Smart arginine-equipped polycationic nanoparticles for p/CRISPR delivery into cells.

An efficient and safe delivery system for the transfection of CRISPR plasmid (p/CRISPR) into target cells can open new avenues for the treatment of various diseases. Herein, we design a novel nonvehicle by integrating an arginine-disulfide linker with low-molecular-weight PEI (PEI1.8k) for the delivery of p/CRISPR. These PEI1.8k-Arg nanoparticles facilitate the plasmid release and improve both membrane permeability and nuclear localization, thereby exhibiting higher transfection efficiency compared to native PEI1.8kin the delivery of nanocomplexes composed of PEI1.8k-Arg and p/CRISPR into conventional cells (HEK 293T). This nanovehicle is also able to transfect p/CRISPR in a wide variety of cells, including hard-to-transfect primary cells (HUVECs), cancer cells (HeLa), and neuronal cells (PC-12) with nearly 5-10 times higher efficiency compared to the polymeric gold standard transfection agent. Furthermore, the PEI1.8k-Arg nanoparticles can edit the GFP gene in the HEK 293T-GFP reporter cell line by delivering all possible forms of CRISPR/Cas9 system (e.g. plasmid encoding Cas9 and sgRNA targeting GFP, and Cas9/sgRNA ribonucleoproteins (RNPs) as well as Cas9 expression plasmid andin vitro-prepared sgRNA) into HEK 293T-GFP cells. The successful delivery of p/CRISPR into local brain tissue is also another remarkable capability of these nanoparticles. In view of all the exceptional benefits of this safe nanocarrier, it is expected to break new ground in the field of gene editing, particularly for therapeutic purposes.

Quince seed mucilage coated iron oxide nanoparticles for plasmid DNA delivery.

This study investigates the potential of iron oxide nanoparticles (Fe3O4) and quince seed mucilage as combined genetic carriers to deliver plasmid DNA (pDNA) through the gastrointestinal system. The samples are characterized by x-ray diffraction (XRD), zeta potential, dynamic light scattering, FT-IR spectroscopy, field emission scanning electron microscopy and vibrating sample magnetometry. The stability of pDNA loading on the nanocarriers and their release pattern are evaluated in simulated gastrointestinal environments by electrophoresis. The XRD patterns reveal that the nanocarriers could preserve their structure during various synthesis levels. The saturation magnetization (Ms) of the Fe3O4cores are 56.48 emu g-1without any magnetic hysteresis. Not only does the loaded pDNA contents experience a remarkable stability in the simulated gastric environment, but also, they could be released up to 99% when exposed to an alkaline environment similar to the intestinal fluid of fish. The results indicate that the synthesized nanoparticles could be employed as efficient low-cost pDNA carriers.

Nucleic Acid Delivery with α-Tocopherol-Polyethyleneimine-Polyethylene Glycol Nanocarrier System.

PURPOSE: Nucleic acid-based therapies are a promising therapeutic tool. The major obstacle in their clinical translation is their efficient delivery to the desired tissue. We developed a novel nanosized delivery system composed of conjugates of α-tocopherol, polyethyleneimine, and polyethylene glycol (TPP) to deliver nucleic acids. METHODS: We synthesized a panel of TPP molecules using different molecular weights of PEG and PEI and analyzed with various analytical approaches. The optimized version of TPP (TPP111 - the 1:1:1 molecular ratio) was self-assembled in water to produce nanostructures and then evaluated in diversified in vitro and in vivo studies. RESULTS: Through a panel of synthesized molecules, TPP111 conjugate components self-assembled in water, forming globular shaped nanostructures of ~90 nm, with high nucleic acid entrapment efficiency. The polymer had low cytotoxicity in vitro and protected nucleic acids from nucleases. Using a luciferase-expressing plasmid, TPP111-plasmid nano-complexes were rapidly up-taken by cancer cells in vitro and induced strong transfection, comparable to PEI. Colocalization of the nano-complexes and endosomes/lysosomes suggested an endosome-mediated uptake. Using a subcutaneous tumor model, intravenously injected nano-complexes preferentially accumulated to the tumor area over 24 h. CONCLUSION: These results indicate that we successfully synthesized the TPP111 nanocarrier system, which can deliver nucleic acids in vitro and in vivo and merits further evaluation.

