Plasmid DNA Complexes Research Articles

Design of Experiments to Tailor the Potential of BSA-Coated Peptide Nanocomplexes for Temozolomide/p53 Gene Co-Delivery

Background: Gene therapy can be viewed as a promising/valuable therapeutic approach directed to cancer treatment, including glioblastoma. Concretely, the combination of gene therapy with chemotherapy could increase its therapeutic index due to a synergistic effect. In this context, bovine serum albumin (BSA)-coated temozolomide (TMZ)-peptide (WRAP5)/p53 gene-based plasmid DNA complexes were developed to promote payload co-delivery. Methods: Design of experiments (DoE) was employed to unravel the BSA-coated TMZ-WRAP5/p53 nanocomplexes with the highest potential by considering the nitrogen to phosphate groups ratio (N/P), and the BSA concentration as inputs and the size, polydispersity index, surface charge and p53-based plasmid complexation capacity (CC) as DoE outputs. Results: The obtained quadratic models were statistically significant (p-value < 0.05) with an adequate coefficient of determination, and the correspondent optimal points were successfully validated. The optimal complex formulation had N/P of 1.03, a BSA concentration of 0.08%, a size of approximately 182 nm, a zeta potential of +9.8 mV, and a pDNA CC of 96.5%. The optimal nanocomplexes are approximately spherical. A cytotoxicity assay showed that these BSA-coated TMZ-WRAP5/p53 complexes did not elicit toxicity in normal brain cells, and a hemolysis study demonstrated the hemocompatibility of the complexes. The complexes were stable in cell culture medium and fetal bovine serum and assured pDNA protection and release. Moreover, the optimal BSA-coated complexes were able of gene transcription and promoted a significant inhibition of glioblastoma cell viability. Conclusions: The reported findings instigate the development of future research to evaluate their potential utility to TMZ/p53 co-delivery. The DoE tool proved to be a powerful approach to explore and tailor the composition of BSA-coated TMZ-WRAP5/p53 complexes, which are expected to contribute to the progress toward a more efficient therapy against cancer and, more specifically, against glioblastoma.

Targeted Gene Delivery to Muscle Cells In Vitro and In Vivo Using Electrostatically Stabilized DNA—Peptide Complexes

Genetic constructs must be delivered selectively to target tissues and intracellular compartments at the necessary concentrations in order to achieve the maximum therapeutic effect in gene therapy. Development of targeted carriers for non-viral delivery of nucleic acids into cells, including those in muscle, which is one of the most challenging tissues to transfect in vivo, remains a topical issue. We have studied ternary complexes of plasmid DNA and an arginine–histidine-rich peptide-based carrier coated with a glutamate–histidine-rich polymer bearing skeletal muscle targeting peptide (SMTP) for the gene delivery to muscle tissue. The relaxation of the ternary complexes after polyanion treatment was assessed using the ethidium bromide displacement assay. The developed polyplexes were used to transfect C2C12 myoblasts in full-media conditions, followed by analysis of their toxic properties using the Alamar Blue assay and expression analysis of lacZ and GFP reporter genes. After delivering plasmids containing the GFP and lacZ genes into the femoral muscles of mdx mice, which are model of Duchenne muscular dystrophy, GFP fluorescence and β-galactosidase activity were detected. We observed that the modification of ternary polyplexes with 10 mol% of SMTP ligand resulted in a 2.3-fold increase in lacZ gene expression when compared to unmodified control polyplexes in vivo. Thus, we have demonstrated that the developed DNA/carrier complexes and SMTP-modified coating are nontoxic, are stable against polyanion-induced relaxation, and can provide targeted gene delivery to muscle cells and tissues. The results of this study are useful for a range of therapeutic applications, from immunization to amelioration of inherited neuromuscular diseases.

