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Polycation-DNA complexes for gene delivery: A comparison of the biopharmaceutical properties of cationic polypeptides and cationic lipids
Abstract
DNA plasmids formed particulate complexes with a variety of cationic polyamino acids and cationic lipids, which were used to transfect mammalian cells in culture. Complexation was studied by assaying for exclusion of ethidium using a fluorometric assay, which indicated that complexation with cationic polyamino acids took place with utilisation of the majority of charged functional groups. The particle sizes and zeta potentials of a range of complexes were determined. Generally polyamino acids formed uniform particles 80-120 nm in diameter in water, but their particle size increased on dilution of the particles in electrolytes or cell culture media. The efficiency of transfection was compared using complexes of pRSVlacZ, a reporter construct which expressed beta-galactosidase under the control of the Rous sarcoma virus promoter. Positively charged DNA/polyamino acid complexes were taken up by cells but required an endosomolytic agent, such as chloroquine, to facilitate transfection. Polyornithine complexes resulted in the highest levels of expression, in comparison with other homopolyamino acids (polyornithine>poly-L-lysine=poly-D-lysine>polyarginine). Copolyamino acids of lysine and alanine condensed DNA but were less active in transfection experiments. Copoly(L-Lys, L-Ala 1:1) was inactive even in the presence of chloroquine. In contrast DNA/cationic lipid complexes transfected cells spontaneously, and chloroquine did not improve the extent of expression, rather it usually reduced efficiency. There was little correlation between comparative efficiencies of lipid complexes between cell lines suggesting that the nature of the cell membrane and differences in mechanisms of internalisation were determinants of efficiency. In an effort to explore better cell culture models for gene delivery, monolayers of Caco-2 cells were transfected in filter culture. As the cells differentiated and formed a polarized monolayer, expression of beta-galactosidase was reduced until at day 27 expression was not significantly different from basal activity. The Caco-2 filter culture model merits further attention as a model of gene delivery to epithelial surfaces, such as would be encountered in the lung after inhalation.
... DNA concentration of the studied samples was determined by measuring the absorbance at 260 nm (A260), where DNA absorbs light most strongly [45]. The spectrophotometric measurements at A260 can be converted from one absorbance unit at 260 nm to DNA concentration expressed in mg/mL, depending on the nature of the chain [46], i.e., A260 = 1 corresponds to 33 μg/mL and to 50 μg/mL for single stranded DNA and double-stranded DNA, respectively. Double-stranded DNA conversion was used for our sample. ...
... As mentioned before, denaturing conditions such as increase of temperature, increase or decrease of pH, and decrease in Na + ion concentration, disrupt double helix DNA structure, unstack the bases and produce the increase in absorbance [12]. It is well known that the salt concentration influences DNA's melting temperature [46]. The temperature dependence of the absorbance of calf-thymus DNA at a concentration of 0.042 mg/mL was determined for an external salt concentration ranging between 0 M and 3 × 10 −2 M NaCl ( Figure 2). ...
... The observed decrease in Tm at low salt concentrations is caused by an increase of the electrostatic repulsions between the negative phosphate sites of DNA strands competing with the stabilization by the cooperative hydrogen bonds. It is well known that the salt concentration influences DNA's melting temperature [46]. The temperature dependence of the absorbance of calf-thymus DNA at a concentration of 0.042 mg/mL was determined for an external salt concentration ranging between 0 M and 3ˆ10´2 M NaCl ( Figure 2). ...
Studies of DNA molecule behavior in aqueous solutions performed through different approaches allow assessment of the solute-solvent interactions and examination of the strong influence of conformation on its physicochemical properties, in the presence of different ionic species and ionic concentrations. Firstly, the conformational behavior of calf-thymus DNA molecules in TE buffer solution is presented as a function of temperature. Secondly, their rheological behavior is discussed, as well as the evidence of the critical concentrations, i.e., the overlap and the entanglement concentrations (C* and Ce, respectively) from steady state flow and oscillatory dynamic shear experiments. The determination of the viscosity in the Newtonian plateau obtained from flow curves η ( ) allows estimation of the intrinsic viscosity and the specific viscosities at zero shear when C[η] < 40. At end, a generalized master curve is obtained from the variation of the specific viscosity as a function of the overlap parameter C[η]. The variation of the exponent s obtained from the power law η~ −s for both flow and dynamic results is discussed in terms of Graessley’s analysis. In the semi-dilute regime with entanglements, a dynamic master curve is obtained as a function of DNA concentration (CDNA > 2.0 mg/mL) and temperature (10 °C < T < 40 °C).
... 349 This can be improved by using chloroquine, an endosomal escape inducer. 382 Comparisons of PEI and PLL also show that PEI has significantly higher transfection efficiency. 383 Still, PLL is a promising delivery vehicle. ...
Over the past decade, in vivo gene replacement therapy has significantly advanced, resulting in market approval of numerous therapeutics predominantly relying on adeno‐associated viral vectors (AAV). While viral vectors have undeniably addressed several critical healthcare challenges, their clinical application has unveiled a range of limitations and safety concerns. This review highlights the emerging challenges in the field of gene therapy. At first, we discuss both the role of biological barriers in viral gene therapy with a focus on AAVs, and review current landscape of in vivo human gene therapy. We delineate advantages and disadvantages of AAVs as gene delivery vehicles, mostly from the safety perspective (hepatotoxicity, cardiotoxicity, neurotoxicity, inflammatory responses etc.), and outline the mechanisms of adverse events in response to AAV. Contribution of every aspect of AAV vectors (genomic structure, capsid proteins) and host responses to injected AAV is considered and substantiated by basic, translational and clinical studies. The updated evaluation of recent AAV clinical trials and current medical experience clearly shows the risks of AAVs that sometimes overshadow the hopes for curing a hereditary disease. At last, a set of established and new molecular and nanotechnology tools and approaches are provided as potential solutions for mitigating or eliminating side effects. The increasing number of severe adverse reactions and, sadly deaths, demands decisive actions to resolve the issue of immune responses and extremely high doses of viral vectors used for gene therapy. In response to these challenges, various strategies are under development, including approaches aimed at augmenting characteristics of viral vectors and others focused on creating secure and efficacious non‐viral vectors. This comprehensive review offers an overarching perspective on the present state of gene therapy utilizing both viral and non‐viral vectors.
... . 마찬가지로 DNA와 복합체를 형성할 수 있 는 양이온의 폴리라이신(polylysine, PLL)은 플라스미드 DNA를 세 포가 흡수할 수 있는 크기(<100-200 nm)로 압축할 수 있다 (Zauner et al., 1998). 이러한 polycation들은 인체 세포에게 독성을 나타내 지 않음과 동시에 DNA가 안정적이고 효과적으로 전달될 수 있는 운 반체로 이용되고 있다 (Bielinska et al., 1996;Pouton et al., 1998 ...
Purpose: This study aimed to extract phyto DNA from the adventitious root of Aloe vera and confirm its role in improving and regenerating skin and antiaging properties caused by blue light.Methods: Phyto DNA was extracted and a complex containing polylysine and Poly (amidoamine) was made. The antioxidant activity was confirmed using the DPPH method. The MTT test was performed on fibroblasts. The expression of SOD1 , FLG , LOR , COL1A1 , and MMP1 was measured using RT–PCR after blue light irradiation on HaCaT cells. The wound-healing test was performed. A phyto DNA complex cream formulation was created. Using this method, skin epidermal water loss was determined after human application.Results: At 0.85% of the cation polymer–phyto DNA complex concentration, 100% of fibroblasts survived. At 0.1% concentration, the antioxidant effect was 93%. After being exposed to blue light, only 40.4% of fibroblasts survived, but after being treated with a 100 μg/mL complex, 80.5% survived. At a mineral–cation-phyto DNA complex ratio of 10:1, the expression of skin barrier factors was highest. About 95.7% of HaCaT cells regenerated at 10:1 ratio of the mineral with phyto DNA complex in comparison to complex with only the minerals without phyto DNA.Conclusion: Phyto DNA from aloe vera’s adventitious root has no cytotoxicity, has excellent DPPH free radical scavenging ability, and can mitigate blue light cytotoxicity. It can improve skin barrier function by increasing the expression of barrier factors via a synergistic action in a specific ratio composition of phyto DNA, cationic polymer, and mineral complexes.
... As shown in Figure 2A, we first examined the polyion complex (PIC) formation between PVIm-Lac and pDNA by agarose gel electrophoresis. As a positive control, pDNA/bPEI complexes at a positive/negative charge ratio of eight exhibited no fluorescence from ethidium bromide staining because the coil-globule transition of the pDNA inhibited ethidium bromide intercalation [22,23]. In the case of all PVIm-Lac, the complete retardation of the pDNA was observed at a positive/negative charge ratio of eight, suggesting the complete formation of DNA/PVIm-Lac PICs. ...
The lactosylated poly(1-vinylimidazole) (PVIm-Lac) with various lactosylated degrees has been synthesized for the co-delivery of zinc ions (Zn) and plasmid DNA (pDNA). The Zn/DNA/PVIm-Lac complex formation has achieved the specific delivery of zinc ions to HepG2 cells. Especially, the resulting hepatocyte-specific delivery of zinc ions has increased the number of insulin receptors on the cell surface. Consequently, the Zn/DNA/PVIm-Lac complexes have suppressed insulin receptor internalization on the surface of the HepG2 cells, expecting to offer unique therapy to inhibit hepatic insulin clearance.
... Since it is widespread knowledge that there is a poor correlation between transfection efficiency in cell culture and in vivo (Pouton et al., 1998), it would be interesting to try these microparticles in vivo to see if they were able to generate immune responses against the DNA-encoded protein. However, the additional development of better in vitro-in vivo extrapolation models would be important. ...
