9240132
Cationic solid lipid nanoparticles loaded by cysteine proteinase genes as a novel anti-leishmaniasis DNA vaccine delivery system: characterization and in vitro evaluations.
PURPOSE
Leishmaniasis is a major health problem in many tropical and sub-tropical countries and development of a safe and easily-available vaccine has high priority. Although several antigens potentially capable of inducing protective immunity have been studied, in the absence of pharmaceutical industry interest they have remained as fine publications only. Amongst them, Cathepsin L-like cysteine proteinases (CPs) have received considerable attention and type I and II CPs have been used in a heterologous prime-boost vaccination regime for experimental visceral leishmaniasis in dogs. Due to the promising results of the mentioned vaccination regime, we aimed to evaluate cationic solid lipid nanoparticles (cSLNs) for in vitro delivery of cpa, cpb and cpb(CTE) intended to be used as a cocktail DNA vaccine in our forthcoming studies.


METHODS
cSLNs were formulated of cetyl palmitate, cholesterol, DOTAP and Tween 80 via melt emulsification method followed by high shear homogenization. Different formulations were prepared by anchoring pDNAs on the surface of cSLNs via charge interaction. The formulations were characterized according to their size and zeta potential as well as pDNA integrity and stability against DNase I treatment. Lipoplexes' cytotoxicity was investigated on COS-7 cells by MTT test. The effect of the DOTAP:pDNA ratio on protection ability and cytotoxicity was also studied. In vitro transfection efficiency was qualified by fluorescent microscopy and quantified using flow cytometry technique.


RESULTS
cSLN-pDNA complexes were formulated with suitable size and zeta potential. Efficiency/cytotoxicity ratio of cSLN-pDNAs formulations was comparable to linear PEI-25KD-pDNAs polyplexes while exhibiting significantly lower cytotoxicity.


CONCLUSION
Tested formulations were able to deliver immunogenic CP genes efficiently. This data proves the ability of this system as a promising DNA vaccine carrier for leishmaniasis to cover the main drawback of naked pDNA delivery that is rapid elimination from the circulation.
32288682
Leishmaniasis: current treatment and prospects for new drugs and vaccines.
Leishmaniasis is a disease that ranges in severity from skin lesions to serious disfigurement and fatal systemic infection. WHO estimates that the disease results in 2 million new cases a year, threatens 350 million people in 88 countries and that there are 12 million people currently infected worldwide. Current treatment is based on chemotherapy, which relies on a handful of drugs with serious limitations such as high cost, toxicity, difficult route of administration and lack of efficacy in endemic areas. Pentavalent antimonials have been the mainstay of antileishmanial therapy for over 70 years with second line drugs, Amphotericin B and Pentamidine, used in case of antimonial failure. Since the introduction of miltefosine at the beginning of this century, no new antileishmanial compounds have been approved for human treatment. Leishmaniasis is considered one of a few parasitic diseases likely to be controllable by vaccination. However, to date no such vaccine is available despite substantial efforts by many laboratories. The development of a safe, effective and affordable antileishmanial vaccine is a critical global public-health priority. This review outlines the current status of vaccine development and looks at the currently available chemotherapy as well as examples of drugs in development and different approaches to antileishmanial drug discovery and identification of novel antiparasitic compounds.
43505842
Immunoregulatory activity of CpG oligonucleotides in humans and nonhuman primates.
Oligodeoxynucleotides (ODN) containing CpG motifs mimic the ability of microbial DNA to activate the innate immune system. The resultant response limits the early spread of infectious organisms while promoting the development of adaptive immunity. CpG ODN show promise as vaccine adjuvants and in the treatment of asthma, allergy, infection, and cancer. Due to evolutionary divergence in CpG recognition between species, CpG ODN that are most active in rodents are poorly immunostimulatory in primates. Thus, evidence that CpG ODN have therapeutic activity in mice must be confirmed in primates. Two distinct types of CpG ODN were identified that stimulate primate PBMC. D-type ODN trigger plasmacytoid DC to secrete IFNalpha, monocytes to mature into functionally active DC, and NK cells to secrete IFNgamma. K-type ODN stimulate B cells and monocytes to proliferate and secrete IgM, IL-10, and/or IL-6. In vivo studies in nonhuman primates indicate that proinflammatory or humoral immune responses can be selectively facilitated by judicious use of these distinct types of ODN.
