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• Gene therapy is a revolutionary approach for treating incurable diseases, leveraging advancements in genomics and molecular biology [1,2]. It involves using nanoparticles to deliver therapeutic genes to target cells' nuclei [3], inducing protein expression for therapeutic outcomes. However, these nanoparticles face rapid clearance by the reticuloendothelial system, triggered by protein adsorption and enzymatic DNA breakdown [4-6]. Stealthy delivery vehicles are needed to protect DNA from degradation and biological interactions [7-9].

  PEGylation, a technique that adds a poly(ethylene glycol) layer, reduces biointerfacial interactions and enhances stealth in biological environments [10-13]. However, it can limit cellular uptake and translocation efficiency [14]. Dynamic surface chemistry offers a solution, promoting cytomembrane affinities while minimizing systemic reactions. Biological structures, often negatively charged, influence gene delivery systems' clearance and degradation [15]. Slightly negative charges on delivery systems can resist rapid clearance, while positive charges aid in endosome escape for gene transfection.

  To overcome this dilemma, a charge conversional polymer (CCP) was strategically elaborated based on carboxylation reaction onto the primary amine groups in the cationic poly{N'-[N-(2-aminoethyl)−2-aminoehtyl]aspartamide} [PAsp(DET)] with anionic 2-propionic-3-methylmaleic anhydride (CDM) (Fig. S1 in Supporting information). It is important to note that CCP possess negative charge at physiological pH 7.4 due to our proposed carboxylation scheme. Conversely, in response to an acidic pH, such as late endosome pH 5.5, PAsp(DET)'s chemical structure and function can be quickly restored by decarboxylation via hydrolyzing the CDM residues. Intriguingly, the functional PAsp(DET) exhibit unique pH-dependent membrane destabilization activities. In contrast to its low activities in destabilizing membranes at physiological pH (7.4), PAsp(DET) exhibits amplified endosome membrane destabilization activities under acidic endosome conditions [11].

  This striking contrast is attributable to PAsp(DET)'s enhanced protonation behavior, which displays mono-protonation at pH 7.4 and double-protonation at pH 5.5 in response to pH gradients [16]. This pH-responsive CCP surrounding is postulated to facilitate the liberation of the encapsulated pDNA from endosomal entrapment and elicit an appreciable intracellular trafficking route in the cytosol's pH for effective gene expression in the intended cells.

  We synthesized PAsp(DET) via ring-opening polymerization (Fig. S1), yielding a polymer with a degree of polymerization (DP) of 108 and an average molecular weight of 26 kDa, confirmed by ¹H-nuclear magnetic resonance spectroscopy (NMR) (Fig. S2 in Supporting information). The polydispersity index (PDI) was 1.10, indicating a narrow molecular weight distribution (Fig. S3 in Supporting information). PAsp(DET) was conjugated with dibenzocyclooctyne-N-hydroxysuccinimide ester (NHS-DBCO) and CDM to form the CCP. ¹H NMR analysis showed 5.6 DBCO groups per CCP molecule (Fig. S4 in Supporting information).

  We also synthesized a block copolymer, N₃-poly(ethylene glycol)-polylysine (PEG-PLys) (Fig. S5 in Supporting information), with an average molecular weight of 19.9 kDa and a PDI of 1.07 (Figs. S6 and S7 in Supporting information). To construct a three-layered structure with CCP as the outer shell, we conjugated CCP to a PEGylated polyplex micelle system via a click reaction (Fig. 1a). The DNA was condensed into a regular bundle by electrostatic interactions with the cationic block copolymer segment, and the PEG layer served as a spacer. pDNA was complexed with PEG-PLys to form template polyplex micelles, and CCP was conjugated using click chemistry between DBCO and N₃ groups. This reaction is efficient and catalyst-free, advantageous for biological applications due to copper toxicity concerns. The three-layered structure of PLys/pDNA core, PEG layer, and CCP shell reduces protein adsorption and reticuloendothelial system (RES) recognition, and prevents premature dissociation in the kidney glomerular basement membrane (GBM), leading to prolonged blood retention and enhanced bioavailability.

