Visualization of the Plasmid DNA Delivery System by Complementary Fluorescence Labeling of Arginine-Rich Peptides.

Cell-penetrating peptides, such as arginine-rich peptides, encapsulate nucleic acid drugs and deliver them to intracellular compartments. Comprehensive tracking of drug delivery systems (DDSs) provides information about the behavior of the drug as well as the fate of the drug carrier after drug release, which is crucial for minimizing side effects. In this study, we labeled peptides designed to carry plasmid DNA with two types of dyes, traditional dye fluorescein and aggregation-induced emission (AIE) dye tetraphenylethylene, and subsequently tracked the DDS through the complementary ON and OFF fluorescence behaviors of the dyes. Traditional fluorescent dyes are susceptible to aggregation-caused quenching during bioimaging, a problem that is mitigated by using AIE dyes. However, by using both of these contrasting fluorescent labels, we were able to clearly visualize the DDS at different stages of its deployment, from drug transport and delivery to carrier dissociation and migration, demonstrating the feasibility of accurate DDS visualization by complementary fluorescence labeling.

Development of Hydrophobic Cell-Penetrating Stapled Peptides as Drug Carriers.

Cell-penetrating peptides (CPPs) are widely used for the intracellular delivery of a variety of cargo molecules, including small molecules, peptides, nucleic acids, and proteins. Many cationic and amphiphilic CPPs have been developed; however, there have been few reports regarding hydrophobic CPPs. Herein, we have developed stapled hydrophobic CPPs based on the hydrophobic CPP, TP10, by introducing an aliphatic carbon side chain on the hydrophobic face of TP10. This side chain maintained the hydrophobicity of TP10 and enhanced the helicity and cell penetrating efficiency. We evaluated the preferred secondary structures, and the ability to deliver 5(6)-carboxyfluorescein (CF) as a model small molecule and plasmid DNA (pDNA) as a model nucleotide. The stapled peptide F-3 with CF, in which the stapling structure was introduced at Gly residues, formed a stable α-helical structure and the highest cell-membrane permeability via an endocytosis process. Meanwhile, peptide F-4 demonstrated remarkable stability when forming a complex with pDNA, making it the optimal choice for the efficient intracellular delivery of pDNA. The results showed that stapled hydrophobic CPPs were able to deliver small molecules and pDNA into cells, and that different stapling positions in hydrophobic CPPs can control the efficiency of the cargo delivery.

Development of delivery carriers for plasmid DNA by conjugation of a helical template to oligoarginine.

Arginine (Arg)-rich peptides can penetrate the cell membrane and deliver nucleic acid-based therapeutics into cells. In this study, a helical template designed with a repeating sequence composed of two l-leucines (l-Leu) and a 2-aminoisobutyric acid (Aib) (l-Leu-l-Leu-Aib) was conjugated to nona-arginine on either the C- or N- terminus, designated as Block 1 and Block 2. Each terminal modification induced helical structure formation and improved the physicochemical properties of peptide/plasmid DNA (pDNA) complexes, resulting in efficient intracellular pDNA delivery. The introduction of a helical template may be effective for the endosomal escape of pDNA and pDNA release from complexes in cells. These results emphasized the potency of a helical template for the development of novel cell-penetrating peptides for pDNA delivery.

Peptide-Mediated Gene Transfer into Marine Purple Photosynthetic Bacteria.

Use of photosynthetic organisms is one of the sustainable ways to produce high-value products. Marine purple photosynthetic bacteria are one of the research focuses as microbial production hosts. Genetic transformation is indispensable as a biotechnology technique. However, only conjugation has been determined to be an applicable method for the transformation of marine purple photosynthetic bacteria so far. In this study, for the first time, a dual peptide-based transformation method combining cell penetrating peptide (CPP), cationic peptide and Tat-derived peptide (dTat-Sar-EED) (containing D-amino acids of Tat and endosomal escape domain (EED) connected by sarcosine linkers) successfully delivered plasmid DNA into Rhodovulum sulfidophilum, a marine purple photosynthetic bacterium. The plasmid delivery efficiency was greatly improved by dTat-Sar-EED. The concentrations of dTat-Sar-EED, cell growth stage and recovery duration affected the efficiency of plasmid DNA delivery. The delivery was inhibited at 4 °C and by A22, which is an inhibitor of the actin homolog MreB. This suggests that the plasmid DNA delivery occurred via MreB-mediated energy dependent process. Additionally, this peptide-mediated delivery method was also applicable for E. coli cells. Thus, a wide range of bacteria could be genetically transformed by using this novel peptide-based transformation method.