Targeted double domain nanoplex based on galactosylated polyethylenimine enhanced the delivery of IL-12 plasmid.

The objective of the present investigation was to design a targeted polyethylenimine (PEI)-based polyplex by conjugating lactose bearing galactose groups on low molecular weight PEI (LMW PEI) grafted to a high molecular weight PEI (HMW PEI) via a succinic acid linker in order to restore the amine content of the whole conjugate used for ligand conjugation. The PEI conjugate was synthesized and characterized in terms of buffering capacity, particle size, zeta potential, plasmid condensation ability, and protection of DNA against degrading enzymes. Also, the transfection efficiency and cytotoxicity were evaluated in the cell line over-expressing asialoglycoprotein receptors (ASGPRs) and compared with the cells lacking the receptors. The results demonstrated the ability of PEI conjugate in condensation of plasmid DNA and protection against enzyme degradation. The PEI conjugate formed nanoparticles of around 75 nm with higher buffering capacity compared with unmodified PEI. The polyplexes prepared by the modified PEI could increase the level of transgene up to four folds in the cells over-expressing the receptor. The results demonstrated the separation of targeting and delivery domains could be considered as a strategy to restore the amine content of the PEI molecule utilized for targeting ligand conjugation.

Orthogonal test design for the optimization of superparamagnetic chitosan plasmid gelatin microspheres that promote vascularization of artificial bone.

The optimal conditions for the preparation of superparamagnetic chitosan plasmid (pReceiver-M29-VEGF165/DH5a) gelatin microspheres (SPCPGMs) were determined. Then, the performance of the SPCPGMs during neovascularization was evaluated in vivo. The SPCPGMs were prepared through a cross-linking curing method and then filled into the hollow scaffold of an artificial bone. Neovascularization at the bone defect position was histologically examined in samples collected 2, 4, 6, and 8 weeks after the operation. The cellular magnetofection rate of superparamagnetic chitosan nanoparticles/plasmid (pReceiver-M29-VEGF165/DH5a) complexes reached 1-3% under static magnetic field (SMF). Meanwhile, the optimal conditions for SPCPGM fabrication were 20% Fe3 O4 (w/v), 4 mg of plasmid, 5.3 mg of glutaraldehyde, and 500 rpm of emulsification rotate speed. Under oscillating magnetic fields (OMFs), 4-6 µg of plasmids was released daily for 21 days. Under the combined application of SMF and OMF, evident neovascularization occurred at the bone defect position 6 weeks after the operation. This result is expected to provide a new type of angiogenesis strategy for the research of bone tissue engineering.

Controlling complexation/decomplexation and sizes of polymer-based electrostatic pDNA polyplexes is one of the key factors in effective transfection.

The delivery of plasmid DNA (pDNA) using polycations has been investigated for several decades; however, obstacles that limit efficient gene delivery still hinder the clinical application of gene therapy. One of the major limiting factors is controlling pDNA binding affinity with polymers to control the complexation and decomplexation of polyplexes. To address this challenge, polycations of α-poly(L-lysine) (APL) and ε-poly(L-lysine) (EPL) were used to prepare variable complexation/decomplexation polyplexes with binding affinities ranging from too tight to too loose and sizes ranging from small to large. APL-EPL/ATP-pDNA polyplexes were also prepared to compare the effects of endosomolytic ATP on complexation/decomplexation and the sizes of polyplexes. The results showed that smaller and tighter polyplexes delivered more pDNA into the cells and into the nucleus than the larger and looser polyplexes. Larger polyplexes exhibited slower cytosolic transport and consequently less nuclear delivery of pDNA than smaller polyplexes. Tighter polyplexes exhibited poor pDNA release in the nucleus, leading to no improvement in transfection efficiency. Thus, polyplexes should maintain a balance between complexation and decomplexation and should have optimal sizes for effective cellular uptake, cytosolic transport, nuclear import, and gene expression. Understanding the effects of complexation/decomplexation and size is important when designing effective polymer-based electrostatic gene carriers.

Fluorination effect to intermediate molecular weight polyethylenimine for gene delivery systems.

