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Shedding PEG palisade by temporal photo-stimulation and intracellular reducing milieu for facilitated intracellular trafficking and DNA releasing Tieyan Wang, Qixian Chen, Hongguang Lu, Wei Li, Zaifen Li, Jianbiao Ma, and Hui Gao Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.6b00355 • Publication Date (Web): 23 Jul 2016 Downloaded from http://pubs.acs.org on July 28, 2016
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Shedding PEG palisade by temporal photo-stimulation and intracellular reducing milieu for facilitated intracellular trafficking and DNA releasing
Tieyan Wanga,1, Qixian Chenb,1, Hongguang Lua, Wei Lia, Zaifen Lic, Jianbiao Maa, Hui Gaoa,*
a
School of Chemistry and Chemical Engineering, Tianjin Key Laboratory of Organic Solar Cells and
Photochemical Conversion, Tianjin University of Technology, Tianjin, Xiqing District, 391 Binshui Xidao, 300384, China b
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, 77 Massachusetts
Avenue, 02139, United States c
School of Science, Tianjin University, Tianjin, Nankai District, 92 Weijin Road, 300072, China
*
Corresponding author email:
[email protected] or
[email protected] 1
These two authors contributed equally to this work.
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ABSTRACT The dilemma of poly(ethylene glycol) surface modification (PEGylation) inspired to elaborate an intracellular-sheddable PEG palisade for the synthetic delivery systems. Here, PEG was attempted to conjugate to polyethylenimine (PEI) through tandem linkages of disulfide-bridge liable to cytoplasmic reduction and azobenzene/cyclodextrin inclusion complex responsive to external photoirradiation. The subsequent investigations revealed that facile PEG detachment could be achieved in endosome upon photo-irradiation, consequently engendering exposure of membrane-disruptive PEI for facilitated endosome escape. The liberated formulation in the cytosol was further subjected to complete PEG detachment relying on disulfide cleavage in the reductive cytosol, thus accelerating dissociation of electrostatically-assembled PEI/DNA polyplex to release DNA by means of polyion exchange reaction with intracellular charged species, ultimately contributing to efficient gene expression.
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■ INTRODUCTION Gene therapy was originated from a novel therapeutic scheme of delivering the programmed genomic sequence to the cells in the pathological site, followed by expression of the encoded functional proteins in the targeted cells to yield therapeutic outcome.1-4 Nevertheless, pertaining to the inherent characters of nucleic acids [including susceptibility to degradation in the biological milieu and reluctance to cell internalization due to its physical characters (micrometer size and negative charge)], it requests manufacture of well-defined gene delivery systems to accommodate nucleic acids (plasmid DNA: pDNA) into condensed structures, meanwhile capable of circumventing the predefined extracellular and intracellular barriers that potentially inhibit transgene, ultimately affording the functionalities required to perform the task of gene delivery and gene expression.5,6 Aiming to address the intrinsic drawbacks of reluctant cell internalization of DNA, synthetic cationic materials (lipids, polymers) were extensively developed to self-assemble with pDNA based on electrostatic interaction,7-14 having validated to be capable of packaging DNA into 100 or sub-100 nanoscaled structures in accompanied with negative charge of DNA being neutralized to be relatively positive. Yet, the physiochemical properties for the surface of the resulted formulation were accused to be non-stealth, susceptible to non-specific interactions in the biological milieu and thus ominous consequences e.g. electrostatic mediated association with charged biological species and protein adsorption provoked severe toxicity or bio-elimination triggered from reticuloendothelial system.15,16 As an acknowledged approach to resolve this issue, poly(ethylene glycol) (PEG) surface modification (PEGylation) advocated tempting utilities in curtailing non-specific interactions by virtue of its excellent hydrophilicity and biocompatibility.17-19 Particularly, PEG camouflage