  Figure 1

  

  Figure 1.  Insights into CCP attachment onto the surface onto complex by pDNA and N₃-PEG-PLys(thiol) with disulfide linkage [PM(ss)]. (a) Schematic illustration of polyplex micelles with dynamic CCP surroundings. (b) Microscopic morphologies of PM(ss) by aqueous AFM measurement. (c) Microscopic morphologies of CCP-PM(ss) by aqueous AFM measurement. (d) Time dependent zeta-potential measurement for PM(ss) at pH 7.4 (close circles) and CCP-PM(ss) at pH 7.4 (square) and pH 5.5 (close square).

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  For pDNA complexation, polyplex micelles were formed at a charge ratio of 2:1 (N₃-PEG-PLys to pDNA), and stabilized with disulfide crosslinking using N₃-PEG-PLys(thiol). The complexation was confirmed by dynamic light scattering (DLS) measurement, showing a z-average size of 99.2 nm and a PDI of 0.18. Uncomplexed polymers were removed by ultracentrifugation, and CCP was conjugated to the polyplex solution overnight at 4 ℃.

  Ultracentrifugation purified the reaction, yielding pure CCP-PM(ss). DLS measurements revealed a larger z-average diameter of 139.1 nm for CCP-PM(ss), indicating CCP attachment to the polyplex micelle. FCS measurements confirmed successful conjugation, showing a significant increase in CCP diffusion time from 150 µs to 2800 µs after the click reaction, matching PM(ss) diffusion time. Atomic force microscope (AFM) imaging showed PEG-PLys condensed plasmid DNA into rod-like nanoparticles (Fig. 1b), while CCP-PM(ss) formed ellipsoid shapes (Fig. 1c), suggesting an additional CCP layer. Zeta potential measurements confirmed the spread of CCP, with a shift from a slight positive charge of +2 mV for PM to a mild negative charge of −5 mV for CCP-PM(ss) (Fig. 1d), which is beneficial for stealth function and prolonged circulation. The CCP's charge conversion function was validated by a positive zeta potential shift to +14 mV at acidic pH 5.5, mimicking late endosome conditions. These results confirm the formation of a well-defined CCP-sheathed polyplex micelle and successful CCP conjugation, as evidenced by increased size, retarded CCP diffusion, and zeta potential changes. Using ultracentrifugation, we determined that each polyplex micelle was conjugated with about 60.3 CCP molecules at a 1:1 molar ratio. This was achieved by estimating the remaining unconjugated Alexa Fluor 647-labeled CCP in the supernatant against a calibration curve.

  The negatively charged CCP corona likely shields the nanoparticle from anionic biological species, preventing premature dissociation through exchange reactions, opsonization, or adsorption. To test this, we exposed PM, PM(ss), and CCP-PM(ss) to heparan sulfate (HS) (Fig. 2a), a component of the kidney's GBM that contributes to poor blood retention of complex systems. Gel electrophoresis showed that PM began to release DNA at HS concentrations of 2.0 mg/mL, while CCP-PM(ss) demonstrated significantly greater resistance to dissociation at the same concentration, highlighting the CCP corona's role in reducing non-specific interactions and enhancing stealth properties.

  Figure 2

  

  Figure 2.  CCP's functionality in terms of stealthiness. (a) Resistance to polyion exchange reactions with anionic heparin sulfate at varied concentrations. (b) Tolerability to DNase I degradation (DNase 1: 0.01 U/mL). Data are presented as mean ± standard deviation (SD) (n = 4). **P < 0.01, ***P < 0.001 (student's t-test). (c) RT-PCR assessment of the blood retention profile of PM, PM(ss), CCP-PM. The amount of pDNA that remained intact on the Y axis was represented on an exponential scale into (d) an inset and on a linear scale in (c).

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  Moreover, the covalent disulfide crosslinking in CCP-PM(ss) provided substantial resistance to structural disassembly, suggesting that the CCP layer can be preserved in the extracellular environment. However, when incubated in a glutathione (GSH)-rich intracellular environment, CCP-PM(ss) became susceptible to dissociation, allowing for pDNA release at high HS concentrations. This indicates that the CCP-PM(ss) structure can respond to intracellular cues, facilitating gene delivery. The anionic CCP sheath was explored for its potential to protect DNA from nuclease degradation by minimizing accessibility to anionic nucleases through electrostatic repulsion. Using DNase I, which is present in the extracellular matrix and human serum, we assessed the resistance of CCP-PM(ss) to enzymatic degradation via real-time reverse transcription polymerase chain reaction (RT-PCR). CCP-PM(ss) demonstrated significantly increased tolerance to nuclease degradation, with substantial DNA persisting for over an hour at physiological DNase I concentrations, compared to control PM(ss) which showed negligible DNA after 30 min (Fig. 2b).

