decomplexation and sizes of polymer-based electrostatic pDNA polyplexes is one of the key factors in effective transfection

Colloids and Surfaces B: Biointerfaces 184 (2019) 110497

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Controlling complexation/decomplexation and sizes of polymer-based electrostatic pDNA polyplexes is one of the key factors in effective transfection

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Kyoungnam Kima,1, Hee Sook Hwangb,1, Min Suk Shimc, Yong-Yeon Choa, Joo Young Leea, ⁎ Hye Suk Leea, Han Chang Kanga, a Department of Pharmacy and BK21PLUS Team for Creative Leader Program for Pharmacomics-based Future Pharmacy, College of Pharmacy, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do 14662, Republic of Korea b Division of Biotechnology, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do 14662, Republic of Korea c Division of Bioengineering, Incheon National University, Incheon 22012, Republic of Korea

A R T I C LE I N FO

A B S T R A C T

Keywords: ATP Complexation/decomplexation Poly(L-lysine) Polymeric gene delivery Size

The delivery of plasmid DNA (pDNA) using polycations has been investigated for several decades; however, obstacles that limit efficient gene delivery still hinder the clinical application of gene therapy. One of the major limiting factors is controlling pDNA binding affinity with polymers to control the complexation and decomplexation of polyplexes. To address this challenge, polycations of α-poly(L-lysine) (APL) and ε-poly(L-lysine) (EPL) were used to prepare variable complexation/decomplexation polyplexes with binding affinities ranging from too tight to too loose and sizes ranging from small to large. APL-EPL/ATP-pDNA polyplexes were also prepared to compare the effects of endosomolytic ATP on complexation/decomplexation and the sizes of polyplexes. The results showed that smaller and tighter polyplexes delivered more pDNA into the cells and into the nucleus than the larger and looser polyplexes. Larger polyplexes exhibited slower cytosolic transport and consequently less nuclear delivery of pDNA than smaller polyplexes. Tighter polyplexes exhibited poor pDNA release in the nucleus, leading to no improvement in transfection efficiency. Thus, polyplexes should maintain a balance between complexation and decomplexation and should have optimal sizes for effective cellular uptake, cytosolic transport, nuclear import, and gene expression. Understanding the effects of complexation/decomplexation and size is important when designing effective polymer-based electrostatic gene carriers.

1. Introduction Polymeric gene carriers such as poly(L-lysine) (PLL), branched or linear polyethylenimine (bPEI or lPEI), and poly(amidoamine) dendrimers have been widely modified and used for gene delivery [1,2]. Polymeric gene delivery systems have been proposed for the efficient protection and delivery of nucleic acids with the potential to treat diseases caused by gene defects and to express additionally or newly functional proteins related to survival, death, homeostasis, etc. [1,3,4]. However, there remain several sequential barriers during gene delivery from administration to the nucleus of the targeted cells, and polymerbased gene delivery systems still face a lack of sufficient gene delivery to achieve optimal therapeutic efficacy [3]. To achieve highly effective gene delivery, the proposed intracellular

rate-limiting steps are related to cellular internalization, endolysosomal escape, cytosol-to-nucleus transport, the nuclear entry of polyplexes, and the unpacking of genes from the polyplexes. Among them, this study mainly focused on unpacking and releasing genetic materials for effective translocation to the nucleus of target cells [4,5]. The complexation and decomplexation between genes and cationic polymers are affected by their polymeric structures, molecular weights, and charge densities because their different binding strengths determine gene packing abilities and complex dissociation rates [4,6]. Additionally, the resultant tightness of the binding between pDNA and polycations in polyplexes in turn affects their sizes. In general, the degree of complexation between pDNA and polycations influences the polyplex sizes, leading to in vitro cell issues as well as in vivo issues: administration routes (e.g., intravenous, intratumoral,

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Corresponding author at: Department of Pharmacy and BK21PLUS Team for Creative Leader Program for Pharmacomics-based Future Pharmacy, College of Pharmacy, The Catholic University of Korea, 43 Jibong-ro, Wonmi-gu, Bucheon-si, Gyeonggi-do 14662, Republic of Korea. E-mail address: [email protected] (H.C. Kang). 1 These authors equally contributed to this work. https://doi.org/10.1016/j.colsurfb.2019.110497 Received 25 June 2019; Received in revised form 22 August 2019; Accepted 7 September 2019 Available online 09 September 2019 0927-7765/ © 2019 Elsevier B.V. All rights reserved.

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[2,21–23]. However, without the aid of other functional solutions, such as the facilitated endosomal release and improved nuclear import of pDNA and/or its polyplexes, it could be difficult for complexation/decomplexation-balanced polyplex systems to reach high transfection efficiencies. First, for improved nuclear import, additional functional components might not be required in the designed mixed polyplexes (i.e., APL-EPL/pDNA complexes) because both APL and EPL have good nuclear import activities, as reported [21]. Second, to avoid sequestration and degradation of the delivered pDNA and polyplexes in endolysosomes, an effective endosomolytic function is generally required in polymer-based gene delivery systems [24]. Fortunately, in APL-EPL/ pDNA complexes, EPL has good endosomolytic function due to its primary amines with pKa 7.6, and their proton buffering capacities in an endolysosomal pH range of pH 5.1-pH 7.4 were 3.4-fold higher than that of APL [21]. In particular, although EPL showed 1.9-fold lower endosomolytic activity than the well-known endosomolytic bPEI25kDa based on the unit mass, which is suitable for weight ratio-based polyplexes, the unit mole-based endosomolytic activities (suitable for N/P ratio-based polyplexes) of the protonatable groups in EPL were 1.5-fold higher than those of bPEI25kDa [21,25,26]. Therefore, APL-EPL/pDNA complexes with a high EPL content might not require additional endosomolytic functions. Nevertheless, we are concerned about low EPL systems in APL-EPL/pDNA complexes due to their deficient endosomolysis. Depending on the contents of EPL in the designed mixed polyplexes, their sufficient or insufficient characteristics might support the introduction of an endosomolytic molecule into the mixed polyplexes. To confer additional endosomolytic properties on APL-EPL/pDNA complexes, bPEI with protonatable secondary and tertiary amines could be considered for the construction of APL-EPL-bPEI/pDNA complexes because bPEI is a gene carrier with a good proton sponge effect, resulting in efficient release from the endolysosomal compartment [27–31]. However, we exclude the use of bPEI because high-molecularweight PEIs display high cytotoxicity and nondegradability [26,30,32]. Additionally, in mixed polyplexes of three polycations (i.e., APL, EPL, bPEI), it is impossible to understand the complexation/decomplexation balance between APL and EPL because bPEI also strongly affects the binding with pDNA and the release of pDNA from the polyplexes. Therefore, we selected ATP, which was found by our group to be nontoxic and show good endosomolytic properties [26]. ATP has been demonstrated to be nontoxic, and its secondary phosphate group has a pKa of 6.1–6.5 for proton buffering in endosomal pH values [25,26]. In particular, the controllable properties of complexation and decomplexation by both APL and EPL could be investigated because ATP is negatively charged like pDNA. Thus, in APL-EPL/ATP-pDNA complexes, the mixed polycation combinations of APL and EPL could control the complexation and release rate of the gene, and ATP could promote the escape of the polyplexes from the endolysosomes into the cytosol. In this study, by adjusting the concentration of APL and EPL in the delivery system, we investigated the effects of complexation/decomplexation and the size of polyplexes on the preparation and intracellular delivery of the polyplexes and their transfection efficiency. To pursue this aim, after APL-EPL/ATP-pDNA polyplexes were formed by introducing ATP into APL-EPL/pDNA polyplexes, their physicochemical characteristics (i.e., sizes and zeta-potentials) and cellular characteristics (i.e., cellular uptake, nuclear uptake, and transfection efficiencies) were evaluated and comprehensively analyzed. Especially, to avoid the concerns regarding molecular weights (MWs) and molecular weight distributions (MWDs) of the two polycations, this study used APL and EPL with similar MWs and MWDs.

