Non-viral Gene Delivery Vectors Research Articles

Smart Supra- and Macro-Molecular Tools for Biomedical Applications.

Stimuli-responsive, “smart” polymeric materials used in the biomedical field function in a bio-mimicking manner by providing a non-linear response to triggers coming from a physiological microenvironment or other external source. They are built based on various chemical, physical, and biological tools that enable pH and/or temperature-stimulated changes in structural or physicochemical attributes, like shape, volume, solubility, supramolecular arrangement, and others. This review touches on some particular developments on the topic of stimuli-sensitive molecular tools for biomedical applications. Design and mechanistic details are provided concerning the smart synthetic instruments that are employed to prepare supra- and macro-molecular architectures with specific responses to external stimuli. Five major themes are approached: (i) temperature- and pH-responsive systems for controlled drug delivery; (ii) glycodynameric hydrogels for drug delivery; (iii) polymeric non-viral vectors for gene delivery; (iv) metallic nanoconjugates for biomedical applications; and, (v) smart organic tools for biomedical imaging.

Nanoparticle Based Gene Therapy Approach: A Pioneering Rebellion in the Management of Psychiatric Disorders.

The neuropsychiatric illnesses have been enigmatic, with no effective treatment to date. The complexity and heterogeneity of psychiatric disorders are daunting for the development of novel treatment modalities. The conventional treatment approaches are less effective and are associated with several side effects, thus creating the need for the development of more innovative strategies. Since psychiatric disorders are known to exhibit genetic linkage, gene therapy has created an interest among the researchers worldwide. The delivery of nucleic acids is a complex process requiring the transport of genetic material across various intracellular and extracellular barriers to reach the target cells eliciting the transfection process. Therefore, the identification or development of the delivery system for nucleic acid delivery still remains the challenge. Viral vectors are quite effective but are associated with toxicity and side effects. With the rapid advancement in the field of nanotechnology, nanosized materials were identified to be the perfect candidate for nonviral vectors in gene delivery. The biggest advantage of nanoparticles is that their surface can be engineered in many possible ways to deliver the drugs directly to the target site. Although gene therapy has already been established as an innovative treatment modality for several neurological diseases, its use in psychiatry still warrants more investigations for its translation into clinical use. The present manuscript discusses the prospects of gene therapy in psychiatric disorders, their benefits, and pitfalls. The review embarks upon the importance of nanoparticle-based gene therapy for effective management of psychiatric disorders.

The cell-penetrating YopM protein-functionalized quantum dot-plasmid DNA conjugate as a novel gene delivery vector

Non-viral gene delivery systems have great potential for safe and efficient gene therapy, while inefficient cellular and nuclear uptake remain as the major hurdles. Novel approaches are needed to enhance the transfection efficiency of non-viral vectors. In accordance with this need, the objective of this study was to construct a non-viral vector that could achieve gene delivery without using additional lipid-based transfection agent. We aimed to impart self-delivery property to a non-viral vector by using the cell and nucleus penetrating properties of YopM proteins from the three Yersinia spp. (Y. pestis, Y. enterocolotica and Y. pseudotuberculosis). Plasmid DNA (pDNA) encoding green fluorescent protein (GFP) was labeled with quantum dots (QDs) via peptide-nucleic acid (PNA) recognition site. Recombinant YopM protein was then attached to the conjugate via a second PNA recognition site. The YopM ̶ QDs ̶ pDNA conjugate was transfected into HeLa cells without using additional transfection reagent. All three conjugates produced GFP fluorescence, indicating that the plasmid was successfully delivered to the nucleus. As control, naked pDNA was transfected into the cells by using a commercial transfection reagent. The Y. pseudotuberculosis YopM-functionalized conjugate achieved the highest GFP expression, compared to other two YopM proteins and the transfection reagent. To the best of our knowledge, YopM protein was used for the first time in a non-viral gene delivery vector.

