Effects of the Physicochemical, Colloidal, and Biological

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Effects of the physicochemical, colloidal, and biological characteristics of different polymer structures between #poly(L-lysine) and #-poly(L-lysine) on polymeric gene delivery Kyoungnam Kim, Kitae Ryu, Yeon Su Choi, Yong-Yeon Cho, Joo Young Lee, Hye Suk Lee, and Han Chang Kang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00097 • Publication Date (Web): 04 Jun 2018 Downloaded from http://pubs.acs.org on June 4, 2018

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Biomacromolecules

Effects of the physicochemical, colloidal, and biological characteristics of different polymer structures between α-poly(L-lysine) and ε-poly(L-lysine) on polymeric gene delivery

Kyoungnam Kim§, Kitae Ryu§, Yeon Su Choi§, Yong-Yeon Cho, Joo Young Lee, Hye Suk Lee, Han Chang Kang*

Department of Pharmacy, College of Pharmacy, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do 14662, Republic of Korea

§

KK, KR, and YSC were equally contributed to this work.

* Correspondence to: Professor Han Chang Kang, Ph.D., Department of Pharmacy, College of Pharmacy, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do 14662, Republic of Korea Tel: +82-2-2164-6533; Fax: +82-2-2164-4059 E-mail: [email protected]

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

Abstract Though α-poly(L-lysine) (APL) has been well-studied in gene delivery, ε-poly(L-lysine) (EPL) with same repeating unit of L-lysine but different structure has been rarely investigated. This study compared various effects of their different structures in gene delivery processes. EPL showed less cytotoxicity and more proton buffering capacity for endosomal release than APL. Also, EPL/pDNA polyplexes represented higher nucleus preference than APL/pDNA polyplexes. However, EPL had weaker affinities with pDNA than APL, leading to formation of larger EPL/pDNA complexes with less compactness and successively faster decomplexation. The resultant difference of their pDNA binding affinity caused lower cellular uptake and lower transfection efficiency of EPL/pDNA complexes than APL/pDNA complexes. Thus, this study confirmed that various effects of gene delivery processes are changed by chemical structure of polymeric gene carriers. Especially, despite of low transfection efficiency of EPL-based polyplexes, the study found potentials of EPL in cytocompatibility, endosomal release, and nuclear import.

Keywords Decomplexation; Endosomal escape; Nuclear localization; Poly(L-lysine); Polymeric gene delivery

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Introduction Polycations, such as poly(lysine) and poly(vinylamine), were first used for DNA complexation in the 1950s 1. However, their potential for nucleic acid delivery had not been shown until the first investigations of poly(L-lysine) (PLL)-grafted asialo-orosomucoid for receptor-mediated cell targeting in the late 1980s 2 and poly(ethyleneimine) (PEI) for endosomal disruption in the mid-1990s 3. Although polymer-based gene delivery vectors still have lower transfection efficiencies than viral vectors, serious concerns regarding the safety (e.g., cytotoxicity, immune reactions and tumor formation) and manufacturing (e.g., difficulty of mass production) of viral vectors

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have spurred screening of their alternatives. Polymers that can

deliver genetic materials are potential vector candidates due to their chemical and physicochemical characteristics (e.g., structures, chemistry, size, charges, and colloidal stabilization) and bio-functional abilities (e.g., cellular targetability, endosomal disruption, cytosolic transport, nuclear import, triggered drug release, and biocompatibility) 4, 9-14. It is believed these bio-functional abilities could be potent strategies for enhancing the therapeutic effects of polymeric gene carriers by overcoming the various extracellular and intracellular barriers in the gene delivery processes. The differences in the sizes, zeta-potentials and complexation/decomplexation behaviors of polymer/gene complexes (polyplexes) mostly influence the colloidal stability, biodistribution, cellular internalization and gene release rate. Although gene-carrying polymers have similar chemical components, molecular weights and degradabilities, their different polymeric structures strongly affect their charge densities as well as the pKas of their chemical components 15, 16. Additionally, polyplexes that have different pKas at endolysosomal pHs escape from the endosome/lysosome at different times. For optimal transfection, it is critical that decomplexation to release genes from their carriers should occur at 3



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the right place and time 14. This means that the transfection efficiencies of polyplexes depend not only on the general polymeric characteristics but also on the polymeric structures. These relationships have been often investigated using representative polycations, such as PEI and PLL, and their derivatives. Two different polymeric structures of PEI (i.e., linear PEI (lPEI) and branched PEI (bPEI)) represented differences in the formation, colloidal stability, and in vitro/in vivo transfection efficiencies of PEI/gene complexes that have the same chemical components 17. Additionally, E. Ramsay and M. Gumbleton compared the transfection efficiency of PLL and poly(L-ornithine) (PLO) polyplexes with similar molecular weights and found that PLL and PLO, which have different side chains, had different transfection efficiencies due to their pDNA affinity 18.

H N

O

O

NH2

OH

n

H N

O n

NH2

NH2 α-poly(L-lysine) (APL)

NH2 L-lysine

ε-poly(L-lysine) (EPL)

Figure 1. Chemical structures of α-poly(L-lysine) (APL) and ε-poly(L-lysine) (EPL).