Cationic Polymer-Mediated CRISPR/Cas9 Plasmid Delivery for Genome Editing.

Delivery of CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated protein-9 (Cas9) represents a major hurdle for successful clinical translation of genome editing tools. Owing to the large size of plasmids that encode Cas9 and single-guide RNA (sgRNA), genome editing efficiency mediated by current delivery carriers is still unsatisfactory to meet the requirement for its real applications. Herein, cationic polymer polyethyleneimine-ß-cyclodextrin (PC), known to be efficient for small plasmid transfection, is reported to likewise mediate efficient delivery of plasmid encoding Cas9 and sgRNA. Whereas PC can condense and encapsulate large plasmids at high N/P ratio, the delivery of plasmid results in efficient editing at two genome loci, namely, hemoglobin subunit beta (19.1%) and rhomboid 5 homolog 1 (RHBDF1) (7.0%). Sanger sequencing further confirms the successful genome editing at these loci. This study defines a new strategy for the delivery of the large plasmid encoding Cas9/sgRNA for efficient genome editing.

A Helix-Stabilized Cell-Penetrating Peptide as an Intracellular Delivery Tool.

Two types of cationic cyclic α,α-disubstituted α-amino acids: ApiC2NH2 (which possesses a lysine mimic side chain) and Api(C2Gu) (which possesses an arginine mimic side chain), were developed. These amino acids were incorporated into an arginine-based peptide sequence [(L-Arg-L-Arg-dAA)3: dAA=ApiC2NH2 or Api(C2Gu)], and the relationship between the secondary structures of the resulting peptides and their ability to pass through cell membranes was investigated. The peptide containing Api(C2Gu) formed a stable α-helical structure and was more effective at penetrating cells than the nonhelical Arg nonapeptide (R9). Furthermore, the peptide was able to deliver plasmid DNA into various types of cells in a highly efficient manner.

Plasmid DNA delivery by arginine-rich cell-penetrating peptides containing unnatural amino acids.

Cell-penetrating peptides (CPPs) have been developed as drug, protein, and gene delivery tools. In the present study, arginine (Arg)-rich CPPs containing unnatural amino acids were designed to deliver plasmid DNA (pDNA). The transfection ability of one of the Arg-rich CPPs examined here was more effective than that of the Arg nonapeptide, which is the most frequently used CPP. The transfection efficiencies of Arg-rich CPPs increased with longer post-incubation times and were significantly higher at 48-h and 72-h post-incubation than that of the commercially available transfection reagent TurboFect. These Arg-rich CPPs were complexed with pDNA for a long time in cells and effectively escaped from the late endosomes/lysosomes into the cytoplasm. These results will be helpful for designing novel CPPs for pDNA delivery.

Exosomes as an Emerging Plasmid Delivery Vehicle for Gene Therapy.

Despite its introduction more than three decades ago, gene therapy has fallen short of its expected potential for the treatment of a broad spectrum of diseases and continues to lack widespread clinical use. The fundamental limitation in clinical translatability of this therapeutic modality has always been an effective delivery system that circumvents degradation of the therapeutic nucleic acids, ensuring they reach the intended disease target. Plasmid DNA (pDNA) for the purpose of introducing exogenous genes presents an additional challenge due to its size and potential immunogenicity. Current pDNA methods include naked pDNA accompanied by electroporation or ultrasound, liposomes, other nanoparticles, and cell-penetrating peptides, to name a few. While the topic of numerous reviews, each of these methods has its own unique set of limitations, side effects, and efficacy concerns. In this review, we highlight emerging uses of exosomes for the delivery of pDNA for gene therapy. We specifically focus on bovine milk and colostrum-derived exosomes as a nano-delivery "platform". Milk/colostrum represents an abundant, scalable, and cost-effective natural source of exosomes that can be loaded with nucleic acids for targeted delivery to a variety of tissue types in the body. These nanoparticles can be functionalized and loaded with pDNA for the exogenous expression of genes to target a wide variety of disease phenotypes, overcoming many of the limitations of current gene therapy delivery techniques.