subcutaneous, intramuscular, and intraperitoneal injection, and transdermal, pulmonary, oral, and intranasal delivery) [7], clearance or extravasation-mediated biodistribution [8–10], and cellular internalization mechanisms (e.g., macropinocytosis, clathrin-mediated endocytosis, caveolae-mediated endocytosis, etc.) [11–14]. The sizes of polyplexes could limit their applications and administration routes. For intravenous injection, polyplexes near or below 100 nm in diameter are more appropriate than polyplexes larger than 500 nm because the former could avoid phagocytosis-mediated clearance [9] and be distributed into physiological organs (e.g., liver, spleen, and bone marrow) and pathological tissues (e.g., solid tumor and inflammatory tissues) by extravasation through fenestrated or sinusoidal capillaries [10], but the latter would rarely be distributed from blood vessels to tissues or organs, resulting in their becoming trapped in lung capillaries or undergoing cellular clearance by phagocytosis [9]. However, other routes could allow sizes from 100 nm to several microns. For example, intratumoral injection could allow the direct delivery of nanosized particles to solid tumors [15], and subcutaneous, intramuscular, and intraperitoneal injection could deliver small nanoparticles (e.g., approximately 100 nm) to the blood stream or deposit large nano- or microparticles as drug depots for sustained drug release [16,17]. In a pulmonary route, particles having several microns in size could be deposited on bronchial tissues, whereas particles having less than 1 μm in size could reach the pulmonary alveoli [18]. Additionally, these nano or microparticles could detour the blood-brain barrier to reach brain tissues using intranasal routes [19,20]. Regarding the size effects of nanoparticles on cellular internalization mechanisms, clathrin- and caveolae-mediated endocytosis have size limitations of 300 nm and 80 nm, respectively, whereas particles from 500 nm to approximately 2 μm undergo micropinocytosis [12–14]. As previously mentioned, various size issues have been considered and investigated from the perspectives of administration, biodistribution, clearance, and cellular entry. However, the effects of polyplex sizes of 100 nm and larger on the proton buffering of endosomolytic molecules, cytosol-to nucleus transport, and nuclear import have rarely been studied. Therefore, controlling the balance between the complexation and decomplexation of polyplexes determines their optimal or suboptimal transfection efficiencies. To balance the decomplexation and complexation of polyplexes, we adopted certain cationic biodegradable polypeptides (i.e., α-poly(L-lysine) (APL) and ε-poly(L-lysine) (EPL)) that have the same repeating unit but different polymeric structures. In our previous study [21], APL and EPL were investigated as gene delivery carriers, and their complexation, decomplexation, cellular uptake, nuclear uptake, and transfection efficiency were compared. The study found that APL has a strong complexation ability with pDNA, moderate cellular uptake, poor endosomolytic function, good nuclear uptake, slow pDNA release, and poor transfection efficiency, whereas EPL has a weak complexation ability with pDNA, poor cellular uptake, good endosomolytic function, good nuclear uptake, fast pDNA release, and poor transfection efficiency [21]. In particular, their different complexation abilities with genes and their different pDNA release characteristics were caused by both their different polymer structures and their different binding affinities with pDNA. The results showed that both APL and EPL-based pDNA complexes suffered from poor transfection efficiencies via the strong pDNA complexation of APL/ pDNA complexes, resulting in very slow pDNA release in the nucleus for gene expression and the weak pDNA complexation of EPL/pDNA complexes linked with the possibility of negatively charged plasma membrane-induced decomplexation before their cellular internalization, respectively [21]. Therefore, using a physical mixture of APL and EPL for pDNA complexes could provide a balance between complexation and decomplexation, leading to improved cellular uptake and optimized pDNA release in the nucleus, resulting in improved transfection efficiencies because of combining the interesting intrinsic characteristics of APL and EPL (i.e., the good endosomolytic activity of EPL and the good nuclear translocating activity of both lysine-based polymers)

2. Materials and methods Materials and methods were minutely explained in the supplementary information due to the limitation of words. This study carried 2

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influenced by the major component or contributor in the designed polyplexes. That is, APL would be a stronger contributor of tight complexation than EPL in APL-rich polyplexes and APL50-EPL50/pDNA polyplexes, whereas EPL would be a stronger contributor of weak complexation than APL in EPL-rich polyplexes. The APL- or EPLmediated binding affinity with pDNA could result in particle sizes of APL100-x-EPLx/pDNA polyplexes below 300 nm, which may not encounter significant size limitations on administration routes, extravasation for biodistribution, and cellular internalization. However, due to positively charged polyplexes originating from excess primary amines of both APL and EPL, the zeta-potentials of APL100-x-EPLx/pDNA polyplexes were 10–15 mV (Fig. 1(b)). The results indicate that intravenous injection of the polyplexes would be unsuitable due to the strong possibility of aggregates with serum components, but other routes might allow them to be used. Nevertheless, surface masking strategies using hydrophilic or anionic shielding materials could allow the designed polyplexes to be used in clinical applications [35,36].

out preparation and physicochemical characterizations (i.e., size, zetapotentials, and heparin-induced decomplexation), and biological characterizations (i.e., transfection efficiency, cellular uptake (CU), nuclear uptake (NU), and intracellular trafficking) of polyplexes. The important equations used in this study are as follows.

