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Biological and Medical Applications of Materials and Interfaces

Remarkable Amplification of Polyethylenimine-Mediated Gene Delivery Using Cationic Poly(phenylene ethynylene)s as Photosensitizers Tiantian Wu, Zhiliang Li, Yajie Zhang, Jinkai Ji, Yun Huang, Hao Yuan, Fude Feng, and Kirk S. Schanze ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.8b07124 • Publication Date (Web): 29 Jun 2018 Downloaded from http://pubs.acs.org on July 1, 2018

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ACS Applied Materials & Interfaces

Remarkable Amplification of PolyethylenimineMediated Gene Delivery Using Cationic Poly(phenylene ethynylene)s as Photosensitizers Tiantian Wu,† Zhiliang Li,‡ Yajie Zhang,§Jinkai Ji,† Yun Huang, ‡Hao Yuan,⁋ Fude Feng,*,† and Kirk S. Schanze*,‡

†

Department of Polymer Science & Engineering, School of Chemistry & Chemical Engineering, Nanjing University, Nanjing 210023, PR China

‡

Department of Chemistry, University of Texas at San Antonio, One UTSA Way, San Antonio, Texas 78249, United States §College of Life Science and Chemistry, Jiangsu Key Laboratory of Biological Functional Molecules, Jiangsu Second Normal University, Nanjing, Jiangsu, PR China 210013 ⁋School of Chemistry and Chemical Engineering, State Key Laboratory of Coordination Chemistry, Nanjing University, Nanjing 210023, PR China

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KEYWORDS: conjugated polymer, poly(phenylene ethynylene), gene delivery, photochemical internalization, supercoiled DNA

ABSTRACT: Conjugated polymers can serve as good photosensitizers in biomedical applications. However, it remains unknown whether they are phototoxic to supercoiled structure of DNA in improving gene delivery by photochemical internalization (PCI) strategy, which complicates the application of conjugated polymers in gene delivery. In this work, we introduced trace amount of cationic poly(phenylene ethynylene)s (cPPEs) into the polymeric shell of branched polyethylenimine (BPEI)/DNA complexes, studied photosensitization of singlet oxygen by cPPEs, and confirmed that supercoiled DNA is undamaged by singlet oxygen generated by photoexcitation of cPPEs. By taking advantage of cPPE-mediated PCI effect, we report that addition of trace amount of cPPEs to the outer shell of BPEI/DNA polyplexes could greatly amplify transfection of gene green fluorescence protein (GFP) on tumor cells with efficiency from 14% to 86% without decreasing cell viabilities, well solving the problem with poor transfection capability of BPEI under low DNA loading condition. Our strategy to employ conjugated polymers as photosensitizing agents in gene delivery system is simple, safe and efficient, and promising for broad application in gene delivery areas.

INTRODUCTION Conjugated polyelectrolytes (CPEs) feature -conjugated backbones and ionic side chains which make them very soluble in water.1-2 In recent years, due to the unique optical properties, increasing


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efforts have been made into the broad applications of CPEs in molecular detection,3 cell imaging,45

drug delivery,6 antimicrobials,7-8 and disease diagnosis.9 Cationic CPEs have been used as gene

carriers or a component of gene carriers, highly dependent on the chemical structures. For example, Wang’s group reported that quaternary ammonium-modified PPV could deliver green fluorescent protein (GFP)-coding plasmid DNA (pGFP) into tumor cells with comparable efficiency relative to polyethylenimine (PEI).10 Most recently, our group reported that diethyltriamine (DET)modified linear regioregular polythiophenes with varying chain lengths had strong DNA binding affinity with CE50, which were defined as the charge excess required to gain a 50% reduction of ethidium bromide (EB) fluorescence in EB exclusion assay, much smaller than BPEI, but failed to achieve expression of GFP that was delivered as cargo.11 In another study, we prepared DETmodified linear non-regioregular polythiophenes with varying molecular weights, geometrical conformation and charge densities, which showed limited DNA condensing capability and poor delivery performance.12 It seems that DNA binding of CPEs is not the major determinant, at least, not sufficient for a satisfactory gene delivery. Cationic polymers are important nonviral gene vectors, with merits such as high cost effectiveness, easy availability and tailorability, facile operation and processing, and significant biocompatibility.13 However, one of the serious concerns with cationic polymers is the low transfection efficiency as compared to viral vectors.14 To improve the expression of target genes, transfection materials including both nucleic acids and polymers have to be loaded maximally, which is very common for the use of cationic polymers such as PEI, polylysine (PLL), PAMAM, chitosan, and a great number of other synthetic materials.15-19 In most cases, the success of gene delivery relies on the too much excess use of exogenous materials, which definitely overburdens cells and causes cytotoxicity.20 PEI has attracted considerable attention in transgene enhancement.