DNA vaccines have emerged to be an alternative to conventional vaccines due to their increased safety compared with recombinant protein vaccines and live/attenuated vaccines. Pulmonary delivery of vaccines could offer additional advantages, because from an immunological point of view, there is a large presence of macrophages for antigen/DNA uptake and it also part of the mucosal immune system, which is important for effective immune protection. Encapsulating the DNA into a particulate delivery system would be advantageous for protection against degrading nucleases. The aim of this project was to produce microparticles encapsulating plasmid DNA with a high loading efficiency, appropriate size suitable for pulmonary delivery and with the maintenance of plasmid DNA conformation. Microparticles were produced using poly(DL-lactide-co-glycolide) as the polymer due to its well-known biodegradable and biocompatibility characteristics, and polyvinyl alcohol (PVA) as the main stabiliser. Plasmid DNA encoding for luciferase protein was used as a model plasmid for studies. Water-in-oil-in-water (w1/o/w2) was used as the method for making particles. Factorial experimental design was used to rapidly optimize a formulation that had the desired properties. Further studies examined the effect of adding various excipients (0.1M NaHCO3, 1% m/m Na2HPO 4) on the loading, diameters, morphology and charge on the microparticles. Analytical (including TGA, NMR and DSC) and cell culture experiments - using a human alveolar cell line (A549) and mouse macrophage/monocyte cell line (J774A.1) - were carried out to analyse their physical and biological properties. The factorial experimental design aided in choosing a platform formulation, with particle diameters of 2-5 μm and loading of 66-72% m/m. Particles without added excipients had low plasmid DNA loading and the DNA had lost most of its original conformation. However, addition of buffers into the aqueous phases, particularly 1% m/v NaH2PO4, reduced the loss of conformation and enhanced loading efficiency. Physicochemical studies gave mixed results on the formulations tested in terms of thermal stability, DCM and PVA levels and performance as a dry powder. Results demonstrated that microparticles made without buffer were more thermally stable, had lower residual DCM and PVA levels in contrast to those made with 1% m/v Na2HPO4 and 0.1M NaHCO3 . The microparticles did not aerosolise well as a dry powder - even with addition of surfactants such as lecithin and DPPC - with diameters between 69-95 μm being measured, which is in contrast to the diameters measured in a 'wet' state (2-5 μm). Cell culture studies demonstrated that entrapped DNA (which was extracted out from the particles and delivered to cells using a commercial agent) was still biologically active, but the A549 cell line was much easier to transfect than the J774A.1 cells. However, the microparticles themselves were poor delivery vehicles of DNA in these models. Overall, these experiments demonstrated the range of factors that need to be considered when trying to create suitable microparticulate carriers for pulmonary delivery of DNA vaccines.
... Ornithine (Orn) is a naturally-occurring non-proteinogenic amino acid contains a primary amine in the pendant group able to provide the positive charge necessary for oligonucleotide complexation. To date, applications of PLO and Orn include: (i) gene silencing by polyplex formation (plasmid [20][21][22], DNA [24], and siRNA [25] complexation), (ii) cell penetration [26], (iii) growth improvement and adhesion of many cell types (including brain-forming cells such as neural progenitors [27][28][29], and (iv) coating materials [30], overall displaying lower immunogenicity than other cationic polypeptides. This synthetic polyamino acid also provides for tighter/stronger oligonucleotide condensation at a lower mass [23,31] and increased resistance to disruption in the presence of an anionic counter species [23]. ...
Gene silencing therapies have successfully suppressed the translation of target proteins, a strategy that holds great promise for the treatment of central nervous system (CNS) disorders. Advances in the current knowledge on multimolecular delivery vehicles are concentrated on overcoming the difficulties in delivery of small interfering (si)RNA to target tissues, which include anatomical accessibility, slow diffusion, safety concerns, and the requirement for specific cell uptake within the unique environment of the CNS. The present work addressed these challenges through the implementation of polyornithine derivatives in the construction of polyplexes used as non-viral siRNA delivery vectors. Physicochemical and biological characterization revealed biodegradability and biocompatibility of our polyornithine-based system and the ability to silence gene expression in primary oligodendrocyte progenitor cells (OPCs) effectively. In summary, the well-defined properties and neurological compatibility of this polypeptide-based platform highlight its potential utility in the treatment of CNS disorders.
... Naked plasmid DNA is known to transfect cells but with lower transfection efficiency. Polyplexes with positive charge improves DNA uptake and often facilitate cellular transfection when used with endosomolytic agents such as chloroquine [80]. Lipopolyamines with lower toxicity than viral vectors are also known to facilitate transfusion but with lower efficiency [81]. ...
Hybrid chemical compounds formed by conjugation of two or more bioactive molecules have shown wide variety of applications in biology, microelectronics as well as material sciences. In particular, the conjugates of steroid framework are known to have broad biological activity profile due to their ability to penetrate the biomembranes and bind to specific hormone receptors. Among the various conjugates of steroids, Steroid Amino Acid Conjugates (SAACs) are attractive because of the possibility of fine tuning of the amphiphilicity with position, orientation and nature of amino acids. The structural details, applications, mechanistic insights and their diverse pharmacological as well as other physicochemical properties of several SAACs are summarized in the present review. This review provides better insight for medicinal chemists to design and explore such novel conjugates which can be used as lead structures in the future drug discovery or as probes to understand the complex biological system.
... Further additions of ELP(-NH 2 ) and ELP(-NHCH 3 ) to CT-DNA solution after the isoelectric point allowed obtaining overcharging around to +24 mV and +18 mV, respectively. This overcharging plateau is in good agreement with the ones obtained for other systems including chitosans [75,83], polyaminoacids [87,88] and cationic polymers presenting dendrimer structures or linear configurations [86]. Finally, the obtained positive f -potential for CT-DNA/ELP(-NH 2 ) and CT-DNA/ELP(-NHCH 3 ) systems suggests that DNA compaction is completely accomplished with pH-responsive ELPs chains [6,75,83], ELP(-NH 2 ) being the compound yielding complexes with the greatest positive net charge at their surface and potentially displaying higher stability. ...
Positively charged elastin-like polypeptides (ELPs) were synthesized for the compaction of genetic material. A recombinant ELP (VPGXG)40 with X = V,M (3:1) was post-modified in two steps to introduce chemoselectively either primary or secondary amine pendant groups at each methionine residue. Positively charged ELPs were characterized by SDS-PAGE, size exclusion chromatography, 1H NMR, potentiometric titrations and dynamic light scattering to assess their purity and determine their degree of functionalization, molecular weight, isoelectric point and thermo-responsive behaviour. Electrostatic complexation between the different ELP derivatives and nucleic acids was studied to determine the stoichiometry of ELPS/nucleic acids complex formation, and to find optimal conditions leading to stable nanoparticles with controlled size and surface potential. The stability of these complexes was investigated in the presence of salts at physiological concentrations and in the presence of surfactant. This study revealed that two regimes of stable nanoparticles in terms of size and charge can be obtained from the electrostatic complexation between the primary amine containing ELP derivative, ELP(-NH2), and plasmid DNA. Resulting complexes were found to be stable to dissociation for charge ratios up to 2.5 under physiological salt concentrations (154 mM NaCl), showing that plasmid DNA was completely condensed by the polycationic ELP and protected against electrolyte-mediated dissociation.
... Alternatively, conjugation of PLL to fusogenic peptides, fatty acids, histidine, and chloroquine improves endosomal escape. They destabilize the endosomal membrane and allow delivery of the nucleic acids into the cytoplasm (Pouton et al. 1998). ...
Gene therapy has emerged as an alternative in the treatment of cancer, particularly in cases of resistance to chemo and radiotherapy. Different approaches to deliver genetic material to tumor tissues have been proposed, including the use of small non-coding RNAs due to their multiple mechanisms of action. However, such promise has shown limits in in vivo application related to RNA’s biological instability and stimulation of immunity, urging the development of systems able to overcome those barriers. In this review, we discuss the use of RNA interference in cancer therapy with special attention to the role of siRNA and miRNA and to the challenges of their delivery in vivo. We introduce a promising class of drug delivery system known as micelle-like nanoparticles and explore their synthesis and advantages for gene therapy as well as the recent findings in in vitro, in vivo and clinical studies.
... PLL can condense elongated pDNA into uniform toroidal and rodshaped structures, and the average size of polyplexes was approximately 200 nm [27] . Previous studies demonstrated that Th1 responses could be induced when large vesicles were phagocytized by macrophages [28] , and pDNA complexes formed by PLL showed better effects in transfection than naked pDNA in vitro [29] . Several studies showed that injection of pDNA can induce substantial expression of protein in vivo and elicit immune responses against the protein expressed [9,30] . ...
Background:
The prevalence of food allergy has been increasing, but treatment is very limited. DNA vaccination has been recognized as a promising method for the treatment of allergic diseases; however, poor immunogenicity has hindered its application.
Methods:
BALB/c mice were intradermally injected with plasmid DNA encoding the peanut protein Ara h 2 (pAra h 2) or pAra h 2 pretreated with poly-L- lysine (PLL) before or after sensitization with Ara h 2 protein. Ara h 2-specific antibodies were measured by ELISA. CD207+ dendritic cells (DCs) and Treg cells in draining lymph nodes were analyzed by flow cytometry after DNA immunization, and cytokine production in splenocytes was also analyzed.
Results:
In the prophylactic study, pretreatment with pAra h 2 or PLL-pAra h 2 resulted in lower levels of Ara h 2-specific IgG1, IgG2a, and IgE after sensitization with Ara h 2 protein, and mice in the PLL-pAra h 2 group had a significantly lower level of antibodies than those in the pAra h 2 group. In the treatment study, intradermal injection with pAra h 2 or PLL-pAra h 2 after Ara h 2 protein sensitization significantly decreased the level of Ara h 2-specific antibodies, and PLL- pAra h 2 had stronger effects than pAra h 2. There were increased numbers of CD207+ DCs and Treg cells in the mice receiving intradermal injection with PLL-pAra h 2, and splenocytes from PLL-pAra h 2-treated mice secreted increased levels of IFN-γ and IL-10.
Conclusions:
Modification of pAra h 2 with PLL improved its prophylactic and therapeutic effects in peanut-allergic mice.
... Cationic lipid is able to resemble traditional pharmaceuticals with little immunogenicity, and has no potential for viral infection. Nevertheless, cationic lipid is limited for clinic applications because of its inevitable toxicity and low transfection efficiency 16,17 . Compared with other non-viral vectors, polycationic carrier is attractive for its better biocompatibility, lower immunogenicity and easier modification 18 . ...
Small interfering RNA (siRNA) has increased the hope for highly-efficient treatment of gene-related diseases. However, the stable and efficient delivery of therapeutic nucleic acids is a prerequisite for the successful clinical translation of RNA interfering therapy. To achieve this, we condensed the low molecular weight polyethyleneimine (PEI, Mw < 2000) with 2,6-pyridinedicarboxaldehyde (PDA) to synthesize a biologically responsive and degradable cationic polymer (abbreviated to PDAPEI) which was utilized as a gene vector for the delivery of a VEGF-A shRNA expression plasmid DNA (pDNA). The resulting electrostatic interaction between PDAPEI and pDNA led to the self-assembly of nanoscale polyplexes with suitable particle size and stable zeta potential. The PDAPEI/pDNA polyplexes demonstrated an outstanding gene transfection and silencing efficiency at 30 w/w ratio, as well as negligible cytotoxicity. Also, the designed polymer showed no stimulation to the innate immune system. Moreover, compared with PEI 25 KDa, the polyplexes accomplished comparatively better anti-angiogenesis efficacy, which resulted in the inhibition of tumor growth in subcutaneous tumor mice models. In conclusion, PDAPEI has great potential to be a gene delivery vector for cancer therapy.
... Other common polycations such as PLL lack this ability to facilitate endosomal escape [113]. One way of addressing this issue is through the introduction of lysosomotropic reagents such as chloroquine to DNA polyplexes [114]. This method has successfully increased the transfection efficiency of PLL based delivery systems [115,116]. ...