endowed appreciable stealthy functionality and colloidal stabilities in the extracellular milieu, giving rising to improved bioavailability of the proposed formulations to the targeted cells.20-23 Furthermore, as noted that the nanoscaled structures internalized into the cells through endocytosis, the proposed PEGylated formulations were further threatened to degrade in the late endosome/lysosome. To the crucial task of retrieving the entrapped formulations from the late endosome/lysosome, PEGylation was conjectured to impede exposure of cationic species thus ensuing interaction and destabilization to the membrane of late endosome/lysosome, ultimately hampering the transgene activity.24,25 To manage this PEG dilemma, we attempted to introduce tandem linkage of redox-responsive disulfide bond and light-regulated azobenzene (Az)/cyclodextrin (CD) inclusion complex between PEG
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and cationic poly(ethyleneimine) (PEI) segment (Fig 1). These linkages are postulated to yield a distinctive PEG shedable palisade. Note that supramolecular nanoparticles or polyplexes have been previously constructed based on cyclodextrin (CD)/adamantine host-guest interaction.26-30 Herein, we adopted this platform to develop a stimulus-responsive polyplex gene delivery system. Presumably, the subsequent introduction of dual-responsive PEG detachment components could result in promotion of the previous platform with enhanced transfection efficiency.31 The azobenzene (Az)/CD inclusion complex has been verified to regulate by photo-irradiation based on photoisomerization of azobenzene,32-35 whereas disulfide linkage could be selectively cleaved in the cell interior. Of note, indeed plenty of delivery systems contrived to compile disulfide linkage for pursuit of intracellularresponsive cleavage with respect to the striking intracellular reducing potential (abundant glutathione: GSH) in contrast to the extracellular milieu.36-38 Nevertheless, the intracellular acidic and digestive late endosome/lysosome was implicated to present relative oxidative potential as compared to the cytosol. Hence, PEG detachment based on strategic cleavage of disulfide-bridge in endosome for the sake of endosome escape should be difficult to accomplish. As a rationale alternative, the strategic use of photo-responsive azobenzene could serve as an intriguing approach to detach PEG for facilitated endosome escape.39,40 Furthermore, subsequent to the proposed formulation being released into the cytosol, PEG which remained tethering onto polyplex was presumed to experience complete detachment by mean of disulfide cleavage due to abundant GSH in the cytosol. This could propel the
Fig 1. Schematic illustration of formulation of polyplex micelle with shedable PEG palisade through stimuliresponsive Az/CD inclusion complexation and redox- responsive disulfide linkage.
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interactions of the resulting formulation with neighboring charged compounds, eventually facilitating DNA release based on exchange reaction for gene expression.
■ RESULTS AND DISCUSSION Synthesis and characterizations. PEI bearing CD residues was synthesized according to a coupling reaction, where the hydroxyl groups of CD was activated by CDI, followed by reaction with amino groups of PEI. According to subsequent 1H-NMR measurement for the product, approximate 9 % of total amino groups of PEI were determined to have been functionalized with CD, namely, 1.26 units of CD was grafted onto each LMW PEI. Furthermore, difunctionalized PEG was synthesized through two-step coupling reaction, where sulfhydryl-terminated PEG was activated by CDI, followed by reaction with excessive cystamine dihydrochloride in presence of triethylamine to yield PEG-SSNH2. Subsequently, the terminal amino group of PEG-SS-NH2 was activated by CDI and reacted with the carboxyl groups of amino benzene acetic acid to yield PEG-SS-Az.. On the other hand, PEG-Az was also synthesized as a control based on a similar procedure using ethanediamine instead of cystamine dihydrochloride. The subsequent 1H-NMR measurement verified the chemical structure of the ultimate PEG-SS-Az and PEG-Az. Details of synthetic schemes and structural information are provided in the Supporting Information.