Autophagy-inhibiting polymer as an effective nonviral cancer gene therapy vector with inherent apoptosis-sensitizing ability

Conventionally, polycations are pharmacological inert used as nonviral gene delivery vectors with the sole function of compacting and protecting nucleic acids. Here, the first autophagy-inhibiting cationic polymer delivering plasmid DNA (pDNA) encoding TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) is prepared for cancer gene therapy. The copolymerization of methacryloyl chloroquine (MACQ) with 2-(dimethylamino)ethyl methacrylate (DMAEMA) not only improves transfection efficacy through hydrophobic modification, but also endows the copolymer with autophagy-blocking capability, which further sensitizes cancer cells to TRAIL induced apoptosis. Importantly, the designed copolymer shows efficient TRAIL expression, autophagy inhibition and enhances TRAIL-induced apoptosis in an autophagy-dependent manner. In contrast, TRAIL gene delivered by the autophagy-blocking-deficient control copolymer without the chlorine atom presents weaker antitumor efficacy, although expressing a similar amount of therapeutic TRAIL protein. Thus, this study demonstrates a conceptually new approach in which the therapeutic outcome of the delivered gene can be inherently strengthened by the delivery vehicle with intrinsic pharmacological activity.

Polyethylenimine-based nanovector grafted with mannitol moieties to achieve effective gene delivery and transfection

Polyethylenimine (PEI), a kind of cationic non-viral gene delivery vector, is capable of stable and efficient transgene expression for gene delivery. However, low transfection efficiency in vivo along with high toxicity limited the further application of gene therapy in the clinic. To enhance gene transfection performance and reduce cytotoxicity of polyethylenimine, branched polyethylenimine-derived cationic polymers BPEI25 k-man-S/L/M/H with different grafting degree with mannitol moieties were prepared and the transfection efficiency was evaluated. Among them, BPEI25 k-man-L showed the best transfection efficiency, lower toxicity, and significantly enhanced long-term systemic transgene expression for 96 h in vivo even at a single-dose administration. The results of cellular uptake mechanism and western-blot experiments revealed that the mannitol modification of BPEI25 k induced and up-regulated the phosphorylation of caveolin-1 and thus enhanced the caveolae-mediated cellular uptake. This class of gene delivery system highlights a paradigmatic approach for the development of novel and safe non-viral vectors for gene therapy.

Effects of Branching Strategy on the Gene Transfection of Highly Branched Poly(β-amino ester)s

Highly branched poly(β-amino ester)s (HPAEs) have emerged as one type of the most viable non-viral gene delivery vectors, both in vitro and in vivo. However, the effects of different branching strategies on the gene transfection performance have not yet been explored. Here, using triacrylate (B3) and diamine (B4) as the branching monomers, a series of HPAEs were synthesized via the “A2 + B3 + C2” and “A2 + B4 + C2” strategies, respectively. Results show that the branching strategy plays a pivotal role in dictating the physiological properties of the HPAE/DNA polyplexes and thus leads to obviously different cell viability and transfection efficiency. Comparatively, HPAEs synthesized via the “A2 + B3 + C2” branching strategy are more favorable for DNA transfection than that synthesized via the “A2 + B4 + C2” strategy. This study may provide new insights into the development of HPAEs based non-viral DNA delivery system.

Carbonized chitosan encapsulated hierarchical porous zeolitic imidazolate frameworks nanoparticles for gene delivery

Abstract Hierarchical mesoporous carbon (MPC) nanomaterials derived from the carbonized chitosan (CTS) encapsulated zeolitic imidazolate frameworks (ZIF-8) is synthesized and applied for gene delivery. The synthesis of ZIF-8 is achieved at room temperature using water as a solvent in the presence of CTS within 60 min. The synthesis method offered a hierarchical porous structure of ZIF-8. The carbonization of the prepared materials leads to the formation of MPC nanomaterials. MPC materials were applied as a non-viral vectors for gene delivery using two oligonucleotides (ONs) called Luciferase-expressing plasmid (pGL3), and splice correction oligonucleotides (SCO). The materials are biocompatible and showed insignificant toxicity. The transfection using MPC with and without cell-penetrating peptides (CPPs) was reported. MPC improved the transfection efficiency of CPPs (PepFect 14 (PF-14), and PF-221) by 10 fold due to the synergistic effect of MCP and CPPs. The reasonable mechanism for the cell transfection using these new vectors was also highlighted.

Efficacy of insulin targeted gene therapy for type 1 diabetes mellitus: A systematic review and meta-analysis of rodent studies.

