Intracellular DNA Release Research Articles

Tumour lysis syndrome.

Tumour lysis syndrome (TLS) represents a critical oncological emergency characterized by extensive tumour cell breakdown, leading to the swift release of intracellular contents into the systemic circulation, outpacing homeostatic mechanisms. This process results in hyperuricaemia (a by-product of intracellular DNA release), hyperkalaemia, hyperphosphataemia, hypocalcaemia and the accumulation of xanthine. These electrolyte and metabolic imbalances pose a significant risk of acute kidney injury, cardiac arrhythmias, seizures, multiorgan failure and, rarely, death. While TLS can occur spontaneously, it usually arises shortly after the initiation of effective treatment, particularly in patients with a large cancer cell mass (defined as ≥500 g or ≥300 g/m2 of body surface area in children). To prevent TLS, close monitoring and hydration to improve renal perfusion and urine output and to minimize uric acid or calcium phosphate precipitation in renal tubules are essential. Intervention is based on the risk of a patient of having TLS and can include rasburicase and allopurinol. Xanthine, typically enzymatically converted to uric acid, can accumulate when xanthine oxidases, such as allopurinol, are administered during TLS management. Whether measurement of xanthine is clinically useful to optimize the use of allopurinol or rasburicase remains to be determined.

Di[12]aneN3-Functionalized Green Fluorescent Protein Chromophore for GFP Luminescence Simulation and Efficient Gene Transfection In Vitro and In Vivo.

Although a plethora of gene carriers have been developed for potential gene therapy, imageable stimuli-responsive gene vectors with fast access to the nucleus, high biocompatibility, and transfection efficiency are still scarce. Herein, we report the design and synthesis of four dendrite-shaped cationic liposomes, MPA-HBI-R/DOPE (R: n-butyl, 1; n-octyl, 2; n-dodecyl, 3; palmyl, 4), prepared via esterification of 4-alkoxybenzylideneimidazolinone containing aliphatic chains of different lengths (HBI-R), the green fluorescent protein (GFP) chromophore, with a di[12]aneN3 unit. Liposomes were fabricated via the self-assembly of MPA-HBI-R, assisted with 1,2-dioleoyl-sn-glycerol-3-phosphorylethanolamine (DOPE). These liposomes (MPA-HBI-R/DOPE) exhibited efficient DNA condensation, pH-responsive degradation, excellent cellular biocompatibility (up to 150 μM), and high transfection efficiency. Molecular docking experiments were also used to verify the optimal interaction between MPA-HBI-R and DNA, as well as the fluorescence enhancements. In particular, MPA-HBI-2/DOPE delivered DNA into the nucleus in less than an hour, and its luciferase transfection activity was more than 10 times that by Lipo2000, across multiple cell lines. The GFP chromophore conjugation allowed trackable intracellular delivery and release of DNA in real time via fluorescence imaging. Furthermore, efficient red fluorescent protein (RFP) transfection in zebrafish, with an efficiency of more than 6 times that by Lipo2000, was also achieved. The results not only realized, for the first time, the combination of gene delivery and GFP-simulated light emission, allowing fluorescent tracking and highly efficient gene transfection, but also offered valuable insights into the use of biomimetic chromophore for the development of the next-generation nonviral vectors.

Self-Assembled Cationic Polypeptide Supramolecular Nanogels for Intracellular DNA Delivery.

Supramolecular nanogels are an emerging class of polymer nanocarriers for intracellular delivery, due to their straightforward preparation, biocompatibility, and capability to spontaneously encapsulate biologically active components such as DNA. A completely biodegradable three‐component cationic supramolecular nanogel was designed exploiting the multivalent host‐guest interaction of cyclodextrin and adamantane attached to a polypeptide backbone. While cyclodextrin was conjugated to linear poly‐L‐lysine, adamantane was grafted to linear as well as star shaped poly‐L‐lysine. Size control of nanogels was obtained with the increase in the length of the host and guest polymer. Moreover, smaller nanogels were obtained using the star shaped polymers because of the compact nature of star polymers compared to linear polymers. Nanogels were loaded with anionic model cargoes, pyranine and carboxyfluorescein, and their enzyme responsive release was studied using protease trypsin. Confocal microscopy revealed successful transfection of mammalian HeLa cells and intracellular release of pyranine and plasmid DNA, as quantified using a luciferase assay, showing that supramolecular polypeptide nanogels have significant potential in gene therapy applications.