Normalized CU (NCU) of sample polyplex=

CU of sample polyplex CU of control polyplex

Normalized NU (NNU) of sample polyplex=

NU of sample polyplex NU of control polyplex

Nuclear Preference (NP)to CU (NP/CU) of sample polyplex NNU of sample polyplex = NCU of sample polyplex Endolysosomal localization of pDNA (%) Colocalized LysoTracker Red and YOYO−1 labeled pDNA pixels = YOYO−1 labeled pDNA pixels in cell × 100

3.2. Transfection efficiency of APL100-x-EPLx/pDNA polyplexes

Nuclear localization of pDNA (%) YOYO−1 labeled pDNA pixels in nucleus × 100 = YOYO−1 labeled pDNA pixels in cell

The transfection efficiencies of polyplexes are strongly influenced by their particle sizes, zeta potentials, cellular uptake, endosomal escape, nuclear uptake, pDNA release in the nucleus, and so on. In vitro cell transfection of APL100-x-EPLx/pDNA polyplexes in serum-free medium might be controlled by cellular uptake, endosomal escape, nuclear uptake, and pDNA release in the nucleus because most APL100-xEPLx/pDNA polyplexes except for EPL/pDNA polyplexes have diameters less than 200 nm and positive zeta-potentials. Thus, prior to monitoring cellular uptake, nuclear uptake, and pDNA release in the nucleus, the transfection efficiencies of the designed APL100-x-EPLx/ pDNA polyplexes were evaluated using luciferase-expressing pDNA (pLuc). As shown in Fig. 2(a) and (b), although the transfection efficiencies of APL100-x-EPLx/pDNA polyplexes were much lower than those of bPEI25kDa/pDNA polyplexes (as a gold standard transfection polyplex), the effects of EPL content in APL100-x-EPLx/pDNA polyplexes on transfection efficiencies were similar in both HepG2 and HEK293 cells. With increasing EPL content in the polyplexes, their transfection efficiencies very gently decreased up to 75% EPL and then dropped very sharply. Namely, APL100-EPL0/pDNA polyplexes had 2.93-fold and 2.69-fold higher transfection efficiencies than APL25-EPL75/pDNA polyplexes in HepG2 and HEK293 cells, respectively, whereas the transfection efficiencies of APL0-EPL100/pDNA polyplexes were 71.8fold and 7.1-fold lower than those of APL25-EPL75/pDNA polyplexes in HepG2 and HEK293 cells, respectively. The transfection efficiencies of APL100-x-EPLx/pDNA polyplexes might be strongly affected by the contents of APL and EPL in the polyplexes because different binding affinities of APL and EPL with pDNA could influence the cellular uptake, endosomal escape, nuclear uptake, and pDNA release of the polyplexes.

3. Results and discussion Prior to examining the transfection effects of decomplexation-tunable, endolysosome-escapable APL100-x-EPLx/ATPz-pDNA polyplexes, we first studied the physicochemical characteristics, transfection efficiencies, cellular uptake, nuclear uptake, and pDNA release (i.e., decomplexation) of APL100-x-EPLx/pDNA polyplexes. Then, APL100-xEPLx/ATPz-pDNA polyplexes were investigated. Here, x represents the mass % of EPL in the total mass of the polycations (i.e., APL and EPL), and z is the dose of ATP in the polyplexes expressed as [ATP] = z nmol/ μg pDNA. 3.1. Particle size and zeta-potentials of APL100-x-EPLx/pDNA polyplexes The particle size of the polyplex is an important factor that is influenced directly by complexation because looser complexation makes larger polyplexes. Additionally, size is a key factor influencing in vitro cell issues (e.g., cellular internalization and colloidal stability) and in vivo issues (e.g., administration routes, clearance, biodistribution, cellular internalization, and colloidal stability). In addition, the zeta-potentials of nanoparticles strongly affect colloidal stability in blood and cellular internalization. For example, in blood, positively charged nanoparticles could form aggregates with negatively charged serum proteins and be cleared by blood cells via nonspecific electrostatic attraction, whereas negatively charged or hydrophilic (e.g., typically, PEGylated) particles could exhibit colloidal stability due to less interaction with serum components [33,34]. However, in the internalization of nanoparticles into cells, cationic systems could take fast adsorptive endocytosis pathways, whereas anionic or hydrophilic systems could use slow fluid-phase endocytosis mechanisms [14,33]. Based on various effects of sizes and zeta-potentials in in vitro cell studies and in vivo studies, we tried to analyze the potentials of the designed APL100-x-EPLx/pDNA polyplexes. As shown in Fig. 1(a), the sizes of APL100-x-EPLx/pDNA polyplexes increased with increasing EPL content in the polyplexes from approximately 40 nm for APL100-EPL0/ pDNA polyplexes (i.e., APL/pDNA polyplexes) to approximately 270 nm for APL0-EPL100/pDNA polyplexes (i.e., EPL/pDNA polyplexes). Polyplexes containing less than 50% EPL in the polyplexes were less than 100 nm in diameter, whereas the sizes of APL25-EPL75/pDNA polyplexes were approximately 160 nm. The findings indicate that the binding tightness between pDNA and polycations could be strongly

3.3. Cellular uptake (CU) and nuclear uptake (NU) of APL100-x-EPLx/ pDNA polyplexes In general, smaller particles mostly exhibit faster cellular internalization rates than larger particles [33]. Additionally, slow fluid phase endocytosis is mainly used for negatively charged particles, whereas positively charged or hydrophobic particles are taken up by fast adsorptive endocytosis, and ligand or antibody-equipped particles follow its corresponding receptor or antigen-mediated endocytosis with fast cellular entry kinetics [14]. Thus, to understand why high EPL contents in APL100-x-EPLx/pDNA polyplexes caused low transfection efficiencies, we first monitored their CU and NU using DNA-intercalating YOYO-1 dye in HepG2 cells. As shown in Fig. 3(a), the histograms of APL100-x-EPLx/pDNA polyplex taken up into cells (i.e., CU) and 3

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Fig. 1. (a) Particle sizes and (b) zeta-potentials of APL100-x-EPLx/pDNA complexes with a fixed N/P 5 (mean ± standard deviation (SD); n = 3).