Diabetes mellitus (DM) is a major worldwide public health challenge, for which gene therapy offers a potential therapeutic approach. To date, no systematic review or meta-analysis has been published in this area, so we examined all relevant published studies on rodents to elucidate the overall effects of gene therapy on bodyweight, intraperitoneal glucose tolerance test (IPGTT), fasting blood glucose, and insulin in animals with type 1 DM. The Cochrane Library, PubMed, Embase, ISI Web of Science, SCOPUS, and Google Scholar were systematically searched for potentially relevant studies. Mean±standard deviation (SD) was pooled using a random-effects model. After the primary search, out of 528 studies identified, 16 studies were in concordance with predefined criteria and selected for the final assessment. Of these, 12 studies used viral manipulation, and 4 employed non-viral vectors for gene delivery. The meta-analysis showed gene therapy with a viral vector decreased mean IPGTT (-12.69 mmol/l, P<0.001), fasting blood glucose (-13.51 mmol/l, P<0.001), insulin (398.28 pmol/l, P<0.001), and bodyweight (24.22 g, P<0.001), whereas non-viral vectors reduced fasting glucose (-29.95 mmol/l, P<0.001) and elevated insulin (114.92 pmol/l, P<0.001). Gene therapy has favorable effects on alleviating type 1 DM related factors in diabetic rodents.

Amino Acid-Linked Low Molecular Weight Polyethylenimine for Improved Gene Delivery and Biocompatibility.

The construction of efficient and low toxic non-viral gene delivery vectors is of great significance for gene therapy. Herein, two novel polycations were constructed via Michael addition from low molecular weight polyethylenimine (PEI) 600 Da and amino acid-containing linkages. Lysine and histidine were introduced for the purpose of improved DNA binding and pH buffering capacity, respectively. The ester bonds afforded the polymer biodegradability, which was confirmed by the gel permeation chromatography (GPC) measurement. The polymers could well condense DNA into nanoparticles and protect DNA from degradation by nuclease. Compared with PEI 25 kDa, these polymers showed higher transfection efficiency, lower toxicity, and better serum tolerance. Study of this mechanism revealed that the polyplexes enter the cells mainly through caveolae-mediated endocytosis pathway; this, together with their biodegradability, facilitates the internalization of polyplexes and the release of DNA. The results reveal that the amino acid-linked low molecular weight PEI polymers could serve as promising candidates for non-viral gene delivery.

Non-Viral in Vitro Gene Delivery: It is Now Time to Set the Bar!

Transfection by means of non-viral gene delivery vectors is the cornerstone of modern gene delivery. Despite the resources poured into the development of ever more effective transfectants, improvement is still slow and limited. Of note, the performance of any gene delivery vector in vitro is strictly dependent on several experimental conditions specific to each laboratory. The lack of standard tests has thus largely contributed to the flood of inconsistent data underpinning the reproducibility crisis. A way researchers seek to address this issue is by gauging the effectiveness of newly synthesized gene delivery vectors with respect to benchmarks of seemingly well-known behavior. However, the performance of such reference molecules is also affected by the testing conditions. This survey points to non-standardized transfection settings and limited information on variables deemed relevant in this context as the major cause of such misalignments. This review provides a catalog of conditions optimized for the gold standard and internal reference, 25 kDa polyethyleneimine, that can be profitably replicated across studies for the sake of comparison. Overall, we wish to pave the way for the implementation of standardized protocols in order to make the evaluation of the effectiveness of transfectants as unbiased as possible.

Mass Spectrometric Detection and Characterization of Metabolites of Gemini Surfactants Used as Gene Delivery Vectors.

Gemini surfactants are a class of lipid molecules that have been successfully used in vitro and in vivo as nonviral gene delivery vectors. However, the biological fate of gemini surfactants has not been well investigated. In particular, the metabolism of gemini surfactants after they enter cells as gene delivery vehicles is unknown. In this work, we used a high-resolution quadrupole-Orbitrap mass spectrometry (Q-Exactive) instrument to detect the metabolites of three model gemini surfactants, namely, (a) unsubstituted (16-3-16), (b) with pyridinium head groups (16(Py)-S-2-S-16(Py)), and (c) substituted with a glycyl-lysine di-peptide (16-7N(GK)-16). The metabolites were characterized, and structures were proposed, based on accurate masses and characteristic product ions. The metabolism of the three gemini surfactants was very different as 16-3-16 was not metabolized in PAM 212 cells, whereas 16(Py)-S-2-S-16(Py) was metabolized primarily via phase I reactions, including oxidation and dealkylation, producing metabolites that could be linked to its observed high toxicity. The third gemini surfactant 16-7N(GK)-16 was metabolized mainly via phase II reactions, including methylation, acetylation, glucose conjugation, palmityl conjugation, and stearyl conjugation. The metabolism of gemini surfactants provides insight for future directions in the design and development of more effective gemini surfactants with lower toxicity. The reported approach can also be applied to study the metabolism of other structurally related gemini surfactants.