21957500
New horizons in adjuvants for vaccine development.
Over the last decade, there has been a flurry of research on adjuvants for vaccines, and several novel adjuvants are now in licensed products or in late stage clinical development. The success of adjuvants in enhancing the immune response to recombinant antigens has led many researchers to re-focus their vaccine development programs. Successful vaccine development requires knowing which adjuvants to use and knowing how to formulate adjuvants and antigens to achieve stable, safe and immunogenic vaccines. For the majority of vaccine researchers this information is not readily available, nor is access to well-characterized adjuvants. In this review, we outline the current state of adjuvant research and development and how formulation parameters can influence the effectiveness of adjuvants.
21181885
Vaccines for leishmaniasis in the fore coming 25 years.
Human vaccination against leishmaniasis using live Leishmania was used in Middle East and Russia (1941-1980). First-generation vaccines, composed by killed parasites induce low efficacies (54%) and were tested in humans and dogs Phase III trials in Asia and South America since 1940. Second-generation vaccines using live genetically modified parasites, or bacteria or viruses containing Leishmania genes, recombinant or native fractions are known since the 1990s. Due to the loss of PAMPs, the use of adjuvants increased vaccine efficacies of the purified antigens to 82%, in Phase III dog trials. Recombinant second-generation vaccines and third-generation DNA vaccines showed average values of parasite load reduction of 68% and 59% in laboratory animal models, respectively, but their success in field trials had not yet been reported. This review is focused on vaccine candidates that show any efficacy against leishmaniasis and that are already in different phase trials. A lot of interest though was generated in recent years, by the studies going on in experimental models. The promising candidates may find a place in the forth coming years. Among them most probably are the multiple-gene DNA vaccines that are stable and do not require cold-chain transportation. In the mean time, second-generation vaccines with native antigens and effective adjuvants are likely to be licensed and used in Public Health control programs in the fore coming 25 years. To date, only three vaccines have been licensed for use: one live vaccine for humans in Uzbekistan, one killed vaccine for human immunotherapy in Brazil and a second-generation vaccine for dog prophylaxis in Brazil.
84506516
Leishmania Cysteine Proteinases: From Gene to Subunit Vaccine
Whole genome sequences of microbial pathogens present new opportunities for clinical application. Presently, genome sequencing of the human protozoan parasite Leishmania major is in progress. The driving forces behind the genome project are to identify genes with key cellular functions and new drug targets, to increase knowledge on mechanisms of drug resistance and to favor technology transfer to scientists from endemic countries. Sequencing of the genome is also aimed at the identification of genes that are expressed in the infectious stages of the parasite and in particular in the intracellular form of the parasite. Several protective antigens of Leishmania have been identified. In addition to these antigens, lysosomal cysteine proteinases (CPs) have been characterized in different strains of Leishmania and Trypanosoma, as new target molecules. Recently, we have isolated and characterized Type I (CPB) and Type II (CPA) cysteine proteinase encoding genes from L. major. The exact function of cysteine proteinases of Leishmania is not completely understood, although there are a few reports describing their role as virulence factors. One specific feature of CPB in Leishmania and other trypanosomatids, is the presence of a Cterminal extension (CTE) which is possibly indicative of conserved structure and function. Recently, we demonstrated that DNA immunization of genetically susceptible BALB / c mice, using a cocktail of CPB and CPA genes, induced long lasting protection against L. major infection. This review intends to give an overview of the current knowledge on genetic vaccination used against leishmaniasis and the importance of CP genes for such an approach.
9892929
A protective cocktail vaccine against murine cutaneous leishmaniasis with DNA encoding cysteine proteinases of Leishmania major.
The protection elicited by the intramuscular injection of two plasmid DNAs encoding Leishmania major cysteine proteinase type I (CPb) and type II (CPa) was evaluated in a murine model of experimental cutaneous leishmaniasis. BALB/c mice were immunized either separately or with a cocktail of the two plasmids expressing CPa or CPb. It was only when the cpa and cpb genes were co-injected that long lasting protection against parasite challenge was achieved. Similar protection was also observed when animals were first immunized with cpa/cpb DNA followed by recombinant CPa/CPb boost. Analysis of the immune response showed that protected animals developed a specific Th1 immune response, which was associated with an increase of IFN-gamma production. This is the first report demonstrating that co-injection of two genes expressing different antigens induces a long lasting protective response, whereas the separate injection of cysteine proteases genes is not protective.