Fluorinated intermediate molecular weight polyethylenimine (FP2ks) with various fluorination degrees was synthesized by conjugation with heptafluorobutyric anhydride and the fluorination effect for gene delivery systems was examined. FP2ks could condense pDNA, forming compact, positively charged, and nano-sized spherical particles. It was thought that their decreased electrostatic interaction with pDNA would be compensated by hydrophobic interaction. The cytotoxicity of FP2ks was increased with the increase of fluorination degree, probably due to the cellular membrane disruption via hydrophobic interaction with FP2ks. The transfection efficiency of highly fluorinated FP2ks was not severely affected in serum condition, assuming their good serum-compatibility. Discrepancy between their higher cellular uptake efficiency and lower transfection efficiency than PEI25k was thought to arise from the formation of compact polyplexes followed by the decreased dissociation of pDNA. It was also suggested that multiple energy-dependent cellular uptake mechanisms and endosome buffering would mediate the transfection of FP2ks.

Glycopolycation-DNA Polyplex Formulation N/P Ratio Affects Stability, Hemocompatibility, and in Vivo Biodistribution.

Genome editing therapies hold great promise for the cure of monogenic and other diseases; however, the application of nonviral gene delivery methods is limited by both a lack of fundamental knowledge of interactions of the gene-carrier in complex animals and biocompatibility. Herein, we characterize nonviral gene delivery vehicle formulations that are based on diblock polycations containing a hydrophilic and neutral glucose block chain extended with cationic secondary amines of three lengths, poly(methacrylamido glucopyranose- block-2-methylaminoethyl methacrylate) [P(MAG- b-MAEMt)-1, -2, -3]. These polymers were formulated with plasmid DNA to prepare polyelectrolyte complexes (polyplexes). In addition, two controls, P(EG- b-MAEMt) and P(MAEMt), were synthesized, formulated into polyplexes and the ex vivo hemocompatibility, or blood compatibility, and in vivo biodistribution of the formulations were compared to the glycopolymers. While both polymer structure and N/P (amine to phosphate) ratio were important factors affecting hemocompatibility, N/P ratio played a stronger role in determining polyplex biodistribution. P(EG- b-MAEMt) and P(MAEMt) lysed red blood cells at both high and low N/P formulations while P(MAG- b-MAEMt) did not significantly lyse cells at either formulation at short and medium polymer lengths. Conversely, P(MAG- b-MAEMt) did not affect coagulation at N/P = 5, but significantly delayed coagulation at N/P = 15. P(EG- b-MAEMt) and P(MAEMt) did not affect coagulation at either formulation. After polymer and pDNA cargo distribution was observed in vivo, P(EG- b-MAEMt) N/P = 5 and P(MAG- b-MAEMt) N/P = 5 both dissociated and deposited polymer in the liver, while pDNA cargo from P(MAG- b-MAEMt) N/P = 15 was found in the liver, lungs, and spleen. The contrast between P(MAG- b-MAEMt) at N/P = 5 and 15 demonstrates that polyplex stability in the blood can be improved with N/P ratio and potentially aid polyplex biodistribution through simply varying the formulation ratios.

Coalesced thermal and electrotransfer mediated delivery of plasmid DNA to the skin.

Efficient gene delivery and expression in the skin can be a promising minimally invasive technique for therapeutic clinical applications for immunotherapy, vaccinations, wound healing, cancer, and peripheral artery disease. One of the challenges for efficient gene electrotransfer (GET) to skin in vivo is confinement of expression to the epithelium. Another challenge involves tissue damage. Optimizing gene expression profiles, while minimizing tissue damage are necessary for therapeutic applications. Previously, we established that heating pretreatment to 43 °C enhances GET in vitro. We observed a similar trend in vivo, with an IR-pretreatment for skin heating prior to GET. Currently, we tested a range of GET conditions in vivo in guinea pigs with and without preheating the skin to ~43 °C. IR-laser heating and conduction heating were tested in conjunction with GET. In vivo electrotransfer to the skin by moderately elevating tissue temperature can lead to enhanced gene expression, as well as achieve gene transfer in epidermal, dermal, hypodermal and muscle tissue layers.

Photo-responsive hollow silica nanoparticles for light-triggered genetic and photodynamic synergistic therapy.