Acid-activated nonviral peptide vector for gene delivery.

Nonviral vector-based gene therapy is a promising strategy for treating a myriad of diseases. Cell-penetrating peptides are gaining increasing attention as vectors for nucleic acid delivery. However, most studies have focused more on the transfection efficiency of these vectors than on their specificity and toxicity. To obtain ideal vectors with high efficiency and safety, we constructed the vector stearyl-TH by attaching a stearyl moiety to the N-terminus of the acid-activated cell penetrating peptide TH in this study. Under acidic conditions, stearyl-TH could bind to and condense plasmids into nanoparticle complexes, which displayed significantly enhanced cellular uptake and transfection efficiencies. In contrast, stearyl-TH lost the capacities of DNA binding and transfection at physiological pH. More importantly, stearyl-TH and the complexes formed by stearyl-TH and plasmids displayed no obvious toxicity at physiological pH. Consequently, the high transfection efficiency under acidic conditions and low toxicity make stearyl-TH a potential nucleic acid delivery vector for gene therapy.

The Development of Functional Non-Viral Vectors for Gene Delivery.

Gene therapy is manipulation in/of gene expression in specific cells/tissue to treat diseases. This manipulation is carried out by introducing exogenous nucleic acids, such as DNA or RNA, into the cell. Because of their negative charge and considerable larger size, the delivery of these molecules, in general, should be mediated by gene vectors. Non-viral vectors, as promising delivery systems, have received considerable attention due to their low cytotoxicity and non-immunogenicity. As research continued, more and more functional non-viral vectors have emerged. They not only have the ability to deliver a gene into the cells but also have other functions, such as the performance of fluorescence imaging, which aids in monitoring their progress, targeted delivery, and biodegradation. Recently, many reviews related to non-viral vectors, such as polymers and cationic lipids, have been reported. However, there are few reviews regarding functional non-viral vectors. This review summarizes the common functional non-viral vectors developed in the last ten years and their potential applications in the future. The transfection efficiency and the transport mechanism of these materials were also discussed in detail. We hope that this review can help researchers design more new high-efficiency and low-toxicity multifunctional non-viral vectors, and further accelerate the progress of gene therapy.

Advances in Targeted Gene Delivery.

Gene therapy has the potential to treat both acquired and inherited genetic diseases. Generally, two types of gene delivery vectors are used - viral vectors and non-viral vectors. Non-viral gene delivery systems have attracted significant interest (e.g. 115 gene therapies approved for clinical trials in 2018; clinicaltrials.gov) due to their lower toxicity, lack of immunogenicity and ease of production compared to viral vectors. To achieve the goal of maximal therapeutic efficacy with minimal adverse effects, the cell-specific targeting of non-viral gene delivery systems has attracted research interest. Targeting through cell surface receptors; the enhanced permeability and retention effect, or pH differences are potential means to target genes to specific organs, tissues, or cells. As for targeting moieties, receptorspecific ligand peptides, antibodies, aptamers and affibodies have been incorporated into synthetic nonviral gene delivery vectors to fulfill the requirement of active targeting. This review provides an overview of different potential targets and targeting moieties to target specific gene delivery systems.

Development and characterization of an improved formulation of cholesteryl oleate-loaded cationic solid-lipid nanoparticles as an efficient non-viral gene delivery system

