Low Molecular Weight Chitosan (LMWC)-based Polyplexes for pDNA Delivery: From Bench to Bedside

Non-viral gene delivery vectors are emerging as a safer alternative to viral vectors. Among natural polymers, chitosan (Ch) is the most studied one, and low molecular weight Ch, specifically, presents a wide range of advantages for non-viral pDNA delivery. It is crucial to determine the best process for the formation of Low Molecular Weight Chitosan (LMWC)-pDNA complexes and to characterize their physicochemical properties to better understand their behavior once the polyplexes are administered. The transfection efficiency of Ch based polyplexes is relatively low. Therefore, it is essential to understand all the transfection process, including the cellular uptake, endosomal escape and nuclear import, together with the parameters involved in the process to improve the design and development of the non-viral vectors. The aim of this review is to describe the formation and characterization of LMWC based polyplexes, the in vitro transfection process and finally, the in vivo applications of LMWC based polyplexes for gene therapy purposes.

Gene delivery using dendrimer/pDNA complexes immobilized in electrospun fibers using the layer-by-layer technique

Tissue engineering is an important branch of regenerative medicine that uses cells, materials (scaffolds), and suitable biochemical and physicochemical factors to improve or replace specific biological functions. In particular, the control of cell behavior (namely, of cell adhesion, proliferation and differentiation) is a key aspect for the design of successful therapeutical approaches. In this study, poly(lactic-co-glycolic acid) (PLGA) fiber mats were prepared using the electrospinning technology (the fiber diameters were in the micrometer range). Furthermore, the electrospun fiber mats thus formed were functionalized using the layer-by- layer (LbL) technique with chitosan and alginate (natural and biodegradable polyelectrolytes having opposite charges) as a mean for the immobilization of pDNA/dendrimer complexes. The polyelectrolyte multilayer deposition was confirmed by fluorescence spectroscopy using fluorescent-labeled polyelectrolytes. The electrospun fiber mats coated with chitosan and alginate were successfully loaded with complexes of pDNA and poly(amidoamine) (PAMAM) dendrimers (generation 5) and were able of releasing them in a controlled manner along time. In addition, these mats supported the adhesion and proliferation of NIH 3T3 cells and of human mesenchymal stem cells (hMSCs) in their surface. Transfection experiments using a pDNA encoding for luciferase showed the ability of the electrospun fiber mats to efficiently serve as gene delivery systems. When a pDNA encoding for bone morphogenetic protein-2 (BMP-2) was used, the osteoblastic differentiation of hMSCs cultured on the surface of the mats was promoted. Taken together, the results revealed that merging the electrospinning technique with the LbL technique, can be a suitable methodology for the creation of biological active matrices for bone tissue engineering.

Correlation of the Physicochemical and Structural Properties of pDNA/Cationic Liposome Complexes with Their in Vitro Transfection

In this study, we characterized the conventional physicochemical properties of the complexes formed by plasmid DNA (pDNA) and cationic liposomes (CL) composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) (50/25/25% molar ratio). We found that these properties are nearly unaffected at the studied ranges when the molar charge ratio (R-+/-) between the positive charge from the CL and negative charge from pDNA is not close to the isoneutrality region (R-+/- = 1). However, the results from in vitro transfection of HeLa cells showed important differences when R-+/- is varied, indicating that the relationships between the physicochemical and biological characteristics were not completely elucidated. To obtain information regarding possible liposome structural modifications, small-angle X-ray scattering (SAXS) experiments were performed as a function of R-+/- to obtain correlations between structural, physicochemical, and transfection properties. The SAXS results revealed that pDNA/CL complexes can be described as being composed of single bilayers, double bilayers, and multiple bilayers, depending on the R-+/- value. Interestingly, for R-+/- = 9, 6, and 3, the system is composed of single and double bilayers, and the fraction of the latter increases with the amount of DNA (or a decreasing R-+/-) in the system. This information is used to explain the transfection differences observed at an R-+/- = 9 as compared to R-+/- = 3 and 6. Close to the isoneutrality region (R-+/- = 1.8), there was an excess of pDNA, which induced the formation of a fraction of aggregates with multiple bilayers. These aggregates likely provide additional resistance against the release of pDNA during the transfection phenomenon, reflected as a decrease in the transfection level. The obtained results permitted proper correlation of the physicochemical and structural properties of pDNA/CL complexes with the in vitro transfection of HeLa cells by these complexes, contributing to a better understanding of the gene delivery process.