Oppositely charged polyions can self-assemble in solution to form colloidal polyion complex (PIC) particles. Such nanomaterials can be loaded with charged therapeutics such as DNA, drugs or probes for application as novel nanomedicines and chemical sensors to detect disease markers. A comprehensive discussion of the factors affecting PIC particle self-assembly and their response to physical and chemical stimuli in solution is described herein. Finally, a collection of key examples of polyionic nanoparticles for biomedical applications is discussed to illustrate the behaviour and demonstrate the potential of PIC nanoparticles in medicine
... In both cases studied (Figs. 3 and 4), the -potential gives the same trend: (i) the initial highly negative -potential (around −40 mV, corresponding to DNA molecules) decreases with the addition of the cationic vector to DNA solution, which is in good agreement with several reports in the literature [36,[40][41][42][43]. It shows that electrostatic interactions between chitosan and DNA take place in the solution and represent the starting of the complexation process of DNA, as presented by number (1) , corresponding to overcharging of DNA. ...
... In addition to the AmB loading, the micelles in this study also contained a pHsensitive protonation, which led to further enhancement of endosomal escape, possibly through a proton sponge mechanism. In addition to AmB, chloroquine and influenza virus hemagglutininbased fusogenic peptides have also been used to enhance the endosomal escape of nanoparticle delivery vehicles (107,108). ...
Nanoparticle-mediated gene delivery is a promising alternative to viral methods; however, its use in vivo, particularly following systemic injection, has suffered from poor delivery efficiency. Although PEGylation of nanoparticles has been successfully demonstrated as a strategy to enhance colloidal stability, its success in improving delivery efficiency has been limited, largely due to reduced cell binding and uptake, leading to poor transfection efficiency. Here we identified an optimized PEGylation scheme for DNA micellar nanoparticles that delivers balanced colloidal stability and transfection activity. Using linear polyethylenimine (lPEI)-g-PEG as a carrier, we characterized the effect of graft length and density of polyethylene glycol (PEG) on nanoparticle assembly, micelle stability, and gene delivery efficiency. Through variation of PEG grafting degree, lPEI with short PEG grafts (molecular weight, MW 500 – 700 Da) generated micellar nanoparticles with various shapes including spherical, rod-like, and worm-like nanoparticles. DNA micellar nanoparticles prepared with short PEG grafts showed comparable colloidal stability in salt and serum-containing media to those prepared with longer PEG grafts (MW 2 kDa). Corresponding to this trend, nanoparticles prepared with short PEG grafts displayed significantly higher in vitro transfection efficiency compared to those with longer PEG grafts. More importantly, short PEG grafts permitted marked increase in transfection efficiency following ligand conjugation to the PEG terminal in metastatic prostate cancer-bearing mice. This study identifies that lPEI-g-PEG with short PEG grafts (MW 500 – 700 Da) is the most effective to ensure shape control and deliver high colloidal stability, transfection activity, and ligand effect for DNA nanoparticles in vitro and in vivo following intravenous administration.
... As a consequence, compaction of DNA becomes necessary for its delivery into cells by receptor-mediated endocytosis. DNA compaction is generally achieved by complexation with high molecular weight polycations (polylysine, polyethylenimine, and polyamidoamine dendrimers) in ratios that can allow retention of electrostatic charge, if desired [76]. For example, a slight excess of positive charge has been found useful for encapsulation of DNA·polylysine particles into folate-targeted anionic liposomes [51,77]. ...
The receptor for folic acid constitutes a useful target for tumor-specific drug delivery, primarily because: (1) it is upregulated in many human cancers, including malignancies of the ovary, brain, kidney, breast, myeloid cells and lung, (2) access to the folate receptor in those normal tissues that express it can be severely limited due to its location on the apical (externally-faring) membrane of polarized epithelia, and (3) folate receptor density appears to increase as the stage/grade of the cancer worsens. Thus, cancers that are most difficult to treat by classical methods may be most easily targeted with folate-linked therapeutics. To exploit these peculiarities of folate receptor expression, folic acid has been linked to both low molecular weight drugs and macromolecular complexes as a means of targeting the attached molecules to malignant cells. Conjugation of folic acid to macromolecules has been shown to enhance their delivery to folate receptor-expressing cancer cells in vitro in almost all situations tested. Folate-mediated macromolecular targeting in vivo has, however, yielded only mixed results, largely because of problems with macromolecule penetration of solid tumors. Nevertheless, prominent examples do exist where folate targeting has significantly improved the outcome of a macromolecule-based therapy, leading to complete cures of established tumors in many cases. This review presents a brief mechanistic background of folate-targeted macromolecular therapeutics and then summarizes the successes and failures observed with each major application of the technology.
... The complexation of DNA, oligonucleotides and siRNA onto the surface of nanoparticles can protect them from degradation and improve transfection [45, 46]. DNA can be condensed on dendrimers [47, 48], cationic peptides [49, 50], cationic polymers51525354555657, cationic lipids [58– 61] and liposomes [39,62636465666768. DNA could be transferred to cells via encapsulation or adsorption onto a particle surface [45, 46, 59,697071. ...
The spherical PLGA nanoparticles (NP) calorimetric investigation is presented in this paper. Such nanoparticles is used for biological active substances (drugs) encapsulating inside of them with the purpose of medicine transferring into the cell. It is clear that without determination of particle stability it is impossible their practical usage. From calorimetric study of PLGA nanoparticles with PLA/PGA ratio 70:30 it was determined the entirety conditions of such particles and the temperature interval, where the particle destructions take place. It was unambiguously shown that for noncoated PLGA NP and for chitosancoated PLGA NP the stability temperature are equal to 370C and less than physiological temperature, which exclude their practical application. Also it was determined that hermiticity destroy temperature depends on heating rate. At the same time it was established that strongly alkaline and acid area (pH2 - pH9) do not destroy noncoated PLGA NP and chitosancoated PLGA NP what gives possibility for their using orally.
The co-delivery system of zinc ions (Zn2+) and plasmid DNA (pDNA) has been designed by the use of poly(1-vinylimidazole) (PVIm) derivatives for myoblast differentiation. Six PVIm derivatives were synthesized, followed by the optimization of the chemical structure. As a result, methylated and carboxymethylated PVIm (CM-PVIm-Me) delivered the highest amount of Zn2+ ions inside C2C12 myoblast cells. The CM-PVIm-Me also delivered pDNA inside the myoblast cells to exhibit pDNA gene expression which was upregulated by the co-delivered Zn2+ ions. The co-delivered Zn2+ ions were localized in the cell nucleus presumably to affect cellular functions. Actually, the myoblast cells treated with the Zn2+/CM-PVIm-Me/pDNA complexes differentiated to myotubes. These results suggest that the Zn2+ ions delivered by the CM-PVIm-Me inside the cells differentiated myoblasts to myotubes. Our co-delivery system of Zn2+ ion and pDNA without zinc transporter can be a unique tool of regenerative medicine for muscular injury.
The unique physico-chemical properties of cationic polymers and their ability to be easily modified make them attractive for many biological applications. As a result there is a vast amount of research focussed on designing novel natural or synthetic cationic polymers with specific biological functionality.
Cationic Polymers in Regenerative Medicine brings together the expertise of leading experts in the field to provide a comprehensive overview of the recent advances in cationic polymer synthesis, modification and the design of biomaterials with different structures for therapeutic applications. Chapters cover recent developments in novel cationic polymer based systems including poly(L-lysine), Poly(N,N-dimethylaminoethyl methacrylate) and cationic triazine dendrimers as well as cationic polymer-coated micro- and nanoparticles and cationic cellulose and chitin nanocrystals. Applications discussed in the book include drug and gene delivery, therapeutics in thrombosis and inflammation as well as gene therapy.
Suitable both for an educational perspective for those new to the field and those already active in the field, the book will appeal to postgraduates and researchers. The broad aspects of the topics covered are suitable for polymer chemists interested in the fundamentals of the materials systems as well as pharmaceutical chemists, bioengineering and medical professionals interested in their applications.
Water-soluble imidazole-phosphate copolymers were obtained by copolymerization of 1-vinylimidazole with vinyl acetate with subsequent phosphorylation under the action of phosphoric acid. The introduction of phosphate units into the vinylimidazole chain unexpectedly increased the basic properties of the polymer, the buffer capacity at pH 5–7 and activity in coordination with DNA oligonucleotides, which is important in gene therapy and genetic engineering. The condensation of silicic acid in the presence of new polymers leads to the appearance of composite nanoparticles, which are a model of silicon transport vesicles in nature, as well as a new precursor of silicon materials.
While DNA and messenger RNA (mRNA) based therapies are currently changing the biomedical field, the delivery of genetic materials remains the key problem preventing the wide introduction of these methods into clinical practice. Therefore, the creation of new methods for intracellular gene delivery, particularly to hard-to-transfect, clinically relevant cell populations is a pressing issue. Here, we report on the design of a novel approach to format 50-150 nm calcium carbonate particles in the vaterite state and using them as a template for polymeric core-shell nanoparticles. We apply such core-shell nanoparticles as safe and efficient carriers for mRNA and pDNA. We prove that such nanocarriers are actively internalized by up to 99% of primary T-lymphocytes and exert minimal toxicity with the viability of >90%. We demonstrate that these nanocarriers mediate more efficient transfection compared with the standard electroporation method (90% vs. 51% for mRNA and 62% vs. 39% for plasmid DNA) in primary human T-lymphocytes as a model of the hard to transfect type that is widely used in gene and cell therapy approaches. Importantly, these polymeric nanocarriers can be used in serum containing basic culture medium without special conditions and equipment, thus having potential for being introduced in clinical development. As a result, we have provided proof-of-principle that our nanosized containers represent a promising universal non-viral platform for efficient and safe gene delivery. This journal is
Efficient delivery of DNA first into the individual cells and then into the cell nucleus are crucial steps in gene delivery. Of the different vectors available for gene transfection, non-viral vectors are attractive although current non-viral vector transfection efficiency needs to be improved significantly. Dendrimers are distinguished from most other non-viral vectors by their precisely controlled topology, uniform molecular weight, and flexibility of functionalization. This thesis examines the development of some 30 new novel compounds, a new series of partial dendrimers (dendrons) as part of a search for DNA carriers. A series of dendrons based on branched lysine with or without lipidic chains were synthesised by systematically modifying the structure of the dendrons by varying the length and number of lipid chains in the core and the amino terminals created by lysine moieties. As some of the dendrons discussed here are amphiphilic, the possibility of self-assembly was studied using the surface tension measurements, NMR and asymmetric flow field-flow fractionation (aFFF). Self-assembly of both complete spherical dendrimers and partial dendrimers or dendrons have been of interest for their applications in supramolecular dendrimer chemistry. Formation of dendron - DNA complexes occurs spontaneously by the addition of cationic dendrons to DNA. These complexes we term "dendriplexes", whose biophysical properties including hydrodynamic diameter, surface charge, DNA condensing ability, DNase protection, cytotoxicity and in-vitro cell transfection ability have been studied here. Although all the dendrons condense the DNA and form compact complexes the degree of compaction increases when the lipidic chain length increases. Dendritic poly(lysine) with 8 terminal amino groups with or without three lipid chains (C10,C14,C18) in the core were studied systematically to determine the influence of lipophilicity of these dendrons on transfection properties. Of this series of dendrons those with three Cl8 carbon chains [(C18) 3(L)7(NH2)8] show higher gene transfer activity compared to their non lipidic analogues or those with shorter hydrocarbon chains, indicating that the lipophilicity of the dendrons plays a role in complex formation and thereby transfection.