Nanoparticle-mediated plasmid delivery is considered a useful tool to introduce foreign DNA into the cells for the purpose of DNA vaccination and/or gene therapy. Cationic solid-lipid nanoparticles (cSLNs) are considered one of the most promising non-viral vectors for nucleic acid delivery. Based on the idea that the optimization of the components is required to improve transfection efficiency, the present study aimed to formulate and characterize cholesteryl oleate-containing solid-lipid nanoparticles (CO-SLNs) incorporating protamine (P) to condense DNA to produce P:DNA:CO-SLN complexes as non-viral vectors for gene delivery with reduced cytotoxicity and high cellular uptake efficiency. For this purpose, CO-SLNs were used to prepare DNA complexes with and without protamine as DNA condenser and nuclear transfer enhancer. The main physicochemical characteristics, binding capabilities, cytotoxicity and cellular uptake of these novel CO-SLNs were analyzed.Positively charged spherical P:DNA:CO-SLN complexes with a particle size ranging from 330.1 ± 14.8 nm to 347.0 ± 18.5 nm were obtained. Positive results were obtained in the DNase I protection assay with a protective effect of the genetic material and 100% loading efficiency was achieved at a P:DNA:CO-SLN ratio of 2:1:7. Transfection studies in human embryonic kidney (HEK293T) cells showed the versatility of adding protamine to efficiently transfect cells, widening the potential applications of CO-SLN-based vectors, since the incorporation of protamine induced almost a 200-fold increase in the transfection capacity of CO-SLNs without toxicity.These results indicate that CO-SLNs with protamine are a safe and effective platform for non-viral nucleic acid delivery.

PLGA cationic nanoparticles, obtained from nano-emulsion templating, as potential DNA vaccines

Polymeric nanoparticles offer advantageous characteristics as gene-delivery vectors such as biocompatibility and biodegradability. With this aim, a smart and innovative strategy was followed here: Cationic PLGA nano-emulsions, prepared by a low energy method, were used as templates to obtain cationic nanoparticles (NPs) able to easily complex with nucleic acids (i.e. plasmid DNA) by electrostatic interactions. The strategy employed to produce stable positively-charged nanoparticles was the use of non-ionic/cationic surfactant mixtures to stabilize template nano-emulsions. This methodology allowed obtaining nanoparticles with reproducible nanometric sizes and positive zeta potential values, appropriate to successfully complex with nucleic acids, resulting in nanometric spherical polyplexes. Nanoparticles, plasmids and polyplexes proved to be biocompatible at the optimal concentration. Therefore, we can conclude that we have designed a novel strategy to efficiently obtain cationic polymeric nanoparticles that can be a promising approach to act as novel non-viral gene-delivery vectors, useful for many applications in gene therapy, such as gene vaccines.

New Water-Soluble Oxyamino Chitosans as Biocompatible Vectors for Efficacious Anticancer Therapy via Co-Delivery of Gene and Drug.

Among the many nonviral gene delivery vectors, chitosan, being a polysaccharide of natural origin, has gained special importance. In this report, chitosan (CS) has been solubilized in water by preparing its O-carboxymethyl derivative, CS(CH2COOH), with an optimum degree of carboxymethylation. This has been further derivatized to get the pyridine-substituted product (py)CS(CH2COOH), where the degree of pyridine substitution (47%) was optimized based on zeta potential measurements. The optimized formulation showed a high gene binding ability, forming nanosized positively charged polyelectrolyte complexes with DNA. These polyplexes were stable to DNase and physiological polyanions such as heparin. They also exhibited minimal toxicity in vitro and showed transfection levels comparable to the commercial standard Lipofectamine 2000 and much higher than polyethylenimine (MW, 25 kDa). Additionally, in this study, a hitherto unknown oxyamine derivative of chitosan has been prepared by phthaloyl protection, tosylation, and Gabriel's phthalimide synthesis. Nearly 40% of the primary alcohols were successfully converted to oxyamino functionality, which was used for forming oxime with the anticancer drug doxorubicin. The pH sensitivity of the oxime ether linkage and stability under biologically relevant conditions were then used to establish the compound as a versatile drug delivery vector. Co-delivery of functional gene (p53) and drug (doxorubicin) was accomplished in vitro and in vivo with the chitosan-pyridine imine vector (py)CS(CH2COOH) and the newly synthesized doxorubicin oxime ether CS(Dox). Complete tumor regression with no tumor recurrence and appreciable survivability point to the in vivo effectiveness and biocompatibility of the designed composite formulation. Overall, the pH sensitivity of the oxime linkage aiding slow and steady drug release, together with the sustained gene expression by pyridine-tethered carboxymethyl chitosan, allows us to generate a nanobiocomposite with significantly high anticancer therapeutic potential.