Poly(L-lysine), especially α-poly(L-lysine) (APL), and its derivatives have been extensively investigated as gene delivery carriers 19-22. Additionally, APL/pDNA polyplexes have strong nuclear localization, which improves gene expression. However, they have disadvantages, such as agglutination of blood and low gene expression. Their deficient endosomolytic and 4


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decomplexation capabilities lead to a low transfection efficiency due to sequestration of pDNA in endolysosomes 23 and in complexes 24, 25. ε-poly(L-lysine) (EPL) has the same monomeric unit as APL (Figure 1), but it has linkages between the α-carboxyl group and ε-amino group of L-lysine. On the other hand, APL has linkages between the α-carboxyl group and α-amino group of Llysine. EPL is a naturally occurring substance that is produced by the metabolism of Streptomyces albus in mass production, and it has been used as a food preservative in Japan, South Korea, the United States and other countries 26, 27. Despite EPL having the same monomer unit as APL, EPL has been rarely investigated in biomedical and pharmaceutical applications, especially as a gene delivery carrier. Our group has discovered that a high transfection efficiency can be obtained by tuning the pH at which the polyplex is released from the endolysosomes and that pH-tunable sulfonamide-based oligomers/polymers loaded in polyplexes affected gene expression but not gene silencing in gene delivery

23, 28

. Despite the recognized significance of the intracellular

location and timing of gene delivery, related investigations have seldom been conducted. Thus, this study aims to perform a comparative study of APL and EPL as gene delivery carriers to understand the physicochemical, colloidal, and biological characteristics of poly(L-lysine)-based pDNA polyplexes.

Experimental Materials α-Poly(L-lysine) (APL) hydrogen bromide (APL·HBr; Molecular weight (MW) 4~15 kDa), branched polyethyleneimine (bPEI25kDa; MW 25 kDa, Mn 10 kDa), heparin sodium salt (from porcine intestinal mucosa; 196 USP units/mg), 3-(4,5-dimethylthiazol-2-yl)-2,55



diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), ethidium bromide (EtBr), 4(2-hydroxy-ethyl)-1-piperazine (HEPES), D-glucose, sodium bicarbonate, Dulbecco’s modified Eagle’s medium (DMEM), Ca2+-free and Mg2+-free Dulbecco’s phosphate buffered saline (DPBS), fetal bovine serum (FBS), penicillin streptomycin antibiotics, trypsin-EDTA solution, Hoechst 33342, and formalin were purchased from Sigma-Aldrich Company (St. Louis, MO). εPoly(L-lysine) (EPL·HCl; MW 3.5~4.7 kDa) was purchased from Zhengzhou Bainafo Bioengineering Company (Henan, RP China). YOYO-1 and LysoTrackerTM Red DND99 were purchased from Invitrogen, Inc. (Carlsbad, CA), and plasmid DNA (pDNA) encoding firefly luciferase (gWiz-Luc and pLuc) was purchased from Aldevron, Inc. (Fargo, ND). The luciferase assay kit was purchased from Promega Corporation, Inc. (Madison, WI).

Cells and cell culture HepG2 cells (human hepatoma cell line) and HEK293 cells (human embryonic kidney cells) were cultured in culture medium at 37°C under 5% CO2-containing humidified air. The culture medium was DMEM supplemented with D-glucose (4.5 g/L) and 10% FBS. FBS-free DMEM was used as the transfection medium.

MW measurements of APL and EPL polymers According to the MW information provided by the manufacturers, the MW of APL used in the study was 4–15 kDa (measured by viscosity), whereas that of EPL was 3.5–4.7 kDa (measured by MALDI-TOF). To directly compare their MWs and avoid any possible concerns about MW-dependent effects, the MWs, molecular weight distributions (MWDs), and polydispersity indexes (PDIs) of APL and EPL were measured using an Agilent 1260 Infinity II 6


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LC system equipped with the gel permeation chromatography (GPC) mode. For GPC analysis, an Ultrahydrogel™ linear column (WAT011545, 10 µm, 7.8 mm × 300 mm, 500–10 M; Waters, Milford, MA, USA) was used, and elution was carried out with 1% formic acid (aq) at a flow rate of 0.6 mL/min at 30 ℃. After preparing 5 mg/mL polymer solutions, the MWs, MWDs, and PDIs of APL and EPL were analyzed by comparison with a MW standard curve of monodispersed polyethylene glycols.

In vitro cytotoxicity test of APL and EPL polymers The in vitro cytotoxicities of APL and EPL polymers were evaluated using an MTTbased cell viability assay. Cells were seeded at 5 × 103 cells per well on a 96-well cell culture plate and were incubated for 24 h in culture medium. After cells were exposed to polymers (0−1600 µg/mL) for 48 h, MTT solution (10 µL, 5 mg/mL) was added to the culture medium (0.1 mL). After an additional 4 h incubation, the culture medium was discarded and DMSO (0.1 mL) was added to dissolve the formazan crystals produced by living cells. After measuring their absorbance at 570 nm, the cell viabilities of the polymers were calculated by the following equation: ! " #$ − &'() Cell Viability of Polymer (%) = , × 100% *+ ! " #$ − &'() where ABSPolymer-treated cells, ABSUntreated cells, and ABSDMSO are the absorbances of polymer-treated cells, untreated cells, and DMSO, respectively.

