Gene Delivery Reagents Research Articles

WS17.04 Developing a non-viral gene therapy strategy for treating lung cystic fibrosis disease

Objective: Currently there is no cure for cystic fibrosis (CF) disease and lung is the most affected organ [ 1 Cooney A Gene. 2018 Nov; 9: 538 Crossref Scopus (68) Google Scholar ]. Gene replacement is a promising strategy to treat this disease. However, the two big challenges that have impeded its success are the absence of: (1) an efficient and safe gene delivery reagent to transfect the target cells, and (2) a stable plasmid that enables high and sustained CF transmembrane conductance regulator (CFTR) expression. Thus, the aim of this work is to develop a non-viral CFTR gene replacement system for lung CF disease to overcome these two challenges.

Machine learning guided structure function predictions enable in silico nanoparticle screening for polymeric gene delivery

Developing highly efficient non-viral gene delivery reagents is still difficult for many hard-to-transfect cell types and, to date, has mostly been conducted via brute force screening routines. High throughput in silico methods of evaluating biomaterials can enable accelerated optimization and development of devices or therapeutics by exploring large chemical design spaces quickly and at low cost. This work reports application of state-of-the-art machine learning algorithms to a dataset of synthetic biodegradable polymers, poly(beta-amino ester)s (PBAEs), which have shown exciting promise for therapeutic gene delivery in vitro and in vivo. The data set includes polymer properties as inputs as well as polymeric nanoparticle transfection performance and nanoparticle toxicity in a range of cells as outputs. This data was used to train and evaluate several state-of-the-art machine learning algorithms for their ability to predict transfection and understand structure-function relationships. By developing an encoding scheme for vectorizing the structure of a PBAE polymer in a machine-readable format, we demonstrate that a random forest model can satisfactorily predict DNA transfection in vitro based on the chemical structure of the constituent PBAE polymer in a cell line dependent manner. Based on the model, we synthesized PBAE polymers and used them to form polymeric gene delivery nanoparticles that were predicted in silico to be successful. We validated the computational predictions in two cell lines in vitro, RAW 264.7 macrophages and Hep3B liver cancer cells, and found that the Spearman's R correlation between predicted and experimental transfection was 0.57 and 0.66 respectively. Thus, a computational approach that encoded chemical descriptors of polymers was able to demonstrate that in silico computational screening of polymeric nanomedicine compositions had utility in predicting de novo biological experiments. Statement of significanceDeveloping highly efficient non-viral gene delivery reagents is difficult for many hard-to-transfect cell types and, to date, has mostly been explored via brute force screening routines. High throughput in silico methods of evaluating biomaterials can enable accelerated optimization and development for therapeutic or biomanufacturing purposes by exploring large chemical design spaces quickly and at low cost. This work reports application of state-of-the-art machine learning algorithms to a large compiled PBAE DNA gene delivery nanoparticle dataset across many cell types to develop predictive models for transfection and nanoparticle cytotoxicity. We develop a novel computational pipeline to encode PBAE nanoparticles with chemical descriptors and demonstrate utility in a de novo experimental context.

TMOD-03. A SWINE MODEL OF GLIOBLASTOMA INDUCED BY SOMATIC GENE MODIFICATION

Abstract Glioblastoma (GBM) is the most common and malignant primary brain tumor. Novel therapeutic development for GBM is desperately needed, as the standard of care universally fails to cure patients and the 5-year survival rate remains extremely low. GBM therapeutic development is hampered by the lack of relevant preclinical models for preclinical studies. To mitigate this problem, we have developed a genetic model of GBM in outbred, immune-proficient swine which have comparable brain size and anatomy to humans. We developed methods for introducing genome engineering tools to minipig brain in vivo by direct injection of gene delivery reagents to the lateral ventricle, altering major signaling pathways frequently changed in human GBM. Using this technique, we have delivered a combination of expression vectors for oncogenes and targeted nucleases to disrupt tumor suppressor genes commonly altered in human GBM. We have altered six major human GBM-associated signaling pathways and modeled molecular GBM subclasses. We have also engineered a secreted tumor reporter that can be used to monitor tumor size through a simple blood test. This somatic cell gene-modification platform we have developed in the minipig allows us to reproduce the genetic heterogeneity seen in GBM and understand the impact of the tumor microenvironment, immune system, and response to therapy. This minipig model of GBM Is being used to test the standard of care against novel therapies in preclinical studies, and biopsy, surgical, imaging, and radiation therapy techniques are being optimized in this mode to improve clinical trial success rates and patient outcomes. Funding for this study is provided by the National Institutes of Health though SBIR grant # 1R43CA235837-01A1.