Gene delivery using layer-by-layer functionalized multi-walled carbon nanotubes: design, characterization, cell line evaluation

Multi-walled carbon nanotubes (MWCNTs) with special nanoneedle structure have emerged as new promising candidates for plasmid and drug delivery. However, the delivery is greatly limited by the high tendency of CNT to form aggregates, the “less dispersion problem,” and CNT cytotoxicity. Here, we described an extensive evaluation of the ability of layer-by-layer modification strategy to reduce CNT size and toxicity, and to shield CNT hydrophobic surfaces. The MWCNTs can be derivatized with carboxylate groups (cMWCNT) and sequentially functionalized with protein, cationic polyethylenimine (PEI), and polysaccharide. The protein coating, characterized by Fourier transform infrared and deconvolution methods, could serve as the hydrophilic, biocompatible matrix and scaffold for sequential conjugation. We found that coated PEI-enhanced electrostatic interactions between plasmid DNA and CNTs. The functionalized cMWCNTs were analyzed by thermogravimetric analysis, dynamic light scattering, and electron microscopy technologies. The conjugation of cMWCNTs–ovalbumin–PEI with oxidized pectin further promoted green fluorescence intensity by balancing the intracellular DNA release and were easier to disperse. Our in-depth study demonstrated that functionalized CNTs can be improved by fine-tuned process parameters of the protein–PEI–polysaccharide modification.

Glutathione-Specific and Intracellularly Labile Polymeric Nanocarrier for Efficient and Safe Cancer Gene Delivery.

Cationic polymers condense nucleic acids into nanosized complexes (polyplexes) that are widely explored for nonviral gene delivery, but their strong electrostatic binding with DNA causes inefficient intracellular gene release and translation and thereby unsatisfactory gene transfection efficiencies. Facilitated intracellular dissociation of polyplexes by making the polymer undergo positive-to-negative/neutral charge reversal can effectively solve these problems, but they must be sufficiently stable during the delivery. Herein, we report the first glutathione (GSH)-specific intracellular labile polyplexes for cancer-targeted gene delivery. The polymers are made from p-(2,4-dinitrophenyloxybenzyl)-ammonium cationic moieties, whose p-2,4-dinitrophenyl ether is cleaved specifically by GSH, rather than other biological thiols, triggering the conversion of the ammonium cation into the carboxylate anion and thus the fast intracellular DNA release of the polyplexes. Furthermore, the polyplexes coated with PEG-functionalized lipids are stable in biological fluids to gain long blood circulation for tumor accumulation. Thus, the efficient tumor accumulation and cell transfection of the polyplexes loaded with the tumor suicide gene tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) give rise to potent antitumor activity similar to that of the first-line chemotherapy drug paclitaxel but with much less adverse effects.

Double-Network Nanogel as a Nonviral Vector for DNA Delivery.

A double-network nanogel, composed of a silane-cross-linked polyethylenimine (PEI) network (i.e., PEI-S) and a pH-responsive poly(2-(hexamethyleneimino) ethyl methacrylate) (PC7A) polymer, was developed for efficient DNA transfection. The chemical cross-linking and hydrophobic interactions in the two networks led to improved stability outside the cell and also pH-triggered intracellular release of DNA. The nanogel with an optimal PEI-S and PC7A weight ratio of 1.3:1 exhibited significantly higher transfection efficiency than Lipofectamine 2000 in multiple cell lines. The nanogel also possessed a small size with a hydrodynamic diameter of 55 nm, low cytotoxicity, and superior stability in serum-containing media. Moreover, besides the PEI-based gene delivery system, we have also demonstrated that addition of the PC7A polymer to several types of cationic polymers commonly used for gene delivery also led to significant transfection enhancement of the resulting nanoparticles, suggesting that the PC7A polymer may be a universal additive that can benefit versatile cationic polymer-based gene delivery systems.