Caracterização de nanopartículas pDNA-protamina e pDNA-protamina-lipossoma e avaliação da eficiência de entrega gênica a células de mamífero.

Em estudos de terapia gênica e vacinação por DNA, a eficiência e a segurança dos vetores que transportam o material genético terapêutico possuem papel fundamental. Vetores não virais são considerados mais seguros, mas menos eficientes em relação aos vetores virais. Em parte, isso se deve à falta de estudos sistemáticos e comparativos no que diz respeito às características físico-químicas desses vetores quando em soluções biológicas e o efeito delas sobre a eficiência de entrega gênica. O objetivo deste trabalho é avaliar o efeito do pH, da força iônica e do tipo tampão de complexação sobre as características físico-químicas de nanopartículas pDNA-protamina e pDNA-protamina-lipofectamina, visando à entrega gênica para diferentes linhagens celulares. Para isso, nanopartículas formadas em diferentes condições foram caracterizadas através de ensaios de espalhamento dinâmico de luz (DLS) e potencial zeta. Os estudos indicaram que o pH, a força iônica, o tipo de tampão e a presença de meio de cultura e soro no ambiente de complexação alteram significativamente o tamanho, a polidispersidade e o potencial zeta das partículas formadas. Finalmente, buscou-se avaliar o efeito dessas características sobre a eficiência de transfecção in vitro de células de macrófagos IC21 e células HeLa. Os estudos de transfecção em células Hela indicam que tanto a composição como as condições de formação das partículas influenciam significativamente a eficiência de transfecção.

Microparticle-mediated gene delivery for the enhanced expression of a 19-KDa fragment of merozoite surface protein 1 of Plasmodium falciparum

The 19 kDa carboxyl-terminal fragment of merozoite surface protein 1 (MSP119) is a major component of the invasion-inhibitory response in individual immunity to malaria. A novel ultrasonic atomization approach for the formulation of biodegradable poly(lactic-co-glycolic acid) (PLGA) microparticles of malaria DNA vaccines encoding MSP119 is presented here. After condensing the plasmid DNA (pDNA) molecules with a cationic polymer polyethylenimine (PEI), a 40 kHz ultrasonic atomization frequency was used to formulate PLGA microparticles at a flow rate of 18 mL h1. High levels of gene expression and moderate cytotoxicity in COS-7 cells were achieved with the condensed pDNA at a nitrogen to phosphate (N/P) ratio of 20, thus demonstrating enhanced cellular uptake and expression of the transgene. The ability of the microparticles to convey pDNA was examined by characterizing the formulated microparticles. The microparticles displayed Z-average hydrodynamic diameters of 1.50-2.10 lm and zeta potentials of 17.8-23.2 mV. The encapsulation efficiencies were between 78 and 83%, and 76 and 85% of the embedded malaria pDNA molecules were released under physiological conditions in vitro. These results indicate that PLGA-mediated microparticles can be employed as potential gene delivery systems to antigen-presenting cells in the prevention of malaria.