Poly(2-(dimethylamino) ethyl methacrylate)-b-poly (hydroxy propyl methacrylate) nanoparticles for guided delivery of MCL-1 CRISPR-Cas9 plasmid and doxorubicin to non-small cell lung cancer

Combining gene therapy with other therapeutic methods, such as chemotherapy, using nanocarriers can lead to higher therapeutic effects through different mechanisms. In the current study, poly(2-(dimethylamino) ethyl methacrylate)-b-poly (hydroxy propyl methacrylate) (PDMAEMA-b-PHPMA) copolymer was synthesized in order to encapsulate either doxorubicin (DOX) or MCL-1 CRISPR.Cas9 plasmid (NP/DOX and NP/CRISPR.Cas9 plasmid). Then, for site-specific targeting against non-small cell lung cancer (SKMES-1 and A549 cell lines) overexpressing nucleolin receptors, AS1411 aptamer was attached to the surface of the prepared platforms through electrostatic interactions. The developed system was extensively evaluated for its transfection efficiency, cellular toxicity, and effectiveness in treating SKMES-1 tumor-bearing C57BL/6 nude mice. The results indicated that the targeted NP/DOX+NP/CRISPR.Cas9 plasmid complex enhanced cellular uptake into target cells, SKMES-1 and A549, through receptor- mediated endocytosis. Furthermore, comparative in vitro cytotoxicity results showed that the cells were exposed to the combination of NP/DOX and NP/CRISPR.Cas9 plasmid complex caused significantly enhanced cytotoxicity compared to treatment with each of them alone. In vivo study revealed that the AS1411-tagged nanoparticles (Apt-NP/DOX+Apt-NP/CRISPR.Cas9 plasmid) had a more pronounced inhibitory effect on tumor growth compared to non-targeted nanoparticles (NP/DOX+NP/CRISPR.Cas9 plasmid), moreover, co-treatment with MCL-1 sgRNA CRISPR.Cas9 plasmid and DOX increased the drug sensitivity of SKMES-1 cells towards DOX, which enhanced the therapeutic effects in mice receiving treatment with Apt-NP/DOX in combination with Apt-CRISPR.Cas9 plasmid nanocomplex. In conclusion, the findings indicated that the combination of common chemotherapy such as DOX and genome editing for MCL-1 through CRISPR.Cas9 system would provide a potential and safe method for treating non-small cell lung cancer which has excellent capacity for the clinical translation.

A photothermal surface modified with polyelectrolyte multilayers for gene transfection and cell harvest

Gene transfection, which involves introducing nucleic acids into cells, is a pivotal technology in the life sciences and medical fields, particularly in gene therapy. Surface-mediated transfection, primarily targeting cells adhering to surfaces, shows promise for enhancing cell transfection by localizing and presenting surface-bound nucleic acids directly to the cells. However, optimizing endocytosis for efficient delivery remains a persistent challenge. Additionally, ensuring efficient and non-traumatic cell harvest capability is crucial for applications such as ex vivo cell-based therapy. To address these challenges, we developed a photothermal platform with enzymatic degradation capability for efficient gene transfection and cell harvest. This platform is based on carbon nanotubes (CNTs) doped with poly(dimethylsiloxane) and modified with polyelectrolyte multilayers (PEMs) containing hyaluronic acid and quaternized chitosan, allowing for substantial loading of poly(ethyleneimine)/plasmid DNA (pDNA) complexes through electrostatic interactions. Upon irradiation of near-infrared laser, the photothermal properties of CNTs enable high transfection efficiency by delivering pDNA into attached cells via a membrane disruption mechanism. The engineered cells can be harvested by treating with a non-toxic hyaluronidase solution to degrade PEMs, thus maintaining good viability for further applications. This platform has demonstrated remarkable efficacy across various cell lines (including Hep-G2 cells, Ramos cells and primary T cells), achieving a transfection efficiency exceeding 95 %, cell viability exceeding 90 %, and release efficiency surpassing 95 %, highlighting its potential for engineering living cells.

Plasmid DNA Complexes in Powder Form Studied by Spectroscopic and Diffraction Methods.