3.4. Decomplexation-induced pDNA release from APL100-x-EPLx/pDNA polyplexes

either APL100-x-EPLx/pDNA polyplex or pDNA taken up into nuclei (i.e., NU) seem to be too complicated for a clear comparison about the CU and NU effects of EPL contents among APL100-x-EPLx/pDNA polyplexes. Therefore, the CU and NU of APL100-x-EPLx/pDNA polyplexes were normalized by those of bPEI25kDa/pDNA polyplexes, and the normalized CU (NCU) and NU (NNU) of bPEI25kDa/pDNA polyplexes were set as unity. The NCUs of APL100-x-EPLx/pDNA polyplexes decreased almost linearly from 0.99 to 0.27 with increasing EPL content (Fig. 3(b)). Compared to the NCU of APL100-EPL0/pDNA polyplexes, APL25-EPL75/ pDNA polyplexes and APL0-EPL100/pDNA polyplexes had 1.90-fold and 3.67-fold lower values, respectively. Similarly, the NNUs of APL100-xEPLx/pDNA polyplexes decreased almost linearly from 1.23 to 0.47 with increasing EPL content (Fig. 3(c)). Interestingly, the NNUs of APL100-EPL0/pDNA polyplexes and APL75-EPL25/pDNA polyplexes were 1.23-fold higher than and very similar to that of bPEI25kDa/pDNA polyplexes, respectively. When calculating the nuclear preference with respect to CU (NP/CU) for estimating the nuclear importing activity of pDNA driven by APL and EPL, the NP/CUs of APL100-EPL0/pDNA polyplexes and APL0-EPL100/pDNA polyplexes were 1.24 and 1.74, respectively (Fig. 3(d)). In particular, 25% and 50% EPL content in APL100-x-EPLx/pDNA polyplexes led to NP/CU values of approximately 1.12–1.15, and 75% EPL resulted in an NP/CU of 1.56. As in our previous results [21], the CUs and NUs of APL100-x-EPLx/pDNA polyplexes suggest that high EPL contents in the polyplexes could cause low CUs but relatively high NP/CUs. Additionally, the results support that both APL and EPL have good nuclear delivery activity of pDNA.

In general, to express the corresponding protein from pDNA, the pDNA delivered with polycation-mediated polyplexes must be released from the polyplexes. However, depending on the binding affinity between pDNA and polycations, pDNA could be dissociated from polycations by competing electrostatic attractions with various negatively charged materials [5] (e.g., glycosaminoglycans in the plasma membrane [37–39], cytosolic RNA in the cytosol [40], and nuclear DNA in the nucleus [41]). If polyplexes have excessively weak complexation, they could release pDNA before entering cells because of interactions with glycosaminoglycans in the plasma membrane. Additionally, if polyplexes have excessively tight complexation, they could hardly release pDNA in the nucleus, resulting in no or very poor gene expression. That is, to obtain optimal transfection efficiencies, polyplexes should release their delivered pDNA at the right place and time [4]. As shown in Fig. 4, decomplexation was performed in 150 mM NaCl aqueous solution, using heparin as a model polyanion to cause pDNA release from polyplexes. Although APL100-x-EPLx/pDNA polyplexes have the same N/P ratio, APL100-EPL0/pDNA polyplexes and APL0EPL100/pDNA polyplexes released a major portion of pDNA at 40 μg/mL and 15 μg/mL heparin, respectively. The results indicate that the binding affinity between APL and pDNA is stronger than that between EPL and pDNA because APL100-EPL0/pDNA polyplexes need more polyanions than APL0-EPL100/pDNA polyplexes for decomplexation. In addition, with increasing EPL content in APL100-x-EPLx/pDNA polyplexes, pDNA release required a smaller amount of heparin. The results

Fig. 2. Transfection efficiencies of APL100-x-EPLx/pDNA (N/P 5) complexes in (a) HepG2 cells and (b) HEK293 cells at 48 h post-treatment (mean ± standard error (SE); n ≥ 8). bPEI25kDa/pDNA (N/P 5) complexes were used as a control polyplex. 4

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Fig. 3. Cellular uptake and nuclear uptake of APL100-x-EPLx/pDNA (N/P 5) complexes in HepG2 cells at 4 h post-treatment: (a) FACS histograms, (b) normalized cellular uptake (NCU), (c) normalized nuclear uptake (NNU), and (d) nuclear preference with respect to cellular uptake (NP/CU). NCU and NNU were expressed as Mean ± SE (n = 3), whereas NP/CU was expressed as Mean (n = 3). bPEI25kDa/pDNA (N/P 5) complexes were used as a control polyplex.

(Fig. 4) with the effects of the CU and NU of APL-rich polyplexes and EPL-rich polyplexes (Fig. 3) on transfection efficiencies (Fig. 2(a)), we drew the three following conclusions. First, APL100-EPL0/pDNA polyplexes and APL75-EPL25/pDNA polyplexes had 1.23-fold and 1.1-fold superior NU to bPEI25kDa/pDNA polyplexes, respectively, but the

support that APL has stronger binding affinity with pDNA than EPL, as previously reported [21]. Moreover, the findings indicate that APL-rich polyplexes and EPL-rich polyplexes could release pDNA slowly and quickly, respectively. Furthermore, through integrating the decomplexation results

Fig. 4. Decomplexation of APL100-x-EPLx/pDNA (N/P 5) complexes after heparin treatment for 1 h in 150 mM NaCl aqueous solution. 5