Synthesis of biodegradable polymer-mesoporous silica composite microspheres for DNA prime-protein boost vaccination

DNA vaccines or proteins are capable of inducing specific immunity; however, the translation to the clinic has generally been problematic, primarily due to the reduced magnitude of immune response and poor pharmacokinetics. Herein we demonstrate a composite microsphere formulation, composed of mesoporous silica spheres (MPS) and poly(d,l-lactide-co-glycolide) (PLGA), enables the controlled delivery of a prime-boost vaccine via the encapsulation of plasmid DNA (pDNA) and protein in different compartments. Method with modified dual-concentric-feeding needles attached to a 40 kHz ultrasonic atomizer was studied. These needles focus the flow of two different solutions, which passed through the ultrasonic atomizer. The process synthesis parameters, which are important to the scale-up of composite microspheres, were also studied. These parameters include polymer concentration, feed flowrate, and volumetric ratio of polymer and pDNA-PEI/MPS-BSA. This fabrication technique produced composite microspheres with mean D[4,3] ranging from 6 to 34 μm, depending upon the microsphere preparation. The resultant physical morphology of composite microspheres was largely influenced by the volumetric ratio of pDNA-PEI/MPS-BSA to polymer, and this was due to the precipitation of MPS at the surface of the microspheres. The encapsulation efficiencies were predominantly in the range of 93-98% for pDNA and 46-68% for MPS. In the in vitro studies, the pDNA and protein showed different release kinetics in a 40 day time frame. The dual-concentric-feeding in ultrasonic atomization was shown to have excellent reproducibility. It was concluded that this fabrication technique is an effective method to prepare formulations containing a heterologous prime-boost vaccine in a single delivery system.

Using DNA as a drug - bioprocessing and delivery strategies

DNA may take a leading role in a future generation of blockbuster therapeutics. DNA has inherent advantages over other biomolecules such as protein, RNA and virus-like particles including safety, production simplicity and higher stability at ambient temperatures. Vaccination is the principal measure for preventing influenza and reducing the impact of pandemics; however, vaccines take up to 8-9 months to produce, and the global production capacity is woefully low. With production times as short as 2 weeks, improved safety and stability, bioprocess engineering developments, and the ability to perform numerous therapeutic roles, DNA has the potential to meet the demands of emerging and existing diseases. DNA is experiencing sharp growths in demand as indicated by its use in gene therapy trials and DNA vaccine related patents. Of particular interest for therapeutic use is plasmid DNA (pDNA), a form of non-genomic DNA that makes use of cellular machinery to express proteins or antigens. The production stages of fermentation and downstream purification are considered in this article. Forward looking approaches to purifying and delivering DNA are reported, including affinity chromatography and nasal inhalation. The place that pDNA may take in the preparation for and protection against pandemics is considered. If DNA therapeutics and vaccines prove to be effective, the ultimate scale of production will be huge which shall require associated bioprocess engineering research and development for purification of this large, unique biomolecule.

DNA molecules and human therapeutics

Nucleic acid molecules are championing a new generation of reverse engineered biopharmaceuticals. In terms of potential application in gene medicine, plasmid DNA (pDNA) vectors have exceptional therapeutic and immunological profiles as they are free from safety concerns associated with viral vectors, display non-toxicity and are simpler to develop. This review addresses the potential applications of pDNA molecules in vaccine design/development and gene therapy via recombinant DNA technology as well as a staged delivery mechanism for the introduction of plasmid-borne gene to target cells via the nasal route.

Process considerations related to the microencapsulation of plasmid DNA via ultrasonic atomization

An effective means of facilitating DNA vaccine delivery to antigen presenting cells is through biodegradable microspheres. Microspheres offer distinct advantages over other delivery technologies by providing release of DNA vaccine in its bioactive form in a controlled fashion. In this study, biodegradable poly(D,L-lactide-coglycolide) (PLGA) microspheres containing polyethylenimine (PEI) condensed plasmid DNA (pDNA) were prepared using a 40 kHz ultrasonic atomization system. Process synthesis parameters, which are important to the scale-up of microspheres that are suitable for nasal delivery (i.e., less than 20 μm), were studied. These parameters include polymer concentration; feed flowrate; volumetric ratio of polymer and pDNA-PEI (plasmid DNA-polyethylenimine) complexes; and nitrogen to phosphorous (N/P) ratio. PDNA encapsulation efficiencies were predominantly in the range 82-96%, and the mean sizes of the particle were between 6 and 15 μm. The ultrasonic synthesis method was shown to have excellent reproducibility. PEI affected morphology of the microspheres, as it induced the formation of porous particles that accelerate the release rate of pDNA. The PLGA microspheres displayed an in vitro release of pDNA of 95-99% within 30 days and demonstrated zero order release kinetics without an initial spike of pDNA. Agarose electrophoresis confirmed conservation of the supercoiled form of pDNA throughout the synthesis and in vitro release stages. It was concluded that ultrasonic atomization is an efficient technique to overcome the key obstacles in scaling-up the manufacture of encapsulated vaccine for clinical trials and ultimately, commercial applications.