Progress of cationic gene delivery reagents for non-viral vector.

Gene delivery systems play a vital role in gene therapy and recombinant protein production. The advantages of using gene delivery reagents for non-viral vector include the capacity to accommodate a large packaging load and their low or absent immunogenicity. Furthermore, they are easy to produce at a large scale and preserve. Gene delivery reagents for non-viral vector are commonly used for transfecting a variety of cells and tissues. It is mainly composed of liposomes and non-liposome cationic polymers. According to the different head structures used, the non-viral cationic transfection reagents include a quaternary ammonium salt, amine, amino acid or polypeptide, guanidine salt, and a heterocyclic ring. This article summarizes these approaches and developments of types and components of transfection reagents and optimization of gene delivery. The optimization of mammalian cell transient recombinant protein expression system and cationic reagents for clinical or clinical trials are also discussed.

TMOD-20. A SWINE MODEL OF GLIOBLASTOMA INDUCED BY SOMATIC GENE MODIFICATION

Abstract Glioblastoma (GBM) is the most common and malignant primary brain tumor. Novel therapeutic development for GBM is needed since the standard of care universally fails to cure patients and the five-year survival rate remains below 10%. GBM therapeutic development is hampered by the lack of relevant large animal models for preclinical studies. To mitigate this problem, we are developing a model of GBM in outbred, immune-proficient swine which have comparable brain size and anatomy to humans. We developed methods for introducing genome engineering tools to minipig brain cells in vivo by direct injection of gene delivery reagents to the lateral ventricle. Using this technique, we have delivered a combination of expression vectors for oncogenes and targeted nucleases to disrupt tumor suppressor genes commonly altered in human GBM to alter six major human GBM-associated signaling pathways in a cohort of minipigs (Ras, Pi3k, p53, Rb/E2F, Pdgf, and the alternative lengthening of telomeres (ALT) pathways). These minipigs are being monitored for tumorigenesis using a secreted reporter, detectable through a simple luminescence-based blood test. Resulting tumors will be examined molecularly to detect the pathway-associated alterations in tumor tissue and determine the resemblance to human GBM. We hypothesize that this somatic cell gene-modification platform we have developed in the minipig will facilitate the efficient production of brain tumors that histologically and genetically resemble human GBM. It will allow the production of tumors that are genetically heterogeneous, of specified molecular subclasses, containing therapeutic targets of interest, and in the context of genetic backgrounds of interest. This minipig model of GBM will be applied towards preclinical therapeutic studies, imaging studies using human clinical grade equipment, and surgical technique development, to improve clinical trial success rates and patient outcomes. Funding for this study is provided by the National Institutes of Health through SBIR grant # 1R43CA235837-01A1.

Dual Gene Delivery Reagents From Antiproliferative Alkylphospholipids for Combined Antitumor Therapy.

Alkylphospholipids (APLs) have elicited great interest as antitumor agents due to their unique mode of action on cell membranes. However, their clinical applications have been limited so far by high hemolytic activity. Recently, cationic prodrugs of erufosine, a most promising APL, have been shown to mediate efficient intracellular gene delivery, while preserving the antiproliferative properties of the parent APL. Here, cationic prodrugs of the two APLs that are currently used in the clinic, miltefosine, and perifosine, are investigated and compared to the erufosine prodrugs. Their synthesis, stability, gene delivery and self-assembly properties, and hemolytic activity are discussed in detail. Finally, the potential of the pro-miltefosine and pro-perifosine compounds ME12 and PE12 in combined antitumor therapy is demonstrated using pUNO1-hTRAIL, a plasmid DNA encoding TRAIL, a member of the TNF superfamily. With these pro-APL compounds, we provide a proof of concept for a new promising strategy for cancer therapy combining gene therapy and APL-based chemotherapy.