Varieties of chemical modifications on the amines of PEI with ligands such as saccharides, peptides and polymers to increase cell uptake or alter intracellular trafficking have been explored with enhanced delivery performance and reduced cytotoxicity.20-23 However, simple and generalized designs for enhancing PEI transgene efficacy, particularly obviating the need of any additional covalent manipulation on PEI, are highly desired to minimize complex factors from polymer engineering, and at the same time improve transgene performance at a low cost of plasmid DNA (pDNA) loading. External triggers have been harnessed in drug and gene delivery, such as ultrasound,24-25 light,26-27 and magnetic field.28 In the previous study, DET-modified polythiophenes added in small quantities were found to enhance transfection capability of star-shaped cationic polyaspartamides on delivery of pGFP into tumor cells, particularly under brief white light exposure which allowed photochemical internalization (PCI) effect to occur.11 We revealed that PCI-induced generation of reactive oxygen species (ROS) by exciting polythiophenes in the outer coating of polyplex nanoparticles contributes to the endolysosome membrane disruption and thereby the increase of overall delivery efficacy at a pGFP dose of 2 μg/mL. That is the first example using CPEs as photosensitizers for promoting endolysosomal escape by PCI effect that normally relies on porphyrins or phthalocyanines conjugated to gene carriers.29-33 However, the PCI effect of CPEs and stability of DNA upon excitation of CPEs need further investigation. Among CPEs, conjugated poly(phenylene ethynylene)s (PPEs) have been extensively studied in photophysics and photochemistry.34-39 As strong photosensitizers, some PPEs were reported with high light-activated biocidal activities which were correlated to the capability of inducing ROS generation.7, 40 In the present approach, we investigated the optical properties of four cationic PPEs that differ in side chains or charge density, and applied them as a component of BPEI/DNA


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polyplexes in trace amounts to boost gene delivery by BPEI in tumor cells. The improved generation efficiency of ROS would be an advantage of PPEs over polythiophenes in enabling strong PCI effect to facilitate successful gene delivery. RESULTS AND DISCUSSION Design of the sequential mix method by assembling cPPEs into the outer layer of BPEI/DNA complexes

Figure 1. Chemical structures of cPPEs.

Four cPPEs were selected (Figure 1). Copolymers P-O-3 and P-C-3 feature branched side chains possessing high density of positively charged primary amines, which makes them well solubilized in water. The structural difference is that the C-linked P-C-3 exists in a molecularly dissolved “free chain” state, while the O-linked P-O-3 tends to form aggregates via interchain - interactions facilitated by the more planar conformation of the backbones.41 Unlike P-O-3, the O-linked



homopolymer PIM-4 with imidazolium solubilizing group-ended side chains is unaggregated in water. The copolymer PIM-2 is aggregated in water because of the lower charge density on the backbones.42

Figure 2. (a) Schematic presentation of the sequential mix method for preparation of cPPE-coated BPEI/DNA polyplexes. Side chains of cPPEs are omitted for clarity. (b) Schematic illustration of singlet oxygen generation by exciting cPPEs. (c) Schematic presentation of in vitro PCI-effective amplification of gene delivery with cPPEs as photosensitizers.


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BPEI has strong DNA binding affinity, making it capable of compacting and condensing plasmid DNA into small nanoparticles by neutralizing the negative charges of DNA. This kind of polyplex has a core-shell like structure in which the condensed DNA resides in the core and excess polycations form the shell.43 In a sequential mix method, cPPE of interest is added in a tiny quantity to the BPEI/pDNA complex, resulting in formation of multicomponent polyplexes that have a cPPE coating (Figure 2a). For BPEI/pDNA, the N/P value refers to the ratio of the moles of the amine groups (including primary, secondary and tertiary amines) on BPEI to those of phosphate ones on DNA. To take into account the contribution of PPE by affording additional charges in cPPE/BPEI/pDNA, the number of the amines, including primary amines and ammonium on each repeat unit of P-O-3, P-C-3, PIM-2 and PIM-4, are counted as 6, 6, 2 and 2, respectively. This means a given N/P would require 3 times of PIM-2 and PIM-4 relative to P-O-3 and P-C-3 in the moles of repeat units. The PPE coating is photoactive, which allows PCI effect mediated by the singlet oxygen photosensitization after the cells internalize the polyplexes. The PCI effect facilitates nuclear transport of internalized particles to achieve enhanced expression of target gene (Figure 2b-c).

Photophysical properties of cPPEs



Figure 3. (a) Normalized UV-Vis absorption spectra of four cPPEs in water. (b) Normalized fluorescence spectra of cPPEs in water. (c) The decreased absorption of ABDA in the presence of various cPPEs as a function of light irradiation time. A0 and A represent the absorption intensity of ABDA before and after light irradiation, respectively. (d) The singlet oxygen quantum yields of cPPEs.

The absorption and fluorescence properties of cPPEs were previously reported.41-42 Briefly, relative to the C-linked P-C-3 absorbing light maximally at 387 nm, the O-linked PPEs (P-O-3, PIM-2 and PIM-4) show longer absorption wavelengths due to the electron-donating property of alkoxyl groups conjugated to the PPE backbones (Figure 3a). In contrast to the unaggregated P-C3 and PIM-4 showing structured emission bands peaked at 414 and 466 nm, respectively, P-O-3