Proton buffering capacity of APL and EPL polymers To evaluate the proton buffering capacity of polymers, the polymer solution (1 mg/mL) 7



in 150 mM NaCl (aq) was adjusted to pH 11 by adding 1 N NaOH (aq). With the addition of 0.1 N HCl (aq), the pH change of the polymer solution was monitored. In the endosomal pH range (pH 5.1-7.4), the proton buffering capacity was calculated by the following equation 29, 30: Proton Buffering Capacity =

∆78.:; _@O

>_kTlmnopq/@&;V #@O r_@O

r_kTlmnopq/@&;V #@O

rJK?sHftLu rIMHLsK _aesvL rJK?sHftLu >LHHIHsK _aesvL

11



For chloroquine pretreatment study, chloroquine (50 µM) at 2 h prior to polyplex addition was added into cells. During nuclear uptakes, concentration of chloroquine was maintained.

To investigate the intracellular trafficking, nuclear localization, and endosomal escaping behavior of YOYO-1-intercalated pDNA delivered with polyplexes, 3 × 104 HepG2 cells were seeded on a dish with a cover-glass bottom and were incubated in complete culture medium for 24 h. One hour before the addition of polyplexes, the culture medium was replaced with a fresh serum-free transfection medium. A polyplex solution (0.2 µg of pDNA in 20 µL) was used to treat cells for 1 h, 2 h, or 4 h. Ten minutes prior to the end of each incubation time, Hoechst 33342 (5 µg/mL for 10 min) was added to polyplex-transfected cells for nuclear staining, and rinsed with DPBS. After nucleus staining, LysoTrackerTM red DND99 (100 nmol for 25 min) for acidic vesicles (i.e., endolysosomes) was treated and rinsed with DPBS. The fluorescence of YOYO-1 in cells were visualized by a laser scanning confocal microscope equipped with excitation lasers (408 nm for the diode, 488 nm for Ar, and 543 nm for He-Ne) and variable band-pass emission filters. Due to the three dimensional structure of the cells, the confocal images were sectioned to every 0.5 µm. After that, the nuclear and lysosomal localization of YOYO-1-intercalated pDNA in a whole cell was quantified from the fluorescence of three regions of interest, and the fluorescence intensities of YOYO-1-intercalated pDNA in the nucleus, lysosome, and whole cell were calculated by using confocal imaging software. For calculate subcellular localization of polyplexes, all experiments were performed in triplicate. Subcellular localization of pDNA (%) =

GHIJKLPMLNML@O U+ !+ x!+$ × 100(%) GHIJKLPMLNML@O U+ ! yz #

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Results and discussion Polymer characteristics: molecular weight, cytotoxicity, and proton buffering capacity Considering the comparative studies between APL and EPL polymers, MW is important for subsequent comparative experiments. The MW information provided by the manufacturer was as follows: 4–15 kDa (measured by viscosity) for APL and 3.5–4.7 kDa (measured by MALDI-TOF) for EPL. Because of the different methods used for the MW measurements, there might be differences in the MWs between APL and EPL. By the viscosity method, APL and the bromide counterion were measured together; however, the chloride ion present in EPL was removed before measuring its MW by MALDI-TOF. To compare the MWs of the APL and EPL polymers by the same measurement method, GPC was used. As shown in Figure S1, APL had Mn = 7.7 kDa, Mw = 11.3 kDa, and PDI = 1.47, whereas EPL had Mn = 7.1 kDa, Mw = 8.8 kDa, and PDI = 1.24. The results indicated that the MWs and MWDs of the APL and EPL polymers used were not significantly different. In general, a negligible cytotoxicity of carrier materials is very significant in polymeric gene delivery because their harmful effects to the viability and pivotal pathways of cells during the delivery could limit their applications for a wide range of drugs. Prior evaluating the structural effects between APL and EPL in polymer-based gene delivery, their cytotoxicities were examined to confirm whether APL and EPL actually had low or negligible cytotoxicity. As shown in Figure 2, the MTT-based cell viability assay of APL and EPL using HepG2 and HEK293 cells showed that EPL was less cytotoxic than APL below 2 mg/mL, although their concentration-dependent cytotoxicity profiles were cell-dependent. In HepG2 cells, the cytotoxicities of APL and EPL gradually increased with the increasing concentration of polymers. EPL had approximately 3.6-fold less cytotoxicity than APL, i.e., the IC50 values of APL and EPL 13


Biomacromolecules

were 0.4 mg/mL and 1.1 mg/mL, respectively (Figure 2(a)). However, the concentration range of EPL for greater than 80% cell viability was 6.5-fold higher than that of APL (i.e., 11 µg/mL for APL and 71 µg/mL for EPL). Although the polymers applied to HEK293 cells had concentrations as high as 0.11 mg/mL for APL and 0.75 mg/mL for EPL, the relative cell viabilities remained above 80%. Based on the IC50 values, EPL had at least 11.7-fold less cytotoxicity than APL because the former’s IC50 value was above 2 mg/mL, while the latter’s one was approximately 0.17 mg/mL (Figure 2(b)).

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HepG2

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0 1

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HEK293

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0 1

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Polymer (µ µ g/mL)

(b)

Figure 2. In vitro cytotoxicity of APL and EPL in (a) HepG2 and (b) HEK293 cells 48 h posttreatment (mean ± standard error (SE); n=12).