Hydrodynamics-Based Transplacental Delivery as a Useful Noninvasive Tool for Manipulating Fetal Genome.

We previously demonstrated that the injection of pregnant wild-type female mice (carrying enhanced green fluorescent protein (EGFP)-expressing transgenic fetuses) at embryonic day (E) 12.5 with an all-in-one plasmid conferring the expression of both Cas9 and guide RNA (targeted to the EGFP cDNA) complexed with the gene delivery reagent, resulted in some fetuses exhibiting reduced fluorescence in their hearts and gene insertion/deletion (indel) mutations. In this study, we examined whether the endogenous myosin heavy-chain α (MHCα) gene can be successfully genome-edited by this method in the absence of a gene delivery reagent with potential fetal toxicity. For this, we employed a hydrodynamics-based gene delivery (HGD) system with the aim of ensuring fetal gene delivery rates and biosafety. We also investigated which embryonic stages are suitable for the induction of genome editing in fetuses. Of the three pregnant females injected at E9.5, one had mutated fetuses: all examined fetuses carried exogenous plasmid DNA, and four of 10 (40%) exhibited mosaic indel mutations in MHCα. Gene delivery to fetuses at E12.5 and E15.5 did not cause mutations. Thus, the HGD-based transplacental delivery of a genome editing vector may be able to manipulate the fetal genomes of E9.5 fetuses.

Intratracheal aerosolization of viral vectors to newborn pig airways.

Gene therapy for airway diseases requires efficient delivery of nucleic acids to the airways. In small animal models, gene delivery reagents are commonly delivered as a bolus dose. However, large animal models are often more relevant for the transition from preclinical studies to human trials. Aerosolizing viral vectors to the lungs of large animals can maximize anatomical distribution. Here, we describe a technique for aerosolization of viral vectors to the airways of newborn pigs. Briefly, a pig is anesthetized and intubated with an endotracheal tube, and a microsprayer is passed through the endotracheal tube. A fine mist is then sprayed into the distal trachea. Widespread and uniform distribution of transgene expression is critical for developing successful lung gene therapy treatments.

New-Generation Benzimidazole-Based Plasmid Delivery Reagents with High Transfection Efficiencies on the Mammalian Cells.

Gene transfer and gene therapy studies require high-efficiency gene delivery reagents. By transferring the piece of DNA that we are interested in, we can alter the expression of certain gene or genes to further characterize its role in the cell function or in the organism's development, metabolism, immune system, etc. Transfection reagents that enable efficient delivery of the DNA to the cells are important tools in the molecular and cellular biology studies. There are chemical products and tools that have been used for transfection of the cells but they are not as efficient as desired or they can induce cytotoxicity. It is crucial to design and generate new transfection reagents to further support the field of biotechnology, molecular studies, cellular biology, and in vitro studies relying on them. The more efficient and the less cytotoxic compounds will be especially useful for the field. We synthesized a new set of benzimidazole-based transfection reagents that have higher efficiency to carry GFP expressing plasmid in to the mammalian cells compared with the commercially available ones with low cytotoxicity. GFP expression levels were tracked by flow cytometry to determine the transfection efficiencies. Benzimidazole-based transfection reagents can be safely used for transfection studies in tissue culture as well as in gene therapy applications due to their high efficiency in the gene transfer to the mammalian cells.