A Fluorescent Vector of Carbon Dot to Deliver Rab13 and Rab14 Plasmids for Promoting Neurite Outgrowth.

The complex processes of neuron differentiation and neuron repair are critical for treating nervous system injuries and neurodegenerative diseases. Neurite outgrowth plays a crucial role in these processes by enabling the formation of connections between neurons and the generation of neuroplasticity to restore the function of the nervous system. In this study, we fabricated functionalized carbon dots (CDs) with distinctive photoluminescence and low cytotoxicity for use as fluorescence imaging probes and nanocarriers to deliver plasmid DNAs to neurons effectively for inducing neurite outgrowth. CDs were prepared through a reflux process in nitric acid solution, and their surface was then modified using polyethylenimine (PEI) to obtain positively charged CDs for increasing the absorption of plasmid DNAs and the efficiency of cell uptake. Experimental results indicated that the fabricated CDs maintained a low cytotoxicity and exhibited a high neuron uptake of up to 97%. An improvement in the plasmid DNA ingestion of neurons resulted in enhanced expression of Rab13-Q67L and Rab14 proteins, which considerably promoted neurite sprouting and elongation. After the fabricated PEI-modified CDs were used to deliver the Rab13-Q67L and Rab14 plasmids, more than 56% of the neurons had a neurite length that was greater than twice the size of their soma. Thus, DNA delivery through functionalized CDs has a high potential for use in gene therapy for neuronal injuries and diseases.

Improved transfer efficiency of supercharged 36 + GFP protein mediate nucleic acid delivery.

The potential of nucleic acid therapeutics to treat diseases by targeting specific cells has resulted in its increasing number of uses in clinical settings. However, the major challenge is to deliver bio-macromolecules into target cells and/or subcellular locations of interest ahead in the development of delivery systems. Although, supercharged residues replaced protein 36 + GFP can facilitate itself and cargoes delivery, its efficiency is still limited. Therefore, we combined our recent progress to further improve 36 + GFP based delivery efficiency. We found that the penetration efficacy of 36 + GFP protein was significantly improved by fusion with CPP-Dot1l or treatment with penetration enhancer dimethyl sulfoxide (DMSO) in vitro. After safely packaged with plasmid DNA, we found that the efficacy of in vitro and in vivo transfection mediated by 36 + GFP-Dot1l fusion protein is also significantly improved than 36 + GFP itself. Our findings illustrated that fusion with CPP-Dot1l or incubation with DMSO is an alternative way to synergically promote 36 + GFP mediated plasmid DNA delivery in vitro and in vivo.

In silico and experimental validation of a new modified arginine-rich cell penetrating peptide for plasmid DNA delivery.

Cell-penetrating peptides (CPPs) attracted great attention because of the capability to deliver various types of cargo molecules across into the cells. In this study, we presented a new arginine rich CPP, named MR, for efficient transporting plasmid DNA. We used a combined bioinformatic-based approach to improve the speed and accuracy of CPP evaluation. MR protein properties, structural models, interaction with DNA, as well as cell localization and membrane interaction were evaluated through multiple servers. Importantly, analysis using different algorithms showed the high CPP prediction confidence of MR. Experimental results also revealed the capacity of this gene delivery system in vitro for efficient plasmid DNA transfection. Additionally, in vitro mechanistically studies together with bioinformatic investigation suggested that MR peptide may internalize into the cell through endocytosis pathways. Moreover, in silico safety analysis such as immunogenicity, allergenicity, toxicity, and hemolysis activity as well as MTT assay also confirmed the safety of MR peptide. This study illustrated that MR peptide could be presented as remarkable potential gene delivery system for promising transport of plasmid DNA towards the therapeutic applications.