Reactive Oxygen Species (ROS)-Responsive Charge-Switchable Nanocarriers for Gene Therapy of Metastatic Cancer.

The application of nonviral gene vectors has been limited by their insufficient transfection efficiency because of poor serum stability, high endosomal entrapment, limited intracellular release, and low accumulation in the targeted organelle. It is still challenging to design gene carriers with properties that can overcome all of the barriers. We previously developed a reactive oxygen species (ROS)-responsive cationic polymer, poly[(2-acryloyl)ethyl( p-boronic acid benzyl) diethylammonium bromide] (B-PDEAEA), which switches the charge at high concentrations of intracellular ROS to promote intracellular DNA release. However, its gene-delivery efficiency has been limited by serum instability and lysosomal trapping, and coating with an anionic PEGylated lipid only showed mild enhancement. Herein, we coated the ROS-responsive B-PDEAEA polymer with two cationic lipids to form ROS-responsive lipopolyplexes with integrated properties to overcome multiple delivery barriers. The surface cationic lipids endowed the nanocarrier with improved serum stability, effective cellular uptake, and lysosomal evasion. The interior B-PDEAEA/DNA polyplexes, which were highly stable in the extracellular environment, but quickly dissociated, released DNA, promoted nuclei localization, and achieved efficient transcription. The mechanisms of the ROS-responsive and charge-switchable properties of B-PDEAEA were quantitatively studied. The transfection efficiency and antitumor activity of lipopolyplexes were studied in vitro and in vivo. We found that the ROS-responsive lipopolyplexes effectively delivered therapeutic genes into cell nuclei and caused high tumor inhibition in mice bearing peritoneal or lung metastases.

Stimulus-Responsive Peptide for Effective Delivery and Release of DNA in Plants.

For efficient gene delivery in plant systems, nonviral vector and DNA complexes require extracellular stability, cell wall/membrane translocation capability, and the ability to mediate both endosomal escape and intracellular DNA release. Peptides make appealing gene delivery vectors due to their DNA-binding, cell-penetrating, and endosome escape properties. However, DNA release within cells has so far been inefficient, which results in poor and delayed gene expression, while the lack of understanding of both internalization and trafficking mechanisms is a further obstacle to the design of efficient peptide gene delivery vectors. Here, we report successful gene delivery into plants using a cellular environment-responsive vector, BPCH7, which is an efficient cell-penetrating peptide with a cyclic DNA-binding domain that is formed by a disulfide bond between two cysteines. The cyclic structure of BPCH7 confers high avidity attachment to DNA in vitro. Following endocytosis into cells, disulfide bond cleavage facilitated by intracellular glutathione induces structural changes within BPCH7 that enable the release of the associated DNA cargo. Comparative studies with BPKH, a cell-penetrating peptide with a linear DNA-binding domain, show that BPCH7 maximized and expedited gene transfer in cells and unveil for the first time the crucial role of plant stomata in the internalization of peptide-DNA complexes.

Stability and binding affinity of DNA/chitosan complexes by polyanion competition

The stability of DNA/chitosan complexes upon exposure to hyaluronic acid, chondroitin sulfate, and heparin, was assessed by fluorescence spectroscopy to quantify DNA release. Only the highly charged heparin was found to release DNA from the complexes. Complex stability upon exposure to heparin increased with the degree of deacetylation and molecular weight of chitosan and with the ratio of chitosan amino groups to DNA phosphate groups (N/P ratio) in the complexes. Isothermal titration microcalorimetry revealed that among polyanions tested, only heparin has a binding affinity to chitosan approaching that of DNA and can therefore release DNA from the complexes. These results also indicate that anionic components with sufficiently high charge density can induce extracellular or intracellular release of DNA, the former negatively affecting delivery efficiency while the latter is required for gene transfer to occur. Our findings also suggest that increased N/P ratio of the complexes can play an important role in preventing premature dissociation of DNA/polycation complexes upon interaction with anionic components in extracellular milieu.

