoplasmic © 2015 American Chemical Society

reductive milieu was utilized to trigger the degradation of disulfide-containing polycation vectors,37−40 considering the equilibrium and reversible nature of thiol−disulfide exchange reactions. In comparison, self-assembling amphiphiles possessing bioresponsive polycation building blocks are more promising.41−43 Extra polyplexes stability can be envisaged due to the multivalent pDNA-binding nature, whereas plasmid release can be actuated via polycation degradation and/or micelle-unimer equilibrium shift. However, reduction-triggered cleavage of disulfide linkages are mainly used in the design of biocleavable polycation amphiphiles, still incurring incomplete plasmid DNA release;41,42 in addition, micellar polyplexes possess extended half-lives relative to the duration of the transfection process. Overall, transfection efficiencies of the previously described degradable cationic vectors rarely exceed that of PEI.25−34,37−44 Thus, it is highly desirable to screen novel design criteria and elucidate elementary cellular transport pathways. Note that the latter relies on in situ monitoring of intracellular trafficking and disassembly of polyplexes.45,46 Previously, the abundance of glutathione (GSH) in cytoplasmic milieu (∼1−10 mM) has been widely exploited to trigger intracellular payload release from disulfide-containing nanocarriers and cationic vectors.37−40,47−52 We then speculate Received: May 22, 2015 Revised: July 9, 2015 Published: August 4, 2015 5959

DOI: 10.1021/acs.macromol.5b01110 Macromolecules 2015, 48, 5959−5968

Article

Macromolecules


Scheme 1. Supramolecular Aggregates of Thiol-Reactive PyCMA-PDMAEMA Cationic Amphiphiles (P1−P4) as Fluorogenic Intracellular Delivery Nanocarriersa

a

Nonfluorescent PyCMA-PDMAEMA amphiphiles (P1−P4) self-assemble into micellar nanoparticles capable of pDNA binding and polyplexes formation. Upon cellular uptake, endolysosomal escape, and exposure to cytoplasmic reductive milieu, Michael addition reactions of hydrophilic thiols (e.g., GSH) with PyCMA moieties within hydrophobic cores trigger disintegration of micellar nanoparticles, and the loss of multivalent interactions then leads to pDNA release; concomitantly, caged PyCMA emission is switched on, allowing for image-guided intracelular pDNA delivery.

that the integration of GSH-involved irreversible Michael addition reactions with self-assembling cationic amphiphiles might offer an excellent gene delivery vector with an intracellular pDNA “clean” release feature if hydrophilic GSH addition can render disintegration of cationic micellar assemblies.53,54 Furthermore, if GSH-relevant Michael addition is fluorogenic,55,56 we can thus facilely monitor the pDNA release process. To test the preceding hypothesis, we synthesized a series of amphiphiles consisting of thiol-reactive hydrophobic coumarin derivative (i.e., PyCMA) and cationic PDMAEMA of varying MWs (Scheme 1). Initially, PyCMAPDMAEMA spontaneously self-assembles into micelles, exhibiting no coumarin emission due to caging by α,βunsaturated ketone in PyCMA. Cationic micellar coronas are capable of effectively binding pDNA to form polyplexes. Upon cellular uptake and endosomal escape, cytoplasmic GSH-

mediated addition reactions with hydrophobic PyCMAcontaining cores will lead to micellar disintegration and pDNA release, the process of which can be in situ monitored due to emission turn-on of coumarin moieties.

■

MATERIALS AND METHODS

Materials. 2-Dimethylaminoethyl methacrylate (DMAEMA) was purified by vacuum distillation and stored at −20 °C prior to use. 2,2′Azobis(2-methylpropionitrile) (AIBN) was recrystallized from 95% ethanol. 2-Pyridinecarboxaldehyde, 2-bromoethanol, and Nile red were purchased from Sigma-Aldrich and used as received. 1,3-Benzenediol, phosphorus oxychloride (POCl3), ethyl acetoacetate, piperidine, N,N′dicyclohexylcarbodiimide (DCC), 4-dimethylaminopyridine (DMAP), glutathione (GSH), and 2-(2-ethoxyethoxy)ethanol (Aladdin) were purchased from Sinopharm Chemical Reagent Co., Ltd. and used as received unless otherwise noted. Fetal bovine serum (FBS), penicillin, streptomycin, and Dulbecco’s modified Eagle’s medium (DMEM) 5960


