Transfection Activity Research Articles

Highly branched poly(β-amino ester)s with narrow molecular weight distribution: Fractionation and gene transfection activity

Highly branched poly(β-amino ester)s (HPAEs) have shown their great promise in gene delivery. However, their broad molecular weight distribution (MWD) poses an additional challenge to the mechanistic understanding of the influence of molecular weight (MW) on their gene transfection activity. Using a stepwise precipitation strategy, HPAEs were fractionated. It is shown that MW has a significant effect on the transfection activity and cytotoxicity of HPAEs. The intermediate MW mediates higher transfection efficiency while maintaining high cell viability. Mechanistic studies show that the intermediate MW confers stronger DNA binding affinity to HPAEs, leading to the formulation of polyplexes with a relatively smaller size and more positive zeta potential. This study not only suggests a simple strategy to fractionate HPAEs with narrow MWD but also provides new insights into understanding the structure-property relationship, which would facilitate the clinical translation of HPAEs in gene therapy.

Capillary-Mediated Vitrification: Preservation of mRNA at Elevated Temperatures

RNA is a fundamental tool for molecular and cellular biology research. The recent COVID-19 pandemic has proved it is also invaluable in vaccine development. However, the need for cold storage to maintain RNA integrity and the practical and economic burden associated with cold chain logistics highlight the need for new and improved preservation methods. We recently showed the use of capillary-mediated vitrification (CMV), as a tool for stabilizing temperature-sensitive enzymes. Here, we demonstrate the use of CMV as a method to preserve mRNA. The CMV process was performed by formulating a green fluorescent protein (GFP)-encoding mRNA with common excipients, applying the solution to a porous support, referred to as the scaffold, and drying the samples under vacuum for 30 min. The CMV preserved samples were stored at 55 °C for up to 100 days or 25 °C for 60 days and analyzed by electrophoresis and for transfection efficiency in a cell-based assay. The 55 °C-stressed mRNA exhibited comparable electrophoresis banding patterns and band intensity when compared to a frozen, liquid control. Additionally, the CMV stabilized mRNA maintained 97.5 ± 8.7% transfection efficiency after 77 days and 78.4 ± 3.9% after 100 days when stored 55 °C and analyzed using a cell-based assay in the CHO-K1 cell line. In contrast, a liquid control exhibited no bands on the electrophoresis gel and lost all transfection activity after being stored overnight at 55 °C. Likewise, after 60 days at 25 °C, the CMV-processed samples had full transfection activity while the activity of the liquid control was reduced to 40.1 ± 4.6%. In conclusion, CMV is a simple formulation method that significantly enhances the thermal stability of mRNA, requires minimal processing time, and could enable formulation of mRNA that can tolerate exposure to temperatures well above 25 °C during shipment and deployment in extreme environments.Graphical abstract

The first selenium containing chitin and chitosan derivatives: Combined synthetic, catalytic and biological studies

Ultrasonic approach to the synthesis of the first selenium-containing derivatives of chitin and chitosan has been developed. The synthetic procedure is simple, provides high yields, does not require harsh conditions, and uses water as the reaction medium. The elaborated chitin and chitosan derivatives and their based nanoparticles are non-toxic and possess high antibacterial and antifungal activity. Their antimicrobial activity exceeds the effect of the classic antibiotics (Ampicillin and Gentamicin) and the antifungal drug Amphotericin B. The obtained selenium-containing cationic chitin and chitosan derivatives exhibit a high transfection activity and are promising gene delivery vectors. Nanoparticles of the synthesized polymers are highly efficient catalysts for the oxidation of 1-phenylethyl alcohol to acetophenone by bromine at room temperature.

ROS responsive polyethylenimine-based fluorinated polymers for enhanced transfection efficiency and lower cytotoxicity.