The significant structural differences between the APL and EPL polymers are the length of the side chain (i.e., -CH-(CH2)4-NH2 for APL versus -CH-NH2 for EPL) and distance between side chains (i.e., -NH-C-CO- for APL versus -NH-C-C-C-C-C-CO- for EPL). In general, the charge density of polymers is a key parameter for cytotoxicity as well as the molecular weight of polymers 31. The APL polymer has a relatively dense and long side chain, while the EPL polymer has a loose and short side chain. In a previous study, EPL was shown to have a lower charge density than that of APL

32

. Focusing on the differences, we suggest that the difference in

cytotoxicity between the APL and EPL polymers could also be due to the difference in the cationic charge density per unit of the backbone length. As a result, the relatively low cytotoxicity of the EPL polymer compared with that of the APL polymer could be due to the low 15


Biomacromolecules

charge density.

10 0.1 N NaOH APL EPL

9 8

pH

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7 6 5 4 0

50

100

150

200

0.1 N HCl (µ µ L)

Figure 3. Acid-base titration of APL and EPL.

After cellular internalization of polymer-based drug delivery carriers, they are often sequestered in acidic endosomal or lysosomal compartments, leading to a low delivery efficiency of their payloads into subcellular organelles. Thus, ability of carriers to escape from endolysosomes was estimated evaluating their proton buffering capacities between pH 5.1 and pH 7.4 based on an acid-base titration method. As is known, the titration profile of APL is similar to that of 0.1 N NaCl within a given pH range (i.e., 0.76 µmol of HCl per 1 mg of lysine and 0.097 µmol of HCl per 1 of µmol lysine) (Figure 3), which indicated that APL did not have a proton buffering capability in endosomal pH ranges. However, the proton buffering capacity of EPL was 2.57 µmol of HCl per 1 mg of lysine and 0.328 µmol of HCl per 1 µmol of lysine, meaning approximately that EPL had a 3.4-fold higher buffering capacity than that of APL. In 16


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Biomacromolecules

particular, EPL had an approximately 1.9-fold lower or 1.5-fold higher proton buffering capacity based on the unit mass (i.e., 1 mg) of the polymer or the unit mole (i.e., 1 µmol amine) of the protonatable groups in the polymer than bPEI25kDa, a well-known endosomolytic polycation 33, 34. These findings could be due to the different polymeric structures of APL and EPL. APL and EPL have different pKa values, approximately 9-10 for APL and 7.6 for EPL 35, indicating that EPL has more protonatable and uncharged primary amines than APL. The different proton buffering capacities of APL and EPL may be supported by the results of proton buffering studies on hyperbranched polylysines that have either α-amine or ε-amine groups 36.

Formation of APL/pDNA and EPL/pDNA polyplexes and their sizes, zeta-potentials, and colloidal stabilities To understand the different gene delivery carrier characteristics between APL and EPL, pDNA was used as a model cargo. In particular, to exclude the MW effects of polycations, APL and EPL with similar MWs (i.e., Mn = 7.7 kDa and PDI = 1.47 for APL and Mn = 7.1 kDa and PDI = 1.24 for EPL; Figure S1) were selected among commercially available poly(L-lysine) candidates. The polyplexes were formed according to the N/P ratio using the nitrogen (N) of the polymers and phosphate (P) of the pDNA. APL had a stronger pDNA condensation ability in comparison to EPL (Figure 4(a)). An N/P ratio of 2 for APL was sufficient to complex with pDNA; on the other hand, an N/P ratio of at least 3 was needed for EPL to complex with pDNA. Additionally, EPL/pDNA polyplexes showed little fluorescence signal in the wells of an agarose gel, meaning that EPL was not able to form completely compact complexes with pDNA. In addition, the particle compactness was examined by a dye quenching assay (Figure 4(b)). APL made more compact complexes with pDNA compared to EPL at the same N/P ratio. The 17


Biomacromolecules

compactness of APL/pDNA was above 80% at all of the tested experimental N/P ratios; however, EPL/pDNA polyplexes with N/P ratios of 3, 5, 7, and 10 showed approximately 58%, 66%, 67%, and 70% compactness, respectively. These findings showed that the long and flexible side chains of APL improved its condensation ability with pDNA compared to EPL.

(a)

APL/pDNA EPL/pDNA

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Figure 4. (a) Gel electrophoresis of APL/pDNA and EPL/pDNA complexes and (b) the compactness of pDNA in their complexes (mean ± standard deviation (SD); n=3). 500 APL EPL

Particle size (nm)

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(a) 30 APL EPL

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Zeta-potential (mV)

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10

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Figure 5. (a) Particle size and (b) zeta-potential of APL/pDNA and EPL/pDNA polyplexes (mean ± SD; n=3). 19