TMOD-17. A SWINE MODEL OF GLIOBLASTOMA INDUCED BY SOMATIC GENE MODIFICATION

Abstract Glioblastoma (GBM) is the most common and malignant primary brain tumor. New, effective treatments for GBM are needed since the standard of care universally fails to cure patients. A major hurdle for GBM therapeutic development is the lack of relevant large animal models with high translational value for preclinical studies. To overcome this, we are developing a model of GBM in outbred, immune-proficient swine which have comparable brain size and anatomy to humans. GBM tumors will be driven by somatic alterations to major signaling pathways frequently altered in human GBM and will express a secreted reporter for tumor growth detectable in the peripheral blood. These alterations and reporter expression cassettes will be induced by delivering transposons and CRISPR/Cas9 gene-editing tools to the subventricular brain cells of live swine by stereotactic injections. We have developed and deployed these gene-editing tools, demonstrating their efficacy in swine cells. We have identified stereotactic coordinates to reproducibly target the lateral ventricles of neonatal swine brains and have demonstrated successful plasmid delivery to cells at these coordinates. Stable, persistent transposon integration and clonal expansion of modified cells has been confirmed by sequencing transposon-genomic DNA junctions in brain tissue six-months post-injection. To induce GBM-like tumors, we have introduced gene delivery and gene editing reagents to alter six major human GBM-associated signaling pathways in a cohort of swine. Resulting tumors will be examined molecularly to detect the pathway-associated transposons and CRISPR/Cas9-induced mutations in tumor tissue and determine the resemblance to human GBM. This somatic cell gene-modification platform will be adaptable, allowing on-demand inclusion of preclinical study-relevant alterations in GBM tumors, and will allow the rapid development tumors histologically and genetically similar to human GBM, which will be valuable for use in pre-clinical therapeutic studies, imaging studies using human clinical grade equipment, and surgical technique development.

Erufosine (ErPC3) Cationic Prodrugs as Dual Gene Delivery Reagents for Combined Antitumor Therapy.

Sixteen cationic prodrugs of the antitumor alkylphospholipid (APL) erufosine were rationally synthesized to provide original gene delivery reagents with improved cytotoxicity profile. The DNA complexation properties of these cationic lipids were determined and associated transfection rates were measured. Furthermore, the self-assembly properties of the pro-erufosine compounds were investigated and their critical aggregation concentration was determined. Their hydrolytic stability under pH conditions mimicking the extracellular environment and the late endosome milieu was measured. Hemolytic activity and cytotoxicity of the compounds were investigated. The results obtained in various cell lines demonstrate that the prodrugs of erufosine display antineoplastic activity similar to that of the parent antitumor drug but are not associated with hemolytic toxicity, which is a dose-limiting side effect of APLs and a major obstacle to their use in anticancer therapeutic regimen. Furthermore, by using lipoplexes prepared from a prodrug of erufosine and a plasmid DNA encoding a pro-apoptotic protein (TRAIL), evidence was provided for selective cytotoxicity towards tumor cells while nontumor cells were resistant. This study demonstrates that the combination approach involving well tolerated erufosine cationic prodrugs and cancer gene therapy holds significant promise in tumor therapy.

Influence of carbonization conditions on luminescence and gene delivery properties of nitrogen-doped carbon dots.

Carbon dots (CDs) have been intensively investigated due to their unique photoluminescence (PL) properties that are improved through surface passivation with nitrogen-containing groups. Recently, gene delivery applications emerged as passivation of CDs may yield positively charged nanoparticles that can interact with negatively charged nucleic acids. However previous work in the field focused on the use of high molecular weight polyamines for CD passivation, posing the problem of the separation of nanoparticles from residual polymer that is harmful to cells. In this work, cationic CDs were prepared by pyrolysis of citric acid/bPEI600 (1/4, w/w) so unreacted low molecular weight reagents could be conveniently eliminated by extensive dialysis. Various reaction conditions and activation modes were evaluated and eleven CDs that exhibited superior solubility in water were produced. All the nanoparticles were characterized with respect to their physical, optical and PL properties and their ability to deliver plasmid DNA to mammal cells was evaluated. Despite their similar physical properties, the CDs displayed marked differences in their gene delivery efficiency. CDs produced under microwave irradiation in a domestic oven were revealed to be superior to all the other nanoparticles produced in this study and compared to the gold standard transfection reagent bPEI25k, with an optimal CD/pDNA w/w ratio that was significantly down shifted, as was the associated cytotoxicity.

Polymer functionalized gold nanoparticles as nonviral gene delivery reagents.