Cationic polymer polyethylenimine (PEI) plays a crucial role in gene delivery. However, high molecular weight PEI leads to higher efficient transfection efficacy and higher cytotoxicity, while low molecular weight PEI exhibits lower transfection performance with lower toxicity. Therefore, effective chemical modification of PEI is required to enhance transfection activity and improve biocompatibility. Here, reactive oxygen species (ROS) responsive PEI-based fluorinated polymers (TKPF) with three degrees of fluorination (TKPF 12.5%, TKPF 25%, and TKPF 50%) were designed and synthesized by crosslinking low molecular weight PEI (PEI 1.8K) with a thioketal (TK) linker and then modifying heptafluorobutyric anhydride onto their surface. Fluorination reduced the positive charge density and endowed hydrophobic and lipophobic characteristics to resist serum interactions. The fluorophilic effect mediated efficient cellular uptake and endosomal escape while ROS-responsive TK linker allowed the polyplex disassembly to decrease the cytotoxicity of the polycations and improve the release of payloads at specific sites. TKPFs attained superior transfection efficiency in multiple cell lines (293TN cells and B16F10 cells) in vitro and showed excellent biocompatibility. TKPFs also exhibited great serum resistance in gene delivery and TKPF 50% transfected nearly 80% cells in the presence of 70% FBS. These results demonstrate that the fluorination and ROS responsiveness combined polycations are excellent gene-delivery vectors with serum-resistant capacity for further application.

Cyclic poly(β-amino ester)s with enhanced gene transfection activity synthesized through intra-molecular cyclization.

Topological structure plays a critical role in gene delivery of cationic polymers. Cyclic poly(β-amino ester)s (CPAEs) are successfully synthesized via sequential Michael addition and free radical initiating ring-closure reaction. The CPAEs exhibit superior gene transfection efficiency and safety profile compared to their linear counterparts.

Epigenetic modifications of tumor necrosis factor-alpha in joint cartilage tissue from osteoarthritis patients - CONSORT.

Background:Osteoarthritis (OA) remains one of the most common osteopathy for centuries, which can be attributed to multiple risk factors including mechanical and biochemical ones. More and more studies verified that inflammatory cytokines play important roles in the progression of OA, such as tumor necrosis factor-alpha (TNF-α). In this study, we aimed to investigate the relationship between epigenetic manifestations of TNF-? and the pathogenesis of OA.Methods:Totally, 37 OA patients’ cartilage was collected through the knee joint and 13 samples of articular cartilage as healthy control was collected through traumatic amputation. Real-time PCR, Western blot and ELISA analysis were performed to observe the expression of target genes and proteins in collected samples.Results:Compared with the healthy control group, TNF-? was over-expressing in cartilage which was collected from OA patients. DNA hypomethylation, histone hyperacetylation and histone methylation were observed in the TNF-? promoter in OA compared with normal patients, and we also studied series of enzymes associated with epigenetics. The results showed that by increasing DNA methylation and decreasing histone acetylation in the TNF-? promoter, and TNF-? over-expression in OA cartilage was suppressed, histone methylation has no significant correlation with OA.Conclusion:In conclusion, the changes of epigenetic status regulate TNF-α expression in the cells, which are pivotal to the OA disease process. These results may give us a better understanding of OA and may provide new therapeutic options.

Design of a new cell penetrating peptide for DNA, siRNA and mRNA delivery.

Delivery systems, including peptide-based ones, that destabilize endosomes in a pH-dependent manner are increasingly used to deliver cargoes of therapeutic interest, such as nucleic acids and proteins into mammalian cells. The negatively charged amphipathic alpha-helicoidal forming peptide named HELP (Helical Erythrocyte Lysing Peptide) is a derivative from the bee venom melittin and was shown to have a pH-dependent activity with the highest lytic activity at pH5.0 at the same time as becoming inactive when the pH is increased. The present study aimed to determine whether replacement in the HELP peptide of the glutamic acid residues by histidines, for which the protonation state is sensitive to the pH changes that occur during endosomal acidification, can transform this fusogenic peptide into a carrier able to deliver different nucleic acids into mammalian cells. The resulting HELP-4H peptide displays high plasmid DNA, small interfering RNA and mRNA delivery capabilities. Importantly, in contrast to other cationic peptides, its transfection activity was only marginally affected by the presence of serum. Using circular dichroism, we found that acidic pH did not induce significant conformational changes for HELP-4H. In summary, we were able to develop a new cationic histidine rich peptide able to efficiently deliver various nucleic acids into cells.