The development of multifunctional carriers incorporating genetic and photodynamic therapy (PDT) for synergistic antitumor treatment has attracted intensive interests very recently. However, most of the currently reported systems employ passive gene release strategies depending on tumor microenvironment, which are negatively affected by the heterogeneity of cancer cells, thus resulting in limited controllability in therapeutic progress. Herein, a novel photo-responsive hollow silica nanoparticle (HNP)-based gene and photosensitizer (PS) co-delivery nanovehicle is designed for dual-wavelength light-triggered synergistic gene and PDT therapy. The resultant HNP conjugated with PDMAEMA polycation through a 405-nm light-cleavable Cou-linker, namely, HNP-Cou-PD, exhibits excellent gene condensation capacity, good biocompatibility, outstanding PS loading ability, and light-triggered gene release properties. HNP-Cou-PD with Chlorin e6 (Ce6) loaded inside the silica cavity and a plasmid encoding caspase-8 gene (CSP8) attached to the PDMAEMA outside layer (Ce6-HNP-Cou-PD/CSP8) has been proven to possess better antitumor effects under the irradiation of pre-405-nm and post-670-nm light both in vitro and in vivo because of the light-triggered intracellular gene release and reactive oxygen species (ROS) generation. Therefore, HNP-Cou-PD designed as a gene and PS co-delivery carrier might have promising applications in the future to precisely treat various types of cancers. STATEMENT OF SIGNIFICANCE: Multifunctional carriers incorporating genetic and photodynamic therapy (PDT) have drawn intense attention very recently, ascribing to their enhanced anticancer effects. However, in the present gene and PDT synergistic system, gene release strategies passively relying on tumor microenvironment often result in no or poor controllability compared with PDT (a spatial and temporal therapeutic modal), which may hinder their synergistic efficacy, especially in an on-demand manner. To resolve this problem, we designed a hollow silica nanoparticle-based dual-wavelength light-responsive gene and photosensitizer (PS) co-delivery platform to achieve photo-triggered gene and PDT synergistic therapy. We believe that our work may have extensive application prospects in precise treatment of various cancers and be of interest to the readership.

Nanoparticle uptake by circulating leukocytes: A major barrier to tumor delivery.

Decades of research into improving drug delivery to tumors has documented uptake of particulate delivery systems by resident macrophages in the lung, liver, and spleen, and correlated short circulation times with reduced tumor accumulation. An implicit assumption in these studies is that nanoparticles present in the blood are available for distribution to the tumor. This study documents significant levels of lipoplex uptake by circulating leukocytes, and its effect on distribution to the tumor and other organs. In agreement with previous studies, PEGylation dramatically extends circulation times and enhances tumor delivery. However, our studies suggest that this relationship is not straightforward, and that particle sequestration by leukocytes can significantly alter biodistribution, especially with non-PEGylated nanoparticle formulations. We conclude that leukocyte uptake should be considered in biodistribution studies, and that delivery to these circulating cells may present opportunities for treating viral infections and leukemia.

Double domain polyethylenimine-based nanoparticles for integrin receptor mediated delivery of plasmid DNA.

The objective of the present study is to conjugate L-thyroxine PEI derivative onto another PEI to compensate the amine content of the whole structure which has been utilized for the ligand conjugation. Since αvß3 integrin receptors are over-expressed on cancer cells and there is binding site for L-thyroxine on these receptors, PEI conjugation by L-thyroxine along with restoring the PEI amine content might be an efficient strategy for targeted delivery using polymeric nanoparticles. The results demonstrated the ability of the PEI conjugate in the formation of nanoparticles with the size of around 210 nm with higher buffering capacity. The conjugated PEI derivative increased the transfection efficiency in the cell lines over-expressing integrin by up to two folds higher than unmodified PEI, whereas in the cell lines lacking the integrin receptors there was no ligand conjugation-associated difference in gene transfer ability. The specificity of transfection demonstrated the delivery of plasmid DNA through integrin receptors. Also, the results of in vivo imaging of the polyplexes revealed that 99mTc-labeled PEI/plasmid DNA complexes accumulated in kidney and bladder 4 h post injection. Therefore, this PEI derivative could be considered as an efficient targeted delivery system for plasmid DNA.

Comparative Analysis of Polyethyleneimine Efficiency for Improvement of Plasmid DNA Bioavailability.