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and PIM-2 exhibit structureless fluorescence that appears at longer wavelengths, correlating with their aggregated states (Figure 3b). The 1O2 photosensitization property of PPEs is crucial for mediating the PCI effect, and the 1O2 generation yields (Δs) of cPPEs in air-saturated D2O containing 10% PBS were examined using Rose Bengal as standard and ABDA as 1O2 trapping agent,44 according to the decrease of ABDA absorption intensity at 400 nm (Figure 3c). As shown in Figure 3d, P-C-3 has a slightly larger Δ than P-O-3 (0.15 vs 0.13), likely due to the aggregation-induced quenching of P-O-3. The Δ of molecularly-dispersed PIM-4 is increased to 0.24, however, noticeably smaller than that of PIM2 at 0.44. PIM-2 seems unusually more effective than PIM-4 in 1O2 photosensitization, inversely correlated to the order of fluorescence quantum yields (Fs). This observation implies that the impact of aggregation effect on Δ is counteracted by other factors such as the degree of substitution on the phenyl units and the difference of chain lengths. The aggregates here are different from a condensed bulky state consisting of static -stacks, but more like loose and dynamic clusters composed of PPE multimers as revealed previously by fluorescence correlation spectroscopy (FCS) for the PPE-type homopolymers and alternating copolymers.45 Characterization of polyplexes



Figure 4. (a) Hydrodynamic diameters and zeta potentials of cPPE-containing BPEI/DNA polyplexes (N/P 8) prepared by the sequential mix method. The columns indicate RHs. Circles and triangles indicate zeta potentials. (b) TEM image of BPEI/DNA polyplexes (N/P 8) containing 2.5 N/P% PIM-2. The scale bar indicates 100 nm.

We prepared cPPE/BPEI/pDNA polyplexes by the sequential mix method at an N/P of 8 containing 0.25-2.5 N/P% of cPPE (or N/P of 0.02-0.2) and 97.5-99.75 N/P% of BPEI (or N/P of 7.8-7.98). The N/P% was calculated according to equation (2) (see Experimental section). The polyplexes containing 0.5 and 2.5 N/P% cPPEs were characterized by dynamic light scattering (DLS), zeta potential and transmission electron microscopy (TEM), with BPEI/pDNA (N/P 8) as control. As shown in Figure 4a, the hydrodynamic diameters (RHs) of polyplexes were 80-90 nm, and they are almost unaffected by the presence of 0.5 or 2.5 N/P% cPPEs. However, significant changes in zeta potentials occurred. The increase of zeta potentials Olinked cPPEs-containing polyplexes in comparison to BPEI/pDNA suggests O-linked cPPEs, particularly the lower content of O-linked PPEs, have strong interactions with BPEI/pDNA


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complex to change the charge density at particle surface. For C-linked P-C-3, it shows a trend of increase of zeta potentials as its content increases. As compared to the relatively ununiform morphology of BPEI/DNA complex, 2.5% cPPE-containing BPEI/DNA polyplexes were visualized as uniform sphere-shaped nanoparticles under transmission electron microscope (TEM) (Figure 4b and S1).

DNA integrity against ROS

Figure 5. (a) Schematic presentation of one-step mix method for preparation of polyplexes. (b-c) Agarose gel analysis for PIM-2-containing BPEI/DNA polyplexes (N/P 8) prepared by sequential mix method or one-step mix method. “+” and “-” in red denote the presence and absence of heparin



treatment, respectively. “+” and “-” in purple denote the presence and absence of light irradiation, respectively. “M” and “N” represent 10 kb DNA ladder and naked plasmid, respectively.

One of premises for PCI effect mediated by photosensitizers is protection of DNA integrity from ROS. Otherwise, DNA damaging by ROS will compromise transgene outcomes.46-47 For this reason, PCI-based strategy need be carefully applied in gene delivery approaches by minimizing the risk of ROS attack, particularly from the highly oxidative 1O2.48-49 For example, the photosensitizer moiety is typically detached from DNA, and in close proximity to or translocated to the endolysosome membrane structure after cellular internalization of polyplexes. In cells, 1O2 has a very short half-life of approx. 0.2 μs and limited diffusion length of 10 nm due to its short lifetime in water.50 Polymeric shell encapsulating DNA may create a barrier for 1O2 to contact DNA strands. In fact, without necessary protection by the polymeric shell, DNA was sensitive to the ROS upon exposure to the cationic polythiophenes under light before formation of nanoparticles, leading to irreversible damage of supercoiled structure and suppressed expression of delivered gene.46 In another study, we confirmed that in a state of nanoparticles, DNA fully compacted by the well-defined cationic polythiophenes was prevented from ROS attack under light exposure, allowing PCI-enabled gene expression at a comparable level to the transfection by BPEI.12 These observations support the hypothesis that the polymeric shell of polyplex nanoassemblies plays a role, in addition to improving stability against degradation by nuclease, in shielding DNA from harmful ROS such as 1O2, which necessitates a close investigation into DNA stability against light-induced ROS from the polyplexes.


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In the sequential mix method (Figure 2), the photosensitizers cPPEs are likely located at the outer surface of polyplex nanoparticles containing 0.5 and 2.5 N/P% cPPEs, according to the aforementioned analysis of zeta potentials. For comparison, we also prepared the polyplexes by a one-step mix method (Figure 5a) in which the mixtures of each cPPE and BPEI are mixed with plasmid such that cPPE chains are randomly distributed in the whole nanoparticles, including both the core and shell regions without location selectivity. In this case, aggregation of cPPEs in a form of -stacks is not favored because of the interchain charge repulsion and dilution of cPPEs by BPEI. Polyplexes prepared by the sequential and one-step mix methods were exposed to LED light (excitation wavelength was 405, 385, 435 and 435 nm for P-O-3, P-C-3, PIM-2 and PIM-4, respectively, 100 mW/cm2, 3 min) to induce ROS by exciting PPE backbones. As control, another group received no light treatment. To check the DNA integrity, we carried out heparin competition assay to completely displace firefly luciferase plasmid DNA (pLuc) from polymers, followed by gel analysis. For the sequential mix method, as shown in Figure 5b-c, the complexed plasmid by BPEI and trace amount of PIM-2 was fully retarded at the bottom of lane without migration before heparin treatment. In the gel, heparin-treated DNA migrated from polymers and the supercoiled structure was clearly visible, similar to the observation with naked DNA. This suggests that the process of polymer binding and heparin treatment did not affect the supercoiled structure of DNA which is generally essential for efficient gene transfer.51-52 Importantly, exactly comparable results were obtained with light irradiation-treated PIM-2-containing samples showing the intense migrated bands of supercoiled DNA at the top, although PIM-2 are 1O2 effective. Similar results were obtained with other cPPEs (Figure S2-S4), comparable to the observation with BPEI/DNA