In silico identification and experimental validation of cellular uptake by a new cell penetrating peptide P1 derived from MARCKS.

Viral vectors for vaccine delivery are challenged by recently reported safety issues like immunogenicity and risk for cancer development, and thus there is a growing need for the development of non-viral vectors. Cell penetrating peptides (CPPs) are non-viral vectors that can enter plasma membranes efficiently and deliver a broad range of cargoes. Our bioinformatic prediction and wet-lab validation data suggested that peptide P1 derived from MARCKS protein phosphorylation site domain is a new potential CPP candidate. We found that peptide P1 can efficiently internalize into various cell lines in a concentration-dependent manner. Receptor-mediated endocytosis pathway is the major mechanism of P1 penetration, although P1 also directly penetrates the plasma membrane. We also found that peptide P1 has low cytotoxicity in cultured cell lines as well as mouse red blood cells. Furthermore, peptide P1 not only can enter into cultured cells itself, but it also can interact with plasmid DNA and mediate the functional delivery of plasmid DNA into cultured cells, even in hard-to-transfect cells. Combined, these findings indicate that P1 may be a promising vector for efficient intracellular delivery of bioactive cargos.

Structure-Activity Relationship of Mono-Ion Complexes for Plasmid DNA Delivery by Muscular Injection.

The structure-activity relationship of mono-ion complexes (MICs) for plasmid DNA (pDNA) delivery by muscular injection is demonstrated. MICs were formed between pDNA and monocationic poly(ethylene glycol) (PEG) macromolecules. As monocationic PEGs, the ω-amide-pentylimidazolium (APe-Im) end-modified PEGs with a stable amide (Am) and hydrolytic ester (Es) bond, that is, APe-Im-Am-PEG and APe-Im-Es-PEG, respectively, are synthesized. The difference between the APe-Im-Am-PEG and APe-Im-Es-PEG was only a spacer structure between a terminal cation and a PEG chain. The resulting pDNA MICs with APe-Im-Am-PEG at a charge ratio (+/-) of 32 or 64 were more stable than those with APe-Im-Es-PEG in the presence of serum proteins. The highest gene expression by muscular injection was achieved using the APe-Im-Am-PEG/pDNA MIC at a charge ratio (+/-) of 32 with a smaller particle diameter of approximately 50 nm, as compared to that charge ratio of 64. Consequently, the pDNA MIC with the monocationic PEG with a stable amide spacer, as compared to a hydrolytic ester spacer, is considered to be suitable for the highest gene expression by muscular injection.

HGF Gene Delivering Alginate/Galactosylated Chitosan Sponge Scaffold for Three-Dimensional Coculture of Hepatocytes/3T3 Cells.

Gene delivery from tissue engineering scaffold is a novel strategy in regulating long-term growth and function of cells in vitro culture. In this study, a hepatocyte growth factor plasmid/polyetherimide (pHGF/PEI) polyplex delivering alginate (AL)/galactosylated chitosan (GC) (pHGF/PEI-AL/GC) sponge scaffold was prepared for the in vitro coculture of hepatocytes/3T3 cells. The pHGF/PEI polyplex released for 6 days in the sponge scaffold with weight ratio of AL/GC being 3:1 and fixed amount of pHGF being 40 µg (24-well scaffold). In addition, the 3T3 cells culturing in the pHGF/PEI-AL/GC sponge scaffold could be continually transfected and expressed the exogenous HGF for 6 days. Furthermore, the albumin secretion and urea synthesis of hepatocytes were significantly enhanced when cocultured with 3T3 cells in the pHGF/PEI-AL/GC sponge scaffold compared with that in the AL/GC sponge without pHGF. In summary, the preparation of AL/GC sponge scaffold delivering pHGF/PEI polyplex is a critical significance for maintaining the long-term survival and function of primary hepatocytes in vitro.