Currently, new functional materials are being created with a strong emphasis on their ecological aspect. Materials and devices based on DNA biopolymers, being environmentally friendly, are therefore very interesting from the point of view of applications. In this paper, we present the results of research on complexes in the powder form based on plasmid DNA (pDNA) and three surfactants with aliphatic chains containing 16 carbon atoms (cetyltrimethylammonium chloride, benzyldimethylhexadecylammonium chloride and hexadecylpyridinium chloride). The X-ray diffraction results indicate a local hexagonal packing of DNA helices in plasmid DNA complexes, resembling the packing for corresponding complexes based on linear DNA. Based on the Fourier-transform infrared spectroscopy results, the DNA conformation in all three complexes was determined as predominantly of A-type. The two relaxation processes revealed by dielectric spectroscopy for all the studied complexes are connected with two different contributions to total conductivity (crystallite part and grain boundaries). The crystallite part (grain interior) was interpreted as an oscillation of the polar surfactant head groups and is dependent on the conformation of the surfactant chain. The influence of the DNA type on the properties of the complexes is discussed, taking into account our previous results for complexes based on linear DNA. We showed that the type of DNA has an impact on the properties of the complexes, which has not been demonstrated so far. It was also found that the layer of pDNA-surfactant complexes can be used as a layer with variable specific electric conductivity by selecting the frequency, which is interesting from an application point of view.

Notch-Targeted Therapeutic in Colorectal Cancer by Notch1 Attenuation Via Tumor Microenvironment-Responsive Cascade DNA Delivery.

The Notch signaling is a key molecular pathway that regulates cell fate and development. Aberrant Notch signaling can lead to carcinogenesis and progression of malignant tumors. However, current therapies targeting Notch pathway lack specificity and induce high toxicity. In this report, a tumor microenvironment-responsive and injectable hydrogel is designed to load plasmid DNA complexes as a cascade gene delivery system to achieve precise Notch-targeted gene therapy of colorectal cancer (CRC). The hydrogels are prepared through cross-linking between phenylboric acid groups containing poly(oligo(ethylene glycol)methacrylate) (POEGMA) and epigallocatechin gallate (EGCG), used to load the complexes between plasmid DNA encoding short hairpin RNAs of Notch1 (shNotch1) and fluorinated polyamidoamine (PAMAM-F) (PAMAM-F/shNotch1). In response to low pH and H2O2 in tumor microenvironment, the hydrogel can be dissociated and release the complexes for precise delivery of shNotch1 into tumor cells and inhibit Notch1 activity to suppress malignant biological behaviors of CRC. In the subcutaneous tumor model of CRC, PAMAM-F/shNotch1-loaded hydrogels can accurately attenuate Notch1 activity and significantly inhibit tumor growth without affecting Notch signal in adjacent normal tissues. Therefore, this therapeutic system can precisely inhibit Notch1 signal in CRC with high responsiveness and low toxicity, providing a promising Notch-targeted gene therapeutic for human malignancy.

Interaction of γ-Polyglutamic Acid/Polyethyleneimine/Plasmid DNA Ternary Complexes with Serum Components Plays a Crucial Role in Transfection in Mice.

Typical examples of non-viral vectors are binary complexes of plasmid DNA with cationic polymers such as polyethyleneimine (PEI). However, problems such as cytotoxicity and hemagglutination, owing to their positively charged surfaces, hinder their in vivo use. Coating binary complexes with anionic polymers, such as γ-polyglutamic acid (γ-PGA), can prevent cytotoxicity and hemagglutination. However, the role of interactions between these complexes and serum components in in vivo gene transfer remains unclear. In this study, we analyzed the contribution of serum components to in vivo gene transfer using PEI/plasmid DNA binary complexes and γ-PGA/PEI/plasmid DNA ternary complexes. In binary complexes, heat-labile components in the serum greatly contribute to the hepatic and splenic gene expression of the luciferase gene. In contrast, serum albumin and salts affected the hepatic and splenic gene expression in the ternary complexes. Changes in physicochemical characteristics, such as increased particle size and decreased absolute values of ζ-potential, might be involved in the enhanced gene expression. These findings would contribute to a better understanding of in vivo non-viral gene transfer using polymers, such as PEI and γ-PGA.

Discovery of a Novel Dual Targeting Peptide for Human Glioma: From In Silico Simulation to Acting as Targeting Ligand.