The particle sizes and zeta-potentials of APL/pDNA and EPL/pDNA polyplexes were examined by a particle size and zeta-potential analyzer. APL/pDNA polyplexes were less than 100 nm in size, whereas EPL/pDNA polyplexes were 200~400 nm in size at N/P ratios of 2 to 10 (Figure 5(a)). In addition, the particle sizes of EPL/pDNA polyplexes decreased and became more stable as the N/P ratio increased from 2 to 10. The size distributions of APL/pDNA and EPL/pDNA polyplexes were exhibited in Figure S2. APL/pDNA polyplexes had relatively higher zeta-potentials than EPL/pDNA polyplexes at all of the tested experimental N/P ratios (Figure 5(b)). Particularly, APL/pDNA polyplexes at a N/P ratio of 1 had a negative zeta potential (−6.0 mV), indicating that polyplex particles were not formed at this N/P ratio. At N/P ratios above 2, the zeta potentials were positive and gradually increased as the N/P ratio increased. In the case of EPL/pDNA polyplexes, the zeta potentials were negative, −18.8 mV and −11.9 mV at an N/P ratio of 1 and 2, respectively. The negative zeta potentials of the polyplexes indicated that the complexes were not completely condensed. The polyplexes started to form positively charged complexes at N/P ratios above 3. These results supported the results of the agarose electrophoresis assay (Figure 4(a)). The time-dependent particle sizes of polyplexes were monitored after a 4 h incubation to identify the colloidal stability of polyplexes (Figure 6). The particle sizes of APL/pDNA were still under 100 nm after a 4 h incubation at all of the experimental N/P ratios. However, the EPL/pDNA polyplexes increased in particle size after 4 h of incubation; 1.9, 2.4, 2.4, and 2.0fold increased particle sizes of EPL/pDNA polyplexes were found at N/P ratios of 3, 5, 7, and 10, respectively. 20


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1000 APL/pDNA (0 hr) APL/pDNA (4 hr) EPL/pDNA (0 hr) EPL/pDNA (4 hr)

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Particle size (nm)

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600

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Figure 6. Changes in the particle size of APL/pDNA and EPL/pDNA polyplexes after 4 h incubation (mean ± SD; n=3).

In addition, the particle compactness of the polyplexes after a 4 h incubation was measured (Figure 7(a)). After a 4 h incubation, we found that APL/pDNA and EPL/pDNA polyplexes had a similar compactness to the previously executed measurements (Figure 4(b)). These results indicated that APL/pDNA polyplexes maintained more compact particles than EPL/pDNA polyplexes even after a 4 h incubation. In addition, APL/pDNA and EPL/pDNA polyplexes sustained pDNA condensed complexes after a 4 h incubation (Figure 7(b)). These findings showed that APL had a higher colloidal stability than EPL, although EPL still interacted with pDNA even after 4 h of incubation. During the formation of polyplexes between pDNA and poly(L-lysine)s, with the same molecular components, the polymeric structure influenced the physicochemical characteristics of the APL/pDNA and EPL/pDNA polyplexes, including the 21


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pDNA condensation ability, particle compactness, particle size, zeta-potential, and colloidal stability. APL/pDNA (4 hr) EPL/pDNA (4 hr)

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(b) Figure 7. (a) Compactness of pDNA in APL/pDNA and EPL/pDNA polyplexes (mean ± SD; n=3) and (b) their gel electrophoresis after 4 h incubation.

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80 60 40 20 0 5

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Figure 8. In vitro cytotoxicity of APL/pDNA and EPL/pDNA polyplexes in (a) HepG2 and (b) HEK293 cells at 48 h posttransfection (mean ± SE; n=6).

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Cytotoxicity test of APL/pDNA and EPL/pDNA polyplexes The cytotoxicities of APL/pDNA and EPL/pDNA polyplexes were examined by the MTT assay in HepG2 and HEK293 cells. As mentioned previously, APL and EPL had negligible cytotoxicity and high IC50 values in HepG2 and HEK293 cells. We found that polyplexes using APL and EPL also had low cytotoxicities (Figure 8). Although the IC50 values of APL and EPL against HepG2 cells were 0.4 mg/mL and 1.1 mg/mL, respectively, APL/pDNA and EPL/pDNA polyplexes have greater than 85% and 90% relative cell viabilities against HepG2 cells, respectively (Figure 8(a)). Although the IC50 values of APL and EPL were 0.17 mg/mL and 2 mg/mL in HEK293 cells, respectively, APL/pDNA and EPL/pDNA polyplexes had negligible cell toxicities at a N/P ratio of 40 (Figure 8(b)). Therefore, APL and EPL polyplexes, which had low cytotoxicities, were used as safe gene delivery systems.

Transfection experiments of APL/pDNA and EPL/pDNA polyplexes The transfection efficiencies of APL/pDNA and EPL/pDNA polyplexes were evaluated in HepG2 and HEK293 cells. In HepG2 cells, APL/pDNA polyplexes had similar transfection efficiencies from N/P ratios of 5 to 40; however, EPL/pDNA polyplexes had increased transfection efficiencies as the N/P ratio increased (Figure 9(a)). In addition, APL/pDNA polyplexes exhibited distinguished transfection efficiencies compared with EPL/pDNA polyplexes over the same N/P ratio ranges. APL/pDNA had approximately 30- to 300-fold higher transfection efficiencies than EPL/pDNA in HepG2 cells. APL/pDNA and EPL/pDNA polyplexes had similar transfection efficiency trends in HEK293 cells (Figure 9(b)). The transfection efficiencies of APL/pDNA polyplexes were higher than those of EPL/pDNA polyplexes. EPL/pDNA polyplexes had approximately 2- to 50-fold decreased transfection 24


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efficiencies at N/P ratio of 40 and 5, respectively. Additionally, EPL/pDNA polyplexes had increased transfection efficiencies in HEK293 cells with increasing N/P ratios, as previously shown. 109

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Figure 9. In vitro transfection efficiency of APL/pDNA and EPL/pDNA polyplexes in (a) HepG2 and (b) HEK293 cells at 48 h posttransfection (mean ± SE; n=8). By using these cell lines, we found that APL/pDNA polyplexes had relatively higher transfection efficiencies compared with EPL/pDNA polyplexes. In addition, the transfection efficiencies of EPL/pDNA polyplexes increased as the content of EPL polymers increased. The transfection experiment results may have been influenced by cellular uptake, nuclear localization, decomplexation of genetic materials, and so on. Therefore, we expected that the differences in transfection efficiencies between APL and EPL polyplexes might be influenced by cellular uptake, nuclear localization, and decomplexation of pDNA.