Improvement of mRNA Delivery Efficiency to a T Cell Line by Modulating PEG-Lipid Content and Phospholipid Components of Lipid Nanoparticles.

(1) Background: T cells are important target cells, since they exert direct cytotoxic effects on infected/malignant cells, and affect the regulatory functions of other immune cells in a target antigen-specific manner. One of the current approaches for modifying the function of T cells is gene transfection by viral vectors. However, the insertion of the exogenous DNA molecules into the genome is attended by the risk of mutagenesis, especially when a transposon-based gene cassette is used. Based on this scenario, the transient expression of proteins by an in vitro-transcribed messenger RNA (IVT-mRNA) has become a subject of interest. The use of lipid nanoparticles (LNPs) for the transfection of IVT-mRNA is one of the more promising strategies for introducing exogenous genes. In this study, we report on the development of LNPs with transfection efficiencies that are comparable to that for electroporation in a T cell line (Jurkat cells). (2) Methods: Transfection efficiency was improved by optimizing the phospholipids and polyethylene glycol (PEG)-conjugated lipid components. (3) Results: Modification of the lipid composition resulted in the 221-fold increase in luciferase activity compared to a previously optimized formulation. Such a high transfection activity was due to the efficient uptake by clathrin/dynamin-dependent endocytosis and the relatively efficient escape into the cytoplasm at an early stage of endocytosis.

ATP-Responsive Multifunctional Supramolecular Polymer as a Nonviral Vector for Boosting Cholesterol Removal from Lipid-Laden Macrophages.

Specific delivery of NCEH1 plasmid is a promising approach to boost the cholesterol removal from lipid-laden macrophages for antiatherosclerosis. Polyethylenimine (PEI) is one of the most efficient gene carriers among nonviral vectors. However, the high transfection activity of PEI is always accompanied by profound cytotoxicity. To tackle the paradox between transfection efficiency and safety, we constructed a novel ATP-responsive multifunctional supramolecular polymer by cross-linking functionalized low-molecular-weight PEI via a boronic ester bond for NCEH1 plasmid delivery. The supramolecular polymer could condense NCEH1 plasmids to form stable nanosized polyplexes when the w/w ratios of the polymer and gene were higher than 2. ATP-triggered degradation of the polymer and pDNA release were characterized by a series of studies, including 1H NMR, 31P NMR, XPS, agarose gel electrophoresis, and ethidium bromide exclusion tests. In addition, the changes in particle size and morphology were observed in the presence of ATP. Interestingly, the supramolecular polymer showed broad spectrum antioxidant activities by measuring the elimination rates of different reactive oxygen species. In addition, the supramolecular polymer displayed a high buffering capability and good cytocompatibility as demonstrated by the results of the buffering capacity, a hemolysis assay, and a cytotoxicity test. Importantly, it was revealed that the supramolecular polymer/NCEH1 plasmid polyplex formulated at a w/w ratio of 20 was most effective in enhancing cholesterol removal from lipid-laden macrophages and reducing the accumulation of lipid droplets as evidenced by transfection study, cholesterol efflux assay, and oil red O staining studies. Collectively, the ATP-responsive multifunctional supramolecular polymer holds great potential for safe and efficient gene delivery for antiatherosclerosis.