We studied the influence of the type and structure of polyethyleneimine on bioavailability and expression of plasmid DNA carrying IGF-1 gene. Polymers with different molecular weights (2.5, 10, 25, and 60 kDa) of linear and branching structure were studied. It was found that the time of polyplex circulation in the blood did not exceed 24 h and the maximum concentration of plasmid DNA was attained with complexes with a molecular weight of 60 kDa. Analysis of liver samples showed that administration of 60-kDa branched polyethyleneimine complex provides DNA protection from degradation for 4 h; in 24 h from the start of the experiment, its concentration was significantly higher than the concentration of other studied polyethyleneimines. The expression of plasmid IGF-1 DNA for this complex attained maximum in 4 h and was equal to 15.50 (7.98; 21.98) arb. units/ml. These results allow us to recommend using polyethyleneimines with branched structure and a molecular weight of 60 kDa for improving plasmid DNA protection and bioavailability.

Transfection efficiencies of α-tocopherylated cationic gemini lipids with hydroxyethyl bearing headgroups under high serum conditions.

Herein, five new α-tocopheryl cationic gemini lipids with hydroxyethyl bearing headgroups (THnS, n = 4, 5, 6, 8, 12) have been synthesized for efficient plasmid DNA (pDNA) delivery into cancer cells. Among these gemini lipid formulations, the lipid with an octamethylene [-(CH2)8] spacer (TH8S) showed the highest transfection efficiency (TE) that was comparable to that of the commercial standard lipofectamine 2000 (L2K) in terms of luciferase expression in HepG2 (liver hepatocellular carcinoma) cells. The addition of the helper lipid DOPE (1,2-dioleoyl phosphatidyl ethanolamine) with cationic lipids in mixed liposomes further enhanced the TE and the optimized molar ratio was 2 : 1 (DOPE : cationic lipid). The optimized co-liposomal formulation of TH8S (DOPE : TH8S = 2 : 1) showed a higher TE in HepG2, A549 (human lung carcinoma) and MCF7 (human breast adenocarcinoma) cells than other optimized co-liposomal formulations and was also significantly more potent than L2K. The comparison of the TE of DOPE-TH8S (2 : 1) with the gemini lipid T8T (the headgroup devoid of the hydroxyl group) further demonstrated the importance of the hydroxyethyl functionality at the level of the headgroup. Relatively good binding efficiency and easy release of pDNA (pGL3) were also observed with DOPE-TH8S (2 : 1) in the ethidium bromide (EB)-exclusion and re-intercalation assay, which may be the plausible reason for high TE. The lipoplexes were also characterized by atomic force microscopy (AFM), dynamic light scattering (DLS), zeta potential and small angle X-ray diffraction experiments. Greater cellular internalization of fluorescein tagged pDNA was also observed with DOPE-TH8S (2 : 1) lipoplexes compared to that with L2K. Retention of the TE of DOPE-TH8S (2 : 1) lipoplexes under high serum conditions was conferred by the presence of the tocopherol backbone and also the hydroxyethyl functionalities. The cellular internalization pathway of the lipoplexes was characterized by performing transfection experiment in the presence of inhibitors of different endocytic pathways and it was found to be caveolae mediated. An MTT based cell viability assay indicated that the lipoplex mediated gene delivery vectors exhibited low toxicity in all the three cancer cell lines studied.

Resistencia plasmídica a colistin por el gen mcr-1 en Enterobacteriaceae en Paraguay / Plasmid-Mediated Colistin Resistance Gene mcr-1 in Enterobacteriaceae in Paraguay

La resistencia a las polimixinas mediada por plásmidos (gen mcr-1) representa una amenaza para la salud pública, puesto que colistina es utilizada en la práctica médica como una de las últimas alternativas para el tratamiento de gérmenes multiresistentes. Este estudio describe la circulaciónde cepas de Enterobacterias que portan este gen de resistencia, aisladas de pacientes hospitalizados, así como también de la comunidad. Los hallazgos de la Red de Vigilancia de la Resistencia a los Antimicrobianos-Paraguay fueron de casi el 5 % (4,7) en cepas remitidas con criterio de sospecha, siendo las especies involucradas Escherichiacoli, Klebsiella pneumoniae y Salmonella Schwarzengrund. Además, por métodos moleculares se confirmaron en todas ellas la portación de otros genes de resistencia (KPC, CTX-M, Qnr B, Qnr S, aac (6`)-Ib-cr) asociados al mcr-1. Palabras claves: Enterobacterias, resistencia, colistina, mcr-1.