The polyion complexes (PICs) between plasmid DNA (pDNA) and succinylated branched polyethylenimine (bPEI-Et-COOH) were formed for in vivo pDNA delivery by muscular injection. Transmission electron microscopy (TEM) observation revealed that the PIC between pDNA and bPEI-Et-COOH with higher succinylated degree formed the particle structure with corona-like shell. Furthermore, confocal laser scanning microscopy (CLSM) observation revealed that pDNAs were successfully delivered inside the cells and that the pDNAs were colocalized with the nuclei of the cells after endosomal escape. Although the pDNA/bPEI-Et-COOH PICs mediated significant gene expression in vitro, the PICs did not mediate gene expression in vivo muscular injection. Consequently, the pDNA transfection by bPEI-Et-COOH was noncorrelative between in vitro and in vivo in spite of low toxicity by succinylation both in vitro and in vivo. The noncorrelative relation between in vitro and in vivo for pDNA transfection by bPEI-Et-COOH muscular injection would be considerable design for pDNA carriers in vivo.
Successful clinical implementation of gene delivery relies on the use of viral or non‐viral based vectors to package and protect the therapeutic nucleic acid. These vehicles must also be able to direct the fate of the cargo once it has entered the cell to ensure that the nucleic acid is functional, and the desired outcome is achieved. Compared to viral vectors, non‐viral vectors have the advantage of incorporating different material types such as lipids, polymers, and peptides to tune overall safety and efficacy. Peptides are especially powerful when used in gene delivery vectors as they are able to increase gene delivery efficacy by introducing new biochemical functionality. This review will discuss the use of peptides as central design components in non‐viral gene delivery vectors. The contribution of the peptide component to the overall functionality of the delivery vehicle will be highlighted, with a focus on peptides as the only vehicle component or peptides in complex assemblies with lipids or polymers.
Abstract
Driven by the rapid development of therapeutic applications for synthetic gene delivery systems, specifically formulations of plasmid DNA with cationic lipids and/or peptides, the characterisation of DNA complexes has become an important area of research. Current efforts to increase their transfection efficiency has led to the development of analytical techniques for a better understanding of how the structure, stability and biological activity of DNA complexes are affected by their environment. The effects of ionic strength and pH on the colloidal stability and structure of synthetic gene delivery systems were investigated through salt-induced aggregation of poly-L-lysine/DNA complexes. A two-syringe pump flow system was used to reproducibly prepare the polyplexes. Analysis of the mean hydrodynamic diameter using dynamic light scattering showed that the aggregation rate and polydispersity of the polyplexes increased with ionic strength and pH. Polyplexes with an average diameter of 100-150 nm aggregated to 1500-3000 nm over 1.5 h. Computer simulations using equations based on the DLVO theory were found to adequately describe the stability of the polyplexes. These simulations form the basis for further work on theoretical predictions of the stability of DNA complexes. The concept of DNA complexes as aggregate structures with fractal dimensions was introduced. Static light scattering revealed fractal dimensions of 2.22 and 1.44 for polyplexes at 50-150 and 1000 mM NaCl, respectively. These values correspond to slow and rapid aggregation regimes, respectively. Biophysical characterisation of a novel receptor-mediated system, Lipofectin/integrin- targeting peptide/DNA, with potential for targeted gene therapy in vivo, was conducted. Under physiological ionic strength, lipopolyplexes at 6.8 charge ratio demonstrated instability (average size > 1000 nm) and decreased in vitro transfection activity. A PicoGreen assay showed complete binding of the DNA at a charge ratio of 3.0-4.0, corresponding to maximum expression. Lipopolyplexes at high DNA concentrations (150 μg/ml) in distilled water and 5% dextrose possessed small sizes (110 ± 35 nm and 145 ± 35 nm, respectively), but aggregated upon contact with salt or serum albumin.
Gemini surfactants (GS) have been highlighted as attractive gene carriers for a few years now; however, key aspects of the role of the GS chemical structure on the DNA-GS complexation and subsequent biological activity remain to be determined. Aiming to elucidate the effects of the GS spacer hydrophobicity, this work was focused on the biophysical characterization of the self-assembly, DNA complexation, cytocompatibility, and DNA transfection of a series of bis-quaternary ammonium GS with fixed side alkyl chains of 14 carbons and varying head-to-head alkyl chain spacers of 4, 6, and 14 carbons (referred to as GS4, GS6, and GS14, respectively). The characterization was carried out by a battery of experimental techniques including UV-vis and fluorescence sprectroscopies, ζ potential, dynamic light scattering (DLS), isothermal titration calorimetry (ITC), and flow cytometry, among others. Overall, the spectroscopic results showed that the self-assembly of the GS was favored with the spacer hydrophobicity since lower values of critical micelle concentration (CMC) were observed for samples with longer spacer chains. On the other hand, the ITC results revealed that the DNA-GS complexation was driven by an initial electrostatic attraction between DNA and GS monomers/micelles followed by complementary hydrophobic interactions which strengthen the DNA-GS binding, the latter being more pronounced for GS with longer spacers. Finally, the biological tests demonstrated that while GS with moderate hydrophobicity (GS4 and GS6) yielded outstanding levels of cytocompatibility and DNA transfection over a range of concentrations, the most hydrophobic sample (GS14) proved to be cytotoxic upon administration to cultured HeLa cells (p < 0.05). In our opinion, the fundamental information here presented might be pivotal not only for understanding the DNA-GS complexation mechanism, but also for developing efficient GS-based carriers for gene therapy.
Polymeric amines are being studied intensively as components of systems for gene delivery in genetic engineering and gene therapy of genetic disorders, including cancer. Despite remarkable achievements in the field, polymeric amines, such as polyethyleneimine, show some disadvantages. Strong interaction between the amine-containing polymer and nucleic acid hampers the release of nucleic acid in the cell cytoplasm. Amine groups can interact with the cell membrane which results in cell death. These limitations of polymeric amines stimulated an investigation of new structures for gene delivery. Imidazole-containing polymers have attracted attention as lesser basic substances, while they are able to interact with polymeric acids. Further development of imidazole-based gene delivery agents requires knowledge about some fundamental aspects of interaction between nucleic acids, and polymeric imidazoles. In this work, we studied the complexation of poly(1-vinylimidazole) and oligomeric DNA. We found that the number of active sites capable of binding with negatively charged phosphate groups is comparable with the number of protonated imidazole units in the case of high molecular weight polymer. The increase in polymer charge by 1-bromopropane quaternizating 1%−5% imidazole units or by decreasing the pH to 6.5−7 considerably increased the ability of poly(1-vinylimidazole) to interact with oligonucleotides. The pH sensitivity of this interaction is interesting for cancer gene therapy because the tumours have a lowered intercellular pH (stable oligonucleotide complex) and a higher extracellular pH which can lead to complex dissociation. Minimal critical length for complexation of quaternized poly(1-vinylimidazole) and DNA is below eight units which corresponds to polymers with amine groups. Fluorescence-tagged poly(1-vinylimidazole) samples were obtained and their potential for monitoring the polymer and polymer-oligonucleotide complex internalization into living cells was demonstrated.
Non-viral delivery systems are relatively safe but inefficient in their current form. The main obstacle in using non-viral gene delivery system approach is to transport the gene of interest in cytoplasm and subsequently entering into cell nucleus. In this research, palmityol-CKKHH and its series have been designed and its ability to form nanoparticle of a stable DNA-lipopeptide complex were evaluated to be used as non-viral gene delivery vehicle. The lipopeptide molecules are composed of alkyl chain of palmitoyl (C-16), and amino acid residues of cysteine (C), lysine (K), and histidine (H). The particle size (nm) and zeta potential () of the complexes were determined with a Zetasizer Nano Series. It was revealed that prolonging incubation time of the complex composing of DNA and Pal-CK 2 H 3 (charge ratio of 1.5) more than 2 hours tend to increase the size up to 300 nm. In addition, increasing DNA concentration up to 40 g (~ 120 nmoles) with lipopeptide (charge ratio of 1.5), the complex size was still relatively stable at less than 400 nm. As the number of lysine residue on lipopeptide is increased, the particle size tends to decreased. However, the particle size is increased as the number of histidine residue on lipopeptide is increased. It was also shown that increasing charge ratio of the nanoparticle complex resulted in an increased zeta potential but lowering the particle size. Transfection efficiency of the nanoparticle on COS-7 had shown that the lipopeptide has potency as non-viral gene delivery vehicle. To conclude, the lipopeptide composing of alkyl chain of palmitoyl and amino acid residues form a nanoparticle and having potential characteristics to be further explored as non-viral gene delivery vehicle.
Non-viral delivery systems are relatively safe but inefficient in their current form. The main obstacle in using non-viral gene delivery system approach is to transport the gene of interest in cytoplasm and subsequently entering into cell nucleus. In this research, palmityol-CKKHH and its series have been designed and its ability to form nanoparticle of a stable DNA – lipopeptide complex were evaluated to be used as non-viral gene delivery vehicle. The lipopeptide molecules are composed of alkyl chain of palmitoyl (C-16), and amino acid residues of cysteine (C), lysine (K), and histidine (H). The particle size (nm) and zeta potential () of the complexes were determined with a Zetasizer Nano Series. It was revealed that prolonging incubation time of the complex composing of DNA and Pal-CK2H3 (charge ratio of 1.5) more than 2 hours tend to increase the size up to 300 nm. In addition, increasing DNA concentration up to 40 g (~ 120 nmoles) with lipopeptide (charge ratio of 1.5), the complex size was still relatively stable at less than 400 nm. As the number of lysine residue on lipopeptide is increased, the particle size tends to decreased. However, the particle size is increased as the number of histidine residue on lipopeptide is increased. It was also shown that increasing charge ratio of the nanoparticle complex resulted in an increased zeta potential but lowering the particle size. Transfection efficiency of the nanoparticle on COS-7 had shown that the lipopeptide has potency as non-viral gene delivery vehicle. To conclude, the lipopeptide composing of alkyl chain of palmitoyl and amino acid residues form a nanoparticle and having potential characteristics to be further explored as non-viral gene delivery vehicle. Keywords: Palmitoyl-CKKHH charge ratio, nanoparticle, particle size and zeta potential.