The hydrophobic tail of a pH-sensitive cationic lipid influences siRNA transfection activity and toxicity in human NK cell lines

The use of natural killer (NK) cells in cell therapy is an attractive next generation strategy for cancer immunotherapy. NK-92 cells (a human NK cell line) have been tested in clinical trial stages, making them an off-the-shelf medicine. Controlling gene expression in NK-92 cells by an artificial delivery system is an available for enhancing NK-92 cell therapy. We report here on the development of a siRNA-loaded lipid nanoparticle (LNP) composed of CL1H6 (CL1H6-LNP), an optimized, pH-sensitive cationic lipid, with efficient gene silencing and low cytotoxicity in NK-92 cells. The hydrophilic head group of the lipid molecule used in preparing these particles largely influences the pKa of the final LNP, and lipids with an amino moiety substituted with a methyl group showed a high gene silencing activity. Compared with myristate and palmitate, the hydrophobic tail of oleate had a high gene silencing activity and cell viability. Analyses of intracellular trafficking indicated that the CL1H6-LNP appeared to escape from the endosomes via membrane fusion, without disrupting the membrane. The mechanism of endosomal escape should contribute to our understanding of efficient gene silencing with a low degree of cytotoxicity. These results therefore suggest that a CL1H6-LNP has promise for delivering siRNA to NK-92 cells.

Dynamic mRNA polyplexes benefit from bioreducible cleavage sites for in vitro and in vivo transfer

Currently, messenger RNA (mRNA)-based lipid nanoparticle formulations revolutionize the clinical field. Cationic polymer-based complexes (polyplexes) represent an alternative compound class for mRNA delivery. After establishing branched polyethylenimine with a succinylation degree of 10% (succPEI) as highly effective positive mRNA transfection standard, a diverse library of PEI-like peptides termed sequence-defined oligoaminoamides (OAAs) was screened for mRNA delivery. Notably, sequences, which had previously been identified as potent plasmid DNA (pDNA) or small-interfering RNA (siRNA) carriers, displayed only moderate mRNA transfection activity. A second round of screening combined the cationizable building block succinoyl tetraethylene pentamine and histidines for endosomal buffering, tyrosine tripeptides and various fatty acids for mRNA polyplex stabilization, as well as redox-sensitive units for programmed intracellular release. For the tested OAA carriers, balancing of extracellular stability, endosomal lytic activity, and intracellular release capability was found to be of utmost importance for optimum mRNA transfection efficiency. OAAs with T-shape topology containing two oleic acids as well-stabilizing fatty acids, attached via a dynamic bioreducible building block, displayed superior activity with up to 1000-fold increased transfection efficiency compared to their non-reducible analogs. In the absence of the dynamic linkage, incorporation of shorter less stabilizing fatty acids could only partly compensate for mRNA delivery. Highest GFP expression and the largest fraction of transfected cells (96%) could be detected for the bioreducible OAA with incorporated histidines and a dioleoyl motif, outperforming all other tested carriers as well as the positive control succPEI. The good in vitro performance of the dynamic lead structure was verified in vivo upon intratracheal administration of mRNA complexes in mice.

Gemini Lipopeptide Bearing an Ultrashort Peptide for Enhanced Transfection Efficiency and Cancer-Cell-Specific Cytotoxicity.