Resistance to polymyxins mediated by plasmids (mcr-1 gene) represents a threat to public health, since colistin is used in medical practice, as one of the last alternatives, for the treatment of multi-resistant germs. This study describes the circulation of strains of Enterobacteria that carry this resistance gene, isolated from hospitalized patients, as well as from the community. The findings of the Red de Vigilancia de la Resistencia a los Antimicrobianos­Paraguay were almost 5% (4.7) in strains submitted with suspicion criteria; the species involved being Escherichia coli, Klebsiella pneumoniae and Salmonella Schwarzengrund. In addition, molecular methods confirmed in all of them the carrying of other resistance genes (KPC, CTX-M, Qnr B, Qnr S, aac (6`)-Ib-cr) associated with mcr-1. Key words: Enterobacteria, resistance, colistin, mcr-1.

Physicochemical stability and transfection efficiency of cationic amphiphilic copolymer/pDNA polyplexes for spinal cord injury repair.

Multiple age-related and injury-induced characteristics of the adult central nervous system (CNS) pose barriers to axonal regeneration and functional recovery following injury. In situ gene therapy is a promising approach to address the limited availability of growth-promoting biomolecules at CNS injury sites. The ultimate goal of our work is to develop, a cationic amphiphilic copolymer for simultaneous delivery of drug and therapeutic nucleic acids to promote axonal regeneration and plasticity after spinal cord injury. Previously, we reported the synthesis and characterization of a cationic amphiphilic copolymer, poly (lactide-co-glycolide)-graft-polyethylenimine (PgP) and its ability to efficiently transfect cells with pDNA in the presence of serum. We also demonstrated the efficacy of PgP as a therapeutic siRhoA carrier in a rat compression spinal cord injury model. In this work, we show that PgP/pDNA polyplexes provide improved stability in the presence of competing polyanions and nuclease protection in serum relative to conventional branched polyethylenimine control. PgP/pDNA polyplexes maintain bioactivity for transfection after lyophilization/reconstitution and during storage at 4 °C for up to 5 months, important features for commercial and clinical application. We also demonstrate that PgP/pDNA polyplexes loaded with a hydrophobic fluorescent dye are retained in local neural tissue for up to 5 days and that PgP can efficiently deliver pß-Gal in a rat compression SCI model.

Gene therapy for patients with advanced solid tumors: a phase I study using gene electrotransfer to muscle with the integrin inhibitor plasmid AMEP.

BACKGROUND: Gene electrotrotransfer describes the use of electric pulses to transfer DNA to cells. Particularly skeletal muscle has potential for systemic secretion of therapeutic proteins. Gene electrotransfer to muscle using the integrin inhibitor plasmid AMEP (Antiangiogenic MEtargidin Peptide) was investigated in a phase I dose escalation study. Primary objective was safety. MATERIAL AND METHODS: Patients with metastatic or locally advanced solid tumors, without further standard treatments available, were treated with once-only gene electrotransfer of plasmid AMEP to the femoral muscle. Safety was monitored by adverse events registration, visual analog scale (VAS) after procedure and magnetic resonance imaging (MRI) of treated muscles. Pharmacokinetics of plasmid AMEP in plasma and urine was determined by quantitative polymerase chain reaction. Response was evaluated by positron emission tomography-computed tomography (PET-CT) scans. RESULTS: Seven patients were enrolled and treated at dose levels from 50 to 250 µg of plasmid AMEP, the study was terminated early due to cessation of plasmid production. Minimal systemic toxicity was observed and only transient mild pain was associated with the delivery of the electric pulses. MRI of the treated muscles revealed discrete intramuscular edema 24 h after treatment. The changes in the muscle tissue resolved within 2 weeks after treatment. Peak concentrations of plasmid AMEP was detected only in plasma within the first 24 hours after injection. Protein AMEP could not be detected, which could be due to the limit of detection. No objective responses were seen. CONCLUSIONS: Gene electrotransfer of plasmid AMEP was found to be safe and tolerable. No objective responses were observed but other DNA drugs may be tested in the future using this procedure.

A phase 1/2a, dose-escalation, safety, pharmacokinetic, and preliminary efficacy study of intraperitoneal administration of BC-819 (H19-DTA) in subjects with recurrent ovarian/peritoneal cancer.