We synthesized a new cationic AB2 miktoarm block copolymer consisting of one poly (ethylene glycol) (PEG) block and two cationic poly (l-lysine) (PLL) blocks, wherein the PLL blocks were conjugated to the PEG blocks with or without a bioreducible linker (disulfide bonds). Bioreducible and non-bioreducible miktoarm block copolymers (mPEG-(ss-PLL)2 and mPEG-PLL2) were prepared for efficient gene delivery as a non-viral gene delivery approach. Both cationic copolymers (bioreducible and nonbioreducible) efficiently formed the nanopolyplexes with plasmid DNA (pDNA) through electrostatic interaction at different weight ratio of polymer and pDNA. Gene condensation ability of the polymers and release of the DNA under reduction condition were measured by gel electrophoresis. Dynamic light scattering (DLS) and field-emission transmission electron microscopy (FE-TEM) were used to measure the average hydrodynamic diameter and morphology of the nanoparticles, respectively. The bioreducible nanopolyplexes showed lower cytotoxicity and higher gene expression than the non-reducible nanopolyplexes in cancer cells.
Cell penetrating peptides (CPPs), and protein transduction domains (PTDs) of viruses and other natural proteins serve as a template for the development of efficient peptide based gene delivery vectors. PTDs are sequences of acidic or basic amphipathic amino acids, with superior membrane trespassing efficacies. Gene delivery vectors derived from these natural, cationic and cationic amphipathic peptides, however, offer little flexibility in tailoring the physicochemical properties of single chain peptide based systems. Owing to significant advances in the field of peptide chemistry, synthetic mimics of natural peptides are often prepared and have been evaluated for their gene expression, as a function of amino acid functionalities, architecture and net cationic content of peptide chains. Moreover, chimeric single polypeptide chains are prepared by a combination of multiple small natural or synthetic peptides, which imparts distinct physiological properties to peptide based gene delivery therapeutics. In order to obtain multivalency and improve the gene delivery efficacies of low molecular weight cationic peptides, bioactive peptides are often incorporated into a polymeric architecture to obtain novel 'polymer-peptide hybrids' with improved gene delivery efficacies. Peptide modified polymers prepared by physical or chemical modifications exhibit enhanced endosomal escape, stimuli responsive degradation and targeting efficacies, as a function of physicochemical and biological activities of peptides attached onto a polymeric scaffold. The focus of this review is to provide comprehensive and step-wise progress in major natural and synthetic peptides, chimeric polypeptides, and peptide-polymer hybrids for nucleic acid delivery applications.
Gene therapy is an efficient strategy for the treatment of cancer. This involves the insertion of a corrected gene to target cells to overexpress and replace the defective ones responsible for disease. The delivery of the selective gene to the desired cells has always been a challenging task. Various viral and nonviral gene delivery vectors have been used extensively. Due to inherent toxicities associated with viral vectors, lipid-based nanocarrier systems are getting wider attraction for the delivery of genes to the target sites. Though they have less entrapment efficiency of DNA or genes, still they are preferred as genes delivery systems because of their easy handling, preparation, and various ways to be adopted for target specificity. Most commonly used cationic lipids for developing lipid-based genes delivery systems are Transfectam, lipofectamine, lipofectin, and transfectace. Many cationic lipid-based genes nanocarriers have been studied in preclinical trials and some of them have been successfully qualified for the clinical trials. They are extensively studied so to engineer safe and effective genes delivery tools.
Cancer has become one of the main causes of death in developed countries. Nanomedicine aims to overcome some problems related to this prevalent disease, particularly the lack of efficient diagnostic and therapeutic tools. Nanocarriers can target cancer by simply being entrapped in tumors or by specific recognition of grafted ligands by cell surface receptors, thus leading to receptor-mediated endocytosis. Among several tumor-selective ligands, folic acid displays high affinity for folate receptors, which are commonly overexpressed in tumor tissues and present significant upregulation. Its characteristics have led researchers to hypothesize that folate-attached anticancer molecules (i.e., toxins, genes, drugs) might enhance their effectiveness and potency against tumor cells compared to nontargeted therapies. Additionally, folate-linked carriers have also shown promising results to design diagnostic tools, which allows an earlier detection of the disease. This chapter focuses on the possibilities that folic acid offers toward recent advances in cancer treatment and diagnostics, and also future perspectives of these folate-targeted therapies to fight against cancer.
The permeabilization of cells has obtained several applications. This chapter deals with these applications and includes cationic cell killing, gene delivery, anticancer effects, delivery of quantum dots, electrofusion, macromolecular biosynthetic processes (poly-ADP ribose, DNA, RNA), temporal order of gene replication and gene expression, visualization of replicons and intermediates of chromosome condensation, linear order and spatial arrangement of chromosomes, genotoxic chromatin changes, among them heavy metal (Cd, Pb, Ni, Hg, Ag) treatment and irradiation (α, γ and UV-B) induced chromatin damages.
Nucleic acid delivery presents unique challenges that could be entirely different compared to small molecules due to their molecular size, electric charge, and significant susceptibility to enzymatic degradation. However, some of the basic principles could be applied to both fields, which means specific carriers have been studied for delivery of small molecule drugs, genetic information, and mediators of RNA interference (RNAi). This chapter focuses on a specific category of polymers that have been evaluated in various strategies of targeted delivery of different types of nucleic acids; however, due to conspicuous role of delivery systems in cancer therapy as well as tissue regeneration, the presented data is mainly related to relevant clinical conditions. The most commonly used natural polymers in nucleic acid delivery will be covered spotlight, and their characteristics analyzed.
Nanoparticles (NPs) play expanding roles in biomedical applications including imaging and therapy, however, their long-term fate and clearance profiles have yet to be fully characterized in vivo. NP delivery via the airway is particularly challenging, as the clearance may be inefficient and lung immune responses complex. Thus, specific material design is required for cargo delivery and quantitative, noninvasive methods are needed to characterize NP pharmacokinetics. Here, biocompatible poly(acrylamidoethylamine)-b-poly(DL-lactide) block copolymer-based degradable, cationic, shell-cross-linked knedel-like NPs (Dg-cSCKs) were employed to transfect plasmid DNA. Radioactive and optical beacons were attached to monitor biodistribution and imaging. The preferential release of cargo in acidic conditions provided enhanced transfection efficiency compared to non-degradable counterparts. In vivo gene transfer to the lung was correlated with NP pharmacokinetics by radiolabeling Dg-cSCKs and performing quantitative biodistribution with parallel positron emission tomography and Čerenkov imaging. Quantitation of imaging over 14 days corresponded with the pharmacokinetics of NP movement from the lung to gastrointestinal and renal routes, consistent with predicted degradation and excretion. This ability to noninvasively and accurately track NP fate highlights the advantage of incorporating multifunctionality into particle design.
DNA, the carrier of genetic information, is a strongly charged stiff polymer. Its structure and properties are of central importance in microbiology and gene- or biotechnological applications. For the biophysicist, it is characterized by a few parameters, such as its length, its bending rigidity or its linear charge density. Because of the straightforward preparation of well-defined DNA samples and the readily available techniques for their manipulation and characterization, DNA is now widely studied by experimental and theoretical physicists as a model system to understand the intricate behavior of charged biopolymers. In this contribution it is demonstrated that many effects that are obtained with complexes of DNA and oppositely charged objects can be rationalized using simple statistical-mechanics and electrostatic concepts.
In somatic gene therapy, nucleic acid is transferred into and subsequently expressed in somatic cells (i.e. not in stem cells) in order to alter or substitute gene functions as a means of prophylactic or therapeutic treatment of patients. The nucleic acid itself is the pharmaceutical drug. For transfer of genes, viral (retro, adeno, and adenovirus-associated viruses), physical (electroporation and bioballistics), and chemical systems are utilized. Chemical and physical systems are united as nonviral systems.
Keywords:
Liposomes;
Somatic;
Transfection;
Vectors
Nonviral vectors for gene delivery are receiving increasing attention for application in a broad variety of gene-mediated therapies for humans. Chemical vectors are attractive to the pharmaceutical industry as alternatives to viral vectors because of compound stability and easy chemical modification. Furthermore, the low cost and consistent standard of production (compared to growth of viruses in bioreactors followed by purification), higher biosafety (less immunogenic as compared to viruses such as adenoviruses), and high flexibility once a formulation has been defined make these compounds very attractive [1,2]. The biosafety of nonviral vectors is higher, as compared to viruses, in not eliciting an acute immune reaction; also, they will not recombine with wild-type viruses and will very rarely insert into the host genome (unless integration sequences are incorporated into the delivered plasmid). However, to date, nonviral vectors are generally less efficient in delivering DNA and expressing proteins as compared to their viral counterparts particularly when used in vivo. The efficiency of nonviral mediated gene delivery can be improved when physical methods such as gene gun [3,4], electroporation [5,6] or hydrodynamic techniques [7,8] are used to enhance delivery.
Biomaterials are substances that are used in prostheses or in medical devices designed for treatment, augmentation, or replacement any tissue, organ or function of the body. Both natural and synthetic materials are used as biomaterials.
which can control the translation of a specific protein, the development of the method for efficient and safe delivery of DNA and RNA has been one of the main objectives in the biological and medical science. Therefore, many chemists has been concentrated their efforts to synthesize the non-viral gene delivery carriers such as cationic lipids,
This chapter elaborates the structure, function, and mechanism of various polymeric vectors and their applicability in gene delivery. Polymers play a vital role in the delivery of DNA to the cell. In contrast to conventional polymeric formulation, where the drug is either simply diffused or released by hydrolysis or esterification of polymer, DNA cannot diffuse easily because of its high molecular weight. Polymeric vectors play a key role in the interaction with cell membranes, in intracellular trafficking, and in the transcription of the transgene in the nucleus. An efficient vector polymer delivers the transgene to the nucleus after bypassing several barriers. Polymeric vectors have gained favor owing to their safety, cost-effectiveness, ease of handling, and manufacturing. Three basic mechanisms of DNA polymer complex formation are condensation of DNA with polymers, encapsulation of DNA into the polymers, and complexation of DNA to the surface of preformed polymeric nanoparticles (NPs) grafted with cationic surfactants or polysaccharides. This chapter discusses in detail the basic concepts related to polymeric vectors, noncondensing polymers, lipidic vectors, and peptides. The chapter also elaborates the use of dendrimers and biopolymers in gene delivery.
Genetic immunization is one of the important findings that provide multifaceted immunological response against infectious diseases. With the advent of r-DNA technology, it is possible to construct vector with immunologically active genes against specific pathogens. Nevertheless, site-specific delivery of constructed genetic material is an important contributory factor for eliciting specific cellular and humoral immune response. Nanotechnology has demonstrated immense potential for the site-specific delivery of biomolecules. Several polymeric and lipidic nanocarriers have been utilized for the delivery of genetic materials. These systems seem to have better compatibility, low toxicity, economical and capable to delivering biomolecules to intracellular site for the better expression of desired antigens. Further, surface engineering of nanocarriers and targeting approaches have an ability to offer better presentation of antigenic material to immunological cells. This chapter gives an overview of existing and emerging nanotechnological approaches for the delivery of genetic materials.