In the present study, we investigated the ability of polyethylene glycol (PEG) functionalized gold nanoparticles to function as nonviral vectors in the transfection of different cell lines, comparing them with commercial lipoplexes. Positively-charged gold nanoparticles were synthesized using polyethylenimine (PEI) as a reducing and stabilizer agent and its cytotoxicity was reduced by its functionalization with PEG. We bound the nanoparticles to three plasmids with different sizes (4-40 kpb). Vector internalization was evaluated by confocal and electronic microscopy. Its transfection efficacy was studied by fluorescence microscopy and flow cytometry. The application of the resulting vector in gene therapy was evaluated indirectly using ganciclovir in HeLa cells transfected to express the herpes virus thymidine kinase. An appropriate ratio between the nitrogen from the PEI and the phosphorous from the phosphate groups of the DNA, together with a reduced size and an elevated electrokinetic potential, are responsible for an increased nanoparticle internalization and enhanced protein expression when carrying plasmids of up to 40 kbp (plasmid size close to the limit of the DNA-carrying capacity of viral vectors). Compared to a commercial transfection reagent, an equal or even higher expression of reporter genes (on HeLa and Hek293t) and a suicide effect on HeLa cells transfected with the herpes virus thymidine kinase gene were observed when using this novel nanoparticulated vector. Nonviral vectors based on gold nanoparticles covalently coupled with PEG and PEI can be used as efficient transfection reagents showing expression levels that are the same or greater than those obtained with commercially available lipoplexes.

Liver Lobe and Strain Difference in Gene Expression After Hydrodynamics-Based Gene Delivery in Mice

Hydrodynamics-based gene delivery (HGD) is a widely recognized technique for delivering exogenous DNA with high efficiency to murine hepatocytes. In this study, we investigated stimulation of exogenous DNA uptake and expression using a commercially available reagent for HGD. We also examined which mouse strain and mouse liver lobe would achieve the best gene delivery performance. Mice were injected with a solution containing reporter plasmid DNA or DNA and a gene delivery reagent. One day after the HGD procedure, liver samples were isolated and subjected to biochemical and histochemical analyses. The reporter plasmid DNA showed the strongest signal when the DNA was dissolved in TransIT-EE Hydrodynamic Delivery Solution (Takara Bio Inc., Shiga, Japan). Evaluation of transgene expression in each hepatic lobe in ICR, C57BL/6N, Balb/cA, and B6C3F1 mice showed that ICR mice exhibited the best gene transfer and that the right median lobe had the highest level of transgene expression. These findings suggest the importance of choice in mouse strains and liver lobes when performing gene-based manipulations of the liver.

Polyethyleneimine is a potent systemic adjuvant for glycoprotein antigens.

Polyethyleneimine (PEI) is an organic polycation used extensively as a gene and DNA vaccine delivery reagent. Although the DNA targeting activity of PEI is well documented, its immune activating activity is not. We recently reported that PEI has robust mucosal adjuvanticity when administered intranasally with glycoprotein antigens. Here, we show that PEI has strong immune activating activity after systemic delivery. PEI administered subcutaneously with viral glycoprotein (HIV-1 gp140) enhanced antigen-specific serum IgG production in the context of mixed Th1/Th2-type immunity. PEI elicited higher titers of both antigen binding and neutralizing antibodies than alum in mice and rabbits and induced an increased proportion of antibodies reactive with native antigen. In an intraperitoneal model, PEI recruited neutrophils followed by monocytes to the site of administration and enhanced antigen uptake by antigen-presenting cells. The Th bias was modulated by PEI activation of the Nlrp3 inflammasome; however its global adjuvanticity was unchanged in Nlrp3-deficient mice. When coformulated with CpG oligodeoxynucleotides, PEI adjuvant potency was synergistically increased and biased toward a Th1-type immune profile. Taken together, these data support the use of PEI as a versatile systemic adjuvant platform with particular utility for induction of secondary structure-reactive antibodies against glycoprotein antigens.

Delivery of small interfering RNAs in human cervical cancer cells by polyethylenimine-functionalized carbon nanotubes