Cellular uptake, nuclear uptake, and nuclear localization study of APL/pDNA and EPL/pDNA polyplexes Cellular uptake and nuclear localization are crucial information about biological pathways, including transfection and transcription in the gene delivery field. To confirm the previous results, polyplexes using YOYO-1-intercalated pDNA were treated to HepG2 cells and analyzed by flow cytometry (Figures 11, 12, and S3). YOYO-1 is commonly used di-intercalator having very high binding ability and strong fluorescence intensity when intercalated to DNA 37, 38. First, as shown in Figure S3, APL/pDNA polyplexes had a similar level of cellular uptake compared to bPEI25kDa/pDNA polyplexes. The cellular uptake values of APL and EPL polyplexes were normalized to those of bPEI25kDa/pDNA polyplexes, which were set at unity. The normalized cellular uptake values (NCU) of APL/pDNA polyplexes at N/P ratios of 5, 7, and 10 were 0.99, 1.02, and 1.05, respectively. In addition, NCUs of APL/pDNA polyplexes were slightly increased as the N/P ratio increased. On the other hand, EPL/pDNA polyplexes had low 26


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cellular uptake values. The NCUs of EPL/pDNA polyplexes at N/P ratios of 5, 7, and 10 were 0.28, 0.30, and 0.43, respectively. EPL/pDNA polyplexes also had increased NCU levels as the N/P ratio increased. While APL/pDNA and EPL/pDNA polyplexes had similar zeta-potentials, EPL/pDNA polyplexes had larger particle sizes with weak compactness than APL/pDNA polyplexes (Figure 5). Due to the colloidal instability and ease of dissociation with pDNA of EPL/pDNA polyplexes on cellular surfaces, the cellular uptake of EPL/pDNA was lower than that of APL/pDNA polyplexes. Therefore, EPL/pDNA polyplexes had low transfection efficiencies along with low cellular uptake. Nuclear uptake of APL/pDNA and EPL/pDNA polyplexes had different tendencies according to the flow cytometry results. EPL/pDNA polyplexes at N/P ratios of 5, 7, and 10 had 0.48, 0.58, and 0.60 levels of normalized nuclear uptake (NNU); the nuclear uptake value of bPEI25kDa/pDNA polyplexes as set to the unity. On the other hand, the NNU values of APL/pDNA polyplexes at N/P ratios of 5, 7, and 10 were 1.15, 1.13, and 1.20, respectively. According to the NCU results, EPL/pDNA polyplexes had lower cellular uptake than APL/pDNA polyplexes. Additionally, EPL/pDNA polyplexes had lower nuclear uptake than APL/pDNA polyplexes. However, the nuclear preference to cellular uptake of EPL/pDNA polyplexes was higher than that of APL/pDNA polyplexes. The nuclear preferences of APL/pDNA polyplexes at N/P ratios of 5, 7, and 10 were 1.16, 1.11, and 1.14, respectively. On the other hand, EPL/pDNA polyplexes at 5, 7, and 10 N/P ratios had nuclear preferences of 1.71, 1.93, and 1.40, respectively. As a result, the trend of nuclear localization of EPL/pDNA polyplexes was higher than that of APL/pDNA polyplexes, although the cellular uptake of EPL/pDNA polyplexes was lower than that of APL/pDNA polyplexes. Second, time-dependent cellular uptake and nuclear uptake of the polyplexes (N/P 5) 27



were monitored by flow cytometry to analyze the time-dependent nuclear localization of the polyplexes. As shown in Figure 10(a), the cellular uptake and nuclear uptake of all polyplexes increased over time. Specifically, the fluorescence intensities representing the cellular uptake and nuclear uptake of the bPEI25kDa/pDNA complexes at 1 h posttransfection were set to 1 for normalization (Figure 10(b)). Time-dependent cellular uptake and nuclear uptake showed that the EPL/pDNA complexes had an approximately 1.8-fold and 1.5-fold higher nuclear localization preference than the bPEI25kDa/pDNA and APL/pDNA complexes, respectively (Figure 10(b)). These results indicate that EPL has an intrinsic nuclear localization ability.

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(a)

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Figure 10. (a) Time-dependent cellular uptake and nuclear uptake of polyplexes (N/P 5) in HepG2 cells (mean ± SE; n=3) and (b) their normalized values.

To confirm endosomal release behavior, the nuclear uptake of YOYO-1-intercalated pDNA delivered with polymers was evaluated by flow cytometry in either the absence or the presence of chloroquine, which is commonly used as an endolysosomal escape agent. As shown in Figure 11, after pretreating with chloroquine (50 µM) at 2 h prior to polyplex addition, YOYO-1-intercalated pDNA delivered with APL escaped from the endolysosomes and then was highly taken up in the nucleus. The presence of chloroquine induced approximately 1.8-fold higher nuclear uptake of APL/pDNA complexes than the absence of chloroquine (Figure 11(b)). However, the nuclear uptake of bPEI25kDa/pDNA and EPL/pDNA complexes was not significantly influenced by chloroquine pretreatment. These results indicate 1) that bPEI25kDa and EPL have intrinsic endosomal escaping ability, unlike APL, and 2) that APL has intrinsic nuclear translocation ability, like EPL.