Grafting PEG onto PEI. Aiming for grafting PEG onto PEI, it is critical to validate the feasibility of inclusion complex formation based on host and guest interaction between azobenzene groups from PEG derivative (PEG-SS-Az) and CD groups from PEI derivative (PEI-CD). To test the feasibility, we separately dissolved PEG-SS-Az (under dark) and PEI-CD in 10 mM HEPES buffer (pH 7.4). The reaction was conducted by mixing the aforementioned solutions under dark at equal molar ratio of azobenzene and CD. Following 30 min reaction, GPC measurement (RI detector) was performed for the reaction solution. The starting materials of PEI-CD and PEG-SS-Az were also taken as reference for GPC measurement. Note that PEG-SS-Az was characterized with single azobenzene moiety per polymer, whereas PEI-CD was characterized with approximate 12.31 units of CD per PEICD. Namely, marked excess availability of CD moieties to Az moieties should be consistent to anticipate the high feasibility of CD-Az host-guest assembly. As a result, the presented GPC trace, characterized with majority of the PEG-SS-Az/PEI-CD assembly and minority of the starting materials of PEG-SS-Az and PEI-CD was obtained (Fig 2a). This observation suggested the successful grafting PEG onto PEI through guest-host interaction of azobenzene group from PEG derivative and CD
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residues from PEI derivative. Meanwhile, UV absorption of PEG-SS-Az solution appeared to be markedly amplified with supplementation of PEI-CD solution (Fig 2b). This absorption amplification has been verified in a precedent study to ascribe to the elevated molar extinction coefficient due to formation of inclusion complex of Az and CD,41,42 again affirming the successful linkage of PEG and PEI through host-guest interaction of Az and CD. In respect to the intriguing photo-responsive character of azobenzene (azobenzene was characterized to undergo photoisomerization from trans- to cis- isomers upon photo-irradiation at 365 nm), the inclusion complex was postulated to fall apart upon photo-irradiation. To verify this photoresponsive property, we also measured the GPC profile of the reaction solution under treatment of photo-irradiation. In accordance with our speculation, the molecular weight of the reaction solution under photo-irradiation (365 nm) was remarkably reduced (Fig 2a). The resulting peaks was determined to be well assigned to the starting components of PEG derivative and PEI derivative (Fig 2a). These results approved the feasibility of facile utilizing external stimuli (photo-responsive strategy) to regulate Az and CD inclusion complex for pursuit of facile PEG detachment from the parental PEI. Additionally, UV spectra of the PEI-CD/PEG-SS-Az/DNA evolved post 15 min irradiation (Fig 2c), characterized by absorbance decline at 325 nm, consistent with the previous observation of photoisomerization of Az from trans- to cis- configuration.39,40 Pertaining to the identical UV spectra of the polyplexes of PEI-CD/DNA and PEI-CD/PEG-SS-Az/DNA prior to and post irradiation, photo-responsive character of azobenzene was determined as an intriguing functional component for pursuit of photo-responsive detachment. Formulation of polyplex micelle. PEG grafted PEI (PEI-CD/PEG-SS-Az) was attempted to complex with pDNA to prepare polyplex micelle through electrostatic interaction at varying N/P ratios,
Fig 2. Verification of inclusion complexation through Az and CD for grafting PEG onto PEI. a): GPC measurement; b) and c) UV absorbance measurement.
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Fig 3. Formulation characterization for polyplex from PEI-CD and polyplex micelle from PEI-CD/PEG-SS-Az. a): Size of both formulations was characterized by DLS measurement; b): Morphology characterization of both formulations was characterized by SEM measurement. The scale bar represents 200 nm.
Fig 4. Zeta potential of polyplex from PEI-CD and polyplex micelle from PEI-CD/PEG-SS-Az under diverse treatments.