Carbon nanotubes are capable of penetrating the cell membrane and are widely considered as potential carriers for gene or drug delivery. Because the C-C and C=C bonds in carbon nanotubes are nonpolar, functionalization is required for carbon nanotubes to interact with genes or drugs as well as to improve their biocompatibility. In this study, polyethylenimine (PEI)-functionalized single-wall (PEI-NH-SWNTs) and multiwall carbon nanotubes (PEI-NH-MWNTs) were produced by direct amination method. PEI functionalization increased the positive charge on the surface of SWNTs and MWNTs, allowing carbon nanotubes to interact electrostatically with the negatively charged small interfering RNAs (siRNAs) and to serve as nonviral gene delivery reagents. PEI-NH-MWNTs and PEI-NH-SWNTs had a better solubility in water than pristine carbon nanotubes, and further removal of large aggregates by centrifugation produced a stable suspension of reduced particle size and improved homogeneity and dispersity. The amount of grafted PEI estimated by thermogravimetric analysis was 5.08% (w/w) and 5.28% (w/w) for PEI-NH-SWNTs and PEI-NH-MWNTs, respectively. For the assessment of cytotoxicity, various concentrations of PEI-NH-SWNTs and PEI-NH-MWNTs were incubated with human cervical cancer cells, HeLa-S3, for 48 h. PEI-NH-SWNTs and PEI-NH-MWNTs induced cell deaths in a dose-dependent manner but were less cytotoxic compared to pure PEI. As determined by electrophoretic mobility shift assay, siRNAs directed against glyceraldehyde-3-phosphate dehydrogenase (siGAPDH) were completely associated with PEI-NH-SWNTs or PEI-NH-MWNTs at a PEI-NH-SWNT/siGAPDH or PEI-NH-MWNT/siGAPDH mass ratio of 80:1 or 160:1, respectively. Furthermore, PEI-NH-SWNTs and PEI-NH-MWNTs successfully delivered siGAPDH into HeLa-S3 cells at PEI-NH-SWNT/siGAPDH and PEI-NH-MWNT/siGAPDH mass ratios of 1:1 to 20:1, resulting in suppression of the mRNA level of GAPDH to an extent similar to that of DharmaFECT, a common transfection reagent for siRNAs. Our results indicate that the PEI-NH-SWNTs and PEI-NH-MWNTs produced in this study are capable of delivering siRNAs into HeLa-S3 cells to suppress gene expression and may therefore be considered as novel nonviral gene delivery reagents.

Poly(β-Amino Ester)-Nanoparticle Mediated Transfection of Retinal Pigment Epithelial Cells In Vitro and In Vivo

A variety of genetic diseases in the retina, including retinitis pigmentosa and leber congenital amaurosis, might be excellent targets for gene delivery as treatment. A major challenge in non-viral gene delivery remains finding a safe and effective delivery system. Poly(beta-amino ester)s (PBAEs) have shown great potential as gene delivery reagents because they are easily synthesized and they transfect a wide variety of cell types with high efficacy in vitro. We synthesized a combinatorial library of PBAEs and evaluated them for transfection efficacy and toxicity in retinal pigment epithelial (ARPE-19) cells to identify lead polymer structures and transfection formulations. Our optimal polymer (B5-S5-E7 at 60 w/w polymer∶DNA ratio) transfected ARPE-19 cells with 44±5% transfection efficacy, significantly higher than with optimized formulations of leading commercially available reagents Lipofectamine 2000 (26±7%) and X-tremeGENE HP DNA (22±6%); (p<0.001 for both). Ten formulations exceeded 30% transfection efficacy. This high non-viral efficacy was achieved with comparable cytotoxicity (23±6%) to controls; optimized formulations of Lipofectamine 2000 and X-tremeGENE HP DNA showed 15±3% and 32±9% toxicity respectively (p>0.05 for both). Our optimal polymer was also significantly better than a gold standard polymeric transfection reagent, branched 25 kDa polyethyleneimine (PEI), which achieved only 8±1% transfection efficacy with 25±6% cytotoxicity. Subretinal injections using lyophilized GFP-PBAE nanoparticles resulted in 1.1±1×103-fold and 1.5±0.7×103-fold increased GFP expression in the retinal pigment epithelium (RPE)/choroid and neural retina respectively, compared to injection of DNA alone (p = 0.003 for RPE/choroid, p<0.001 for neural retina). The successful transfection of the RPE in vivo suggests that these nanoparticles could be used to study a number of genetic diseases in the laboratory with the potential to treat debilitating eye diseases.

Encapsulation of CdTe quantum dots in polyvinylpyrrolidone nanoparticle for live cell imaging

Highly luminescent and water-soluble CdTe quantum dots encapsulated in polyvinylpyrrolidone nanoparticles (QDs/PNs) have been synthesised by one-pot aqueous route. Fluorescence of the as-prepared QDs/PNs was tunable from 539 to 606 nm and photoluminescent quantum yield could reach up to 47%. High quality of QDs/PNs was also observed by narrow full-width at half-maximum as small as 36 nm in the fluorescence spectra. Using fluorescence microscopy, we demonstrated uptake of QDs/PNs by 293 T human renal epithelial cells. Our results suggest that such nanoparticles are potentially powerful tools for fluorescent gene delivery reagent.