complexes lacking 1O2 generation capability (Figure S5). The excellent performance of 1O2 resistance indicates that the sequential mix method is safe enough for stabilizing DNA when exciting cPPEs, ensuring the integrity of DNA during intracellular trafficking even if oxidative stress occurs. Unlike the sequential strategy, the one-step mix method does not guarantee the distance between cPPEs and DNA, and emergence of unwound DNA with slower migration in gels is expected if 1

O2 is generated randomly across the whole polyplex structures. Interestingly, like the observation

with sequential mix method, the migrated DNA strands displaced from cPPE-containing polyplexes by one-step mix formulation remained in a supercoiled state, independent on the cPPE type, cPPE content and light irradiation treatment (Figure 5b-c). It seems the supercoiled DNA can survive from 1O2 regardless of the formulation method. To confirm the capability of DNA destruction by light-induced ROS with polymeric shell absent, we carried out heparin assay under the same conditions except that DNA was complexed with cPPEs alone at N/P of 0.04 and 0.2 in the absence of BPEI. Such tiny quantities of cPPEs were not sufficient to neutralize or compact DNA. Accordingly, without light treatment, plasmid DNA retained its supercoiled structure before and after heparin displacement, as visualized at the same migration bands (Figure S6). It reveals that, like BPEI, cPPEs did not cause unwinding of DNA supercoils. In sharp contrast, upon exposure to light, unwound DNA bands showed up at a lower migration rate with concomitant weakening of supercoiled DNA migration. Light irradiation of PO-3, P-C-3 and PIM-4 induced comparable DNA unwinding, and the more cPPE, the stronger the DNA unwinding. In particular, PIM-2 is much more efficient, leading to a percentage of unwound DNA up to 55% and 83% for N/P 0.04 and 0.2, respectively. The ROS effect on damaging DNA agrees well with the order of Δs (Figure 3d).


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The difference in DNA unwinding between cPPE containing BPEI/DNA polyplexes and cPPE/DNA complexes is evident. The latter ones lack polymer coatings on DNA, and serious alteration of the DNA structure take place in a 1O2 dependent manner, arising from the photodamage by DNA-bound cPPEs. We raised a question why the former ones effectively suppress such photodamage and perfectly preserve the supercoiled DNA structure upon excitation of cPPEs, particularly PIM-2. Undoubtedly, supercoiled DNA, in densely condensed or loosely compacted state, is susceptible to photodamage by high level of accessible 1O2.46 From the above results, we inferred that the PEI-dominant polymeric shell serves as a barrier to slow down oxygen diffusion through the self-assembled nanostructures due to the interaction between PEI and oxygen molecules via H-bonding.53-54 The limited oxygen permeability gives rise to less efficient 1O2 photosensitization, since the triplet-triplet energy transfer from cPPE backbones to oxygen molecules is strongly oxygen concentration dependent.55 To ensure sufficient 1O2 available for endolysosomal membrane destabilization, in the present work we are focused on the sequential method to formulate polyplexes with cPPEs more accumulated at the outer shells.

Cell uptake, intracellular ROS generation and photocytotoxicity of polyplexes



Figure 6. CLSM images for HeLa cells incubated with 2.5 N/P% PIM-4-containing BPEI/DNA polyplexes (N/P 8) over a period of 0.5, 2 and 4 h.

To gain direct information for cell uptake of polyplexes, we covalently labeled DNA pLuc with rhodamine dye using a commercial kit (see Experimental section). As polymers P-C-3, P-O-3 and PIM-2 are undetectable at a low dosage by confocal laser scanning microscopy (CLSM), we used PIM-4 as a model cPPE to image the localization of cPPE relative to DNA. The 2.5 N/P% PIM-4 polyplexes were incubated with HeLa cells at an increased DNA loading of 10 μg/mL in serumfree medium to visualize the weakly fluorescent PIM-4 over a period of 0.5, 2, and 4 h before CLSM imaging. Generally, cationic nanoparticles facilitate cell uptake via endocytosis process.10,


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56

As shown in Figure 6, PIM-4 and rhodamine-labeled pLuc (Rho-pLuc) were imaged in green

and red channels, respectively. As incubation time extended, green and red puncta that likely indicates the endolysosome entrapment of internalized particles increasingly accumulated in the cells. The co-localization of PIM-4 and Rho-pLuc was seen across the cells, in good agreement with the notion that cPPEs form composites with BPEI/DNA, not freely dispersed in solution. This point is supported by the difference from cell uptake of free PIM-4 using the same procedure except PIM-4 alone at the same dose was applied instead of polyplexes. In this case, free PIM-4 was rapidly internalized at early time (0.5 h), and moved to peripheral regions of the nuclei at longer incubation time (2 and 4 h) (Figure S7). The rapid entry of PIM-4 to cells relative to polyplexes is ascribed to the small-sized free PIM-4 in favor of diffusion motion and cell binding.