Redox sensitive cationic pullulan for efficient gene transfection and drug retention in C6 glioma cells

Thiolated cationic pullulan was synthesized by conjugating pullulan with polyethyleneimine (PEI) and mercaptosuccinic acid (MSA). The formed conjugate was oxidized to obtain disulfide linked cationic pullulan (PPMSS). PPMSS exhibited good buffering capacity and nanoplexes formulated were of size less than 150nm. Nanoplexes formed with PPMSS are redox sensitive and susceptible to reductive cleavage by dithiothreitol (DTT) ensuring the intracellular release of DNA. In vitro, cytotoxic evaluation studies of polymers in C6 cell lines established its non-toxic nature. The studies using endocytosis inhibitors revealed the uptake pathways of nanoplexes. Further, the plasmid and polymer tracking studies indicated the successful unpacking of DNA from the nanoplexes and its nuclear localization. The gene transfection efficiency was established by the p53 gene expression studies. Furthermore, the ability of the polymer to inhibit efflux pumping in cancer cells has also been elucidated in terms of P-gp inhibition studies and drug retention kinetics using the anticancer drug, doxorubicin (DOX). Our results also suggest that greater retention of DOX was accompanied by the reduction of disulfide linkage by a ubiquitous intracellular stimulus, glutathione. Thus simultaneous gene and drug delivery using redox sensitive cationic polymers may have a promising potential in cancer therapy.

Acid-labile poly(amino alcohol ortho ester) based on low molecular weight polyethyleneimine for gene delivery.

A series of acid-labile poly(amino alcohol ortho ester) (POEeis) were synthesized through ring-opening polymerization between diglycidyl ethers with ortho ester bonded and low molecular weight polyethyleneimine by various feed molar ratios. The obtain POEei 1 and POEei 2 exhibited clear kinetic of degradation and condensed plasmid DNA into nanoparticles of suitable sizes (250-300 nm) and positive zeta potentials (+20-30 mV) while protecting DNA from enzymatic digestion. Further, these polymers have uniform distribution of abundant hydroxyl groups, which could improve their water solubility, biocompatibility, and lower protein adsorption. Significantly, ortho ester groups in POEeis main-chains could hydrolyze rapidly at acidic endosomal pH, resulting in intracellular DNA release and diminished material toxicity. MTT assay revealed that all the polymers exhibited much lower cytotoxicity than 25 kDa PEI in the human neuroblastoma SH-SY5Y cells. Moreover, the transfection efficiency of POEei 1 was higher than 25 kDa PEI in serum-free medium or 10% serum medium.

Harmonizing the Intracellular Kinetics toward Effective Gene Delivery Using Cancer Cell-Targeted and Light-Degradable Polyplexes.

The success of nonviral gene delivery is often restricted by the multiple cellular barriers that posed inconsistent requirements for vector design. High molecular weight (MW) and cationic charge density are required for polycations to enable effective gene encapsulation, which, however, also lead to high toxicity, restricted intracellular cargo release, and poor serum resistance. We herein developed cross-linked polyethylenimine (PEI) with built-in UV-responsive domains (NP-PEI), which can effectively condense DNA while rapidly de-cross-link upon light triggers to promote intracellular DNA release and reduce material toxicity. HA coating of the polyplexes further enhanced their serum stability by shielding the surface positive charges and enabled cancer cell targeting to potentiate the transfection efficiencies. Thus, the polyplexes afforded high transfection efficiencies in serum upon light irradiation, outperforming PEI 25k by 1-2 orders of magnitude. This study therefore provides a useful strategy to overcome the critical barriers against nonviral gene delivery.