where PEI-CD was used as a control. According to gel electrophoresis characterization, pDNA was capable of being fully neutralized in both formulations starting from N/P ratio of 2 (Fig S5 in
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Supporting Information). According to DLS measurement, pDNA could be efficiently condensed by both PEI-CD/PEG-SS-Az and PEI-CD into nanoscaled structures with approximate diameter of 115125 nm for PEI-CD-based polyplexes and 131-145 nm for PEI-CD/PEG-SS-Az/DNA polyplex micelles (Fig 3a). In consistency, SEM morphology characterization verified the nanoscaled structures exhibiting as uniform spherical nanoparticles (Fig 3b). Note that the formulation from PEI-CD/PEGSS-Az displayed relative larger diameter than the formulation from PEI-CD. A plausible reason to explain this relative larger size of PEG-included sample may be the existence of PEG palisade and also unfavorable high PEG crowdedness accompanied with the process of pDNA condensation restricting DNA condensation as compared to PEI-CD-based polyplexes. Still, zeta potential measurement confirmed successful PEG grafting onto PEI (Fig 4), thereby corresponding to a core-shell structure where PEG was serving as external shielding shell, as suggested by the zeta potential of the formulation from PEI-CD/PEG-SS-Az being substantially minimized to be neutral as compared to the polyplex from PEI-CD (Fig 4). Of note, photo-irradiation and GSH treatment for the formulation from PEI-CD/PEG-SS-Az implied to induce a facile PEG detachment, as evidenced by pronounced jump of zeta potential post treatment to become positive charge (comparable to the zeta potential of PEI-CD). Specifically, the zeta potential of samples with single treatment, either with light irradiation or with GSH, also induced an increase zeta potential but not comparable to that of PEI-CD-based polyplexes, indicating the partial detachment of PEG (Fig 4). PEGylation for reduced non-specific interactions and consequence. One of the important merits of PEGylation lies in its appreciable performance in reducing nonspecific interactions with biological species and biological structures.43,44 In consequence, PEGylation can be anticipated to not only improve the colloidal stability of the formulation thus bioavailability to the targeted cells but also reduce disruptive potency of the formulation to the cytoplasm membrane thus cytotoxicity. To verify the impacts of PEGylation, we compared the resistance of polyplex micelle from PEI-CD/PEG-SS-Az and polyplex from PEI-CD against exchange reaction with anionic dextran sulfate. Here, the samples of polyplex micelle from PEI-CD/PEG-SS-Az and polyplex from PEI-CD were incubated at varying concentrations of dextran sulfate, the dissociation behaviors of polyplex micelle and polyplex to release pDNA through polyion exchange reaction were determined by gel electrophoresis of the reaction solution. Apparent dissociation to release DNA was observed to start from dextran sulfate concentration at S/P ratio of 20 (defined as the molar ratio between the sulfur from dextran sulfate and
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the phosphate from pDNA) for the sample of polyplex (Fig 5). Yet, the polyplex micelle displayed markedly improved resistance to dissociation from dextran sulfate even at concentration as high as S/P ratio of 60 (Fig 5). This result approved the functional role of PEGylation in reducing non-specific interactions of anionic biological species, thus suggesting improved stealth function in the extracellular milieu. Most likely, this reduced electrostatic interaction was given by the charge-masking effect due to the existence of PEG palisade. Following this rule, we could also anticipate reduced interactive potency
Fig 5. Resistance of polyplex from PEI-CD and polyplex micelle from PEI-CD/PEG-SS-Az to polyion exchange reaction by incubation with varying concentration of dextran sulfate.
Fig 6. Cell cytotoxicity and transfection efficiency of diverse formulations in HUVEC. a): cytotoxicity of diverse formulations in HUVEC, where cytotoxicity was expressed in terms of cell viabilities; b): transfection efficiency of diverse polyplex formulations in HUVEC, where pDNA encoding luciferase was used as the reporter gene.