The process by which viruses destabilize endosomal membranes in an acidification dependent manner has been mimicked with synthetic peptides that are able to disrupt liposomes, erythrocytes, or endosomes of cultured cells. Peptides containing the 20 amino-terminal amino acid sequence of influenza virus hemagglutinin as well as acidic derivatives showed erythrocyte lysis activity only when peptides were elongated by an amphipathic helix or by carboxyl-terminal dimerization. Interestingly, peptides consisting of the 23 amino-terminal amino acids of influenza virus hemagglutinin were also active in erythrocyte lysis. When peptides were in corporated into DNA complexes that utilize a receptor-mediated endocytosis pathway for uptake into cultured cells, either by ionic interaction with positively charged polylysine-DNA complexes or by a streptavidin-biotin bridge, a strong correlation between pH-specific erythrocyte disruption activity and gene transfer was observed. A high-level expression of luciferase or interleukin-2 was obtained with optimized gene transfer complexes in human melanoma cells and several cell lines.
We have developed a high-efficiency nucleic acid delivery system that uses receptor-mediated endocytosis to carry DNA macromolecules into cells. We accomplished this by conjugating the iron-transport protein transferrin to polycations that bind nucleic acids. Human transferrin, as well as the chicken homologue conalbumin, has been covalently linked to the small DNA-binding protein protamine or to polylysines of various sizes through a disulfide linkage. These modified transferrin molecules maintain their ability to bind their cognate receptor and to mediate efficient iron transport into the cell. The transferrin-polycation molecules form electrophoretically stable complexes with double-stranded DNA, single-stranded DNA, and modified RNA molecules independent of nucleic acid size (from short oligonucleotides to DNA of 21 kilobase pairs). When complexes of transferrin-polycation and a bacterial plasmid DNA containing the gene for Photinus pyralis luciferase are supplied to eukaryotic cells, high-level expression of the luciferase gene occurs, demonstrating transferrin receptor-mediated endocytosis and expression of the imported DNA. We refer to this delivery system as "transferrinfection."
Complexes containing plasmid DNA, transferrin-polylysine conjugates, and polylysine-conjugated peptides derived from the N-terminal sequence of the influenza virus hemagglutinin subunit HA-2 have been used for the transfer of luciferase or beta-galactosidase marker genes to K562 cells, HeLa cells, and BNL CL.2 hepatocytes. These DNA complexes mimic the entry of viruses into cells, as they contain functions for (i) the packaging of the nucleic acid with polylysine, (ii) the attachment to the cell and receptor-mediated endocytosis with transferrin as a ligand, and (iii) the release from endosomes by using membrane-disrupting influenza peptides. The presence of these influenza peptide conjugates in the DNA complexes renders the complexes active in membrane disruption in a liposome leakage assay and results in a substantial augmentation of the transferrin-polylysine-mediated gene transfer.
We have previously described a gene delivery system based upon the receptor-mediated endocytosis of DNA complexed with transferrin-polycation conjugates. This delivery system has been found to be very effective for both the internalization and the expression of genetic material in cells that have many transferrin receptors. Upon scrutinization of the parameters involved in this method, which we have termed transferrinfection, we note two important features of the process: the polycation in polycation-transferrin conjugates, as expected, serves to attach the transferrin moiety to the DNA and, in addition, the polycation functions to condense the DNA into a doughnut structure. Electron microscopic analysis of a range of poorly active to highly active transferrinfection samples reveals a strong correlation between DNA condensation and cellular DNA uptake. Furthermore, we demonstrate that the transfection activity of the DNA complex can be increased by addition of free polycation as long as a sufficient quantity of polycation-transferrin conjugates remains in the complex to ensure its binding to the cellular receptor.
Our understanding of the molecular mechanisms that govern gene expression has been facilitated by the ability to introduce recombinant DNA molecules into heterologous cellular systems both in vitro and in vivo. One approach to defining DNA sequences important in the regulation of gene expression is to place controlling elements (e.g., promoter/enhancer sequences) upstream of a DNA coding sequence, introduce these constructs into transgenic animals or cells in culture, and analyze the levels of gene product produced by the introduced construct. Ideally, such a reporter gene should encode a product that is stable, innocuous to the cell or organism in which it is being expressed, and should be readily detectable, even when present in small quantities.
We are developing efficient methods for gene transfer into tissue culture cells. We have previously shown that coupling of a chimeric adenovirus with polylysine allowed the construction of an adenovirus-polylysine-reporter-gene complex that transferred the transporter gene with great efficiency into HeLa cells. We have now explored simpler, biochemical means for coupling adenovirus to DNA/polylysine complexes and show that such complexes yield virtually 100% transfection in tissue culture cell lines. In these methods adenovirus is coupled to polylysine, either enzymatically through the action of transglutaminase or biochemically by biotinylating adenovirus and streptavidinylating the polylysine moiety. Combination complexes containing DNA, adenovirus-polylysine, and transferrin-polylysine have the capacity to transfer the reporter gene into adenovirus-receptor- and/or transferrin-receptor-rich cells.
RNA and DNA expression vectors containing genes for chloramphenicol acetyltransferase, luciferase, and beta-galactosidase
were separately injected into mouse skeletal muscle in vivo. Protein expression was readily detected in all cases, and no
special delivery system was required for these effects. The extent of expression from both the RNA and DNA constructs was
comparable to that obtained from fibroblasts transfected in vitro under optimal conditions. In situ cytochemical staining
for beta-galactosidase activity was localized to muscle cells following injection of the beta-galactosidase DNA vector. After
injection of the DNA luciferase expression vector, luciferase activity was present in the muscle for at least 2 months.
We have developed a high-efficiency nucleic acid delivery system that uses receptor-mediated endocytosis to carry DNA macromolecules into cells. We accomplished this by conjugating the iron-transport protein transferrin to polycations that bind nucleic acids. Human transferrin, as well as the chicken homologue conalbumin, has been covalently linked to the small DNA-binding protein protamine or to polylysines of various sizes through a disulfide linkage. These modified transferrin molecules maintain their ability to bind their cognate receptor and to mediate efficient iron transport into the cell. The transferrin-polycation molecules form electrophoretically stable complexes with double-stranded DNA, single-stranded DNA, and modified RNA molecules independent of nucleic acid size (from short oligonucleotides to DNA of 21 kilobase pairs). When complexes of transferrin-polycation and a bacterial plasmid DNA containing the gene for Photinus pyralis luciferase are supplied to eukaryotic cells, high-level expression of the luciferase gene occurs, demonstrating transferrin receptor-mediated endocytosis and expression of the imported DNA. We refer to this delivery system as "transferrinfection."
A general and efficient transfection procedure, based on compacted lipopolyamine-coated plasmids, has been developed. The active species is obtained by simple addition of excess synthetic lipospermine solution to the DNA and binds within minutes to the cell membrane. This technique has been developed on endocrine cells of the intermediate lobe of the pituitary as a general tool for physiological work on primary cells; it is not toxic and does not interfere with physiological regulations in melanotrope cells. A variety of eukaryotic cell cultures also have been transfected with success for transient and stable expression.
We report the results of a double-blind, placebo-controlled trial in nine cystic fibrosis (CF) subjects receiving cationic liposome complexed with a complementary DNA encoding the CF transmembrane conductance regulator (CFTR), and six CF subjects receiving only liposome to the nasal epithelium. No adverse clinical effects were seen and nasal biopsies showed no histological or immuno-histological changes. A partial restoration of the deficit between CF and non-CF subjects of 20% was seen for the response to low Cl- perfusion following CFTR cDNA administration. This was maximal around day three and had reverted to pretreatment values by day seven. In some cases the response to low Cl- was within the range for non-CF subjects. Plasmid DNA and transgene-derived RNA were detected in the majority of treated subjects. Although these data are encouraging, it is likely that transfection efficiency and the duration of expression will need to be increased for therapeutic benefit.
Cationic lipids are widely used for gene transfer in vitro and show promise as a vector for in vivo gene therapy applications. However, there is limited understanding of the cellular and molecular mechanisms involved. We investigated the individual steps in cationic lipid-mediated gene transfer to cultured cell lines. We used DMRIE/DOPE (a 1:1 mixture of N-[1-(2,3-dimyristyloxy) propyl]-N,N-dimethyl-N-(2-hydroxyethyl)ammonium bromide (DMRIE) and dioleoyl phosphatidylethanolamine (DOPE) as a model lipid because of its efficacy and because it is being used for clinical trials in humans. The data show that cationic lipid-mediated gene transfer is an inefficient process. Part of the inefficiency may result from the fact that the population of lipid-DNA complexes was very heterogeneous, even under conditions that have been optimized to produce the best transfection. Inefficiency was not due to inability of the complex to enter the cells because most cells took up the DNA. However, in contrast to previous speculation, the results indicate that endocytosis was the major mechanism of entry. After endocytosis, the lipid-DNA aggregated into large perinuclear complexes, which often showed a highly ordered tubular structure. Although much of the DNA remained aggregated in a vesicular compartment, there was at least a small amount of DNA in the cytoplasm of most cells. That observation plus results from direct injection of DNA and lipid-DNA into the nucleus and cytoplasm indicate that movement of DNA from the cytoplasm to the nucleus may be one of the most important limitations to successful gene transfer. Finally, before transcription can occur, the data show that lipid and DNA must dissociate. These results provide new insights into the physical limitations to cationic lipid-mediated gene transfer and suggest that attention to specific steps in the cellular process may further improve the efficiency of transfection and increase its use in a number of applications.
Gene transfer vectors based on adeno-associated virus (AAV) are emerging as highly promising for use in human gene therapy by virtue of their characteristics of wide host range, high transduction efficiencies, and lack of cytopathogenicity. To better define the biology of AAV-mediated gene transfer, we tested the ability of an AAV vector to efficiently introduce transgenes into nonproliferating cell populations. Cells were induced into a nonproliferative state by treatment with the DNA synthesis inhibitors fluorodeoxyuridine and aphidicolin or by contact inhibition induced by confluence and serum starvation. Cells in logarithmic growth or DNA synthesis arrest were transduced with vCWR:beta gal, an AAV-based vector encoding beta-galactosidase under Rous sarcoma virus long terminal repeat promoter control. Under each condition tested, vCWR:beta Gal expression in nondividing cells was at least equivalent to that in actively proliferating cells, suggesting that mechanisms for virus attachment, nuclear transport, virion uncoating, and perhaps some limited second-strand synthesis of AAV vectors were present in nondividing cells. Southern hybridization analysis of vector sequences from cells transduced while in DNA synthetic arrest and expanded after release of the block confirmed ultimate integration of the vector genome into cellular chromosomal DNA. These findings may provide the basis for the use of AAV-based vectors for gene transfer into quiescent cell populations such as totipotent hematopoietic stem cells.