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Figure 11. (a) Nuclear uptake of the bPEI25kDa/pDNA, APL/pDNA, and EPL/pDNA complexes (N/P 5) in either the absence or the presence of chloroquine (CHQ; 50 µM; 2 h) at 4 h posttransfection and (b) their normalization results. The normalized nuclear uptake is expressed as the mean ± SE (n=3).

Although the nuclear uptake profile of APL/pDNA and EPL/pDNA polyplexes was confirmed by FACS analysis, images of polyplex-containing HepG2 cells were analyzed by confocal microscopy. As shown in Figure S4(a) and S4(b), YOYO-1-intercalated pDNA delivered with all polyplexes was spread throughout the cells at 1 h and 2 h posttransfection. However, unlike the strongly orange-colored colocalization of the YOYO-1-intercalated pDNA delivered with the bPEI25kDa/pDNA and APL/pDNA complexes in lysosomes, the YOYO-1intercalated pDNA delivered with the EPL/pDNA complexes weakly overlapped with LysoTrackerTM Red DND99. More detailed analysis of their colocalization efficiencies in the nucleus, lysosomes, and elsewhere (e.g., cytosol) clearly showed that the polyplexes’ intracellular distributions were strongly affected by polymers. At 1 h posttransfection, approximately 31%, 17%, and 52% of the YOYO-1-intercalated pDNA delivered by the bPEI25kDa/pDNA complexes; approximately 36%, 30%, and 34% of the YOYO-1-intercalated pDNA delivered by the APL/pDNA complexes; and approximately 52.5%, 11.5%, and 36% of the YOYO-1-intercalated pDNA delivered by the EPL/pDNA complexes existed in the nucleus, lysosomes, and cytosol, respectively (Figure S4(c)). At 2 h posttransfection, these percentages were approximately 34%, 18%, and 48% of the YOYO-1-intercalated pDNA delivered by the bPEI25kDa/pDNA complexes; approximately 38%, 33%, and 29% of the YOYO-1-intercalated pDNA delivered by the APL/pDNA complexes; and approximately 58%, 12%, and 30% of the 32


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YOYO-1-intercalated pDNA delivered by the EPL/pDNA complexes, respectively (Figure S4(c)). These results show that the reduced cytosol populations caused increased nuclear populations, although the lysosomal populations of all polyplexes were not significantly increased. At 4 h posttransfection, we detected more reduced cytosolic populations but much increased nuclear populations compared to those at 2 h (Figure 12(a)). In detail, the respective percentages were approximately 41%, 22%, and 37% of the YOYO-1-intercalated pDNA delivered by the bPEI25kDa/pDNA complexes; approximately 45%, 39%, and 16% of the YOYO1-intercalated pDNA delivered by the APL/pDNA complexes; and approximately 67%, 15%, and 18% of the YOYO-1-intercalated pDNA delivered by the EPL/pDNA complexes (Figure 12(b)). Nuclear localization of the APL/pDNA complexes was slightly higher (i.e., 1.1-fold) than that of the bPEI25kDa/pDNA complexes, but the APL/pDNA complexes showed 1.8-fold more endosomal sequestration than the bPEI25kDa/pDNA complexes (39% versus 22%, respectively). Interestingly, the EPL/pDNA complexes had approximately 1.6-fold and 1.5-fold higher nuclear localization efficiencies than the bPEI25kDa/pDNA and APL/pDNA complexes, respectively, whereas the endolysosomal localization efficiencies of the EPL/pDNA complexes were approximately 1.8-fold and 2.5-fold lower than those of the bPEI25kDa/pDNA and APL/pDNA complexes, respectively. Furthermore, among the polyplexes that escaped from endolysosomes were 52.3% of the bPEI25kDa/pDNA complexes, 73.1% of the APL/pDNA complexes, and 79.1% of the EPL/pDNA complexes. That is, the ratio of polyplexes in the nucleus to polyplexes in the cytosol was approximately 1.1, 2.7, and 3.8 for the bPEI25kDa/pDNA, APL/pDNA, and EPL/pDNA complexes, respectively. These results clearly show 1) that EPL has 1.08-fold better endosomal escape capability than bPEI25kDa, 2) that APL has 1.28-fold worse endosomal escape capability than bPEI25kDa, 3) that EPL possesses 1.64-fold and 1.50-fold better nuclear 33



translocation ability than bPEI25kDa and APL, respectively, and 4) that APL possesses 1.10-fold better nuclear translocation ability than bPEI25kDa. These results showed strong correlations with flow cytometry results which was previously performed (Figures 10, 11, and S3).

(a)

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Figure 12. (a) Intracellular distributions of the bPEI25kDa/pDNA, APL/pDNA, and EPL/pDNA complexes and (b) their colocalization efficiencies in the nucleus, lysosomes, and elsewhere (e.g., cytosol) at 4 h posttransfection. All polyplexes were prepared at N/P 5. YOYO-1 (green), Hoechst 33342 (blue), and LysoTrackerTM Red DND99 (red) were used to stain the pDNA, nucleus, and lysosomes, respectively, in (a). All experiments were performed triplicate, and representative images are shown in (a). In (b), the colocalization efficiency (%) is expressed as the mean ± SE. Sectioned images are shown in Figure S5.