Article

Macromolecules


Scheme 2. Synthetic Routes Employed for the Preparation of (a) Thiol-Reactive α,β-Unsaturated Ketone-Caged Coumarin Derivative, (E)-7-(2-Hydroxyethoxy)-3-(3-(pyridin-2-yl)acryloyl)-2H-chromen-2-one (PyCMA−OH, 4) and (b) PyCMATerminated Poly(2- (dimethylamino) ethyl methacrylate) Cationic Amphiphiles, PyCMA-PDMAEMA

(44 g, 128 mmol) in acetonitrile (150 mL), dry DMF (12.6 g, 128 mmol), and freshly distilled dry POCl3 (22.6 g, 146 mmol) were added dropwise with stirring in an ice-water bath. The undissolved salt was filtered, washed with cold acetonitrile, and recrystallized in water. The mixture was filtered, washed with cold water, and dried overnight in a vacuum oven, giving 2,4-dihydroxybenzaldehyde (1) as a white solid (34 g; yield, 61.6%). A solution of 1 (10 g, 72.4 mmol), 2-bromoethanol (8.75 g, 70 mmol), and K2CO3 (19.35 g, 140 mmol) in acetone was heated to reflux at 70 °C. After being stirred overnight under reflux, the reaction mixture was cooled to room temperature and filtered to remove the salt. After removing all of the solvents, the residues were dissolved in CH2Cl2, washed with saline, dried over anhydrous MgSO4, filtered, and then concentrated on a rotary evaporator. The crude product was subjected to further purification by silica gel column chromatography using ethyl acetate and petroleum ether (v/v = 1:2) as the eluent, affording 2-hydroxy-4-(2- hydroxyethoxy)benzaldehyde (2; 4.2 g; yield, 32.9%) as a white powder. 1H NMR (CDCl3, ppm, TMS, Figure

were purchased from GIBCO and used as received. Ethidium bromide (EtBr), 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT), and branched polyethylenimine (Mw = 25 kDa, denoted as PEI25k) were purchased from Aldrich and used as received. Green fluorescent protein (GFP)-expressing plasmid DNA (pDNA) and luciferase-expressing pDNA were purchased from Aldevron and used as received. Dialysis tube (MWCO = 14 kDa) was purchased from Shanghai Green Bird Technology Development Co., Ltd. and stored in 1 mM ethylenediaminetetraacetic acid (EDTA) aqueous solution prior to use. All solvents were of analytical grade and used as received unless otherwise noted. Water was deionized with a Milli-Q SP reagent water system (Millipore) to a specific resistivity of 18.4 MΩ·cm. Sample Synthesis. Synthetic routes of (E)-7-(2-hydroxyethoxy)3-(3-(pyridin-2- yl)acryloyl)-2H-chromen-2-one precursor (PyCMA− OH, 4) and PyCMA-terminated PDMAEMA amphiphiles (PyCMAPDMAEMA, P1−P4) are shown in Scheme 2. Synthesis of 2-Hydroxy-4-(2-hydroxyethoxy)benzaldehyde (2; Scheme 2)..57,58 To a well-cooled (0−5 °C) solution of resorcinol 5961


Article

Macromolecules

Table 1. Structural Parameters of PyCMA-Terminated PDMAEMA Cationic Amphiphiles, PyCMA-PDMAEMAn, Used in This Study

a

entry

samples

Mn (kDa)a

Mn (kDa)b

Mw/Mnb

cmc (g/L)c

IC50 (μg/mL)d

P1 P2 P3 P4

PyCMA-PDMAEMA5 PyCMA-PDMAEMA11 PyCMA-PDMAEMA18 PyCMA-PDMAEMA42

1.1 1.9 3.2 6.8

2.0 3.0 6.8

1.06 1.09 1.10

0.010 0.013 0.035 0.082

71 61 52

Calculated from 1H NMR results. bDetermined by GPC using THF as the eluent. cDetermined by surface tensiometry at room temperature. Determined by cell viability against HepG2 cells via MTT assay.