31004129
Protective vaccination against experimental canine visceral leishmaniasis using a combination of DNA and protein immunization with cysteine proteinases type I and II of L. infantum.
Leishmania infantum is known to be associated with visceral leishmaniasis in Iran and canids are natural reservoirs. Control of disease in dogs appears to be one of the most effective approaches for interrupting the domestic cycle of the disease. In search for successful vaccine strategies, we evaluated the cysteine proteinases (CPs) type I and II using a heterologous prime-boost regime for vaccination against experimental visceral leishmaniasis in dogs. Following vaccination and challenge, dogs were followed for 12 months. Ten dogs vaccinated by prime/boost with DNA/recombinant CPs (in combination with CpG ODN and Montanide 720) remained free of infection in their bone morrow. In contrast, three out of four dogs in the control groups had infection in their bone marrow. The peripheral lymphocytes from protected animals had generally higher proliferation responses to F/T antigen, recombinant CPA (rCPA) and recombinant CPB (rCPB) than controls. During post-challenge period, the difference in stimulation index is significant (p<0.05) on months 11 and 12 to F/T antigens, all months for rCPA and 5, 7, 9, 11 and 12 months for rCPB. Analysis of cytokine mRNA level suggested that vaccinated dogs had elevated IFN-gamma mRNA in peripheral blood mononuclear cells (PBMC), whereas there was a consistent increase in the level of IL-10 in the control groups and some vaccinated dogs. The level of total IgG and IgG2, but not IgG1, to rCPA and rCPB was significantly higher in the vaccinated group (p<0.05) than the control groups. We also showed that with the exception of one dog, all dogs in the vaccinated group in comparison to control dogs had strong DTH responses. We propose that the combination of DNA and recombinant protein vaccination using CPs could be instrumental to control (VL) in dogs.
20327563
Leishmania infantum: prime boost vaccination with C-terminal extension of cysteine proteinase type I displays both type 1 and 2 immune signatures in BALB/c mice.
The aims of current study are to describe the immunogenicity and protective efficacy of prime boost vaccine using C-terminal extension (CTE) of cysteine proteinase type I of Leishmania infantum in BALB/c mice. Group I as vaccinated group primed with 100 microg of pcDNA-CTE and 3 weeks later boosted with combination of 30 microg rCTE, 50 microg of CpG and Montanide 720. Groups II and III were served as control groups. Although, this vaccination regimen did not protect mice against the infectious challenge but it was highly immunogenic. IgG2a has been raised strongly against rCTE in contrast to IgG1 and remains high at every time point under study. By analysis of CTE synthetic peptides (CTE100) before challenge, both IgG1 and IgG2a were produced and for all overlapping synthetic peptides (CTE 1-8) IgG1 raised significantly. This statue is changed at 7 weeks after challenge and only CTE2 and CTE3 have shown to induce considerable amount of IgG1. In all groups, the level of IL-5 started to increase with high concentration shortly passing only 3 weeks after infectious challenge. In compare with two control groups, the vaccinated group produced significantly higher level of IL-5 at 7 weeks post-infection. The parasite burden of all groups is similar at 4 weeks post-challenge in both liver and spleen. In contrast, at 8 weeks post challenge, the spleen of the vaccinated group showed significantly higher level of parasite load in compare with two control groups. This study demonstrated that immunization with CTE display both type 1 and 2 immune signatures in experimental murine model of L. infantum infection.
205280246
Lipid-based delivery of CpG oligonucleotides enhances immunotherapeutic efficacy.