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contents. For example, the sizes of APL100-x-EPLx/ATP4-pDNA polyplexes were 1364% larger for EPL0, 475% larger for EPL25, 375% larger for EPL50, 211% larger for EPL75, and 117% larger for EPL100 than those of APL100-x-EPLx/pDNA polyplexes. In addition, high ATP doses (e.g., 4 < [ATP] (nmol/μg pDNA) ≤ 24) in APL100-x-EPLx/ATPz-pDNA polyplexes caused the polyplexes to have similar sizes within a range of 400 nm–600 nm. Although the average sizes of APL100-x-EPLx/ATPzpDNA polyplexes with 4 < [ATP] (nmol/μg pDNA) ≤ 24 were still approximately 12.0-fold larger for EPL0, 7.6-fold larger for EPL25, 5.2fold larger for EPL50, 2.9-fold larger for EPL75, and 2.0-fold larger for EPL100 than those of APL100-x-EPLx/pDNA polyplexes, the average sizes were approximately 88% for EPL0, 161% for EPL25, 139% for EPL50, 139% for EPL75, and 171% for EPL100 compared to those of APL100-xEPLx/ATP4-pDNA polyplexes. These results might be strongly affected by complexation (i.e., the binding affinity of polycations with pDNA). That is, when adding ATP to APL-rich polyplexes (e.g., too tight-to-tight polyplexes), the introduction of ATP could intervene in the tight complexation between pDNA and APL, reducing the binding affinities between polycations and pDNA and forming moderately tight-to-loose polyplexes (e.g., moderately tight polyplexes for low-ATP APL-rich polyplexes and loose polyplexes for high-ATP APL-rich polyplexes). However, the addition of ATP could occupy existing spaces in EPL-rich polyplexes (e.g., loose-totoo loose polyplexes) and thereby weaken the binding affinities with pDNA. These different binding affinities of polycations with pDNA and internal free volumes in APL100-x-EPLx/pDNA polyplexes might allow the formation of APL100-x-EPLx/ATPz-pDNA polyplexes having diameters of mostly 50–300 nm at low ATP doses but mostly 400–600 nm at high ATP doses. As expected, the zeta potentials of APL100-x-EPLx/ATPz-pDNA polyplexes decreased with increasing ATP doses because ATP is negatively charged (Fig. 5(b)). When 0 < [ATP] (nmol/μg pDNA) ≤ 4 was added, most APL100-x-EPLx/ATPz-pDNA polyplexes had zeta-potentials of 5–15 mV. However, the zeta-potentials of APL100-x-EPLx/ATP8-pDNA polyplexes and APL100-x-EPLx/ATP16-pDNA polyplexes mostly approached to 0 mV, whereas [ATP] =24 nmol/μg pDNA formed APL100x-EPLx/ATP24-pDNA polyplexes with weak negative zeta-potentials (e.g., −5 mV). Based on integrating size (Fig. 5(a)) and zeta-potential (Fig. 5(b)) data, low-ATP APL100-x-EPLx/ATPz-pDNA polyplexes were positively charged 50–300 nm particles, and high-ATP APL100-x-EPLx/ATPz-pDNA polyplexes were neutral-to-negatively charged 400–600 nm particles. Thus, the applications of the designed APL100-x-EPLx/ATPz-pDNA polyplexes in blood could be limited due to the positive zeta-potentials of low-ATP polyplexes and the large sizes of high-ATP polyplexes. Nevertheless, as mentioned above, routes other than intravenous injection could allow the designed polyplexes to be administered, and the designed polyplexes could be applied as macrophage-targeted therapies and drug depots.

former’s transfection efficiency was 6.3-fold and 7.3-fold lower than the latter’s, respectively. The results indicate that APL-rich polyplexes could effectively deliver pDNA to the nucleus but might release pDNA ineffectively (very slowly) in the nucleus. Second, APL0-EPL100/pDNA polyplexes and APL25-EPL75/pDNA polyplexes had lower CUs (i.e., 3.70-fold and 1.92-fold, respectively) but higher NP/CU ratios (i.e., 1.74-fold and 1.56-fold, respectively) than bPEI25kDa/pDNA polyplexes. These findings indicate that EPL-rich polyplexes might deliver pDNA ineffectively into cells, unlike APL-rich polyplexes, but the EPL-rich polyplexes already internalized in the cells might effectively deliver pDNA to the nucleus. In addition, after delivering pDNA into cells, EPLrich polyplexes could have higher nuclear delivery efficiency of pDNA than APL-rich polyplexes. Third, although the nuclear delivery of pDNA by APL0-EPL100/pDNA polyplexes was only 2.15-fold lower than that of APL25-EPL75/pDNA polyplexes, APL0-EPL100/pDNA polyplexes had unexpectedly lower transfection efficiencies (i.e., 71.8-fold lower) than APL25-EPL75/pDNA polyplexes. When transfection efficiency (Fig. 2(a)) was divided by NNU (Fig. 3(c)), the transfection efficiencies per unit mass of pDNA in the nucleus were 5.2 × 107 RLU/mg protein for bPEI25kDa/pDNA polyplexes, 6.67 × 106 RLU/mg protein for APL100EPL0/pDNA polyplexes, 7.03 × 106 RLU/mg protein for APL75-EPL25/ pDNA polyplexes, 6.44 × 106 RLU/mg protein for APL50-EPL50/pDNA polyplexes, 3.46 × 106 RLU/mg protein for APL25-EPL75/pDNA polyplexes, and 8.30 × 105 RLU/mg protein for APL0-EPL100/pDNA polyplexes. These results indicate that the gene expression levels of pDNA delivered by polycations are not linearly correlated with the amount of pDNA delivered into the nucleus but could be affected by the cellular internalization kinetics, endosomal escaping kinetics, and nuclear import kinetics of pDNA delivered by polycations, as well as pDNA release rates from polyplexes. Based on the transfection efficiencies, CUs, and NUs of the designed polyplexes, APL100-EPL0/pDNA polyplexes, APL75EPL25/pDNA polyplexes, APL50-EPL50/pDNA polyplexes, APL25-EPL75/ pDNA polyplexes, and APL0-EPL100/pDNA polyplexes could be called too tight polyplexes, tight polyplexes, moderately tight polyplexes, loose polyplexes, and too loose polyplexes, respectively, as expressed in Fig. 4. 3.5. Particle sizes and zeta-potentials of APL100-x-EPLx/ATPz-pDNA polyplexes From the investigation of physicochemical characteristics, transfection efficiencies, CUs, NUs, and decomplexation of APL100-x-EPLx/ pDNA polyplexes, we understand that the lower transfection efficiencies of APL-rich polyplexes and EPL-rich polyplexes than those of bPEI25kDa/pDNA polyplexes resulted from different reasons: poor endosomal release and poor decomplexation of APL-rich polyplexes but poor complexation and poor cellular uptake of EPL-rich polyplexes. Nevertheless, APL-rich polyplexes still had higher transfection efficiencies than EPL-rich polyplexes. Thus, to enhance their transfection efficiencies, we selected APL, EPL, and ATP as major components of the designed polyplexes because the balance of contents between APL and EPL could control the complexation and decomplexation with pDNA; both EPL and ATP could enhance the facilitated endosomal escape of polyplexes; and both APL and EPL could improve the nuclear delivery of pDNA. As a result, APL100-x-EPLx/ATPz-pDNA polyplexes were manufactured by introducing ATP from 0 nmol to 24 nmol per 1 μg pDNA. As shown in Fig. 5(a), APL100-x-EPLx/ATPz-pDNA polyplexes were larger than APL100-x-EPLx/pDNA polyplexes regardless of the dose of ATP. Additionally, with increasing ATP dose, the particle sizes of APL100-x-EPLx/ATPz-pDNA polyplexes generally increased. However, the effect of ATP on the relative size increase of APL100-x-EPLx/ATPzpDNA polyplexes over APL100-x-EPLx/pDNA polyplexes does not seem to be strictly linear. The sizes of APL100-x-EPLx/ATPz-pDNA polyplexes increased greatly at low ATP doses (e.g., 0 < [ATP] (nmol/μg pDNA) ≤ 4), but the degree of increase sharply decreased with increasing EPL