d

S1a): δ 11.46 (s, 1 H), 9.73 (s, 1 H), 7.44 (d, 1 H), 6.56 (m, 1 H), 6.44 (d, 1 H), 4.14 (m, 2 H), 3.99 (m, 2 H), 2.01 (s, 1H). Synthesis of 3-Acetyl-7-(2-hydroxyethoxy)-2H-chromen-2-one (3; Scheme 2). Ethyl acetoacetate (1.43 g, 11 mmol) and 2 (1.78 g, 9.8 mmol) were dissolved in absolute EtOH (15 mL), and then piperidine (0.1 g) and AcOH (0.1 mL) were added as catalysts. The mixture was heated to reflux for 12 h and then was cooled to room temperature to obtain a bright yellow precipitate. The solid was collected by suction filtration and washed with cold absolute EtOH. The crude product was further purified by recrystallization from absolute EtOH, giving 3acetyl-7-(2-hydroxyethoxy)-2H-chromen-2-one (3; 1.82 g; yield, 75.3%) as a bright yellow crystal. 1H NMR (CDCl3, ppm, TMS, Figure S1b): δ 8.49 (s, 1 H), 7.55 (d, 1 H), 6.93 (m, 1 H), 6.85 (d, 1H), 4.19 (t, 1H), 4.04 (t, 1H), 2.71 (s, 3 H), 2.00 (br, 1 H). Synthesis of (E)-7-(2-Hydroxyethoxy)-3-(3-(pyridin-2-yl)acryloyl)2H-chromen-2-one (4; Scheme 2). Compound 3 (1.44 g, 5.8 mmol) and 2-pyridinecarboxaldehyde (1.24 g, 11.6 mmol) were dissolved in EtOH/CH3CN (40 mL; v/v = 1:1); piperidine (0.4 g) was then added as a catalyst. The mixture was heated to reflux for 24 h, and the solvents were removed under reduced pressure. The crude product was purified by recrystallization from absolute EtOH, affording (E)-7(2-hydroxyethoxy)-3-(3-(pyridin- 2-yl)acryloyl)-2H-chromen-2-one (4) as a yellow solid (0.83 g; yield, 42.6%). 1H NMR (CDCl3, ppm, TMS, Figure S1c): δ 8.72 (d, 1 H), 8.58 (s, 1 H), 8.34 (d, 1 H), 7.85 (d, 1H), 7.76 (m, 1 H), 7.62 (t, 2H), 7.30 (m, 1 H), 6.97 (m, 2 H), 4.23 (t, 2 H), 4.07 (t, 2H), 2.00 (br, 1 H). HRMS (m/z): calcd for C19H15NO5, 337.10; found, 338.10184 [M + H]+ (Figure S2). Synthesis of PyCMA-PDMAEMAn (Scheme 2). Typical procedures employed for the RAFT synthesis of cationic PDMAEMA homopolymers are as follows.59 Using the preparation of PDMAEMA5 as an example, into a reaction tube equipped with a magnetic stirring bar, DMAEMA (2.0 g, 12.7 mmol), 4-cyano-4(phenylcarbonothioylthio)pentanoic acid (586 mg, 2.1 mmol), AIBN (49 mg, 0.3 mmol), and 1,4-dioxane (4 mL) were charged. The reaction tube was carefully degassed by three freeze−pump−thaw cycles and then sealed under vacuum. After thermostatting at 70 °C in an oil bath and stirring for 6 h, the reaction tube was quenched into liquid nitrogen and opened and the reaction mixture was diluted with THF and then precipitated into an excess of petroleum ether. The preceding dissolution−precipitation cycle was repeated three times. PDMAEMA5 was obtained as reddish liquid (1.54 g; yield, 59.6%). PyCMA-PDMAEMA5 (P1) was prepared by the esterification of PDMAEMA5 with PyCMA−OH (4) in the presence of DCC and DMAP. In a typical procedure, PDMAEMA5 (100 mg, 0.1 mmol) was dissolved in anhydrous toluene (10 mL), and azeotropic distillation was carried out at 50 °C under reduced pressure to remove most of the solvent. Compound 4 (40 mg, 0.12 mmol) and dry CH2Cl2 (10 mL) were added. After cooling to 0 °C in an ice-water bath, a CH2Cl2 solution (10 mL) containing DCC (24 mg, 0.12 mmol) and DMAP (2.4 mg, 0.02 mmol) mixture was added dropwise over 1 h. The reaction mixture was stirred at room temperature for 24 h. After removing insoluble salt by filtration, the filtrate was concentrated on a rotary evaporator and then precipitated into an excess of cold diethyl ether. The preceding dissolution−precipitation cycle was repeated three times. After drying in a vacuum oven overnight at room temperature, PyCMA-PDMAEMA5 (P1) was obtained as a yellowish powder (80 mg; yield, 57%).