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from PEGylation to the negative charged plasma membrane, thereby conducing to lowered disruptive potency to the cytoplasm membrane. In this regard, we evaluated the cytotoxicity of HUVEC in presence of polyplex micelle from PEI-CD/PEG-SS-Az and polyplex from PEI-CD. Severe toxicity was confirmed for the control sample of polyplex from PEI-CD, whereas negligible cytotoxicity was confirmed for polyplex micelle from PEI-CD/PEG-SS-Az (Fig 6a). These results approved our strategic use of stimuli-responsive PEG grafting to PEI for construction of PEG shielding corona to pursue improved stealth behavior and safety profile. Stimuli-responsive PEG detachment for facilitated intracellular trafficking. Improved resistance to exchange reaction with biological species by PEGylation in the extracellular milieu is supposed to render improved bioavailability to the targeted cells. Note that the nanoparticles was identified to internalize into the cells through endocytosis pathway. Hence, the internalized polyplex micelles, subsequence to cellular uptake, are subjected to entrapment into acidic late endosome and digestive lysosome. Therefore, polyplex micelles are requested to afford adequate facilities to retrieve
Fig 7. Insight into intracellular trafficking behaviors of polyplex micelle from PEI-CD/PEG-SS-Az. Left: intracellular distribution of polyplex in HUVEC at 6 h post transfection. Cell nuclei were stained into blue, late endosomes/lysosomes were stained into green, and formulations were stained into red. Right: impact of photoirradiation on intracellular distribution of polyplex micelle from PEI-CD/PEG-SS-Az in HUVEC.
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Fig 8. Impact of photo-irradiation and GSH on cell destabilization potency of polyplex micelle from PEG-SSAz/PEI-CD.
from this entrapment. Learning from the mechanism that natural viruses adopt to exert endosome escape, e.g. adenovirus was dissected to experience structural transformation in acidic endosome to expose the membrane-disrupting components for facilitating escape from endosome entrapment.45 Here, we could conjecture that our proposed strategy of PEG detachment based on dissemble of inclusion complex of azobenzene/CD responsive to photo-irritation, could facilitate exposure of cationic PEI, accordingly eliciting readily interaction with anionic endosome membrane for its destabilization. To verify this speculation, the zeta potential change of polyplex micelle from PEICD/PEG-SS-Az upon photo-irradiation was recorded to identify the behavior of PEG detachment. In accordance to our hypothesis, the zeta potential of polyplex micelle from PEI-CD/PEG-SS-Az was observed to undergo pronounced jump to be a remarkable positive charge of + 22.6 mV upon photoirradiation (Fig 4), which was in good agreement of the result of photo-irradiation-mediated PEG detachment in GPC measurement. Furthermore, the remarkable positive charge of the resulting formulation upon photo-irradiation was assumed to stimulate readily association with anionic endosome membrane. To our interest, CLSM observation captured distinctive intra-endosome distribution profiles of polyplex micelle from PEI-CD/PEG-SS-Az prior to and post photo-irradiation. In this observation, HUVEC was seeded in a collagen-coated dish, followed by 24 h incubation. The solution of polyplex micelle from PEI-CD/PEG-SS-Az was added into the medium for 6 h incubation.
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Note that Cy5-labeled DNA was used for prepare polyplex micelle (red), whereas the late endosome and lysosome was stained into green by Cell Light late endosome GFP. As shown in Fig 7, prior to photo-irradiation, the polyplex was observed to fully occupy the entire cavity of endosome, presenting as red micrometer-sized subcellular organelles (Fig 7). In contrast, under 15 min photo-irritation at 365 nm and 1 h post incubation, the resulting formulations appeared distinct propensity to intensively associate with endosome membrane, thus representing as circles aligning the endosome membrane (Fig 7). In light of striking membrane disruptive potency of cationic PEI species, these membraneassociating formulations would commit potent disruption to the endosome membrane, eventually giving rise to the release of the formulations from endosome entrapment to the cytosol. The evaluation of membrane destabilization potency of polyplex micelle prior to and post irradiation approved polyplex micelle upon photo-irradiation presented drastic membrane disruption potency (Fig 8). Hence, the proposed system delineated a tempting endosome escape strategy by transform its physiochemical structure to adhere and disrupt endosome membrane upon external stimuli. To our best knowledge, this could be first direct observation to capturing the consequence of PEG detachment to induce adhesion of cationic complex to the endosome membrane, which could provide important implication in understanding the mechanism of endosome escape