Shedding PEG Palisade by Temporal Photostimulation and Intracellular Reducing Milieu for Facilitated Intracellular Trafficking and DNA Release.

The dilemma of poly(ethylene glycol) surface modification (PEGylation) inspired us to develop an intracellularly sheddable PEG palisade for synthetic delivery systems. Here, we attempted to conjugate PEG to polyethylenimine (PEI) through tandem linkages of disulfide-bridge susceptible to cytoplasmic reduction and an azobenzene/cyclodextrin inclusion complex responsive to external photoirradiation. The subsequent investigations revealed that facile PEG detachment could be achieved in endosomes upon photoirradiation, consequently engendering exposure of membrane-disruptive PEI for facilitated endosome escape. The liberated formulation in the cytosol was further subjected to complete PEG detachment relying on disulfide cleavage in the reductive cytosol, thus accelerating dissociation of electrostatically assembled PEI/DNA polyplex to release DNA by means of polyion exchange reaction with intracellularly charged species, ultimately contributing to efficient gene expression.

Low Molecular Weight PEI-Based Vectors via Acid-Labile Ortho Ester Linkage for Improved Gene Delivery.

A series of novel pH-sensitive gene delivery vectors (POEI 1, 2, and 3) are synthesized through Michael addition from low molecular weight PEI (LMW PEI) via acid-labile ortho ester linkage with terminal acrylates (OEAc) by various feed molar ratios. The obtained POEI 1 and POEI 2 can efficiently condense plasmid DNA into nanoparticles with size range of 200-300 nm and zeta-potentials of about +15 mV while protecting DNA from enzymatic digestion compared with POEI 3. Significantly, ortho ester groups of POEI main-chains can make an instantaneous degradation-response to acidic endosomal pH (≈5.0), resulting in accelerated disruption of polyplexes and intracellular DNA release. MTT assay reveals that all POEIs exhibit much lower cytotoxicity in different cells than branched PEI (25 KDa). As expected, POEI 1 and POEI 2 perform improved gene transfection in vitro, suggesting that such polycations might be promising gene vectors based on overcoming toxicity-efficiency contradiction.

Polyelectrolyte Complexes of Low Molecular Weight PEI and Citric Acid as Efficient and Nontoxic Vectors for in Vitro and in Vivo Gene Delivery.

Gene transfection mediated by the cationic polymer polyethylenimine (PEI) is considered a standard methodology. However, while highly branched PEIs form smaller polyplexes with DNA that exhibit high transfection efficiencies, they have significant cell toxicity. Conversely, low molecular weight PEIs (LMW-PEIs) with favorable cytotoxicity profiles display minimum transfection activities as a result of inadequate DNA complexation and protection. To solve this paradox, a novel polyelectrolyte complex was prepared by the ionic cross-linking of branched 1.8 kDa PEI with citric acid (CA). This system synergistically exploits the good cytotoxicity profile exhibited by LMW-PEI with the high transfection efficiencies shown by highly branched and high molecular weight PEIs. The polyectrolyte complex (1.8 kDa-PEI@CA) was obtained by a simple synthetic protocol based on the microwave irradiation of a solution of 1.8 kDa PEI and CA. Upon complexation with DNA, intrinsic properties of the resulting particles (size and surface charge) were measured and their ability to form stable polyplexes was determined. Compared with unmodified PEIs the new complexes behave as efficient gene vectors and showed enhanced DNA binding capability associated with facilitated intracellular DNA release and enhanced DNA protection from endonuclease degradation. In addition, while transfection values for LMW-PEIs are almost null, transfection efficiencies of the new reagent range from 2.5- to 3.8-fold to those of Lipofectamine 2000 and 25 kDa PEI in several cell lines in culture such as CHO-k1, FTO2B hepatomas, L6 myoblasts, or NRK cells, simultaneously showing a negligible toxicity. Furthermore, the 1.8 kDa-PEI@CA polyelectrolyte complexes retained the capability to transfect eukaryotic cells in the presence of serum and exhibited the capability to promote in vivo transfection in mouse (as an animal model) with an enhanced efficiency compared to 25 kDa PEI. Results support the polyelectrolyte complex of LMW-PEI and CA as promising generic nonviral gene carriers.