Cationic gemini lipopeptides are a relatively new class of amphiphilic compounds to be used for gene delivery. Through the possibility of incorporating short peptides with cell-penetrating functionalities, these lipopeptides may be advantageous over traditional cationic lipids. Herein, we report the design, synthesis, and application of a novel cationic gemini lipopeptide for gene delivery. An ultrashort peptide, containing four amino acids, arginine–cysteine–cysteine–arginine, serves as a cationic head group, and two α-tocopherol moieties act as hydrophobic anchoring groups. The new lipopeptide (ATTA) is incorporated into the conventional liposomes, containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and 1,2-dioleoyl-sn-glycerol-3-phosphoethanolamine (DOPE), at different molar ratios. The formulated liposomes are characterized and screened for better transfection efficiency. Transfection activity in multiple human cell lines from cancerous and noncancerous origins indicates that the inclusion of an optimal ratio of ATTA in the liposomes substantially enhances the transfection efficiency, superior to that of a traditional liposome, DOTAP–DOPE. Cytotoxicity of ATTA-containing formulations against multiple cell lines indicates potentially distinct activity between cancer and noncancer cell lines. Furthermore, lipoplexes of the ATTA-containing formulations with anticancer therapeutic gene, plasmid encoding tumor necrosis factor-related apoptosis-inducing ligand (pTRAIL), induce obviously more cytotoxicity than conventional formulations. The results indicate that arginine-rich cationic lipopeptide appears to be a promising ingredient in gene delivery vector formulations to enhance transfection efficiency and cell-selective cytotoxicity.

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.

Non-structural Proteins of Severe Fever With Thrombocytopenia Syndrome Virus Suppress RNA Synthesis in a Transcriptionally Active cDNA-Derived Viral RNA Synthesis System.

Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease caused by the tick-borne SFTS bunyavirus (SFTSV) resulting in a high fatality rate up to 30%. SFTSV is a negative-strand RNA virus containing three single-stranded RNA genome segments designated as L, M, and S, which respectively, encode the RNA-dependent RNA polymerase (RdRp), glycoproteins Gn and Gc, and nucleoprotein (N) and non-structural proteins (NSs). NSs can form inclusion bodies (IBs) in infected and transfected cells. A previous study has provided a clue that SFTSV NSs may be involved in virus-like or viral RNA synthesis; however, the details remain unclear. Our work described here reveals that SFTSV NSs can downregulate virus-like RNA synthesis in a dose-dependent manner within a cDNA-derived viral RNA synthesis system, i.e., minigenome (−) and minigenome (+) systems based on transfection, superinfection, and luciferase reporter activity determination; meanwhile, NSs also show a weak inhibitory effect on virus replication. By using co-immunoprecipitation (Co-IP) and RT-PCR combined with site-directed mutagenesis, we found that NSs suppress virus-like RNA or virus replication through interacting with N but not with RdRp, and the negative regulatory effect correlates closely with the IB structure it formed but is not associated with its role of antagonizing host innate immune responses. When the cytoplasmic structure of IB formed by SFTSV NSs was deprived, the inhibitory effect of NSs on virus-like RNA synthesis would weaken and even disappear. Similarly, we also evaluated other bandavirus NSs that cannot form IB in neither infected nor transfected cells, and the results showed that the NSs of Heartland bandavirus (HRTV) did not show a significant inhibitory effect on virus-like RNA synthesis within a minigenome system. Our findings provide experimental evidence that SFTSV NSs participate in regulating virus-like or viral RNA synthesis and the negative effect may be due to the NSs–N interaction.

Integrating disulfides into a polyethylenimine gene carrier selectively boosts significant transfection activity in lung tissue enabling robust IL-12 gene therapy against metastatic lung cancers