The process by which viruses destabilize endosomal membranes in an acidification-dependent manner has been mimicked with synthetic peptides that are able to disrupt liposomes, erythrocytes, or endosomes of cultured cells. Peptides containing the 20 amino-terminal amino acid sequence of influenza virus hemagglutinin as well as acidic derivatives showed erythrocyte lysis activity only when peptides were elongated by an amphipathic helix or by carboxyl-terminal dimerization. Interestingly, peptides consisting of the 23 amino-terminal amino acids of influenza virus hemagglutinin were also active in erythrocyte lysis. When peptides were incorporated into DNA complexes that utilize a receptor-mediated endocytosis pathway for uptake into cultured cells, either by ionic interaction with positively charged polylysine-DNA complexes or by a streptavidin-biotin bridge, a strong correlation between pH-specific erythrocyte disruption activity and gene transfer was observed. A high-level expression of luciferase or interleukin-2 was obtained with optimized gene transfer complexes in human melanoma cells and several cell lines.
The application of cationic liposome reagents has advanced DNA and mRNA transfection research in vitro, and data are accumulating which show their utility for in vivo gene transfer. However, chemical structure-activity data leading to a better mechanistic understanding of their biological activity is still limited. Most of the cationic lipid reagents in use today for this application are formulated as liposomes containing two lipid species, a cationic amphiphile and a neutral phospholipid, typically dioleoylphosphatidylethanolamine (DOPE). The studies reported here examine the effects of some systematic chemical structural changes in both of these lipid components. Cationic and neutral phospholipids were formulated together as large multilamellar vesicles (MLV) or small sonicated unilamellar vesicles (SUV) in water, and each formulation was assayed quantitatively in 96-well microtiter plates under 64 different assay conditions using COS.7 cells and an RSV-beta-galactosidase expression plasmid. The cationic lipid molecules used for these studies were derived from a novel series of 2,3-dialkyloxypropyl quaternary ammonium compounds containing a hydroxyalkyl moiety on the quaternary amine. A homologous series of dioleylalkyl (C18:1) compounds containing increasing hydroxyalkyl chain lengths on the quaternary amine were synthesized, formulated with 50 mol % DOPE, and assayed for transfection activity. The order of efficacy was ethyl > propyl > butyl > pentyl > 2,3-dioleyloxypropyl-1-trimethyl ammonium bromide (DOTMA). DOTMA, which is commercially available under the trademark Lipofectin Reagent, lacks a hydroxyalkyl moiety on the quaternary amine. A homologous series of hydroxyethyl quaternary ammonium derivatives with different alkyl chain substitutions were synthesized, formulated with 50 mol % DOPE, and assayed in the transfection assay. The order of transfection efficacy was dimyristyl (di-C14:0) > dioleyl (di-C18:1) > dipalmityl (di-C16:0) > disteryl (di-C18:0). The addition of 100 microM chloroquine in the transfection experiment enhanced the activity of the dioleyl compound by 4-fold and decreased the activity of the dimyristyl compound by 70%. For each of the compounds and formulations examined in this report, large multilamellar vesicles (MLV; diameter 300-700 nm) were more active than small unilamellar vesicles (SUV; diameter 50-100 nm). The neutral phospholipid requirements for transfection activity in COS.7 cells with these cationic lipid molecules were examined.(ABSTRACT TRUNCATED AT 400 WORDS)
Adenovirus uncoating is a stepwise process which culminates in the release of the viral DNA into the nucleus through the nuclear pore complexes and dissociation of the capsid. Using quantitative biochemical, immunochemical and morphological methods, we demonstrate that inhibitors of the cystine protease, L3/p23, located inside the capsid block the degradation of the capsid-stabilizing protein VI, and prevent virus uncoating at the nuclear membrane. There was no effect on virus internalization, fiber shedding and virus binding to the nuclear envelope. The viral enzyme (dormant in the extracellular virus) was activated by two separate signals, neither of which was sufficient alone; virus interaction with the integrin receptor (inhibited with RGD peptides) and re-entry of the virus particle into a reducing environment in the endosome or the cytosol. Incorrectly assembled mutant viruses that lack the functional protease (ts1) failed at releasing fibers and penetrating into the cytosol. The results indicated that L3/p23 is needed not only to assemble an entry-competent virus but also to disassemble the incoming virus.
To understand how DNA is released from cationic liposome/DNA complexes in cells, we investigated which biomolecules mediate release of DNA from a complex with cationic liposomes. Release from monovalent[1,2-dioleoyl-3(1)-1(trimethylammonio)propane] or multivalent (dioctadecylamidoglycylspermine) lipids was quantified by an increase of ethidium bromide (EtBr) fluorescence. Plasmid sensitivity to DNAse I degradation was examined using changes in plasmid migration on agarose gel electrophoresis. Physical separation of the DNA from the cationic lipid was confirmed and quantified on sucrose density gradients. Anionic liposomes containing compositions that mimic the cytoplasmic-facing monolayer of the plasma membrane (e.g. phosphatidylserine) rapidly released DNA from the complex. Release occurred near a 1/1 charge ratio (-/+) and was unaffected by ionic strength or ion type. Water soluble molecules with a high negative linear charge density such as dextran sulfate or heparin also released DNA. However, ionic water soluble molecules such as ATP, tRNA, DNA, poly(glutamic acid), spermidine, spermine, or histone did not, even at 100-fold charge excess (-/+). On the basis of these results, we propose that after the cationic lipid/DNA complex is internalized into cells by endocytosis it destabilizes the endosomal membrane. Destabilization induces flip-flop of anionic lipids from the cytoplasmic-facing monolayer, which laterally diffuse into the complex and form a charge neutral ion pair with the cationic lipids. This results in displacement of the DNA from the cationic lipid and release of the DNA into cytoplasm. This mechanism accounts for a variety of observations on cationic lipid/DNA complex-cell interactions.
To develop a nonviral gene delivery system for treatment of diseases, our strategy is to construct DNA complexes with short synthetic peptides that mimic the functions of viral proteins. We have designed and synthesized two peptides which emulate viral functions - a DNA condensing agent, YKAK(8)WK, and an amphipathic, pH-dependent endosomal releasing agent, GLFEALLELLESLWELLLEA. The active gene delivery complex was constructed step-wise through a spontaneous self-assembly process involving oppositely charged, electrostatic interactions. To assemble DNA-peptide complexes with different overall net charges, only the negative charges of DNA phosphate, the positive charges of the 10 epsilon-amino groups of YKAK(8)WK and the negative charges of the 5 gamma-carboxyl groups of GLFEALLELLESLWELLLEA were considered. In the first step, negatively charged DNA was rapidly-mixed with an excess of YKAK(8)WK to form positively charged DNA-YKAK(8)WK complexes, which gave little gene transfer. In the second step and to form the active complex,the cationic DNA complex was rapidly mixed with spontaneously incorporated through electrostatic interactions. Transfection using these complexes of CMV-luc, YKAK(8)WK and GLFEALLELLESLWELLLEA gave high-levels of gene expression in a variety of cell lines. These simple DNA complexes, which contain only three molecularly defined components, have general utility for gene delivery and can replace viral vectors and cationic lipids for some applications in gene therapy.
Adenovirus uncoating is a stepwise process which culminates in the release of the viral DNA into the nucleus through the nuclear pore complexes and dissociation of the capsid. Using quantitative biochemical, immunochemical and morphological methods, we demonstrate that inhibitors of the cystine protease, L3/p23, located inside the capsid block the degradation of the capsid‐stabilizing protein VI, and prevent virus uncoating at the nuclear membrane. There was no effect on virus internalization, fiber shedding and virus binding to the nuclear envelope. The viral enzyme (dormant in the extracellular virus) was activated by two separate signals, neither of which was sufficient alone; virus interaction with the integrin receptor (inhibited with RGD peptides) and re‐entry of the virus particle into a reducing environment in the endosome or the cytosol. Incorrectly assembled mutant viruses that lack the functional protease (ts1) failed at releasing fibers and penetrating into the cytosol. The results indicated that L3/p23 is needed not only to assemble an entry‐competent virus but also to disassemble the incoming virus.
Cell surface receptors are good candidates to selectively target drugs, oligonucleotides or even genes by making use of their specific ligands. A large number of mammalian cells express cell surface sugar-binding proteins, also called “membrane lectins”. Therefore, sugars may be used as specific recognition signals to specifically deliver biological active components. Tens of membrane lectins with different sugar specificities have been characterized; some of them actively carry their ligands to intracellular compartments, including endsomes, lysosomes and, in some cases, Golgi apparatus.
This paper reviews the current information regarding the use of human adenoviruses as vehicles for transfer of genetic material into mammalian cells. The clinical aspects and genetic program or molecular biology of natural, wild-type adenovirus infection will be discussed in order to provide a background for understanding the pharmacology and toxicology of adenoviral gene transfer vectors. The use of adenovirus itself and adenovirus-based vectors as vaccines is reviewed as well as the use of adenoviral vectors for gene transfer. Adenoviral vectors provide an important potential in vivo delivery vehicle for immunization strategies as well as genetic therapy for correction of genetic and acquired diseases.
The etiology of cystic fibrosis (CF), current therapies, and recent experimental molecular based therapies are briefly described including the use of recombinant human deoxyribonuclease I (rhDNase), nonsteroidal antiinflammatory drugs (ibuprofen), the sodium channel blocking diuretic amiloride, and uridine triphosphate (UTP), a chloride secretagogue. The original approach to CF gene therapy using recombinant replication defective adenovirus and its potential benefits as well as shortcomings are also briefly discussed. More recently, the use of nonviral cationic liposome plasmid complexes to deliver cystic fibrosis transmembrane conductance regulator (CFTR) cDNA has been successfully demonstrated in both transgenic mice and in human patients. The formulations and delivery protocols employed by various investigators are discussed in detail and summarized for comparison in a table. While all investigators show transfection and expression of the transgene to some extent, the experimental conditions vary widely, greatly frustrating attempts to establish common modalities or underlying mechanisms. The numerous safety studies both in experimental animals and in human volunteers are also discussed in detail. Again, the absence of standard protocols as well as a lack of rigorous toxicity study design and analysis precludes generalizations as to the innocuousness of cationic liposomes and cationic liposome plasmid complexes. Suitably designed toxicity, pharmacokinetic and metabolic studies of these new ‘drug’ modalities, and clearer definitions of the expected outcomes of efficacy and toxicity are desirable.