Single step purification of plasmid DNA using peptide ligand affinity chromatography

Single step affinity chromatography was employed for the purification of plasmid DNA (pDNA), thus eliminating several steps compared with current commercial purification methods for pDNA. Significant reduction in pDNA production time and cost was obtained. This chromatographic operation employed a peptide-monolith construct to isolate pDNA from Escherichia coli (E. coli) impurities present in a clarified lysate feedstock. Mild conditions were applied to avoid any degradation of pDNA. The effect of some important parameters on pDNA yield was also evaluated with the aim of optimising the affinity purification of pDNA. The results demonstrate that 81% of pDNA was recovered and contaminating gDNA, RNA and protein were removed below detectable levels. © 2008 Elsevier B.V. All rights reserved.

Rapid production of a plasmid DNA encoding a malaria vaccine candidate via amino-functionalized poly(GMA-co-EDMA) monolith

Malaria is a global health problem; an effective vaccine is urgently needed. Due to the relative poverty and lack of infrastructure in malaria endemic areas, DNA-based vaccines that are stable at ambient temperatures and easy to formulate have great potential. While attention has been focused mainly on antigen selection, vector design and efficacy assessment, the development of a rapid and commercially viable process to manufacture DNA is generally overlooked. We report here a continuous purification technique employing an optimized stationary adsorbent to allow high-vaccine recovery, low-processing time, and, hence, high-productivity. A 40.0 mL monolithic stationary phase was synthesized and functionalized with amino groups from 2-Chloro-N,N- diethylethylamine hydrochloride for anion-exchange isolation of a plasmid DNA (pDNA) that encodes a malaria vaccine candidate, VR1020-PyMSP4/5. Physical characterization of the monolithic polymer showed a macroporous material with a modal pore diameter of 750 nm. The final vaccine product isolated after 3 min elution was homogeneous supercoiled plasmid with gDNA, RNA and protein levels in keeping with clinical regulatory standards. Toxicological studies of the pVR1020-PyMSP4/5 showed a minimum endotoxin level of 0.28 EU/m.g pDNA. This cost-effective technique is cGMP compatible and highly scalable for the production of DNA-based vaccines in commercial quantities, when such vaccines prove to be effective against malaria. © 2008 American Institute of Chemical Engineers.

Development of a pilot-scale bacterial fermentation for plasmid-based biopharmaceutical production using a stoichiometric medium

The recognition of the potential efficacy of plasmid DNA (pDNA) molecules as vectors in the treatment and prevention of emerging diseases has birthed the confidence to combat global pandemics. This is due to the close-to-zero safety concern associated with pDNA vectors compared to viral vectors in cell transfection and targeting. Considerable attention has been paid to the potential of pDNA vectors but comparatively less thought has been given to the practical challenges in producing large quantities to meet current rising demands. A pilot-scale fermentation scheme was developed by employing a stoichiometrically-designed growth medium whose exceptional plasmid yield performance was attested in a shake flask environment for pUC19 and pEGFP-N1 transformed into E. coliDH5α and E. coliJM109, respectively. Batch fermentation of E. coliDH5α-pUC19 employing the stoichiometric medium displayed a maximum plasmid volumetric and specific yield of 62.6 mg/L and 17.1 mg/g (mg plasmid/g dry cell weight), respectively. Fed-batch fermentation of E. coliDH5α-pUC19 on a glycerol substrate demonstrated one of the highest ever reported pilot-scale plasmid specific yield of 48.98 mg/g and a volumetric yield of 0.53 g/L. The attainment of high plasmid specific yields constitutes a decrease in plasmid manufacturing cost and enhances the effectiveness of downstream processes by reducing the proportion of intracellular impurities. The effect of step-rise temperature induction was also considered to maximize ColE1-origin plasmid replication.