3.6. Transfection efficiencies of APL100-x-EPLx/ATPz-pDNA polyplexes The effects of endosomolytic ATP in APL100-x-EPLx/ATPz-pDNA polyplexes on their transfection efficiencies have been investigated by an [ATP]-dependent gene expression study with 0 ≤ [ATP] (nmol/μg pDNA) ≤ 24. As shown in Fig. 6, APL100-x-EPLx/ATPz-pDNA polyplexes showed three different transfection efficiency patterns with increasing ATP doses: 1) a “down” pattern, 2) a “down and up” pattern, and 3) an “up” pattern. In HepG2 cells, the transfection efficiencies of APL100EPL0/ATPz-pDNA polyplexes, which had a “down” pattern, dropped to 7.1-fold lower, whereas the “up”-patterned transfection efficiencies of APL50-EPL50/ATPz-pDNA polyplexes, APL25-EPL75/ATPz-pDNA polyplexes, and APL0-EPL100/ATPz-pDNA polyplexes increased 6.3-fold, 19.6-fold, and 50.4-fold, respectively (Fig. 6(a)). APL75-EPL25/ATPzpDNA polyplexes had “down and up”-patterned transfection efficiencies that decreased 2.0-fold and then increased 5.1-fold, and their transition 6

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Fig. 5. (a) Particle sizes and (b) zeta-potentials of APL100-EPL0/ATPz-pDNA, APL75-EPL25/ATPz-pDNA, APL50-EPL50/ATPz-pDNA, APL25-EPL75/ATPz-pDNA, and APL0-EPL100/ATPz-pDNA (N/P 5) complexes with various ATP concentrations (0 ≤ z ≤ 24 nmol/μg pDNA) (mean ± SD; n = 3).

activated in too tight-to-tight polyplexes but more activated in moderately tight-to-too loose polyplexes because ATP could be slowly or less protonated in the former polyplexes but quickly or more protonated in the latter polyplexes. The results are supported by our previous result that ATP caused greatly enhanced transfection efficiencies in loose bPEI25kDa/ATP-pDNA polyplexes, in contrast to too tight APL/ATPpDNA polyplexes [26].

point was 8 nmol ATP/μg pDNA (Fig. 6(a)). Similarly, in HEK293 cells, the “down”-patterned transfection efficiencies of APL100-EPL0/ATPzpDNA polyplexes dropped 2.7-fold, whereas the “up”-patterned transfection efficiencies of APL25-EPL75/ATPz-pDNA polyplexes and APL0EPL100/ATPz-pDNA polyplexes increased 2.8-fold and 47.9-fold, respectively (Fig. 6(b)). Additionally, APL75-EPL25/ATPz-pDNA polyplexes and APL50-EPL50/ATPz-pDNA polyplexes had “down and up”patterned transfection efficiencies that decreased 2.9-fold and 4.6-fold, respectively, and then increased 2.6-fold and 4.8-fold, respectively, and their transition points were 16 and 8 nmol ATP/μg pDNA, respectively (Fig. 6(b)). Based on the results in Fig. 6, the effects of ATP dose on the transfection efficiencies of APL100-x-EPLx/ATPz-pDNA polyplexes seem to be influenced by the degree of complexation, such as in tight polyplexes and loose polyplexes. Interestingly, when adding ATP into too tight polyplexes (i.e., APL100-EPL0/ATPz-pDNA polyplexes) or loose-totoo loose polyplexes (i.e., APL25-EPL75/ATPz-pDNA polyplexes and APL0-EPL100/ATPz-pDNA polyplexes), their transfection efficiencies decreased or increased, respectively, with increasing ATP doses. In the case of tight polyplexes (i.e., APL75-EPL25/ATPz-pDNA polyplexes), different ATP amounts tuned or decreased tightness between polycations and pDNA in polyplexes, resulting in low-ATP tight polyplexes and high-ATP moderately tight polyplexes, which exhibited reduced transfection efficiencies and improved transfection efficiencies, respectively. The findings suggest that endosomolytic ATP might be less

3.7. CU and NU of APL100-x-EPLx/ATPz-pDNA polyplexes To understand the effects of ATP on the CUs and NUs of APL100-xEPLx/ATPz-pDNA polyplexes compared to those of APL100-x-EPLx/pDNA polyplexes, we adopted [ATP] = 16 nmol/μg pDNA in APL25-EPL75/ ATPz-pDNA polyplexes having “up”-patterned transfection efficiencies because the transfection efficiencies of APL25-EPL75/ATP16-pDNA polyplexes were approximately 20-fold higher than those of APL25EPL75/pDNA polyplexes. As control polyplexes, APL100-EPL0/ATPzpDNA polyplexes with “down”-patterned transfection efficiency and APL0-EPL100/ATPz-pDNA polyplexes with “up”-patterned transfection efficiency were selected, and their transfection efficiencies at [ATP] = 16 nmol/μg pDNA were 7.1-fold lower or 11.1-fold higher, respectively, than those at [ATP] = 0. In Fig. 7(a), the CUs and NUs of the polyplexes were expressed as their histograms, but their comparison was not easy, as shown in Fig. 3(a). Therefore, their NCUs, NNUs, and NP/CUs were calculated in Fig. 7(b)–(d), respectively. 7

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Fig. 6. Transfection efficiencies of APL100-EPL0/ATPz-pDNA, APL75-EPL25/ATPz-pDNA, APL50-EPL50/ATPz-pDNA, APL25-EPL75/ATPz-pDNA, and APL0-EPL100/ATPzpDNA (N/P 5) complexes having various ATP concentrations (0 ≤ x ≤ 24 nmol/μg pDNA) in (a) HepG2 cells and (b) HEK293 cells at 48 h post transfection (Mean ± SE; n ≥ 8).