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Figure 7. (a) Fluorescence of HeLa cells treated with polyplexes and DCFH-DA, imaged under fluorescent microscope. The symbol “*” indicates the 2.5 N/P% cPPE in PBEI/DNA (total N/P 8). (b) MTT assay results for cell viabilities of HeLa cells treated by LED light (excitation wavelength was 405, 385, 435 and 435 nm for P-O-3, P-C-3, PIM-2 and PIM-4, respectively. 100 mW/cm2, 3 min).

The ROS generation in the polyplexes-treated HeLa cells upon exposure to LED light (excitation wavelength was 405, 385, 435 and 435 nm for P-O-3, P-C-3, PIM-2 and PIM-4, respectively.

100

mW/cm2,

3

min)

was

detected

using

a

ROS

probe,

2,7-

dichlorodihydrofluorescein diacetate (DCFH-DA). DCFH-DA is reactive to transient 1O2 and oxidized to fluorescent product dichlorofluorescein (DCF).57 The plasmid encoding luciferase (pLuc) was used instead of pGFP to avoid possible fluorescence interference with DCF from GFP expression. As shown in Figure 7a, in contrast to the nonfluorescent cells lacking light irradiation, the light-treated cells were visualized by bright DCF fluorescence, indicating intracellular ROS generation capability of trace quantities of cPPEs in polyplexes. The cPPEs exhibits undetectable background signals under fluorescent microscope by excitation at 488 nm. On the one hand, cPPEs are light active at desired time in both solution and intracellular environment. On the other hand, it is necessary to maintain a balance between cytotoxicity and ROS level required for PCI effect. As control, the cells treated by BPEI/pLuc complexes, were fluorescently invisible before and after light exposure. To evaluate the photocytotoxicity of cPPEs-containing polyplexes (N/P 8), we determined cell viabilities by MTT assay. The contents of cPPEs in polyplexes were 0, 0.25, 0.5, 1.0 and 2.5 N/P%,


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respectively. The polyplexes-treated cells received LED light irradiation (excitation wavelength was 405, 385, 435 and 435 nm for P-O-3, P-C-3, PIM-2 and PIM-4, respectively. 100 mW/cm2, 3 min), and cell viabilities were calculated after 24 h of incubation. As shown in Figure 7b, no significant difference in cell viabilities was observed, which means the short period of light irradiation, enough effective for intracellular ROS generation by each of the four cPPEs, is safe for the cells.

In vitro transfection by polyplexes



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Figure 8. (a) Fluorescence of HeLa cells expressing GFP after PCI-assisted transfection by PIM2-containing BPEI/pGFP polyplexes (2 μg/mL pGFP). The N/P% of PIM-2 was in a range of 0.25% to 2.5%. (b-c) Flow cytometric analysis about transfection efficiency (TE) and mean fluorescence intensity (MFI) in HeLa cells after transfection by cPPEs-containing polyplexes (2 μg/mL pGFP). The N/P% of cPPEs was 0.5% or 2.5%. BPEI/pGFP polyplexes were used as control.

To resolve the cytotoxicity issue associated with excess use of BPEI and DNA in conventional transfection, we performed in vitro transfection using very low quantities of cPPEs (0.25-2.5 N/P%) that amplify gene transfer capability of BPEI/DNA polyplexes at a low dose of DNA (2 μg/mL) through ROS-dependent PCI effect. Under such transfection-suppressed condition, BPEI had poor transfection performance in delivering pGFP into HeLa cells, as seen from the weak GFP fluorescence under fluorescent microscope after 36 h of transfection (Figure 8a). To allow PCI effect to work, cPPEs-containing BPEI/pGFP polyplexes prepared by the sequential mix method were incubated with HeLa cells for 4 h in serum-free medium, followed by exposure to LED light (excitation wavelength was 405, 385, 435 and 435 nm for P-O-3, P-C-3, PIM-2 and PIM-4, respectively. 100 mW/cm2, 3 min) and additional 36 h incubation in serum-supplemented medium. Interestingly, all of PCI-effective samples exhibit enhanced GFP fluorescence in the cells with no sign of cytotoxicity (Figure 8a). To quantify transfection efficiency (TE), we examined the percentage of transfected cells by flow cytometric analysis. As shown in Figure 8b and Figure S8, only approx. 15% cells were GFP positive after transfection by BPEI alone, nearly independent on light treatment. In contrast, at 0.5 N/P% cPPE loading, the TE significantly rose to 52, 58, 86, and 71% for P-O-3, P-C-3, PIM-2 and