  This enhanced resistance to nuclease degradation suggests that the CCP corona reduces nuclease accessibility and interactions with other anionic proteins, potentially aiding in evading RES identification and increasing stealth for extended blood retention. RT-PCR measurements confirmed that CCP-PM(ss) had significantly prolonged blood circulation times compared to PM and PM(ss), with CCP-PM(ss) maintaining approximately 10 times more intact DNA in the blood after 30 min post-injection (Figs. 2c and d).

  Despite the potential for electrostatic repulsion between the CCP corona and negatively charged species on the cell membrane, which could hinder cytomembrane absorption, our studies showed that CCP-PM had significantly higher cellular uptake than PM (Fig. 3a). This was confirmed using the INCELL Analyzer, which measured intracellular Cy5 fluorescence. The results suggest that the CCP corona does not hinder cellular uptake and may even enhance it, possibly due to the repulsion preventing polyion exchange reactions that would otherwise limit cellular absorption.

  Figure 3

  

  Figure 3.  Cell transfection activities of a varieties of gene delivery systems. (a) Time-dependent cellular uptake activities in Huh-7 cells. (b) Gene expression activities in Huh-7 cells. Scale bar: 50 µm. Data are presented as mean ± SD (n = 4). P < 0.05, ***P < 0.001 (student's t-test). RFU: relative fluorescence intensity; RLU: relative luminescence intensity.

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  The potential for structural dissociation of CCP-PM was considered in the context of prolonged systemic retention and increased cellular uptake. Strategies such as the introduction of a hydrophobic cholesteryl moiety or disulfide crosslinking have been shown to stabilize the polyplex core and enhance cellular uptake. Our CCP-PM(ss), with its favorable CCP surroundings and disulfide structural crosslinking, exhibited the highest cellular uptake efficiencies, explaining that the CCP corona and structural stabilization contribute to improved gene expression at the targeted cells (Fig. 3b).

  After endocytosis, polyplex micelles are trapped in acidic late endosomes and lysosomes. To escape this, CCP-PM must undergo charge conversion [17], which was confirmed by zeta-potential measurements showing a shift to + 18.0 mV after incubation at pH 5.5, as well as the quantitative measurement of the amine groups (Fig. S8 in Supporting information), indicating strong membrane disruption activity. This restored PAsp(DET) structure [18], which is crucial for endosome escape (Figs. 4a and b). CCP-PM(ss)'s membrane destabilization activities were measured at pH 7.4 and 5.5, showing significant activity at endosome pH (Fig. 4c), crucial for retrieving polyplexes from endosome entrapment. Intracellular distribution studies, using plasmid DNA stained red, late endosomes/lysosomes in green, and cell nuclei in blue, revealed that while most PMs were enclosed in endosomes/lysosomes, CCP-PMs disrupted membranes and escaped, as seen by fragmented yellow color indicating interactions with the endosome/lysosome membranes (Fig. 4a).

  Figure 4

  

  Figure 4.  Insights on the functional role of CCP in aiding endosome escape. (a) Intracellular distributions of PM(ss) and CCP-PM(ss) based on confocal laser scanning microscopy (CLSM) observation, blue: nuclei, red: PM(ss) or CCP-PM(s), green: late endosomes and lysosomes. Scale bar: 20 µm. (b) The quantified colocalization degree of pDNA and late endolysosomes (n = 16). ***P < 0.001 (student's t-test). (c) Assessment of membrane destabilization activities of CCP-PM(ss) at varied concentrations when incubated at pH 7.4 and 5.5 (n = 4). Data are presented as mean ± SD.

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  This confirms the CCP corona's ability to rupture membranes and aid in endosome escape. The unique protonation behavior of PAsp(DET) ensures safety, as it is mono-protonated at physiological pH 7.4 and less disruptive, while double-protonated at pH 5.5 for effective endosome disruption. Consequently, enhanced cellular uptake and favorable intracellular trafficking led to a three-fold increase in gene expression for CCP-PM over PM (Fig. 3b). Cell viability experiments showed no toxicity for PM(ss) or CCP-PM(ss), supporting the system's safety (Fig. S9 in Supporting information).