There has been significant interest in the potential of cytosine-guanine (CpG) containing oligodeoxynucleotides (ODN) as an immunotherapy for malignant, infectious and allergic diseases. While human trials have yielded promising results, clinical use of free CpG ODN still faces several challenges which limit their effectiveness. These include suboptimal in vivo stability, toxicity, unfavorable pharmacokinetic/biodistribution characteristics, lack of specificity for target cells and the requirement for intracellular uptake. To overcome these challenges, optimized lipid-based delivery systems have been developed to protect the CpG ODN payload, modify their circulation/distribution so as to enhance immune cell targeting and facilitate intracellular uptake. Ultimately, lipid-mediated delivery has the capacity to increase the immunopotency of CpG ODN and enhance their prophylactic or therapeutic efficacy in a range of diseases. Lipid-encapsulation provides a feasible strategy to optimize the immunostimulatory activity and immunotherapeutic efficacy of CpG ODN, thereby allowing their full clinical potential to be realized.
12195897
Cytotoxicity of Solid Lipid Nanoparticles as a Function of the Lipid Matrix and the Surfactant
AbstractPurpose. Assessment of the in vitro cytotoxicity of solid lipid nanoparticles (SLNs) as a function of lipid matrix (Dynasan 114, Compritol ATO 888), and stabilizing surfactant (poloxamers, Tween 80, soya lecithin, and sodium dodecyl sulphate). Comparison with other colloidal carriers should determine their potential use in the clinic.
Methods. SLNs were produced by high pressure homogenisation. Cytotoxicity was assessed by measuring the viability of HL60 cells and human granulocytes after incubation with SLNs. Particle internalisation was quantified by chemiluminescence measurements.
Results. The nature of the lipid had no effect on viability; distinct differences were found for the surfactants. Binding to the SLN surface reduced markedly the cytotoxic effect of the surfactants, e.g., up to a factor of 65 for poloxamer 184. The permanent HL60 cell line— differentiated from cells with granulocyte characteristics by retinoic acid treatment—yielded results identical to freshly isolated human granulocytes. In general, the SLNs showed a lower cytotoxicity compared to polyalkylcyanoacrylate and polylactic/glycolic acid (PLA/ GA) nanoparticles.
Conclusions. Because the results are identical when using human granulocytes, differentiated HL60 cells can be used as an easily accessible in vitro test system for i.v. injectable SLN formulations. The SLNs appear suitable as a drug carrier system for potential intravenous use due to their very low cytotoxicityin vitro.
30906617
Lipid nanoparticles for parenteral delivery of actives.
The present review compiles the applications of lipid nanoparticles mainly solid lipid nanoparticles (SLN), nanostructured lipid carriers (NLC) and lipid drug conjugates (LDC) in parenteral delivery of pharmaceutical actives. The attempts to incorporate anticancer agents, imaging agents, antiparasitics, antiarthritics, genes for transfection, agents for liver, cardiovascular and central nervous system targeting have been summarized. The utility of lipid nanoparticles as adjuvant has been discussed separately. A special focus of this review is on toxicity caused by these kinds of lipid nanoparticles with a glance on the fate of lipid nanoparticles after their parenteral delivery in vivo viz the protein adsorption patterns.
673903
Effect of cationic lipid and matrix lipid composition on solid lipid nanoparticle-mediated gene transfer.
This investigation is focused on the enhancement of in vitro transfection activity by optimizing cationic lipid and matrix lipid composition of solid lipid nanoparticles (SLN). For this purpose SLN were formulated by using two different matrix lipids and six different cationic detergents. These 12 formulations were tested for physical parameters such as particle size, zeta potential and DNA-binding capacity, and also for their biological properties such as cytotoxicity and in vitro transfection efficiency. The SLN were produced by hot high-pressure homogenization, all formulations were physically stable and showed a highly positive surface charge (+34 to +45 mV). In vitro cytotoxicity measurements on COS-1 cells revealed that cytotoxicity is strongly dependent on the cationic lipid used. SLN made from one-tailed cationic detergents were highly cytotoxic. In contrast the two-tailed cationic lipids were all well tolerated. Transfection activity seems to be determined by both the cationic lipid and the matrix lipid used. Here, the combination of cetylpalmitate and N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride led to significantly higher transfection efficiencies than in all other tested combinations. These results indicate that well tolerated and highly efficient in vitro transfection could be achieved with SLN whenever selecting good combinations of two-tailed cationic lipids and matrix lipids.
40472083
Solid lipid nanoparticles: formulation factors affecting cell transfection capacity.