pathway for the synthetic gene delivery carriers and also design concept for development of efficient gene delivery carriers capable of retrieving DNA from endosome entrapment. Polyplex released to the cytosol, was requested to dissemble to liberate DNA for facilitating the machinery of gene expression.46,47 To achieve this objective, complete PEG detachment is preferable in respect that the liberation of DNA was largely relied on polyion exchange reaction of polyplex formulation with intracellular charged species. In this regard, disulfide linkage was believed to supply another opportunity to accomplish complete PEG detachment from the formulation with respect to GSH abundance in cytoplasm. Noteworthy was the zeta potential measurement for the polyplex micelle from PEI-CD/PEG-SS-Az upon photo-irradiation suggesting incomplete PEG detachment (Fig 4). Yet, further incubation in GSH led to complete PEG detachment, as evidenced by fully recovered zeta potential value for polyplex micelle from PEI-CD/PEG-SS-Az under sequential photo irradiation and GSH treatment (Fig 4). Besides, resistance to exchange reaction by gel electrophoresis also concluded the most readily dissociation behaviors of polyplex micelle with sequential photo-irradiation and GSH treatment rather than only photo-irradiation (Fig 9). These results support the necessity of both
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Fig 9. Resistance of polyplex from PEI-CD and polyplex micelle from PEI-CD/PEG-SS-Az to polyion exchange reaction by incubation with varying concentration of dextran sulfate.
disulfide and inclusion complex to seek adequate PEG detachment for the functionalities of structural transformation responsive to external and internal stimuli for facilitated intracellular trafficking. Ultimately, the proposed formulation of polyplex micelle from PEI-CD/PEG-SS-Az was attempted in cell transfection evaluation. In consistent with the previous research, the transfection efficiency of PEGylated formulation was remarkably lower than PEI (Fig 6b),40,48 which can be explained by the impeded affinity to the biological structure thereby inferior activity in cellular uptake and endosome escape. Nevertheless, the transfection activity was observed to remarkably stimulate
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under photo-irritation or GSH stimulus, and the dual-stimulus vectors even capable of achieving as high as the magnitude of PEI (Fig 6b). Most probably, the photo-irritation encouraged PEG detachment from the polyplex micelle in endosome, thereby eliciting readily exposure of cationic PEI who committed disruption to the endosome membrane. Following this way, the formulation could be released into the cytosol. By virtue of disulfide cleavage in GSH rich cytosol, the formulation (no PEG) in the cytosol was susceptible to dissociation to liberate DNA, eventually facilitating the machinery of gene expression. Pertaining to practical applications of this UV-specified functional system, DNA vaccination could be a particular valid approach by intradermal injection of polyplex micelle solution, followed by temporal irritation treatment for promotion of gene expression. ■ CONCLUSIONS The proposed formulation was manufactured to possess an intriguing stimuli-responsive PEG palisade regulated by external photo-irradiation and endogenous species. Appreciable safety profile was achieved by virtue of PEGylation in the extracellular milieu. Once internalized into the cell through endocytosis, external photo-irradiation inspired structural transformation of the proposed formulation to exert disruption to endosome membrane, consequently retrieving its entrapment from endosome. Ultimately, release of DNA from the formulation for efficient gene expression could be accelerated based on further structural transformation responsive to the intracellular redox. Hence, the novel chemistry strategies in the proposed system should be highlighted to address the issues encountered in development of gene or drug delivery systems to achieve facilitated intracellular trafficking. ■ MATERIAL AND METHODS Material. Branched polyethylenimine (PEI, 600 Da) and 4-amino benzene acetic acid were obtained from Aladdin Industrial Corporation (Shanghai, China). β-CD was purchased from Tianjin Chemical Reagent Co. Ltd (Tianjin, China). Nitrosobenzene was obtained from Tokyo Chemical Industry (Shanghai, China). 1,10-carbonyldiimidazole (CDI) was purchased from Best reagent co., Ltd. (Chengdu, China). Methoxy poly(ethylene glycol) mercaptan (mPEG-SH, 2,000 Da) was obtain from Shanghai ZZBIO Co. Ltd (Shanghai, China). Cystamine dihydrochloride was purchased from Sahn chemical technology co., Ltd (Shanghai, China). Dimethyl sulfoxide (DMSO), dimethylformamide (DMF), tetrahydrofuran (THF), ethylenediamine, triethylamine dichloromethane (DCM) and other reagents were purchased from Tianjin Chemical Reagent Co. Ltd (Tianjin, China). Prior to use, THF was dried over sodium, and further distilled with benzophenone (anhydrous indicator).