  In conclusion, the facie elaboration of CCP as the surface of nanomedicine, as a superior alternative to the classical PEGylation, appeared to be an intriguingly multifaceted corona when interactions with varied biological compartments. In the extracellular milieu, the elaborated negatively charged surroundings could represent as a spatial insulating palisade, capable of affording notably stealth function towards various anionic biological components, thereby contributing to persistent retention in the bloodstream. What is more, the CCP surface coating appeared to be capable of significantly stimulating favorable intracellular behaviors, particularly facilitating translocation of the encapsulated biovulnerable payloads out of the hostile endolysosome entrapment to the cytosol due to its facile charge conversion into highly positive charge, which elicit highly effective gene expression in targeted cells. Moreover, controlled positive charge densities of CCP surrounding when residing in the endolysosomal and cytoplasmic compartments represent an optimal balance in terms of safety concerns. Namely, the positive charge following translocation into cytosol was modulated to be limited so as for not provoking cytotoxic consequence. Therefore, the benefits of CCP surface functionalization offer valuable insights for designing synthetic gene delivery systems. This approach is also commendable for translating the progress of genomic information to usefulness in therapeutic applications by encapsulating delicate therapeutic nucleic acids such as messenger RNA and small interfering RNA.

  Declaration of competing interest

  The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

  CRediT authorship contribution statement

  Changgui Tong: Writing – original draft, Validation, Methodology, Investigation, Formal analysis. Yan Zhao: Investigation, Formal analysis. Sheng Lin: Methodology, Investigation, Formal analysis, Data curation. Yong Zhang: Writing – review & editing, Validation, Supervision, Data curation. Qixian Chen: Writing – review & editing, Supervision, Investigation, Funding acquisition, Conceptualization. Yue Wang: Investigation.

  Acknowledgments

  This research was financially supported by National Key Research and Development Program (No. 2022YFD1700200), National Natural Science Foundation of China (No. 32171330), Training Program of the National Natural Science Foundation of China (Nos. 2021-ZLLH-14, 2021-ZLLH-05).

  Supplementary materials

  Supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.cclet.2024.110796.

  
  
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  Figure 1  Insights into CCP attachment onto the surface onto complex by pDNA and N₃-PEG-PLys(thiol) with disulfide linkage [PM(ss)]. (a) Schematic illustration of polyplex micelles with dynamic CCP surroundings. (b) Microscopic morphologies of PM(ss) by aqueous AFM measurement. (c) Microscopic morphologies of CCP-PM(ss) by aqueous AFM measurement. (d) Time dependent zeta-potential measurement for PM(ss) at pH 7.4 (close circles) and CCP-PM(ss) at pH 7.4 (square) and pH 5.5 (close square).

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  Figure 2  CCP's functionality in terms of stealthiness. (a) Resistance to polyion exchange reactions with anionic heparin sulfate at varied concentrations. (b) Tolerability to DNase I degradation (DNase 1: 0.01 U/mL). Data are presented as mean ± standard deviation (SD) (n = 4). **P < 0.01, ***P < 0.001 (student's t-test). (c) RT-PCR assessment of the blood retention profile of PM, PM(ss), CCP-PM. The amount of pDNA that remained intact on the Y axis was represented on an exponential scale into (d) an inset and on a linear scale in (c).

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  Figure 3  Cell transfection activities of a varieties of gene delivery systems. (a) Time-dependent cellular uptake activities in Huh-7 cells. (b) Gene expression activities in Huh-7 cells. Scale bar: 50 µm. Data are presented as mean ± SD (n = 4). P < 0.05, ***P < 0.001 (student's t-test). RFU: relative fluorescence intensity; RLU: relative luminescence intensity.

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  Figure 4  Insights on the functional role of CCP in aiding endosome escape. (a) Intracellular distributions of PM(ss) and CCP-PM(ss) based on confocal laser scanning microscopy (CLSM) observation, blue: nuclei, red: PM(ss) or CCP-PM(s), green: late endosomes and lysosomes. Scale bar: 20 µm. (b) The quantified colocalization degree of pDNA and late endolysosomes (n = 16). ***P < 0.001 (student's t-test). (c) Assessment of membrane destabilization activities of CCP-PM(ss) at varied concentrations when incubated at pH 7.4 and 5.5 (n = 4). Data are presented as mean ± SD.

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