Mixing of poly(U) with polylysine in solutions of low ionic strength resulted in conversion of the polynucleotide to its double-stranded form. The amount of poly(U) converted depended on the amount of polylysine added, complete conversion being achieved by about lysine: phosphate = 0.7. The melting temperature of the double-helical structure in complexes with high molecular weight polylysines was approximately 33°, independent of lysine:phosphate ratio and polylysine DP. In complexes with the reversibly bound cations pentalysine and Mg2+, Tm increased with increasing cation concentration. Neither polyarginine nor protamine was effective in converting poly(U) to its ordered form. At lysine: phosphate ratios near unity, poly(U)-polylysine complexes formed aggregates with greatly enhanced circular dichroism ((εl - εr) = >100). Very high molecular weight polylysines (DP = 300, 500) were relatively ineffective in producing these large-magnitude spectra. At lysine:phosphate ratios well above 1.0, the complexes were once again soluble, probably due to binding of excess polylysine. In light of the fact that complexes of polylysine with both poly(U) and DNA are capable of forming aggregates with greatly enhanced circular dichroism (CD) spectra, complexes were prepared with a number of other polynucleotides of various secondary structures. Aggregates were formed in electroneutral mixtures with all polynucleotides; however, enhanced CD spectra were observed only in the cases of the double-stranded polynucleotides examined (DNA, [poly(U)]2, poly(I)·; poly(C), and poly(A)· poly(U)). None of the single- (poly(A), poly(C), and TMV RNA) or triple-stranded (poly(A)·2poly(U), [poly(I)]3) polynucleotides gave complexes with enhanced spectra. Interestingly, though, poly(I) appeared to form aggregates with large-magnitude CD in high concentrations of NaCl, without added polylysine.
In a mixture of DNA and polylysine at very high ionic strength (1.50 M NaCl) there appeared to be little or no interaction between the oppositely charged polymers. As the salt concentration was gradually lowered, in a linear dialysis gradient, aggregated complexes were formed which had a circular dichroism spectrum much larger in magnitude than and much different in shape from the sum of the spectra of the individual components. The region of salt concentrations in which the aggregate formation occurred varied with the molecular weight of the polylysine used and with the lysine: phosphate ratio in the mixture, but was generally in the range 0.7-1.0 M NaCl. Up to lysine : phosphate ratios around 0.75, addition of more polylysine led to the incorporation of more of the DNA into the aggregates with anomalous circular dichroism (CD) spectra. Beyond this point, although additional complex may still have been formed, the size of the aggregates became very large and the magnitude of the CD spectrum was reduced due to "shadowing" of the optically active material. It was found that very long polylysines (DP = 300, 500) formed aggregated complexes having lower magnitude CD spectra than those formed with intermediate sizes (DP = 26, 75). Annealed complexes of DNA with polyarginine and protamine had only slightly altered circular dichroism spectra, never approaching the magnitude of those characteristic of DNA-polylysine complexes.
An ability to generate a well defined lipid-based carrier system for the delivery of plasmid DNA in vivo requires the characterization of factors governing DNA/lipid interactions and carrier formation. We report that a hydrophobic DNA/lipid complex can be formed following addition of cationic lipids to DNA in a Bligh and Dyer monophase consisting of chloroform/methanol/water (1:2.1:1). Subsequent partitioning of the monophase into a two-phase system allows for the extraction of DNA into the organic phase. When using monovalent cationic lipids, such as dimethyldioctadecylammonium bromide, dioleyldimethylammonium chloride, and 1,2-dioleyl-3-N,N,N-trimethylaminopropane chloride, greater than 95% of the DNA present can be recovered in the organic phase when the lipid is added at concentrations sufficient to neutralize DNA phosphate charge. When the polyvalent cationic lipids 2,3-dioleyloxy-N-[2(sperminecarboxamido)ethyl]-N,N-dimethyl- 1- propanaminium trifluoroacetate and diheptadecylamidoglycyl spermidine are used, efficient extraction of the DNA into the organic phase is also achieved when the charge ratio between lipid and DNA is approximately equal. Formation of the hydrophobic DNA complex can only be achieved with cationic lipids. In the absence of added cations or in the presence of excess Ca2+, L-lysine, or poly(L-lysine), 100% of the DNA is recovered in the aqueous fraction. The monovalent cationic lipid/DNA complexes can also be prepared in the presence of detergent; however, low concentrations of NaCl (< 1 mM) lead to dissociation of the complex. Importantly, these results clearly demonstrate that cationic lipid binding does not lead to DNA condensation. The methods described, therefore, enable DNA/lipid complexes to be characterized in the absence of DNA condensation.(ABSTRACT TRUNCATED AT 250 WORDS)
The association structures formed by cationic liposomes and DNA-plasmids have been successfully employed as gene carriers in transfection assays. In the present study such complexes was studied by cryo-TEM (cryo-transmission electron microscopy). Cationic liposomes made up by DOPE (dioleoylphosphatidylethanolamine) and various amounts of three different cationic surfactants were investigated. The cryo-TEM analysis suggests that an excess of lipid in terms of charge, leads to entrapment of the DNA molecules between the lamellas in clusters of aggregated multilamellar structures. With increasing amounts of DNA free or loosely bound plasmids were found in the vicinity of the complexes. The importance of the choice of surfactant, as reported from many transfection assays, was not reflected in changes of the type of DNA-vesicle association. A tendency towards polymorphism of the lipid mixtures is reported and its possible implications are discussed.
Structures formed during interaction of cationic liposomes and plasmid DNA were studied by freeze-fracture electron microscopy and their morphology was found to be dependent on incubation time and DNA concentration. These structures were formed with liposomes composed of DC-Chol and DOPE after 30 min incubation at DNA:lipid concentrations encompassing maximal transfection activity. They resembled liposome complexes (meatballs) and additionally bilayer-covered DNA tubules (spaghetti), whereby the DNA-tubules were found to be connected to the liposome complexes as well as occurring free in the suspension. At later times and higher DNA-to-liposome ratios the complexes grow larger while their membranes become discontinuous, allowing the self-encapsulation of the DNA. The relative transfection potency of the various morphologically distinct structures is discussed.
Synthetic gene transfer vectors could be an attractive alternative to biological vehicles for gene therapy. In an effort to improve the previously developed lipopolyamine-mediated transfection technique, various amphiphilic DNA-binding molecules have been synthesized. Besides Transfectam, several lipospermines display very high gene delivery levels. The structure-activity relationship obtained points to the central role played by the polyamine headgroup in condensing the plasmid and binding it to the cell surface, provided the hydrophobic moiety is capable to generate nonmicellar mesomorphic structures. It also highlights other favorable (albeit more speculative) properties shared by protonable lipospermines as compared to quaternary ammonium-bearing lipids, such as their ability to act as a buffer and their strong affinity for chromatin. The former property may prevent the pH decrease along the degradative lysosomial pathway. The ability to bind to chromatin even in the presence of endogeneous polyamines should have two consequences: a nuclear tropism of the transfecting particles and plasmid uncoating in the nucleus by competitive dilution of the lipopolyamine into an ocean of DNA.
Complexes formed between cationic liposomes and nucleic acids represent a highly efficient vehicle for delivery of DNA and RNA molecules into a large variety of eukaryotic cells. By using fluorescence, gel electrophoresis, and metal-shadowing electron microscopy techniques, the factors that affect the, yet unclear, interactions between DNA and cationic liposomes as well as the structural features of the resulting complexes have been elucidated. A model is suggested according to which cationic liposomes bind initially to DNA molecules to form clusters of aggregated vesicles along the nucleic acids. At a critical liposome density, two processes occur, namely, DNA-induced membrane fusion, indicated by lipid mixing studies, and liposome-induced DNA collapse, pointed out by the marked cooperativity of the encapsulation processes, by their modulations by DNA-condensing agents, and also by their conspicuous independence upon DNA length. The DNA collapse leads to the formation of condensed structures which can be completely encapsulated within the fused lipid bilayers in a fast, highly cooperative process since their exposed surface is substantially smaller than that of extended DNA molecules. The formation of the transfecting DNA-liposome complexes in which the nucleic acids are fully encapsulated within a positively-charged lipid bilayer is proposed, consequently, to be dominated by mutual effects exerted by the DNA and the cationic liposomes, leading to interrelated lipid fusion and DNA collapse.
Although most research on gene therapy has focused on the use of recombinant viruses to deliver genes to cells in vivo, progress also has been made toward developing nonviral, pharmaceutical formulations of genes for in vivo human therapy. Various methods for nonviral gene therapy have been proposed. Some approaches are aimed at developing "artificial viruses" that attempt to mimic the process of viral infection using synthetic materials. Others apply the theory and methods of advanced, particulate drug delivery to deliver DNA to select somatic targets. These approaches employ DNA complexes containing lipid, protein, peptide, or polymeric carriers as well as ligands capable of targeting the DNA complex to cell-surface receptors on the target cell and ligands for directing the intracellular trafficking of DNA to the nucleus. Nonviral systems have been used to deliver genes to the lung, liver, endothelium, epithelium, and tumor cells and have been shown to be generally safe. More than a dozen clinical trials are currently underway using nonviral systems for disease indications including cystic fibrosis and cancer. Future advances in nonviral systems will be based on an emerging appreciation of the biological constraints on the fate and function of DNA within the body and within the cell.
Direct gene transfer for the treatment of human diseases requires a vector which can be administered efficiently, safely and repeatedly. Cationic liposomes represent one of the few examples that can meet these requirements. Currently, more than a dozen cationic liposome formulations have been reported. These liposomes bind and condense DNA spontaneously to form complexes with high affinity to cell membranes. Endocytosis of the complexes followed by disruption of the endosomal membrane appears to be the major mechanism of gene delivery. The effectiveness and safety of this DNA delivery method has been established in many studies. Based on these results, two human gene therapy clinical trials using cationic liposomes have been conducted and more trials will be started in the near future. The simplicity, efficiency and safety features have rendered the cationic liposome an attractive vehicle for human gene therapy.
Long-term expression of a reporter gene has previously been reported in skeletal and cardiac muscles after direct injection of naked plasmid DNA. In this study, we have shown that the direct injection of free plasmid DNA into mouse melanoma BL6 solid tumor can also result in a high level of transfection. THe average amount of chloramphenicol acetyltransferase (CAT) expressed by injecting 30 micrograms plasmid DNA containing a CAT gene into a single BL6 tumor was 1.9 +/- 1.0 ng, which is comparable to that reported in the skeletal muscle. Cationic liposomes, Lipofectamine and DC-chol/DOPE, inhibited gene expression in a dose-dependent manner. Transgene expression by free DNA persisted for at least 10 days. The size of tumor did not seem to affect the gene expression, but proper choice of a diluent solution for DNA was an important factor. Genes driven by the CMV promoter were expressed much more efficiently than genes driven by the SV40 or T7 promoter. Optimal dosage of injected DNA was from 30 to 70 micrograms per tumor. Other mouse melanomas, human melanomas and cervical carcinomas are also able to express directly injected plasmid DNA, but the transfection efficiency is lower than the BL6 tumor. Direct injection of free plasmid DNA is a simple and effective approach and might be a potential method for cancer gene therapy.
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