Functional properties and biodistribution of poly(triethylenetetramine/cystamine bisacrylamide) and poly(triethylenetetramine/cystamine bisacrylamide)- poly(ethylene glycol) mixtures formed with nucleic acid

The clinical success of non-viral gene delivery reagents is hampered by their inefficient cellular transgene delivery, which is largely influenced by carrier properties that are currently undefined and misunderstood. In an attempt to further define and understand the requirements for a safe and efficient non-viral gene delivery reagent, research labs often engineer and evaluate many putative products with subtle physiochemical differences in order to delineate requirements for improved in vitro and in vivo success. The synthesis of many putative reagents is often time-intensive, laborious and costly. In a previous manuscript published by our lab, different amounts of poly(triethylenetetramine/cystamine bisacrylamide) (p(TETA/CBA) and its pegylated counterpart, poly(triethylenetetramine/cystamine bisacrylamide)- poly(ethylene glycol) (p(TETA/CBA)-g-PEG) were mixed together to easily identify optimal reagent properties and candidates in vitro, while avoiding the synthesis of many putative candidates for study. This report uses the aforementioned facile approach to evaluate reagent properties of products that were obtained via one-pot synthesis, which improved synthetic ease. As such, synthesis time was reduced from 6days to 3days and had comparable or improved transfection and viability compared to previous works. Moreover, this synthesis resulted in higher molecular weight products than were used in the previous study and allow for lower polymer doses to be used for complexation, which is useful for systemic delivery that is used herein. The physiochemical properties of the formulations derived using these novel reagents was studied prior to investigating their in vivo biodistribution profiles in a murine colon adenocarcinoma model. Interestingly, negatively charged complexes exhibited greater passive tumor accumulation compared to positively charged complexes following their systemic administration. These studies warrant further investigation for the use of negatively charged drug and gene delivery reagents for passive tumor targeting, and they substantiate the use of polycation/PEG-polycation mixtures for facile product evaluation in order to elucidate design and formulation mandates for the clinical success of non-viral gene delivery formulations.

The influence of the structural orientation of amide linkers on the serum compatibility and lung transfection properties of cationic amphiphiles

Understanding the structural parameters of cationic amphiphiles which can influence gene transfer efficiencies of cationic amphiphiles continues to remain important for designing efficient liposomal gene delivery reagents. Previously we demonstrated the influence of structural orientation of the ester linker (widely used in covalently tethering the polar head and the non-polar tails) in modulating in vitro gene transfer efficiencies of cationic amphiphiles. However, our previously described cationic amphiphiles with ester linkers failed to deliver genes under in vivo conditions. Herein we report on the development of a highly serum compatible cationic amphiphile with circulation stable amide linker which shows remarkable selectivity in transfecting mouse lung. We also demonstrate that reversing structural orientation of the amide linker adversely affects both serum compatibility and the lung selective gene transfer property. Dynamic laser light scattering and atomic force microscopic studies revealed smaller average hydrodynamic sizes of the liposomes of transfection efficient lipid than those for the liposomes of transfection incompetent analog (148 ± 1 nm vs 214 ± 4 nm). Average surface potential of the liposomes of transfection competent amphiphiles were found to be significantly higher than that for the liposomes of transfection incompetent analog (10.7 ± 5.4 mV vs 2.8 ± 1.3 mV, respectively). Findings in fluorescence resonance energy transfer and dye entrapment experiments support lower rigidity and higher biomembrane fusogenicity of the liposomes of the transfection efficient amphiphiles. Importantly, cationic lipoplexes of the novel amide-linker based amphiphile exhibited higher mouse lung selective gene transfer properties than DOTAP, one of the widely used commercially available liposomal lung transfection kits. In summary, the present findings demonstrate for the first time that amide linker structural orientation profoundly influences the serum compatibility and lung transfection efficiencies of cationic amphiphiles.