Polyethylenimine-associated cerium oxide nanoparticles: A novel promising gene delivery vector

AimsThe development of highly efficient and low toxic non-viral gene delivery vectors is the most challenging issues for successful application of gene therapy. A particular focus has been on understanding structure-activity relationships for transfection activity and toxicity of polyethylenimine (PEI). During the last decade, the use of cerium oxide nanoparticles (CeO2-NPs) in biomedicine has attracted much attention due to their pH-dependent antioxidant activity. CeO2-NPs provide protection normal cells from various forms of reactive oxygen species, but possess innate cytotoxicity and apoptosis to cancer cells. The purpose of this study was to design a new class of gene carriers by low molecular weight PEI (B-PEI 10 kDa) coordination onto CeO2-NPs. Main methodsB-PEI 10 kDa was conjugated to CeO2-NPs by Epichlorohydrin linker. Transfection efficiency, cytotoxic and apoptotic effects of pDNA-PEI-CeO2 NPs were evaluated on WEHI 164 cancer cells and normal L929 cells lines. Key findingsPEI-CeO2 NPs was able to condense the pDNA at carrier/plasmid (C/P) weight ratios of 0.5. The size and zeta potential of pDNA-PEI-CeO2 NPs were 124 ± 7 nm and 22 ± 2 mV, respectively. The transfection efficacy of synthesized pDNA-PEI-CeO2 NPs improved and the cytotoxicity was decreased compared to pDNA-PEI. Moreover, pDNA-PEI-CeO2 NPs induced more apoptosis than unmodified PEI and CeO2-NPs control groups. pDNA-PEI-CeO2 NPs displayed more transfection, cytotoxicity, and apoptosis in WEHI 164 cancer cells than normal L929 cells. SignificanceIn conclusion, PEI-CeO2 nanocarriers could act as a potential candidate for gene and drug delivery to cancerous and tumor cells.

Polyethylenimine-based Formulations for Delivery of Oligonucleotides.

Polyethyleneimine (PEI) is well-known as a non-viral gene delivery vector, especially for oligonucleotide delivery. However, its clinical applications are significantly limited due to its high cationic charge, lack of specificity, and interaction with the proteins and nontarget cells in the biological fluids, resulting in high cytotoxicity, poor stability and low transfection efficiency for oligonucleotides transporting. It has been shown that the molecular weight (MW) of PEI, degree of branching, N/P ratio, buffer capacity, oligonucleotide structure, culture medium pH, serum, presence or absence of and method of preparation make a significant difference in the cytoxicity, stability, and transfection efficiency for the PEI-based oligonucleotides delivery systems. Ligands, hydrophobic, hydrophilic, and amphiphilic modification of PEI have been investigated to reduce the cytoxicity and improve the stability, the transfection efficiency, and therapeutic effect. Moreover, various intelligent modifications of PEI, such as pH-responsive (hydrazone bond) and redox sensitive linkers (disulfide bond) can control oligonucleotides release and have attracted much attention. In general, more efficient oligonucleotide delivery can be achieved by the introduction of modifications to PEI and by optimization of parameters of PEI or PEI-based formulations.

Interparticle Interactions in Dilute Solutions of Polyelectrolyte Complex Micelles.

The application of dilute solutions of polyelectrolyte complex (PEC) micelles for delivering therapeutic nucleic acids into disease sites has gained momentum. This Letter reports a detailed characterization of PEC micelles in dilute solutions including their internal structures and the determination of the interparticle interactions. The polymer concentration ranges from 0.1 to 0.5 wt %, a regime where micelle-micelle interactions are infrequent. We employ synchrotron small-angle X-ray scattering (SAXS) to simultaneously probe the morphology, internal structure, and radius of gyration (Rg) of the self-assemblies formed by charged diblock polyelectrolytes and homopolyelectrolytes. The emerging appearance of the structure factor in SAXS profiles with the increasing polymer concentration demonstrates the presence of the repulsive intermicellar correlations, which is further confirmed by the differences between the "reciprocal Rg" estimated by Guinier approximation and the "real space Rg" determined by pair distribution functions. We find that the soft corona chains tethered on the surface of phase-separated complex domains are compressed when micelles come closeto thepointwhere a hard-sphere interaction takes over. These findings contribute to the fundamental understanding of the structure and space-filling constraints in the complexation-driven self-assemblies and advance the rational design of cationic polymer-based nonviral gene delivery vectors.