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Polyplex preparation. Aiming to grafting PEG to PEI, aliquot of PEI-CD and PEG-SS-Az or PEG-Az solution at equal molar ratio was mixed in a light-shielded cuvette under vortexing for 30 s, followed by another 30 min incubation at room temperature. Furthermore, the aforementioned reaction mixture was diluted to a targeted concentration for complexation with pDNA solution (50 µg/mL) at targeted N/P ratio (defined as the molar ratio of amino groups from the polymer to the phosphate groups from pDNA) under vortexing for 30 s, followed by 30 min incubation prior to usage. Note that all the solutions of formulations were freshly prepared for all the investigations. Physiochemical characterizations. PEI-CD/DNA and PEI-CD/PEG-SS-Az/DNA polyplexes were prepared at N/P ratio of 10 according to the aforementioned procedure. Molecular weights and polydispersity of the prepared polymers were characterized by gel permeation chromatography (GPC, Waters 2414 system, Milford, MA) equipped with a refractive index detector, using THF as a mobile phase at a flow rate of 1.0 mL/min at 35 °C. The GPC was calibrated with a series of polyethylene glycol with monodisperse. All sample solutions were filtered through a 0.45 µm filter prior to analysis. The pDNA concentration was adjusted at 25 µg/mL for Dynamic Light Scattering (DLS) measurement. Of note, three times independent measurements were conducted for each sample at 20 °C under 90° detection angle. On the other hand, the morphologies of PEI-CD/DNA and PEI-CD/PEG-SS-Az/DNA polyplexes were characterized by scanning electron microscope (SEM) measurement by following the procedures as described previously. Gel retardation assay. The charge neutralizing behaviors of PEI-CD and PEI-CD/PEG-SS-Az to DNA were examined by gel retardation assay. Polyplexes of PEI-CD/PEG-SS-Az/DNA and PEICD/DNA at varying N/P ratios (0.5, 1, 2, 3, 5, 10 and 20) were prepared as aforementioned, where the concentration of DNA was adjusted to 25 µg/mL. To evaluate the stability of polyplexes against exchange reaction with charged molecules, dextran sulfate (25 kDa) was employed as a counter polyanion. Aiming to evaluate the impact of redox on the stability of polyplex micelle from PEICD/PEG-SS-Az, the solution of polyplex micelle was supplemented with GSH (0.5 mM), followed by incubation at 37 °C for 2 h. Then, all the samples were incubated at varying concentration of dextran sulfate. Following 6 h reaction, the reaction solution was injected into an agarose gel for electrophoresis at 80 V for 90 min, and DNA was visualized by subsequent immersion of gel in ethidium bromide solution (0.5 µg/mL).