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PIM-4, respectively. The TEs follows an order PIM-2 > PIM-4 > P-C-3 > P-O-3, correlating well with the photosensitizing capability (Figure 3d) and revealing that PIM-2 is the best PCI inducer giving rise to approx. 6-fold enhancement of TE as compared to BPEI alone. However, when cPPE loading was elevated from 0.5% to 2.5%, although with TEs still much greater than transfection by BPEI alone, transfection was less efficient. It should be of note that enhancement of GFP expression in terms of mean fluorescence intensity (MFI) follows the same order of cPPEs, but more significant (Figure 8c). In particular, in comparison to pure BPEI-mediated transfection, a 13-fold MFI enhancement was achieved for transfection by 0.5 N/P% PIM-2 polyplexes, which means a greater number of survived gene copies were successfully delivered into nuclei. The above results evidence that PCI strategy is very effective for promoting the overall gene transfer performance under cell-friendly conditions. In fact, without light treatment, transfection by trace cPPEs-containing polyplexes can also lead to increased number of GFP-positive cells and enhanced MFI (Figures 8b-c and S8), however, both of which were reduced by approx. half relative to PCI-effective transfections. In consideration of possible intrinsic fluorescence of cPPEs that may give rise to false positive results in flow cytometric analysis, we used pLuc instead of pGFP to exclude fluorescence of expressed proteins and repeated transfection process. Figures S9 and S10 confirmed that the contribution of intrinsic cPPE fluorescence, including both 0.5 and 2.5 N/P% cPPE composites and all of the four cPPEs, was negligible. The MFI values were nearly 2 orders of magnitude lower as compared to pGFP transfections, close to the background noise. Hence, we could definitely rule out the interference from flow cytometric analysis to guarantee the reliability of transfection data.



We pointed out that the polymeric shell plays a vital role in shielding DNA from ROS attack; otherwise, the supercoiled structure of DNA is damaged and thereby detrimental to gene transfer process. To check the quality of irradiated DNA, we transfected HeLa cells with light-irradiated 0.5 or 2.5 N/P% PIM-2 containing BPEI/pGFP polyplexes using the same transfection procedure in dark or under PCI condition. Exactly as expected, enhanced gene transfer performance was present (Figure S11), comparable to the above transfection lacking pre-irradiation operation. The results demonstrate that the DNA structure encapsulated by polymers is in a perfect state and 1O2 insensitive. It should be noted that shorter time of light irradiation (100 mW/cm2, 1 min) to excite cPPEs did not impair cell viability (Figure S12), while promoting pGFP delivery in lesser extent with PIM-2 as example (Figure S13). Extended light irradiation (100 mW/cm2, 5 min) led to significantly reduced cell viabilities, arising from the over generation of ROS. In this case, PCI strategy is not applicable. Therefore, a balance between PCI effect and ROS-induced cytotoxicity need be carefully controlled by appropriate light irradiation time. In the optimal PCI treatment condition, 3 min of light irradiation (100 mW/cm2) for cPPE excitation results in minimal impairment of cytotoxicity and maximal transfection efficiency.

Imaging of transfected cells by CLSM


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Figure 9. CLSM images for HeLa cells incubated with 0.5 N/P% PIM-2 polyplexes for 36 h, with or without exposure to light irradiation. Green, red and blue channels denote fluorescence from GFP, rhodamine, and Hoechst 33258, respectively. BPEI/pGFP complex was used as control.

To check the intracellular distribution of DNA after 36-h transfection, we imaged the cells transfected by BPEI/pGFP polyplexes containing 0.5 N/P% PIM-2 with or without involving PCI effect. Transfection by BPEI/pGFP complex was used as control. Rho-pGFP was introduced in the polyplexes to visualize the location of DNA in red, and nuclei was stained in blue by Hoechst 33258. As shown in Figure 9, expressed GFP was normally visible in green across the cells, not in puncta-like vesicles. The cells almost completely expressed GFP after transfection by PIM-2containing polyplexes. Evidently, PIM-2-containing polyplexes outperform BPEI/pGFP



complexes in both the number of green-positive cells and brightness of green emission, consistent with the flow cytometry results. The red puncta accumulated in the perinuclear region indicated comparable uptakes of pGFP for PIM-2 containing/PIM-2 free polyplexes, which also reflects that the transfection enhancement is not strongly dependent on the cell entry, but possibly on other factors that need be further investigated. At this stage, it is difficult to identify the gene copies transported into nuclei and responsible for successful gene expression, due to the limitation of CLSM in resolution and sensitivity.

CONCLUSION In summary, we coupled photosensitizing capability of different cationic PPEs in aqueous medium and BPEI/DNA complexes to obtain photoactive nanoassemblies. The introduction of trace amount of cPPEs into the polymeric shell of polyplexes by a sequential method did not significantly change the hydrodynamic sizes, but affected the morphologies and zeta potentials. We investigated the relationship between polymeric shell and stability of encapsulated DNA against highly oxidative 1O2 upon excitation of cPPEs, and illustrated the role of polymeric shell in perfectly preventing supercoiled DNA from photodamage, paving a way for applying conjugated polymers-based PCI strategy to gene delivery researches. This finding is of significance that a broad range of photosensitizers are applicable even with strong photosensitizing capability, alleviating frequently-raised concerns of DNA integrity in the process of PCI effect. Importantly, PCI-assisted gene delivery can be achieved under lowered BPEI and DNA loading conditions. For example, BPEI (N/P 8) showed very low transfection efficiency (14%) on HeLa cells at a DNA loading of 2 μg/mL. Under the same DNA loading condition, PCI-effective 0.5


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N/P% PIM-2 could improve the transfection efficiency of BPEI up to 86% and enhance MFI of expressed GFP in cells by 13-fold, without sacrificing cell viabilities. Our data indicate that the PCI effect by light harvesting cPPEs is Δ dependent, and thereby can be more useful for optimizing gene delivery system with stronger macromolecular photosensitizers.