Plasmid DNA Delivery Using Cell-Penetrating Peptide Foldamers Composed of Arg-Arg-Aib Repeating Sequences.

Arginine (Arg)-rich cell-penetrating peptides (CPPs) are promising tools for plasmid DNA (pDNA) delivery. α-Aminoisobutyric acid (Aib) is known to stabilize peptide helical secondary structures and has been used as a building block for foldamers. In the current study, we prepared Aib CPP foldamers, (Arg-Arg-Aib)n (n = 1-6) and examined their pDNA transfection abilities. Transfection efficiencies of peptide/pDNA complexes are dependent on peptide chain length, with longer peptides (n ≥ 4) exhibiting better transfection abilities than an Arg nonapeptide. Furthermore, Aib CPP foldamers achieve prolonged transfection abilities compared with commercially available transfection reagents, which is probably because of the high resistance of the peptides to enzymatic degradation, thereby protecting the encapsulated pDNA for a long period. The obtained results demonstrated promising features of Aib CPP foldamers as pDNA delivery carriers.

Cell-Penetrating Peptides Using Cyclic α,α-Disubstituted α-Amino Acids with Basic Functional Groups.

In the delivery of cell-impermeable molecules, cell-penetrating peptides (CPPs) have been attracting increasing attention as intracellular delivery tools. In the present study, we designed four types of cyclic α,α-disubstituted α-amino acids (dAAs) with basic functional groups on their five-membered rings and different chiralities at the α-position and introduced them into arginine (Arg)-rich peptides. The evaluation of cell-penetrating abilities indicated that these peptides exhibited better cell permeabilities than an Arg nonapeptide. Furthermore, peptides containing dAAs delivered plasmid DNA (pDNA) better than a commercially available transfection reagent with a longer incubation time. These results demonstrate that the introduction of cyclic dAAs with basic functional groups into Arg-rich peptides is an effective strategy for the design of CPPs as a pDNA delivery tool.

Structure-biocompatibility and transfection activity relationships of cationic polyaspartamides with (dialkylamino)alkyl and alkyl or hydroxyalkyl side groups.

A series of 14 cationic derivatives of poly(aspartic acid) i.e. cationic polyaspartamides with different (dialkylamino)alkyl and alkyl or hydroxyalkyl side groups was synthesized by nucleophilic addition on polysuccinimide. The resulting polyaspartamides have moderate amphiphilic properties. Relationships between the structure and ratio of side groups and in vitro properties of polyaspartamides, including their cytotoxic and membrane-damaging activity towards human cell lines, primary skin fibroblasts and erythrocytes, were established and discussed. Cationic polyaspartamides vary in their DNA-binding, condensing and nuclease-protecting characteristics depending on the concentration ratio of (dialkylamino)alkyl and alkyl or hydroxyalkyl side groups. Effective cell transfection was achieved upon polyaspartamide-mediated plasmid DNA delivery in serum-free medium in the presence of chloroquine. Effect of serum proteins adsorption onto polyaspartamide based polyplexes, and the role of concentration of polyplexes in culture medium in their colloidal stability and transfection process were demonstrated. Synthesized polyaspartamides are biocompatible and long-acting gene carriers, which are applied to cells after dilution and without washing, thus providing transfection level comparable to that of commercial transfection reagent.

Near-infrared light-sensitive liposomes for enhanced plasmid DNA transfection.

Near-infrared (NIR) light-responsive liposomes are attractive carriers for targeted and controlled drug delivery to the superficial organ or tissue (e.g., skin). This work describes the development of NIR-responsive liposomes by incorporating gold nanostars within liposomes composed of Phospholipon 90 g and cholesterol. Following cellular delivery, photothermal effect around the gold nanostar upon NIR stimulation induces microcavitation and liposome phase transition which consequently triggers the release of encapsulated molecules. Taking GFP plasmid as an example, we demonstrate enhanced gene transfection into fibroblasts following NIR treatment.