Bioreducible polyethylenimines (SSPEIs) are promising non-viral carriers for cancer gene therapy. However, the availability of significant gene transfection activity by SSPEIs remains a challenge. Herein, an essential step was taken to ascertain whether or not the disulfide bonds of SSPEIs play a critical role in promoting significant gene transfection activity in different tissues. Initially, a disulfide-linked linear polyethylenimine (denoted as SSLPEI) consisting of one 5.0 kDa LPEI main chain and three disulfide-linked 5.7 kDa LPEI grafts was designed and prepared to possess similar molecular weight with commercialized 25 kDa LPEI as a positive control. The SSLPEI could induce superior in vitro transfection activity in different cells to the LPEI control as well as low cytotoxicity. Notably, such enhanced in vitro transfection effect by the SSLPEI was more marked in type-II alveolar epithelial cells compared to different cancer cells. In a Balb/c nude mouse model bearing SKOV-3 tumor, the SSLPEI caused parallel level of transgene expression with the LPEI control in the tumor but significantly higher level in the mouse lung. Furthermore, the SSLPEI and LPEI groups afforded an identical antitumor efficacy against the SKOV-3 tumor via intravenous delivery of a shRNA for silencing VEGF expression in the tumor. However, via intravenous delivery of an interleukin-12 (IL-12) gene into metastatic lung cancers in a C57BL/6 mouse model, the SSLPEI group exerted markedly higher IL-12 expression level in the mouse lung and peripheral blood as compared to the LPEI group, thereby boosting IL-12 immunotherapy against the lung metastasis with longer medium survival time. The results of this work elicit that the disulfide bonds of SSPEIs play a pivotal role in enhancing gene transfection activity selectively in the lung tissue rather than solid tumor, enabling high translational potential of SSPEIs for non-viral gene therapy against metastatic lung cancers.

Different Biological Activities of Histidine-Rich Peptides Are Favored by Variations in Their Design.

The protein transduction and antimicrobial activities of histidine-rich designer peptides were investigated as a function of their sequence and compared to gene transfection, lentivirus transduction and calcein release activities. In membrane environments, the peptides adopt helical conformations where the positioning of the histidine side chains defines a hydrophilic angle when viewed as helical wheel. The transfection of DNA correlates with calcein release in biophysical experiments, being best for small hydrophilic angles supporting a model where lysis of the endosomal membrane is the limiting factor. In contrast, antimicrobial activities show an inverse correlation suggesting that other interactions and mechanisms dominate within the bacterial system. Furthermore, other derivatives control the lentiviral transduction enhancement or the transport of proteins into the cells. Here, we tested the transport into human cell lines of luciferase (63 kDa) and the ribosome-inactivating toxin saporin (30 kDa). Notably, depending on the protein, different peptide sequences are required for the best results, suggesting that the interactions are manifold and complex. As such, designed LAH4 peptides assure a large panel of biological and biophysical activities whereby the optimal result can be tuned by the physico-chemical properties of the sequences.

Dexamethasone Conjugates: Synthetic Approaches and Medical Prospects.

Dexamethasone (DEX) is the most commonly prescribed glucocorticoid (GC) and has a wide spectrum of pharmacological activity. However, steroid drugs like DEX can have severe side effects on non-target organs. One strategy to reduce these side effects is to develop targeted systems with the controlled release by conjugation to polymeric carriers. This review describes the methods available for the synthesis of DEX conjugates (carbodiimide chemistry, solid-phase synthesis, reversible addition fragmentation-chain transfer [RAFT] polymerization, click reactions, and 2-iminothiolane chemistry) and perspectives for their medical application as GC drug or gene delivery systems for anti-tumor therapy. Additionally, the review focuses on the development of DEX conjugates with different physical-chemical properties as successful delivery systems in the target organs such as eye, joint, kidney, and others. Finally, polymer conjugates with improved transfection activity in which DEX is used as a vector for gene delivery in the cell nucleus have been described.

Antisense microRNA185 loaded liposome for efficient inhibition of the hepatic endogenous microRNA185 level