According to similar protocols, P2, P3, and P4 were also synthesized. Their molecular parameters are summarized in Table 1. Determination of Critical Micellization Concentration. The critical micellization concentration (cmc) values of P1−P4 were determined by surface tensiometry. Equilibrium surface tensions were measured using a JK99B tensiometer with a platinum plate. The measuring accuracy of the device as reported by the manufacturer is ±0.1 mN/m. The reported surface tension values were averaged from five successive measurements at a temperature of 37.0 ± 0.2 °C. The cmc values of P1−P4 are summarized in Table 1. Reactivity of 4 and P2 toward Thiol-Containing Compounds. The reactivity of PyCMA−OH (4) was investigated by 1H NMR and HPLC methods using 2-mercaptoethanol as a model thiol-containing compound (Figure S5). 1H NMR results suggested the disapperance of resonance signals characteristic of unsatuated double bond and HPLC elution profiles exhibited a shorter elution time after treating with 2-mercaptoethanol, suggesting the complete Michael addition reaction of 4 with 2-mercaptoethanol (monitored at λ = 365 nm). The reactivity of P2 was also examined by 1H NMR analysis (Figure S6), exhibiting a similar reaction propensity as compared to small molecule 4. Agarose-Gel Retardation Assay. In parallel, the pDNA binding abilities of P1−P3 before and after treating with GSH (10 mM) were investigated by agarose-gel retardation assay. The polyplexes solutions were prepared under a predetermined N/P charge ratio. Electrophoresis was carried out on 1% agarose gel with a current of 60 V for 40 min in TAE buffer solution (40 mM Tris-HCl, 1 vol % acetic acid, and 1 mM EDTA). The retardation of pDNA complexes was visualized by staining with ethidium bromide. The final pDNA content was 0.2 μg/well. Ethidium Bromide Replacement Assay..34,43 Compound P2 micellar solution (0.2 g/L) in 10 mM PBS buffer (pH 7.4) was mixed with pDNA to form polyplexes at an N/P ratio of 10. The polyplexes were mixed with EtBr (final concentration, 0.1 mg/mL) and incubated at room temperature for 15 min. After that, the ternary solution was divided into two aliquots. One aliquot was treated with GSH (final concentration, 10 mM). Subsequently, fluorescence spectra of the mixture solutions were recorded on a fluorescence spectrometer with the excitation and emission wavelengths at 510 and 590 nm, respectively. Cellular Uptake Process of PyCMA-PDMAEMA11 (P2) Micelles Loaded with Nile Red Observed by Confocal Laser Scanning Microscopy (CLSM). HepG2 cells were plated onto Petri dishes (In Vitro Scientific, 35 mm dish with 20 mm bottom well) at a density of 1 × 106 cells per dish and cultured in Dulbecco’s modified Eagle medium (DMEM) supplement with 10% fetal bovine serum (FBS), penicillin (100 units per mL), and streptomycin (100 μg mL−1) for 24 h at 37 °C in CO2/air (5:95). Nile-red-loaded P2 micellar solution was added to the Petri dishes at a final concentration of 0.05 g/L. After being incubated for 1 and 8 h, the cells were washed with PBS for three times. The fluorescence images were then taken on the microscope. The blue fluorescence from activated PyCMA moieties was observed using a 405 nm laser with the emission channel set to be 420−470 nm. Fluorescence of Nile red was observed using a 592 nm laser with the emission channel set to be 620−700 nm. Late endosomes and lysosomes of the cells were stained with LysoTracker Green (200 nM) after 30 min of incubation before imaging and observed using a 488 nm laser, and the emission wavelength was read 5962