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Cytotoxicity and transfection efficiency. Human umbilical vein endothelial cells (HUVEC) were seeded in 24-well collagen-coated plates at a density of 20,000 cells/well with 400 µL of EGMTM plus Medium incubated 24 h (37 °C, CO2 concentration 5%). After the culture medium was replaced with fresh medium, each formulation containing 1 µg pDNA/well was added to each well. At 24 h post incubation, the medium was replaced with 400 µL of fresh medium, followed by another 24 h incubation. Luciferase expression was determined using a Luciferase assay system (Promega, Madison, WI) and a GloMaxTM 96 microplate luminometer (Promega, Madison, WI) following the manufacture’s protocol (n = 4). The quantity of protein in the cell lysates was determined according to a MicroBCA™ Protein Assay Reagent Kit following the manufacture’s protocol. Cell viability was determined using a Cell Counting Kit-8 (Dojindo, Kumamoto, Japan) following the manufacture’s protocol. Cell membrane destabilization activity by INCELL Analyzer. The membrane destabilizing activity of the formulations was assessed by measuring intracellular membrane-impermeable YOYO PRO1 from the cells contacting with varying concentration of CCP. 5,000 HUVEC were plated on 96well plates and incubated overnight in 100 µL of EGMTM medium. The cell culture medium was changed to 100 µL of formulation solutions in 10 mM HEPES buffer (pH 7.4) with 150 mM NaCl together with extra component of YOYO PRO1 (1 µM). After the incubation at 37 °C for 30 min, the fluorescent intensity of YOYO PRO1 in each nucleus (stained by 2.5 µg/mL Hoechst 33342) was quantitatively evaluated using a fluorescence microscope equipped with image-analysis software (IN Cell Analyzer 1000: GE Healthcare UK Ltd., Buckinghamshire, UK). Intracellular distribution. Herein, pDNA was labeled with Cy5 using the Label IT Nucleic Acid Labeling Kit (Mirus, Madison, WI) according to the manufacture’s protocol. HUVEC was seeded on a 35 mm collagen-coated glass base dish (Iwaki, Japan) at a density of 10,000 cells/well with 1 mL of EGMTM medium. Following 24 h incubation (37 °C, CO2 concentration 5%), both Cell Light GFP
Late-endosome and Cell Light GFP Lysosome at dosage of 10 µL were added to the culture medium with the aim of staining late-endosome/lysosome. Following another 48 h incubation, the cells were washed by 1 mL PBS and replaced with 1 mL fresh medium. The polyplex micelle solution of PEGSS-Az/CD-PEI (pDNA concentration: 33.3 µg/mL) containing 4 µg Cy5-labeld pDNA was applied to the cells. Following 6 h incubation, Hoechst 33342 solution (1 mg/mL, 5 µL) was added for nuclei staining. The cells were washed twice with 1 mL PBS and replaced with 1 mL fresh medium. The
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CLSM observation was performed using LSM 780 (Carl Zeiss, Germany) with 63 × objective (CApochromat, Carl Zeiss, Germany) at excitation wavelengths of 488 nm (Ar laser) and 633 nm (He-Ne laser) for GFP and Cy5, respectively. On the other hand, at 6 h post transfection, the cells were subjected to photo-irradiation (365 nm) for 15 min, followed by 1 h incubation. The intracellular distribution of the polyplex micelle was observed according to the same CLSM specification. ■ ASSOCIATED CONTENT
* Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.bioconjchem.xxxxxxx Synthesis schemes for the compounds, their structural information and gel retardation assay of the prepared polyplex micelles. ■ AUTHOR INFORMATION Corresponding Author *Phone: +86 22 6021 4251. Fax: +86 22 6021 4251. E-mail:
[email protected] or
[email protected]. Present Addresses a
School of Chemistry and Chemical Engineering, Tianjin Key Laboratory of Organic Solar Cells and
Photochemical Conversion, Tianjin University of Technology, Tianjin, Xiqing District, 391 Binshui Xidao, 300384, China b
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, 77 Massachusetts
Avenue, 02139, United States c
School of Science, Tianjin University, Tianjin, Nankai District, 92 Weijin Road, 300072, China
Notes The authors declare no competing financial interest. ■ ACKNOWLEDGEMENT The authors thank National Natural Science Foundation of China (21374079), Program for New Century Excellent Talents in University (NCET-11-1063), 131 talents program of Tianjin, and Program for Prominent Young College Teachers of Tianjin Educational Committee for financial support. ■ REFERENCES: [1] Naldini, L. (2015) Gene therapy returns to centre stage. Nature 526, 351-360.
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Table of Contents Graphic For
Shedding PEG palisade by temporal photo-stimulation and intracellular reducing milieu for facilitated intracellular trafficking and DNA releasing
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