studies showed that ATP- or polyATP-incorporated bPEI25kDa polyplexes with negative zeta-potentials had similar cellular uptakes to those of positively charged bPEI25kDa/pDNA polyplexes [25,26]. Thus, the slower cellular entries of APL100-x-EPLx/ATP16-pDNA polyplexes with larger sizes and negatively charged zeta-potentials might be overcome by ATP, resulting in improved cellular entries of APL100-xEPLx/ATP16-pDNA polyplexes. Second, the effects of ATP on the nuclear delivery of pDNA with the designed polyplexes were monitored. Although the increased NNU levels of APL100-x-EPLx/ATP16-pDNA polyplexes might be expected due to the increased NCU levels of the polyplexes (Fig. 7(b)), the NNUs of the polyplexes were dependent on the EPL content. Interestingly, the NNUs of APL100-EPL0/ATP16-pDNA polyplexes were 1.16-fold lower than those of APL100-EPL0/pDNA polyplexes, whereas APL25-EPL75/ATP16pDNA polyplexes and APL0-EPL100/ATP16-pDNA polyplexes had 1.29fold and 1.40-fold higher NNUs than their corresponding ATP-free polyplexes, respectively, as expected (Fig. 7(c)). The changes in the amount of pDNA delivered into the nucleus directly reflected the transfection efficiencies of the polyplexes (Fig. 6(a)). However, APL100EPL0/ATP16-pDNA polyplexes exhibited higher NCU but lower NNU values than APL100-EPL0/pDNA polyplexes. This unusual relation between the NCU and NNU of polyplexes might be explained by the following NP/CU. Third, when delivering pDNA with ATP-incorporated polyplexes,

First, the reduced NCU levels of ATP-incorporated polyplexes were expected because the introduction of ATP resulted in larger polyplexes with neutral or slightly negative zeta-potentials (Fig. 5). However, contrary to our expectations, the NCUs of APL100-EPL0/ATP16-pDNA polyplexes, APL25-EPL75/ATP16-pDNA polyplexes, and APL0-EPL100/ ATP16-pDNA polyplexes were approximately 1.08-fold, 1.58-fold, and 1.63-fold higher than those of their corresponding ATP-free polyplexes, respectively (Fig. 7(b)). Although the reasons for the increased NCU levels are unclear, the phenomena could be caused by three factors (i.e., sizes, zeta-potentials, ATP) compensating for each other. The size of nanoparticles is a key factor for cellular uptake efficiency and kinetics. Many studies have reported that certain optimal sizes have the fastest cellular entry kinetics and that either larger or smaller particles exhibit less efficient cellular entry [42–45]. In addition, the adsorptive endocytosis of positively charged particles results in faster cellular entry than the fluid-phase endocytosis of negatively charged particles [45]. Based on these facts, the larger sizes and slightly negative zeta-potentials of APL100-x-EPLx/ATP16-pDNA polyplexes could result in slower cellular internalization than that of smaller, positively charged APL100x-EPLx/pDNA polyplexes. According to the effects of ATP on cellular entry, it is known that extracellular ATP triggers fast endocytosis of proteins and nanoparticles via stimulating purinergic receptors [46]. Additionally, if ATP binds to purinergic receptors, ATP-decorated particles could be internalized via the receptors. In particular, our previous 8

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Fig. 7. Cellular uptake and nuclear uptake of APL100-EPL0/ATP0-pDNA, APL100-EPL0/ATP16-pDNA, APL25-EPL75/ATP0-pDNA, APL25-EPL75/ATP16-pDNA, APL0EPL100/ATP0-pDNA, and APL0-EPL100/ATP16-pDNA (N/P 5) complexes in HepG2 cells at 4 h post-treatment: (a) FACS histograms, (b) normalized cellular uptake (NCU), (c) normalized nuclear uptake (NNU), and (d) nuclear preference with respect to cellular uptake (NP/CU). NCU and NNU were expressed as Mean ± SE (n = 3), whereas NP/CU was expressed as Mean (n = 3). bPEI25kDa/pDNA (N/P 5) complexes were used as a control polyplex.

pDNA delivered by APL25-EPL75/pDNA polyplexes and APL25-EPL75/ ATP16-pDNA polyplexes was distributed throughout the cell, like the pDNA delivered by bPEI25kDa/pDNA polyplexes. In addition, the intracellular intensities of pDNA delivered by APL25-EPL75/pDNA polyplexes and APL25-EPL75/ATP16-pDNA polyplexes were not markedly different from that of pDNA delivered by bPEI25kDa/pDNA polyplexes. To examine the amount or percentage of pDNA localized in the nucleus or the endolysosomes in more detail, a colocalization analysis mode in the analytical software of a confocal microscope was used. As shown in Fig. 8(b), bPEI25kDa/pDNA polyplexes delivered 30.9% of the pDNA into the nucleus, while 12.6% of the pDNA delivered was still sequestrated in the endolysosomes. For the designed polyplexes, 31.6% and 14.8% of pDNA delivered by APL25-EPL75/ATP0-pDNA polyplexes were localized in the nucleus and the endolysosomes, respectively, whereas APL25-EPL75/ATP16-pDNA polyplexes resulted in 36.4% and 8.7% of pDNA localized in the nucleus and the endolysosomes, respectively. The localization results suggest that the confocal microscopy results (Fig. 8) could strongly support the flow cytometry results (Fig. 7) because the NNUs of APL25-EPL75/pDNA polyplexes and APL25-EPL75/ ATP16-pDNA polyplexes were 1.02 and 1.18, respectively, and the values were not remarkably different from the results estimated by flow cytometry. APL25-EPL75/pDNA polyplexes had insufficient endosomolytic activity, resulting in 1.17-fold more pDNA entrapped in the endolysosomes than in bPEI25kDa/pDNA polyplexes. Interestingly, APL25-EPL75/ATP16-pDNA polyplexes showed 31% less pDNA sequestration in the endolysosomes than bPEI25kDa/pDNA polyplexes. These results indicate that the introduction of endosomolytic ATP into the designed polyplexes could help the polyplexes to escape effectively from the endolysosomes. APL and EPL are naturally synthesized poly(amino acid)s that can be enzymatically degraded or nonspecifically hydrolyzed in cells and bodies. Interestingly, although the two polymers have the same