Article


Macromolecules

Figure 1. (a, b) TEM images and (c, d) AFM images recorded for (a, c) P2 micelles and (b, d) P2/pDNA polyplexes at an N/P ratio of 10. AFM images of P2/pDNA polyplexes upon treating with 10 mM GSH for (e) 30 and (f) 200 min. (g) Hydrodynamic diameter distributions, f(Dh), recorded for micellar solutions (0.2 g/L) of P1, P2, P1/pDNA, and P2/pDNA polyplexes at an N/P ratio of 10, respectively. (h) ζ potential recorded for P2/pDNA polyplexes at varying N/P ratios. from 515 to 560 nm and expressed as green. Cellular uptake of P2 micelles and P2/Cy5-labeled pDNA polyplexes were observed similarly. The intracellular distribution of P2/Cy5-labeled pDNA polyplexes was quantitatively calculated by the co-localization ratio of blue/green and red/green fluorescence pixels with LysoTracker Green pixels. In Vitro Cytotoxicity Assay. HepG2 cell line was employed to evaluate the in vitro cytotoxicity of polymers via a MTT assay. HepG2 cells were first cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine’ serum (FBS), penicillin (100 units/mL), and streptomycin (100 μg/mL) at 37 °C in a CO2/air (5:95) incubator for 2 days. For cytotoxicity assay, HepG2 cells were seeded into 96-well plates at a density of 5000 cells/ well. Afterward, the cells were incubated for 24 h, then DMEM was replaced with fresh media, and the cells were treated with polymer solutions with varying concentrations; the treated cells were incubated in a humidified environment with 5% CO2 at 37 °C for 48 h. The MTT reagent (in 20 μL of PBS, 5 mg/mL) was added to each well. The cells were further incubated for 4 h at 37 °C. The medium in each well was then removed and replaced by 180 μL of DMSO. The plate was gently agitated for 15 min before the absorbance at 570 nm was recorded by a microplate reader (Thermo Fisher). Each experiment condition was done in quadruplicate, and the data are shown as the mean value plus a standard deviation (±SD). In Vitro Transfection and Gene Expression. HepG2 cells were seeded at a density of 5 × 103 cells/well in DMEM media supplemented with 10% FBS in 96-well plates and cultured for 24 h. Before adding polyplexes, the serum-containing culture medium was replaced with 100 μL of serum-free medium. The polyplexes were then added to reach a content of 200 ng of DNA/well at an N/P ratio of 10 and the mixture was incubated for 4 h at 37 °C. The culture medium was then replaced with 200 μL of fresh complete medium, and the cells were incubated for an additional 48 h. Transfection experiments were performed in triplicate. After incubation, the medium was removed and the cells were rinsed once with PBS. For the determination of GFP-expressing pDNA gene transfection, the GFPpositive cells were observed using fluorescence microscopy. For the