Intranasal Administration of Novel Chitosan Nanoparticle/DNA Complexes Induces Antibody Response to Hepatitis B Surface Antigen in Mice.

The generation of strong pathogen-specific immune responses at mucosal surfaces where hepatitis B virus (HBV) transmission can occur is still a major challenge. Therefore, new vaccines are urgently needed in order to overcome the limitations of existing parenteral ones. Recent studies show that this may be achieved by intranasal immunization. Chitosan has gained attention as a nonviral gene delivery system; however, its use in vivo is limited due to low transfection efficiency mostly related to strong interaction between the negatively charged DNA and the positively charged chitosan. We hypothesize that the adsorption of negatively charged human serum albumin (HSA) onto the surface of the chitosan particles would facilitate the intracellular release of DNA, enhancing transfection activity. Here, we demonstrate that a robust systemic immune response was induced after vaccination using HSA-loaded chitosan nanoparticle/DNA (HSA-CH NP/DNA) complexes. Furthermore, intranasal immunization with HSA-CH NP/DNA complexes induced HBV specific IgA in nasal and vaginal secretions; no systemic or mucosal responses were detected after immunization with DNA alone. Overall, our results show that chitosan-based DNA complexes elicited both humoral and mucosal immune response, making them an interesting and valuable gene delivery system for nasal vaccination against HBV.

Efficient Condensation of DNA into Environmentally Responsive Polyplexes Produced from Block Catiomers Carrying Amine or Diamine Groups

The intracellular delivery of nucleic acids requires a vector system as they cannot diffuse across lipid membranes. Although polymeric transfecting agents have been extensively investigated, none of the proposed gene delivery vehicles fulfill all of the requirements needed for an effective therapy, namely, the ability to bind and compact DNA into polyplexes, stability in the serum environment, endosome-disrupting capacity, efficient intracellular DNA release, and low toxicity. The challenges are mainly attributed to conflicting properties such as stability vs efficient DNA release and toxicity vs efficient endosome-disrupting capacity. Accordingly, investigations aimed at safe and efficient therapies are still essential to achieving gene therapy clinical success. Taking into account the mentioned issues, herein we have evaluated the DNA condensation ability of poly(ethylene oxide)113-b-poly[2-(diisopropylamino)ethyl methacrylate]50 (PEO113-b-PDPA50), poly(ethylene oxide)113-b-poly[2-(diethylamino)ethyl methacrylate]50 (PEO113-b-PDEA50), poly[oligo(ethylene glycol)methyl ether methacrylate]70-b-poly[oligo(ethylene glycol)methyl ether methacrylate10-co-2-(diethylamino)ethyl methacrylate47-co-2-(diisopropylamino)ethyl methacrylate47] (POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47), and poly[oligo(ethylene glycol)methyl ether methacrylate]70-b-poly{oligo(ethylene glycol)methyl ether methacrylate10-co-2-methylacrylic acid 2-[(2-(dimethylamino)ethyl)methylamino]ethyl ester44} (POEGMA70-b-P(OEGMA10-co-DAMA44). Block copolymers PEO113-b-PDEA50 and POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) were evidenced to properly condense DNA into particles with a desirable size for cellular uptake via endocytic pathways (R(H) ≈ 65-85 nm). The structure of the polyplexes was characterized in detail by scattering techniques and atomic force microscopy. The isothermal titration calorimetric data revealed that the polymer/DNA binding is endothermic; therefore, the process in entropically driven. The combination of results supports that POEGMA70-b-P(OEGMA10-co-DEA47-co-DPA47) condenses DNA more efficiently and with higher thermodynamic outputs than does PEO113-b-PDEA50. Finally, circular dichroism spectroscopy indicated that the conformation of DNA remained the same after complexation and that the polyplexes are very stable in the serum environment.