the effects of ATP on NU per CU of the pDNA delivered were evaluated. APL100-EPL0/pDNA polyplexes, APL25-EPL75/pDNA polyplexes, and APL0-EPL100/pDNA polyplexes exhibited NP/CU values of 1.12, 1.40, and 1.74, respectively, whereas the NP/CU ratios of their ATP-incorporated counter polyplexes with [ATP] = 16 nmol/μg pDNA were 0.90, 1.14, and 1.50, respectively (Fig. 7(d)). The results indicate that APL100-EPL0/ATP16-pDNA polyplexes, APL25-EPL75/ATP16-pDNA polyplexes, and APL0-EPL100/ATP16-pDNA polyplexes had NP/CU values of approximately 80%, 81%, and 86%, respectively, of those of their ATPfree counter polyplexes. The results indicate that APL100-EPL0/ATP16pDNA polyplexes and APL25-EPL75/ATP16-pDNA polyplexes had more CU but less NU than APL100-EPL0/pDNA polyplexes and APL25-EPL75/ pDNA polyplexes, respectively. These results were very interesting and unexpected because endosomolytic ATP helps to avoid enzymatic loss of pDNA in the lysosomes, which was expected to result in increased NP/CU values. However, the lower NP/CU values might be caused by the formation of larger polyplexes with the incorporation of ATP because APL100-x-EPLx/ATP16-pDNA polyplexes being much larger than APL100-x-EPLx/pDNA polyplexes could contribute to slower cytosolic transport. Nevertheless, in contrast to APL, the nuclear translocating activity of EPL could mitigate the reduced extent of NP/CU by larger polyplexes. 3.8. Intracellular localization of APL100-x-EPLx/ATPz-pDNA polyplexes In addition, to view the intracellular trafficking and distribution of polyplexes, the intracellular localization of YOYO-1-intercalated pDNA in HepG2 cells was detected by a confocal microscope. Among APL100-xEPLx/pDNA polyplexes and APL100-x-EPLx/ATP16-pDNA polyplexes tested in a quantitative assay using flow cytometry (Fig. 7), APL25EPL75/pDNA polyplexes and APL25-EPL75/ATP16-pDNA polyplexes were selected as model polyplexes. Additionally, bPEI25kDa/pDNA polyplexes were used as a control polyplex. As shown in Fig. 8(a), the 9

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Fig. 8. (a) Intracellular distribution of APL25-EPL75/ATP0-pDNA and APL25-EPL75/ATP16-pDNA (N/P 5) complexes prepared using YOYO-1-intercalated pDNA in HepG2 cells at 4 h post-transfection and (b) the colocalization (%) of pDNA delivered by polyplexes in the nucleus and endolysosomes (mean ± SE; n = 5). bPEI25kDa/pDNA (N/P 5) complexes were used as a control polyplex.

polyplexes, and too loose polyplexes (Fig. 4) and ranged from small to large particles (Fig. 1(a)). In particular, among APL-EPL/pDNA polyplexes, the smaller and tighter polyplexes delivered more pDNA into the cells than the larger and looser polyplexes (Fig. 3(b)), and the different cellular delivery of pDNA consequently affected the nuclear delivery of pDNA (Fig. 3(c)) as well as the transfection efficiencies (Fig. 2). However, although certain polyplexes delivered more pDNA into the cells, phenomena such as increased nuclear delivery of pDNA and better transfection efficiencies are not indispensably generated due to polyplexes’ poor endosomolysis and poor pDNA release-mediated interference (combining Figs. 2–4). For enhanced transfection of APL-EPL/pDNA polyplexes, the addition of endosomolytic ATP into the polyplexes was considered to remedy deficient endosomolysis because both APL and EPL could control the size, complexation/decomplexation, and nuclear pDNA delivery of their polyplexes. The incorporation of negatively charged ATP into polyplexes affected their sizes and zeta potentials and resulted in larger and negatively charged APL-EPL/ATP-pDNA polyplexes compared to

repeating unit of L-lysine and good nuclear importing activities, their structural differences lead to different protonation capacities (i.e., different pKa values) of primary amines, resulting in different protonbuffering pH ranges and different binding affinities with pDNA. The primary amines of APL/pDNA polyplexes are used only for binding with pDNA, whereas the primary amines in EPL/pDNA polyplexes play roles as both phosphate-holding groups in pDNA and proton-buffering groups in the endolysosomes (i.e., showing good endosomolytic activity). Based on the different uses of primary amines in APL and EPL, APL/ pDNA polyplexes form smaller and tighter particles with very poor pDNA release ability in the nucleus, whereas EPL/pDNA polyplexes become larger and looser particles that could dissociate before entering cells. The opposite extremes cause both polyplexes to exhibit poor transfection efficiencies. In this study, mixtures of APL and EPL were used to prepare APLEPL/pDNA polyplexes. Depending on the ratio of APL and EPL and the pDNA release rate, these polyplexes could be divided into too tight polyplexes, tight polyplexes, moderately tight polyplexes, loose 10

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APL-EPL/pDNA polyplexes due to the application of a fixed complexation ratio (N/P 5) of polycation and pDNA (Fig. 5). Although the APL-EPL/ATP-pDNA polyplexes were anionic, the ATP-incorporated polyplexes did not show any damage to their cellular and nuclear delivery of pDNA (Fig. 7 (b) and (c)). With ATP-mediated quick endosomal escape of the polyplexes (Fig. 8), the transfection efficiencies of APL-EPL/ATP-pDNA polyplexes were improved as designed (Fig. 7). However, for all preparation conditions of APL-EPL/ATP-pDNA polyplexes, some but not all showed improved transfection. With the addition of ATP, a large size increase in polyplexes might cause very slow cytosolic transport to the nucleus (Fig. 7(d)), canceling out the positive effects of the ATP-induced quick endosomal release on transfection (Fig. 6). Thus, for enhanced transfection, polyplexes should maintain a balance between complexation and decomplexation and should be optimally sized for cellular uptake, cytosolic transport, and nuclear import.

[9] [10] [11] [12] [13]

[14]

[15]

[16]

4. Conclusions

[17]

In this study, APL and EPL were used to prepare APL-EPL/pDNA polyplexes. Because of the unique characteristics of APL and EPL, the formed APL-EPL/pDNA polyplexes vary in tightness (i.e., complexation/decomplexation) and size, and the resulting effects on these two factors successively influenced cellular uptake rates, endosomal release rates, cytosolic transport, nuclear import rates, and pDNA release rates, ultimately establishing their transfection efficiencies. Additionally, when endosomolytic ATP is introduced into APL-EPL/pDNA polyplexes, the resultant APL-EPL/ATP-pDNA polyplexes show improved transfection efficiencies. Nevertheless, various unexpected factors (e.g., sizes, zeta-potentials, and tightness) could cancel out improved transfection efficiencies. Thus, understanding complexation/decomplexation and the related size changes is important in designing effective polymerbased gene carriers.

[18] [19]

[20] [21]

[22]

[23]

[24]

Acknowledgements

[25]

This study was supported by the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT and ME) (NRF2017M3A9F5028608 for the Bio & Medical Technology Development Program, NRF-2017R1A4A1015036, and 22A20130012250 for BK21PLUS) and the Research Fund, 2018 of The Catholic University of Korea.

[26] [27]

[28] [29]

Appendix A. Supplementary data [30]

Supplementary material related to this article can be found, in the online version, at doi:https://doi.org/10.1016/j.colsurfb.2019.110497.

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