Receptor-mediated transcytosis (RMT) is a more specific, highly efficient, and reliable approach to crossing the blood-brain-barrier (BBB) and releasing the therapeutic cargos into the brain parenchyma. Here, we introduced and characterized a human/mouse-specific novel leptin-derived peptide using in silico, in vitro and in vivo experiments. Based on the bioinformatics analysis and molecular dynamics (MD) simulation, a 14 amino acid peptide sequence (LDP 14) was introduced and its interaction with leptin-receptor (ObR) was analyzed in comparison with an well known leptin-derived peptide, Lep 30. MD simulation data revealed a significant stable interaction between ligand binding domains (LBD) of ObR with LDP 14. Analyses demonstrated suitable cellular uptake of LDP 14 alone and its derivatives (LDP 14-modified G4 PAMAM dendrimer and LDP 14-modified G4 PAMAM/pEGFP-N1 plasmid complexes) via ObR, energy and species dependent manner (preferred uptake by human/mouse cell lines compared to rat cell line). Importantly, our findings illustrated that the entry of LDP 14-modified dendrimers in hBCEC-D3 cells not only is not affected by protein corona (PC) formation, as the main reason for diminishing the cellular uptake, but also PC per se can enhance uptake rate. Finally, fluorescein labeled LDP 14-modified G4 PAMAM dendrimers efficiently accumulated in the mice brain with lower biodistribution in other organs, in our in vivo study. LDP 14 introduced as a novel and highly efficient ligand, which can be used for drugs/genes delivery to brain tissue in different central nervous system (CNS) disorders.

Production and Purification of Adeno-Associated Viral Vectors (AAVs) Using Orbitally Shaken HEK293 Cells.

Here, we describe methods for the production of adeno-associated viral (AAV) vectors by transient transfection of HEK293 cells grown in serum-free medium using orbital shaken bioreactors and the subsequent purification of vector particles. The protocol for expression of AAV components is based on polyethyleneimine (PEI)-mediated transfection of a three-plasmid system and is specified for production in milliliter-to-liter scales. After PEI and plasmid DNA (pDNA) complex formation, the diluted cell culture is transfected without a prior concentration step or medium exchange. Following a 7-day batch process, cell cultures are further processed using a set of methods for cell lysis and vector recovery. Methods for the purification of viral particles are described, including immunoaffinity and anion-exchange chromatography, ultrafiltration, as well as digital PCR to quantify the concentration of vector particles.

Retracted: Influenza A Virus Production in Mouse Lung Is Inhibited by Inhalation of Aerosol Polyethylenimine/Short Hairpin RNA Plasmid Complexes.

[This retracts the article DOI: 10.1155/2022/7404813.].

Selectively Fluorinated PAMAM-Arginine Conjugates as Gene Delivery Vectors.

Polyamidoamine (PAMAM) dendrimers are among the most studied cationic polymers as non-viral gene delivery vectors. However, an "ideal" PAMAM-based gene delivery vector is still missing due to the high manufacturing costs and non-negligible cytotoxicity associated with the use of high-generation dendrimers, whereas low-generation dendrimers are far from displaying efficient gene transfection. In order to cover this gap in the literature, in this study, we propose the functionalization of the outer primary amines of PAMAM G2 and PAMAM G4 with building blocks bearing fluorinated moieties along with a guanidino functional group. We have designed and synthetized two fluorinated arginine (Arg)-based Michael acceptors which were straightforwardly "clicked" to PAMAM dendrimers without the need for coupling reagents and/or catalysts. The obtained conjugates, in particular, derivative 1 formed starting from the low-cost PAMAM G2 and a building block bearing two trifluoromethyl groups, were able to efficiently complex plasmid DNA, had negligible cytotoxicity, and showed improved gene transfection efficiency as compared to undecorated PAMAM dendrimers and a corresponding unfluorinated PAMAM-Arg derivative, with derivative 1 being two orders of magnitude more efficient than the gold standard branched polyethylenimine, bPEI, 25 kDa. These results highlight the importance of the presence of trifluoromethyl moieties for both gene transfection and a possible future application in 19F magnetic resonance imaging.

A comparison of three different delivery methods for achieving CRISPR/Cas9 mediated genome editing in Cichorium intybus L.