BACKGROUND: H19 is a paternally imprinted, oncofetal gene expressed in various embryonic tissues and in 85% of the ovarian tumors. H19-DTA (BC-819) is a DNA plasmid that drives the expression of the diphtheria toxin gene under the regulation of the H19 promoter sequence and therefore is a potential treatment for various tumors that overexpress the H19 gene, among them-ovarian cancer. OBJECTIVE: To assess the safety and efficacy of intra-peritoneal (IP) instillations of H19-DTA (BC-819) plasmid in treating ovarian/peritoneal cancer patients with advanced recurrent disease. METHODS: A phase 1-2A multi-centric trial included 14 eligible patients who were either platinum-refractory or platinum-resistant with positive H19 expression. Patients were treated IP with escalating weekly doses of BC-819 for a maximum of 6-9 weeks. Dose-limiting toxicities (DLT) were assessed after the first course of treatment for each patient and each subsequent cohort was enrolled once each subject had completed the first course of treatment and its 4-week follow-up period. The occurrence of adverse events (AEs) and response to treatment were assessed after the induction course and then periodically. RESULTS: During the study, no DLTs were observed. Only 5 grade 1 and 2 AEs, which occurred in 4 patients were considered as possibly related to BC-819. The best tumor response seen was stable disease. Median survivals of 3.2, 5.3 and 6.5 months were observed for the 60, 120 and 240 mg cohorts, respectively. CONCLUSIONS: BC-819 can be considered safe and well tolerated in intraperitoneal doses up to 240 mg. Hybridization of intraperitoneal chemotherapy with the biological treatment of BC-819 should be further evaluated in phase 2 and 3 studies.

Cationic Polyarginine Conjugated Mesoporous Bioactive Glass Nanoparticles with Polyglycerol Coating for Efficient DNA Delivery.

Mesoporous bioactive glass (MBG) is a type of material with high biological activity and excellent biocompatibility. Because of its high specific surface area and adjustable surface morphology, MBG is usable for loading and delivering molecules. In our previous report, MBG particles were used as gene vectors and showed good transfection rate. In this paper, MBG, prepared through a sacrificial liquid template method in sol­gel process, was covered with polyglycerol (PG) and the resulting MBG-PG was further functionalized with octaarginine (Arg8. More specifically, MBG-PG-Arg8 particles were synthesized by PG functionalization of MBG through ring-opening polymerization of glycidol on the MBG surface, followed by multistep organic transformations (­OH→ ­OTs (tosylate)→ ­N3 in the PG layer and click conjugation of the Arg8 terminated with propargyl glycine. MBG-PG-Arg8 was successfully taken up by cells more efficiently due to the cellpenetrating property of Arg8, and thus showed higher plasmid DNA loading and cell transfection efficiency than MBG modified with amino groups. This novel arginine-functionalized MBG may be a good candidate as a vector for gene delivery with higher efficiency.

Quantitative study of effects of free cationic chains on gene transfection in different intracellular stages.

Previously, we revealed that in the application of using cationic polymer chains, polyethylenimine (PEI), to condense anionic plasmid DNA chains (pDNA) to form the DNA/polymer polyplexes, after all the pDNAs are complexed with PEI, further added PEIs exist individual chains and free in the solution mixture. It is those uncomplexed polycation chains that dramatically promote the gene transfection. In the current study, we studied how those free cationic chains with different lengths and topologies affect the intracellular trafficking of the polyplexes, the translocation of pDNA through the nuclear membrane, the transcription of pDNA to mRNA and the translocation of mRNA from nucleus to cytosol in HepG2 cells by using a combination of the three-dimensional confocal microscope and TaqMan real-time PCR. We found that free branched PEI chains with a molar mass of 25,000g/mol and a total concentration of 1.8×10(-6)g/mL promote the overall gene transfection efficiency by a factor of ~500 times. Our results quantitatively reveal that free chains help little in the cellular uptake, but clearly reduce the lysosomal entrapment of those internalized polyplexes (2-3 folds); assist the translocation of pDNA through nuclear membrane after it is released from the polyplexes in the cytosol (~5 folds); enhance the pDNA-to-mRNA transcription efficiency (~4 folds); and facilitate the nucleus-to-cytosol translocation of mRNA (7-8 folds). The total enhancement of those steps agrees well with the overall efficiency, demonstrating, for the first time, how free cationic polymer chains quantitatively promote the gene transfection in each step in the intracellular space.