MicroRNA185 (miR185), an endogenous noncoding RNA with 23 nucleotides, is one of key posttranscriptional modulators of cholesterol metabolism in hepatic cells. The antisense inhibitor of miR185 (miR185i) could decrease cholesterol level in vivo, providing a promising agent for anti-atherosclerosis strategy. In this work, a novel LipomiR185i was constructed by thin film hydration method and post-PEGylation as DOPE: DOTAP: Chol: DSPE-PEG2000 at the molar ratio of 1:1:1:0.1 with a nitrogen-to-phosphate ratio of 3, through the optimization of three cationic lipids (DOTAP, DODMA and DLin-MC3-DMA), six helper lipids (PC-98T, HSPC, DOPE, DMPC, DPPC and DSPC), different amounts and incorporation approaches of DSPE-PEG2000 and nitrogen-to-phosphate ratio. LipomiR185i was characterized with a particle size of 174 ± 11 nm, a zeta potential of 7.0 ± 3.3 mV, high encapsulation efficiency and transfection activity. It could protect miR185i from the rapid degradation by nucleases in serum, enhance cellular uptake and promote lysosomal escape in HepG2 cells. LipomiR185i could accumulate in the liver and remain for at least two weeks. More importantly, LipomiR185i significantly down-regulated the hepatic endogenous miR185 level in vitro and in vivo without significant tissue damage at 14 mg⋅kg−1. The construction of LipomiR185i provides a potential anti-atherosclerotic nanodrug as well as a platform for delivering small RNAs to the liver efficiently and safely.

Mannosylated Cationic Copolymers for Gene Delivery to Macrophages.

Macrophages are desirable targets for gene therapy of cancer and other diseases. Cationic diblock copolymers of polyethylene glycol (PEG) and poly-L-lysine (PLL) or poly{N-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} (pAsp(DET)) are synthesized and used to form polyplexes with a plasmid DNA (pDNA) that are decorated with mannose moieties, serving as the targeting ligands for the C type lectin receptors displayed at the surface of macrophages. The PEG-b-PLL copolymers are known for its cytotoxicity, so PEG-b-PLL-based polyplexes are cross-linked using reducible reagent dithiobis(succinimidyl propionate) (DSP). The cross-linked polyplexes display low toxicity to both mouse embryonic fibroblasts NIH/3T3 cell line and mouse bone marrow-derived macrophages (BMMΦ). In macrophages mannose-decorated polyplexes demonstrate an ≈8 times higher transfection efficiency. The cross-linking of the polyplexes decrease the toxicity, but the transfection enhancement is moderate. The PEG-b-pAsp(DET) copolymers display low toxicity with respect to the IC-21 murine macrophage cell line and are used for the production of non-cross-linked pDNA-contained polyplexes. The obtained mannose modified polyplexes exhibit ca. 500-times greater transfection activity in IC-21 macrophages compared to the mannose-free polyplexes. This result greatly exceeds the targeting gene transfer effects previously described using mannose receptor targeted non-viral gene delivery systems. These results suggest that Man-PEG-b-pAsp(DET)/pDNA polyplex is a potential vector for immune cells-based gene therapy.

GALA-Modified Lipid Nanoparticles for the Targeted Delivery of Plasmid DNA to the Lungs.

This study describes the development of lipid nanoparticles (LNPs) for the efficient and selective delivery of plasmid DNA (pDNA) to the lungs. The GALA peptide was used as a ligand to target the lung endothelium and as an endosomal escape device. Transfection activity in the lungs was significantly improved when pDNA was encapsulated in double-coated LNPs. The inner coat was composed of dioleoylphsophoethanolamine and a stearylated octaarginine (STR-R8) peptide, while the outer coat was largely a cationic lipid, di-octadecenyl-trimethylammonium propane, mixed with YSK05, a pH-sensitive lipid, and cholesterol. Optimized amounts of YSK05 and GALA were used to achieve an efficient and lung-selective system. The optimized system produced a high gene expression level in the lungs (>107 RLU/mg protein) with high lung/liver and lung/spleen ratios. GALA/R8 modification and the double-coating design were indispensable for efficient gene expression in the lungs. Despite the fact that NPs prepared with 1-step or 2-step coating have the same lipid amount and composition and the same pDNA dose, the transfection activity was dramatically higher in the lungs in the case of 2-step coating. Surprisingly, 1-step or 2-step coatings had no effect on the amount of nanoparticles that were delivered to the lungs, suggesting that the double-coating strategy substantially improved the efficiency of gene expression at the intracellular level.