Root chicory (Cichorium intybus L. var. sativum) is used to extract inulin, a fructose polymer used as a natural sweetener and prebiotic. However, bitter tasting sesquiterpene lactones, giving chicory its known flavour, need to be removed during inulin extraction. To avoid this extraction and associated costs, recently chicory variants with a lower sesquiterpene lactone content were created by inactivating the four copies of the germacrene A synthase gene (CiGAS-S1, -S2, -S3, -L) which encode the enzyme initiating bitter sesquiterpene lactone biosynthesis in chicory. In this study, different delivery methods for CRISPR/Cas9 reagents have been compared regarding their efficiency to induce mutations in the CiGAS genes, the frequency of off-target mutations as well as their environmental and economic impacts. CRISPR/Cas9 reagents were delivered by Agrobacterium-mediated stable transformation or transient delivery by plasmid or preassembled ribonucleic complexes (RNPs) using the same sgRNA. All methods used lead to a high number of INDEL mutations within the CiGAS-S1 and CiGAS-S2 genes, which match the used sgRNA perfectly; additionally, the CiGAS-S3 and CiGAS-L genes, which have a single mismatch with the sgRNA, were mutated but with a lower mutation efficiency. While using both RNPs and plasmids delivery resulted in biallelic, heterozygous or homozygous mutations, plasmid delivery resulted in 30% of unwanted integration of plasmid fragments in the genome. Plants transformed via Agrobacteria often showed chimerism and a mixture of CiGAS genotypes. This genetic mosaic becomes more diverse when plants were grown over a prolonged period. While the genotype of the on-targets varied between the transient and stable delivery methods, no off-target activity in six identified potential off-targets with two to four mismatches was found. The environmental impacts (greenhouse gas (GHG) emissions and primary energy demand) of the methods are highly dependent on their individual electricity demand. From an economic view - like for most research and development activities - employment and value-added multiplier effects are high; particularly when compared to industrial or manufacturing processes. Considering all aspects, we conclude that using RNPs is the most suitable method for genome editing in chicory since it led to a high efficiency of editing, no off-target mutations, non-transgenic plants with no risk of unwanted integration of plasmid DNA and without needed segregation of transgenes.

Star-shaped poly(l-lysine) with polyester bis-MPA dendritic core as potential degradable nano vectors for gene delivery

Efficient initiation from ammonium trifluoroacetate salts (TFA) dendritic end-groups yields well-defined poly(l-lysine) star polypeptides with 8, 16 and 32 arms. Hydrolytic core degradation and plasmid DNA complexation is demonstrated.

Comparison of in-situ versus ex-situ delivery of polyethylenimine-BMP-2 polyplexes for rat calvarial defect repair via intraoperative bioprinting

Gene therapeutic applications combined with bio- and nano-materials have been used to address current shortcomings in bone tissue engineering due to their feasibility, safety and potential capability for clinical translation. Delivery of non-viral vectors can be altered using gene-activated matrices to improve their efficacy to repair bone defects. Ex-situ and in-situ delivery strategies are the most used methods for bone therapy, which have never been directly compared for their potency to repair critical-sized bone defects. In this regard, we first time explore the delivery of polyethylenimine (PEI) complexed plasmid DNA encoding bone morphogenetic protein-2 (PEI-pBMP-2) using the two delivery strategies, ex-situ and in-situ delivery. To realize these gene delivery strategies, we employed intraoperative bioprinting (IOB), enabling us to 3D bioprint bone tissue constructs directly into defect sites in a surgical setting. Here, we demonstrated IOB of an osteogenic bioink loaded with PEI-pBMP-2 for the in-situ delivery approach, and PEI-pBMP-2 transfected rat bone marrow mesenchymal stem cells laden bioink for the ex-situ delivery approach as alternative delivery strategies. We found that in-situ delivery of PEI-pBMP-2 significantly improved bone tissue formation compared to ex-situ delivery. Despite debates amongst individual advantages and disadvantages of ex-situ and in-situ delivery strategies, our results ruled in favor of the in-situ delivery strategy, which could be desirable to use for future clinical applications.