DNA decomplexation of APL/pDNA and EPL/pDNA polyplexes Heparin-induced DNA decomplexation mimics an anionic intracellular environment. As shown in Figure 13, the decomplexation rate of polyplexes was delayed with the increasing N/P ratio. pDNA of the EPL/pDNA polyplexes was exposed to heparin concentrations of 25 µg/mL (N/P ratio = 5) and 50 µg/mL (N/P ratio = 7). Additionally, pDNA of the APL/pDNA polyplexes at N/P ratios of 5 and 7 was exposed to heparin concentrations of 50 µg/mL and greater than 50 µg/mL, respectively. These results indicated that EPL/pDNA polyplexes dissociated from pDNA more easily than APL/pDNA polyplexes.

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Figure 13. Heparin-induced decomplexation of APL/pDNA and EPL/pDNA polyplexes after incubation in heparin-containing 150 mM NaCl (aq) at 37°C for 1 h.

DNA release from polyplexes is important for gene delivery processes as well as cellular uptake and nuclear localization. Despite the identical chemical compositions of APL and EPL, the length between the backbone and side chain may influence the pDNA decomplexation rate of polyplexes. Facilitation of DNA release from EPL polyplexes could be due to the shorter side chain of EPL compared to APL. The decomplexation rate of APL and EPL polyplexes also influenced the nuclear localization of DNA

25

. EPL facilitated decomplexation of pDNA from

polyplexes; therefore, EPL/pDNA polyplexes were found in the nucleus more frequently than APL/pDNA polyplexes after cellular uptake. In addition, we found that EPL had a higher proton buffering capacity that APL, which contributed to endosomal escape (Figures 3 and 11). As a result, the enhanced nuclear localization of EPL/pDNA polyplexes in comparison to APL/pDNA polyplexes could be explained by the decomplexation of pDNA and proton endosomal escape abilities of EPL/pDNA. 36


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Although APL has been extensively studied as a gene delivery carrier, EPL has rarely been studied in this respect. According to comparison studies with APL, EPL may have limited application as a gene delivery carrier based on its low transfection efficiency (Figure 9). In addition, EPL has a lower pDNA condensation ability (Figure 4) and lower polyplex stability (Figure 6) than those of APL. However, EPL could aid other gene carriers that are incapable of endosomal release and nuclear import because EPL has a strong endosomal escape ability (Figures 3 and 11) and strong nuclear import ability (Figures 10, 12, and S4). In addition, EPL might help other gene carriers that complex with genes too tightly and have slow gene release because of the improved pDNA decomplexation ability of EPL in comparison with that of APL (Figure 13). Thus, the compensatory abilities of EPL may have a high impact and extend its applications in the gene delivery area. In particular, the lower cytotoxicity of EPL, compared with that of APL, could allow EPL to be used in various applications in the biomedical and pharmaceutical fields (Figures 2 and 8). As a result, we focused on EPL as a new candidate for a safe and efficient gene delivery.

Conclusions In this study, we carried out comparative studies between APL and EPL as gene delivery carriers and furthered our understanding of their physicochemical, colloidal, and biological characteristics. The different chemical structures of APL and EPL affected gene condensation, polyplex size, zeta-potential, colloidal stability, cellular uptake, transfection efficiency, and decomplexation from pDNA. In particular, APL had greater condensability with pDNA than EPL due to its longer and more flexible side chains. Unlike APL, EPL had a higher proton buffering 37



capacity, which might improve its endosomal escaping effect. APL/pDNA had a much higher transfection efficiency than EPL/pDNA polyplexes. However, EPL/pDNA polyplexes had a stronger nucleus preference than APL/pDNA polyplexes. Furthermore, EPL, having facilitating pDNA decomplexation and endosomal escaping activity may be able to positively impact the nuclear localization and control of the pDNA release time of polymeric gene carriers. As a result, APL and EPL, which have the same monomers but different structures, have different properties as gene delivery carriers, and each characteristic could be useful for designed gene delivery systems.

ASSOCIATED CONTENTS Supporting Information The supporting information is available free of charge on ACS Publications website at DOI: GPC results of APL hydrogen bromide and EPL hydrogen chloride; Size distribution results of APL/pDNA complexes and EPL/pDNA complexes; N/P-dependent cellular uptake, nuclear uptake, and nuclear preference of polyplexes at 4 h posttransfection; Time-dependent intracellular distributions and colocalization of polyplexes at 1 h or 2 h posttransfection; Sectioned confocal images of polyplex-transfected HepG2 cells.

AUTHOR INFORMATION Corresponding author 38


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*E-mail: [email protected]. Phone: +82-2-2164-6533. Fax: +82-2-2164-4059 Notes The authors declare no competing financial interest. §

K. Kim, K. Ryu, and Y. S. Choi contributed to this work equally.

ACKNOWLEDGEMENTS This study was supported by the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT) (NRF-2017M3A9F5028608 for the Bio & Medical Technology Development Program; NRF-2017R1A4A1015036; NRF-2015R1A1A05001459). Also, the study was supported by BK21PLUS grant of NRF funded by the Korean government (ME) (22A20130012250) and by the Research Fund, 2017 of The Catholic University of Korea.

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