EXPERIMENTAL SECTION Materials and Methods. All reagents were commercially available and used as received without further purification unless otherwise stated. Branched polyethylenimine (BPEI, 25 kDa) was purchased from Sigma Aldrich. Polymers P-O-3, P-C-3, PIM-2 and PIM-4 were synthesized as reported previously.41-42 Fetal bovine serum (FBS) was purchased from Biological Industries. The Luciferase reporter plasmid DNA (pLuc) and Green Fluorescent Protein reporter plasmid DNA (pGFP) were amplified in DH5α strain of Escherichia coli DH5α and purified by EndoFree Plasmid Mega Kit (QIAGEN, Germany). Particle sizes and ζ-potential values of polyplexes were measured on Zetasizer nanoseries (Nano zs90, Malvern Instruments Ltd., U.K.). UV-vis absorption spectra and fluorescent emission spectra were taken on a Shimadzu UV-2600 spectrophotometer and Hitachi F-7000 fluorimeter, respectively. Transmission electron microscopy (TEM) image were collected on JEM-1011(JEOL, Ltd., Japan). In vitro cell images were acquired by an inverted fluorescence microscope (Nikon TE2000-U, Japan) or confocal laser scanning microscope (LSM 710, Zeiss, Germany). LED lamp (pE-4000, CoolLED Ltd., U.K.) was used as light source. Flow cytometric analysis was performed on BD LSRFortessa (Becton Dickinson Asia Ltd) and results were analyzed using FlowJo software (Tree Star, Ashland, OR).



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Determination of singlet oxygen quantum yields (ΦΔs) of cPPE polymers. Singlet oxygen quantum yields of cPPE polymers were measured in air-saturated D2O containing 10% PBS. Tetrasodium α, α’-(anthracene-9,10-diyl) bis(methylmalonate) (ABDA) and Rose Bengal (RB) were used as singlet oxygen trapping agent and actinometer (ΦΔ 0.75 in water),44 respectively. The ΦΔ values were calculated by the following equation: ΦΔ= ΦΔ, RB × KcPPE × IRB / KRB × IcPPE

(1)

where K and I represent the decomposition rate constant and the rate of light absorption at selective wavelengths, respectively. Preparation of polyplexes. The green fluorescent protein plasmid (pGFP) and the firefly luciferase plasmid (pLuc) were used. Polyplexes consisted of DNA and cationic polymers were prepared at N/P 8. The BPEI/DNA polyplexes were prepared by gently adding DNA to BPEI solution in the same volume and allowed to stand for 20 min at room temperature. For the sequential method, cPPE-containing BPEI/DNA polyplexes were prepared by gently adding a volume of cPPE into the same volume of BPEI/DNA complex solution. cPPE accounts for 0.020.2 N/P, equivalent to 0.25-2.5 N/P% of the total N/P. The N/P% is calculated according to the following equation:

N/P% =

N/P of cPPE in polyplex total N/P in polyplex

100%

(2)

For the one-step mix method, cPPE-containing BPEI/DNA polyplexes were prepared by gently adding a volume of DNA to the same volume of pre-mixed cPPE and BPEI. cPPE accounts for 0.02-0.2 N/P, equivalent to 0.25-2.5 N/P% of the total N/P according to equation (2).


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Determination of hydrodynamic diameters and ζ-potentials of polyplexes. The Zetasizer nanoseries was equipped with dynamic light scattering (unless stated otherwise, analysis is usually performed at an angle of 90°) and a standard capillary cell, respectively. The measurements were performed in triplicate (3 ×30 times). For data analysis, the viscosity (0.8905 mPas) and refractive index (1.333) of pure water at 25 °C was used. The hydrodynamic diameters of polyplexes were calculated from the Stokes-Einstein equation and calibrated with protein microspheres as nanosphere size standards. The ζ-potentials of polyplexes were measured in a disposable folded capillary cell (DTS 1070, Malvern Instruments Ltd., U.K.) containing 1 mM KCl, and calculated from the obtained electrophoretic mobility by applying the Smoluchowski equation. Examination of particle morphologies by transmission electron microscopy (TEM). The polyplexes were negatively stained by 0.8% uranyl acetate for 4 min on 150-mesh copper grids with carbon-coated formvar support (Beijing Zhongjingkeyi Technology Co., Ltd, China). After removal of the excess uranyl acetate, the samples were dried over 20 min at room temperature before being subjected to TEM imaging on JEM-1011. Heparin displacement assay. Heparin sodium salt of 10 IU per µg of polymer complexed pLuc was applied. If needed, polymer/pLuc polyplexes were pretreated by LED light irradiation (100 mW/cm2, 3 min) at selective wavelengths. Heparin was incubated with polyplexes at a final DNA concentration of 100 µg/ml at room temperature for 20 min. Agarose gel stained by ethidium bromide (EB) was run in 1× TAE buffer at 120 V for 60 min, and imaged by gel documentation system. Measurement of In Vitro ROS. HeLa cells were cultured in DMEM (high glucose) medium containing 10% FBS under 5% CO2 at 37 °C. Cells were seeded in 96-well plates at a density of