Dual EGFR- and TfR-targeted gene transfer for sodium iodide symporter gene therapy of glioblastoma

Sodium iodide symporter (NIS) gene transfer for active accumulation of iodide in tumor cells is a powerful theranostic strategy facilitating both diagnostic and therapeutic application of radioiodide. In glioblastoma (GBM), the blood-brain barrier (BBB) presents an additional delivery barrier for nucleic acid nanoparticles. In the present study, we designed dual-targeted NIS plasmid DNA complexes containing targeting ligands for the transferrin receptor (TfR) and the epidermal growth factor receptor (EGFR), thus providing the potential for active transport across the BBB followed by targeting of tumor cells. Invitro 125I transfection studies confirmed TfR- and EGFR-dependent transfection efficiency and NIS-specific iodide uptake of dual-targeted polyplexes. Invivo gene transfer in mice bearing orthotopic U87 GBM xenografts was assessed at 48h after intravenous polyplex injection by positron emission tomography (PET) imaging using 18F-labeled tetrafluoroborate (TFB) as tracer. The tumoral 18F-TFB uptake of mice treated with dual-targeted polyplexes (0.56%± 0.08% ID/mL) was significantly higher compared with mice treated with EGFR-mono-targeted (0.33%± 0.03% ID/mL) or TfR-mono-targeted (0.27%± 0.04% ID/mL) polyplexes. In therapy studies, application of 131I induced a superior therapeutic effect of the dual-targeted therapy, demonstrated by a significant delay in tumor growth and prolonged survival.

A Model of Transcriptional Activation of DNA by Loosening of Chromatin Structure

As a model of transcriptional activation of DNA by loosening of chromatin structure, we have developed a polymeric complex of plasmid DNA and a cationic polymer (SRC-Y) bearing peptide moieties that are phosphorylatable by p60c-Src. After phosphorylation, the diffusion constant of DAPI-intercalated plasmid DNA in SRC-Y/DNA particles increased, concomitantly with initiation of transcription, meaning that the access of preinitiation complex, which recruits RNA polymerase II to promoter sequences, to chromatin-packaged DNA for transcriptional initiation is governed by DNA strand mobility.

Fabrication and Characterization of pcDNA3.1(+) Location within Chitosan/Nanoparticles Complexes for Enhanced Gene Delivery.

Chitosan nanoparticles (CSNP) are becoming a popular alternative for delivering nucleic acids to tissues for gene transfer (gene therapy). The size and morphology of these biodegradable nano-carriers are adjustable, and their positive charge allows them to interact strongly with negatively charged nucleic acids. This study aimed to fabricate and characterize pcDNA3.1 (+) plasmid (pDNA) and CSNP complexes and determine the plasmid location in these vehicles. The characteristics of the pDNA/CSNP complex after production were investigated by SEM, XRD, DLS, TGA, and FTIR. The capacity of CSNP to form complexes with pDNA was investigated by labeling free plasmids with the fluorescent intercalating dye OliGreen. The stability of pDNA/CSNP in the presence of chitosanase was evaluated. Surface-Enhanced Raman Spectroscopy (SERS) for pDNA localization was performed, and absorption rate in BALB/c mice was assessed by real-time PCR. The optimum pDNA/CSNP ratio for plasmid complex formation was established to be 1:2 (w.w) by measuring spectroscopy. At these optimum complex formation ratios, spectroscopy, and gel digest experiments, SERS indicated that a part of the pDNA was present on the complex outer surface. The findings of plasmid absorption in mouse thigh tissue by real-time PCR revealed that the rate of gene uptake was significantly greater at a dose of 1:2 (w.w) of pDNA/CSNP than in other groups (P< 0.001). The findings of this study reveal exactly pDNA fits into polymer nanostructured delivery systems, allowing the formulation to be adjusted for selective distribution. This understanding will aid future research into the system's functioning in vitro and in vivo.

Systematic Study of Enzymatic Degradation and Plasmid DNA Complexation of Mucus Penetrating Star-Shaped Lysine/Sarcosine Polypept(o)ides with Different Block Arrangements.

8-Arm star polypep(o)ides comprising cationic polylysine and hydrophilic polysarcosine blocks with a degree of polymerization (DP) of 30 per block are synthesized. Two different block sequences with polylysine as the inner and polysarcosine as the outer block and vice versa are obtained in addition to a statistical copolymer. Analysis of the enzymatic hydrolysis by the proteolytic enzyme trypsin demonstrates a strong dependence on structural arrangements. While polypept(o)ide disintegration is detectible after 24h by Size Exclusion Chromatography (SEC), significant hydrolysis of the lysine blocks is only monitored after 48h by fluorescamine labeling of the produced lysine and clearly accelerated in structures with more accessible polylysine blocks. All structures are capable of complexing plasmid DNA and form gene nanomedicines at sizes around or below 200nm as determined by Dynamic Light Scattering (DLS), Nanoparticle Tracking Analysis (NTA), and Transition Electron Microscopy (TEM). The polyplex formation is slightly enhanced for both block structures over the random copolypept(o)ide. Moreover, it is demonstrated that the polyplexes can transport through mucus. The results highlight the importance of structural control in compartmentalized polymeric gene vector candidates with hydrophilic domains for potential mucosal delivery.