determination of luciferase-expressing pDNA gene transfection, the medium was removed and the cells were rinsed with PBS. The cells in each well were treated with 200 μL of cell lysis buffer followed by freeze−thaw cycles three times to ensure complete lysis. The cell lysate was transferred into a 200 μL vial and centrifuged for 5 min at 12,000 rpm, and the supernatant was collected for luminescence measurements. Luciferase gene expression was evaluated using the Luciferase Assay System (Promega Co., Madison, WI, USA). Bioluminescence signals were measured using a 96-well microplate luminometer with an exposure time of 10 s. The amount of total cellular protein was determined by the BCA protein assay kit (Pierce, Waltham, MA, USA). The final results were reported in terms of relative light units (RLU) per milligram of protein. PEI25k was employed as a positive control at an N/P ratio of 3. Characterization. All nuclear magnetic resonance (NMR) spectra were recorded on a Bruker AV300 NMR spectrometer (resonance frequency of 300 MHz for 1H) operated in the Fourier transform mode. CDCl3 was used as the solvent. Molecular weights and molecular weight distributions were determined by GPC using a series of three linear Styragel columns HR2, HR4, and HR5 and an oven temperature of 45 °C. A Waters 1515 pump and a Waters 2414 differential refractive index detector (set at 30 °C) were used. The eluent was THF at a flow rate of 1.0 mL min−1. A series of six polystyrene standards with molecular weights ranging from 800 to 400000 g mol−1 were used for calibration. Dynamic light scattering (DLS) measurements were conducted on a Malvern Zetasizer Nano ZS. All data were averaged over three consecutive measurements. All samples were filtered through 0.45 μm Millipore Acrodisc-12 filters to remove the dust. Transmission electron microscopy (TEM) observations were conducted on a Hitachi H-800 electron microscope at an acceleration voltage of 200 kV. The samples for TEM observations were prepared by placing 10 μL of the micellar solution (0.5 g L−1) on copper grids coated with thin films of Formvar and carbon successively. AFM measurements were performed on a Digital Instrument Multimode Nanoscope IIID operating in the tapping mode under ambient conditions. Silicon cantilever (RFESP) with resonance frequency of ∼80 kHz and spring constant of ∼3 N/m was 5963


Article


Macromolecules

Figure 2. (a) Evolution of scattered light intensity of P2 micellar solution (0.2 g/L) upon addition of 10 mM GSH at pH 7.4. (b) Hydrodynamic diameter distributions, f(Dh), recorded for P2 micelles before and after treating with GSH for 60 min. (c) Time-dependent evolution of OD600 during formation and dissociation (adding GSH) of P2/pDNA polyplexes at N/P ratio of 10. Evolution of (d) fluorescence emission spectra (λex = 370 nm) and (e) emission intensity changes at 416 nm recorded for P2/pDNA polyplexes upon treating with GSH at pH 7.4. (f) Macroscopic images recorded for P2/pDNA polyplexes before and after treating with GSH under ambient light (left) and UV irradiation (right). used. The set-point amplitude ratio was maintained at 0.7 to minimize sample deformation induced by the tip. The samples were prepared by dip coating 0.05 g/L aqueous micellar solutions onto the surface of freshly cleaved mica. Confocal laser scanning microscopy (CLSM) images were acquired using a Leica TCS SP5 microscope. HPLC analysis was performed with a Shimadzu HPLC system, equipped with a LC-20AP binary pump, a SPD-20A UV−vis detector, and a Symmetry C18 column. All UV−vis spectra were acquired on a TU1910 double-beam UV−vis spectrophotometer (Puxi General Instrumental Co., Beijing, China). Fluorescence spectra were recorded on an F-4600 (Hitachi) spectrofluorometer. The slit widths were set at 5 nm for both excitation and emission.

terminal moiety and hydrophilic PDMAEMA segment, P1−P4 exhibit amphiphilic features with critical micellization concentrations (cmcs) in the range of 10−80 mg/L (Table 1), and longer cationic blocks render higher cmc values. Taking PyCMA-PDMAEMA11 (P2) as an example, it self-assembles into spherical micelles in aqueous media, as evidenced by both TEM and AFM observations (Figure 1a,c). Possessing a pKa of ∼7.3, PDMAEMA homopolymers with high MWs have been utilized as gene delivery vectors.20−23,60,61 Although P2 unimers are of low MW (

Cytoplasmic Reactive Cationic Amphiphiles for Efficient ... - NSFC

Recommend Documents