Inorganic-organic composite particle as a staged delivery platform for plasmid DNA-based biopharmaceuticals

Infectious diseases such as SARS, influenza and bird flu may spread exponentially throughout communities. In fact, most infectious diseases remain major health risks due to the lack of vaccine or the lack of facilities to deliver the vaccines. Conventional vaccinations are based on damaged pathogens, live attenuated viruses and viral vectors. If the damage was not complete, the vaccination itself may cause adverse effects. Therefore, researchers have been prompted to prepare viable replacements for the attenuated vaccines that would be more effective and safer to use. DNA vaccines are generally composed of a double stranded plasmid that includes a gene encoding the target antigen under the transcriptional directory and control of a promoter region which is active in cells. Plasmid DNA (pDNA) vaccines allow the foreign genes to be expressed transiently in cells, mimicking intracellular pathogenic infection and inducing both humoral and cellular immune responses. Currently, because of their highly evolved and specialized components, viral systems are the most effective means for DNA delivery, and they achieve high efficiencies (generally >90%), for both DNA delivery and expression. As yet, viral-mediated deliveries have several limitations, including toxicity, limited DNA carrying capacity, restricted target to specific cell types, production and packing problems, and high cost. Thus, nonviral systems, particularly a synthetic DNA delivery system, are highly desirable in both research and clinical applications.

Binding mechanism of affinity ligands for purification of plasmid DNA

Plasmid DNA for therapeutic and vaccination purposes must be highly purified. The high selectivity of affinity chromatography makes it ideal for the isolation of pDNA from complex biological feed stocks. Affinity chromatography makes use of the biological function and/or individual chemical structure of the interacting molecules. However, the success of any affinity purification protocol is dependent on the availability of suitable ligands. In this study, surface plasmon resonance (SPR) based Biacore system has been employed for the detection and quantification of the binding between lac operon (lacO) sequence contained in a pDNA and synthetic peptides based on the DNA-binding domain of the lac repressor protein, lad. The equilibrium dissociation constant (K D) and association and dissociation rate constants (ka, kd) for the interaction between plasmid DNA and designed peptides were determined.

Performance of R-N(R′)-R″ functionalised poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) monolithic sorbent for plasmid DNA adsorption

High-throughput plasmid DNA (pDNA) manufacture is obstructed predominantly by the performance of conventional stationary phases. For this reason, the search for new materials for fast chromatographic separation of pDNA is ongoing. A poly(glycidyl methacrylate-co-ethylene glycol dimethacrylate) (GMA-EGDMA) monolithic material was synthesised via a thermal-free radical reaction, functionalised with different amino groups from urea, 2-chloro-N,N-diethylethylamine hydrochloride (DEAE-Cl) and ammonia in order to investigate their plasmid adsorption capacities. Physical characterisation of the monolithic polymer showed a macroporous polymer having a unimodal pore size distribution pivoted at 600 nm. Chromatographic characterisation of the functionalised polymers using pUC19 plasmid isolated from E. coli DH5α-pUC19 showed a maximum plasmid adsorption capacity of 18.73 mg pDNA/mL with a dissociation constant (KD) of 0.11 mg/mL for GMA-EGDMA/DEAE-Cl polymer. Studies on ligand leaching and degradation demonstrated the stability of GMA-EGDMA/DEAE-Cl after the functionalised polymers were contacted with 1.0 M NaOH, which is a model reagent for most 'cleaning in place' (CIP) systems. However, it is the economic advantage of an adsorbent material that makes it so attractive for commercial purification purposes. Economic evaluation of the performance of the functionalised polymers on the grounds of polymer cost (PC)/mg pDNA retained endorsed the suitability of GMA-EGDMA/DEAE-Cl polymer.