Fusogenic charge-reversal vector: a viropexis-mimicking system for gene delivery

Gene therapy is known highly effective for treatment of many diseases; however, its wide use has been severely bottlenecked by lack of safe and effective delivery vectors. Cationic polymers are safe nonviral gene vectors with great potential for large-scale applications, and widely used to condense the large macromolecules into cationic polymer/DNA complexes (polyplexes) nanoparticles, protecting them from degradation and facilitating their cellular internalization. However, once inside the cells, unzipping the cationic polymer/DNA complexes is against the strong electrostatic interaction and thus intracellular release of free DNA for transcription is the main barrier to efficient DNA transfection. In a recent online publication of Advanced Materials, Professor Youqing Shen from Zhejiang University reports a very motivating design of reactive oxygen species (ROS)-labile charge-reversal polymer-based fusogenic lipidic polyplexes (FLPPs), which are promising to successfully overcome these problems.

Reversibly cross-linked polyplexes enable cancer-targeted gene delivery via self-promoted DNA release and self-diminished toxicity.

Polycations often suffer from the irreconcilable inconsistency between transfection efficiency and toxicity. Polymers with high molecular weight (MW) and cationic charge feature potent gene delivery capabilities, while in the meantime suffer from strong chemotoxicity, restricted intracellular DNA release, and low stability in vivo. To address these critical challenges, we herein developed pH-responsive, reversibly cross-linked, polyetheleneimine (PEI)-based polyplexes coated with hyaluronic acid (HA) for the effective and targeted gene delivery to cancer cells. Low-MW PEI was cross-linked with the ketal-containing linker, and the obtained high-MW analogue afforded potent gene delivery capabilities during transfection, while rapidly degraded into low-MW segments upon acid treatment in the endosomes, which promoted intracellular DNA release and reduced material toxicity. HA coating of the polyplexes shielded the surface positive charges to enhance their stability under physiological condition and simultaneously reduced the toxicity. Additionally, HA coating allowed active targeting to cancer cells to potentiate the transfection efficiencies in cancer cells in vitro and in vivo. This study therefore provides an effective approach to overcome the efficiency-toxicity inconsistence of nonviral vectors, which contributes insights into the design strategy of effective and safe vectors for cancer gene therapy.

Concurrent Targeting of KRAS and AKT by MiR-4689 Is a Novel Treatment Against Mutant KRAS Colorectal Cancer.

KRAS mutations are a major cause of drug resistance to molecular-targeted therapies. Aberrant epidermal growth factor receptor (EGFR) signaling may cause dysregulation of microRNA (miRNA) and gene regulatory networks, which leads to cancer initiation and progression. To address the functional relevance of miRNAs in mutant KRAS cancers, we transfected exogenous KRASG12V into human embryonic kidney 293 and MRC5 cells with wild-type KRAS and BRAF genes, and we comprehensively profiled the dysregulated miRNAs. The result showed that mature miRNA oligonucleotide (miR)-4689, one of the significantly down-regulated miRNAs in KRASG12V overexpressed cells, was found to exhibit a potent growth-inhibitory and proapoptotic effect both in vitro and in vivo. miR-4689 expression was significantly down-regulated in cancer tissues compared to normal mucosa, and it was particularly decreased in mutant KRAS CRC tissues. miR-4689 directly targets v-ki-ras2 kirsten rat sarcoma viral oncogene homolog (KRAS) and v-akt murine thymoma viral oncogene homolog 1(AKT1), key components of two major branches in EGFR pathway, suggesting KRAS overdrives this signaling pathway through inhibition of miR-4689. Overall, this study provided additional evidence that mutant KRAS functions as a broad regulator of the EGFR signaling cascade by inhibiting miR-4689, which negatively regulates both RAS/mitogen-activated protein kinase (MAPK) and phosphoinositide 3-kinase (PI3K)/AKT pathways. These activities indicated that miR-4689 may be a promising therapeutic agent in mutant KRAS CRC.