10,000 cells/well and cultured for 12-24 h until 65% confluence. The medium was replaced and cells were incubated with polyplexes-containing medium at a final pLuc concentration of 2 µg/ml for 4 h. After washing step, cells were incubated with fresh culture medium containing 20 µM DCFH-DA for 20 min in dark. The cells were washed immediately with PBS, irradiated by LED light (excitation wavelength was 405, 385, 435 and 435 nm for P-O-3, P-C-3, PIM-2 and PIM-4, respectively. 100 mW/cm2, 3 min), and imaged by inverted fluorescent microscope. As control, no irradiation on the cells was applied before imaging. MTT assay. Polyplexes containing 0, 0.25, 0.5, 1.0, and 2.5 N/P% cPPEs were prepared at a total N/P of 8 by sequential method. HeLa cells were cultured in DMEM (high glucose) medium containing 10% FBS and grown in 5% CO2 at 37 °C. First, cells were seeded in 96-well plates at a density of 15,000 cells/well and cultured for 12-24 h to reach 80% confluence. The medium was replaced with fresh polyplexes-containing medium at a final DNA concentration of 2 µg/ml for 4 h, treated by LED light (excitation wavelength was 405, 385, 435 and 435 nm for P-O-3, P-C-3, PIM-2 and PIM-4, respectively. 100 mW/cm2, 3 min), and then 100 μl of fresh culture medium containing 20% FBS was added. After incubation for 24 h, cells were washed with PBS and incubated with 100 μl of fresh culture medium contained MTT (0.15 mg/ml) for another 4 h at 37 °C. The supernatant was discarded, and 150 μl of DMSO was added. The 96-well plates were shaken for 20 min to dissolve the formazan. The optical density of the solution in each well was detected by microplate reader (Tecan Infinite M200 Pro) at 570 nm. The cell viability was expressed as the ratio of optical density at 570 nm to the control group. As control, no irradiation on the cells was applied. In vitro Transfection of HeLa cells. HeLa cells were seeded in 96-well plates at a density of 12,000 cells/well and cultured for 12-24 h to have approx. 70% confluence. The medium was


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replaced with a mixture containing 60 μl of serum-free medium and 40 μl of polyplexes. The cells were incubated at 37 °C in 5% CO2 for 4 h, treated by LED light (excitation wavelength was 405, 385, 435 and 435 nm for P-O-3, P-C-3, PIM-2 and PIM-4, respectively. 100 mW/cm2, 3 min), and then 100 μl of fresh culture medium containing 20% FBS was added. After 36 h of incubation, the cells were subjected to flow cytometric analysis and fluorescence imaging. As control, the cells were transfected using the same procedure except lack of light irradiation. Analysis of cellular uptake and transfection by confocal laser scanning microscopy (CLSM). For cellular uptake, plasmid DNA was covalently labeled by rhodamine dye using Label IT CXRhodamine Labeling kit (Mirus) according to the manufacturer’s instruction. The naked DNA and rhodamine labeled DNA were mixed at a ratio of 9:1, followed by formation of polyplexes with polymers at a total N/P of 8. HeLa cells were cultured in DMEM (high glucose) supplemented with 10% FBS and grown in a humidified 5% CO2 milieu at 37 °C. Cells were seeded in 35 mm glass-bottom culture dishes at a density of 12× 104 cells per well and cultured for 12-24 h to have a confluence of 80%. The medium was replaced with 500 µl of polyplexescontaining fresh medium at a final DNA concentration of 10 µg/ml. After incubation for 0.5, 2 or 4 h, the cells were washed with PBS and imaged by CLSM. Rhodamine and PIM-4 were excited at 543 and 488 nm, respectively, with emission collected at 560-700 nm and 498-525 nm ranges, respectively. For investigation of transfected cells, HeLa cells were seeded in 35 mm glass-bottom culture dishes at a density of 8× 104 cells per well and cultured for 12-24 h to reach approx. 70% confluence. The medium was replaced with fresh growth medium containing polyplexes with 0.5 N/P% PIM-2 and rhodamine labeled pGFP at a final DNA concentration of 2 µg/ml. After incubation for 4 h, cells were treated by light irradiation (435 nm, 100 mW/cm2, 3 min). The same



volume of fresh culture medium containing 20% FBS was added. After 36 h of incubation, the cells were washed with PBS, fixed by 4% paraformaldehyde, and stained by Hoechst 33258 before subjected to imaging by CLSM. Rhodamine, Hoechst 33258 and GFP were excited at 543, 405 and 488 nm, respectively, with emissions collected at 560-700 nm, 415-475 nm and 498-550 nm ranges, respectively. As control, the cells were transfected using the same procedure except lack of light irradiation before imaging by CLSM.

ASSOCIATED CONTENT Supporting Information. The Supporting Information is available free of charge on the ACS Publications website. TEM images, electrophoresis analysis, CLSM images, flow cytometric analysis, cell viability and transfection data.

AUTHOR INFORMATION Corresponding Author *Email: [email protected] *Email: [email protected]

ORCID Fude Feng: 0000-0002-5348-5959 Kirk S. Schanze: 0000-0003-3342-4080


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Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We’re grateful to Prof. Wei Wang (Nanjing University) for help with DLS and ζ-potential measurements and Intermediate Chemistry Laboratory (Nanjing University) for help with CLSM. We thank National Natural Science Foundation of China (Grant No. 21474046), the 1000 Young Talent Program for financial support. We acknowledge support from the Welch Foundation through the Welch Chair at the University of Texas at San Antonio (Grant No. AX-004520110629).

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