Enhanced Gene Delivery Triggered by Dual pH/Redox Responsive Host-Guest Dimerization of Cyclooligosaccharide Star Polycations.

A robust strategy is reported to build perfectly monodisperse star polycations combining a trehalose-based cyclooligosaccharide (cyclotrehalan, CT) central core onto which oligoethyleneimine radial arms are installed. The architectural perfection of the compounds is demonstrated by a variety of physicochemical techniques, including NMR, MS, DLS, TEM, and GPC. Key to the strategy is the possibility of customizing the cavity size of the macrocyclic platform to enable/prevent the inclusion of adamantane motifs. These properties can be taken into advantage to implement sequential levels of stimuli responsiveness by combining computational design, precision chemistry and programmed host-guest interactions. Specifically, it is shown that supramolecular dimers implying a trimeric CT-tetraethyleneimine star polycation and purposely designed bis-adamantane guests are preorganized to efficiently complex plasmid DNA (pDNA) into transfection-competent nanocomplexes. The stability of the dimer species is responsive to the protonation state of the cationic clusters, resulting in dissociation at acidic pH. This process facilitates endosomal escape, but reassembling can take place in the cytosol then handicapping pDNA nuclear import. By equipping the ditopic guest with a redox-sensitive disulfide group, recapturing phenomena are prevented, resulting in drastically improved transfection efficiencies both in vivo and in vitro.

The Effect of Microcirculation Disturbance of Peribiliary Vascular Plexus on Hepatic Bile Duct Epithelial Cells in Rats Undergoing Liver Transplantation and Postoperative Rejection Reduction by Nanocarrier Mediation.

This study was to investigate the effect of microcirculation disturbance of PVP on HBD epithelial cells in rats undergoing liver transplantation and to explore the postoperative rejection reduction by nanocarriers mediation. For this aim, adult male rats weighing 210-250 g, were fed cleanly and were subjected to liver transplantation. 3 days after the surgery, the rats were randomly divided into three groups based on different intervention factors: group A (HAL), group B (HAMI combined with HAL), and group C (control). The three groups of rats were divided into three subgroups according to the duration of the University of Wisconsin (UW) solution (UW time) used to preserve the donor organs for transplantation, which were 2h, 8h, and 16h, respectively. In addition, the RNA sequence of rat class-II transactivator (CIITA) the rat was searched, and the target interference sequence was designed concerning the RNA. Results showed that the carrier nanoparticles were spherical without obvious oxygen vacancies, the distribution was relatively tight and concentrated, and the main particle size was 50-140 nm. As the mass ratio of HGPAE to DNA increased, the mobility speed of the nanocarrier/shRNA plasmid complex decreased due to the decrease in surface charge. When the mass ratio reached 90:1, the mobility of the complex was completely blocked, suggesting that the DNA was completely compounded. The counts of PCNA, CK-19, F-VIII-Ag, VEGFA, VEGFB, and VEGFC in the 3 groups all showed a downward trend with the increase of UW time; the count in group B was lower than that of groups A and C. In the PCNA count statistics, there was no obvious difference between group A and group B at UW8h, but there were differences in contrast to group C (p<0.05). It was concluded that the blood supply of the microcirculation of the PVP was extremely important for the transplanted liver tissue. When the blood vessels around the HBD of the rat were completely ischemic, the HBD epithelial cells became the most important target of damage, and the proliferation and changes of the HBD epithelial cells can be directly observed. In addition, the nanocarrier-mediated genes were applied to discuss postoperative rejection. The expression of class-II MHC-II gene in nanocarrier CIITA-shRNA was inhibited, which interfered with the recipient's immune recognition of the graft, thereby reducing the intensity of the rejection reaction and relieving the rejection reaction.