Since solid lipid nanoparticles (SLNs) were introduced as non-viral transfection systems, very few reports of their use for gene delivery have been published. In this work different formulations based on SLN-DNA complexes were formulated in order to evaluate the influence of the formulation components on the "in vitro" transfection capacity. SLNs composed by the solid lipid Precirol ATO 5, the cationic lipid DOTAP and the surfactant Tween 80, and SLN-DNA complexes prepared at different DOTAP/DNA ratios were characterized by studying their size, surface charge, DNA protection capacity, transfection and cell viability in HEK293 cultured cells. The incorporation of Tween 80 allowed for the reduction of the cationic lipid concentration. The formulations prepared at DOTAP/DNA ratios 7/1, 5/1 and 4/1 provided almost the same transfection levels (around 15% transfected cells), without significant differences between them (p>0.05). Other assayed formulations presented lower transfection. Transfection activity was dependent on the DOTAP/DNA ratio since it influences the DNA condensation into the SLNs. DNA condensation is a crucial factor which conditions the transfection capacity of SLNs, because it influences DNA delivery from nanoparticles, gene protection from external agents and DNA topology.
205319800
Solid lipid nanoparticles as potential tools for gene therapy: in vivo protein expression after intravenous administration.
Naked plasmid DNA is a powerful tool for gene therapy, but it is rapidly eliminated from the circulation after intravenous administration. Therefore, the development of optimized DNA delivery systems is necessary for its successful clinical use. Solid lipid nanoparticles (SLNs) have demonstrated transfection capacity in vitro, but their application for gene delivery has not been conveniently investigated in vivo. We aimed to evaluate the capacity of SLN-DNA vectors to transfect in vivo after intravenous administration to mice. The SLNs, composed of Precirol ATO 5, DOTAP and Tween 80 were complexed with the plasmid pCMS-EGFP which encodes the enhanced green fluorescent protein (EGFP). The resulting systems were characterized in vitro showing a mean particle size of 276 nm, superficial charge of +28 mV, the ability to protect the plasmid and transfection capacity in culture cells. The intravenous administration in mice led to transfection in hepatic tissue and spleen. Protein expression was detected from the third day after administration, and it was maintained for at least 1 week. This work shows for the first time the capacity of SLN-DNA vectors to induce the expression of a foreign protein after intravenous administration, supporting the potential of SLNs for gene therapy.
205320359
pDNA condensation capacity and in vitro gene delivery properties of cationic solid lipid nanoparticles.
Cationic solid lipid nanoparticles (SLN) are promising nonviral gene delivery carriers suitable for systemic administration. The objective of this study was to investigate the relationship between the composition of cationic SLN and their ability to condense plasmid DNA (pDNA) and to transfer it in neuroblastoma cells. The SLN were prepared by using stearic acid and stearylamine as lipid core along with Esterquart 1 (EQ1) or Protamine obtaining two samples (SLN-EQ1 and SLN-Protamine, respectively). The cationic SLN were freeze-dried after preparation and their physical-chemical properties, including the surface composition and the transfection efficiency were investigated. The results showed that the two samples had similar size, zeta potential and pDNA binding properties but SLN-Protamine were able to condense pDNA more efficaciously than SLN-EQ1 forming smaller and less positive complexes. SLN-Protamine:pDNA complexes demonstrated to be less cytotoxic and more efficient in the transfection of Na1300 cell line than SLN-EQ1:pDNA. These findings were attributed to the different surface composition of the two samples and in particular to the localization of the Protamine on the surface of the particle while EQ1 in the lipid core. In conclusion the results here suggest that not only the z-potential but also the surface composition may affect the pDNA condensation proprieties and thus the transfection efficiency of nonviral gene nanocarriers.
8665920
Cationic solid-lipid nanoparticles can efficiently bind and transfect plasmid DNA.
The suitability of cationically modified solid-lipid nanoparticles (SLN) as a novel transfection agent was investigated. SLN were produced by hot homogenisation using either Compritol ATO 888 or paraffin as matrix lipid, a mixture of Tween 80 and Span 85 as tenside and either EQ1 (N,N-di-(beta-steaorylethyl)-N,N-dimethylammonium chloride) or cetylpyridinium chloride as charge carrier. The resulting particles were approximately 100 nm in size and showed zeta potentials around +40 mV at pH 7.4. DNA binding was tested by agarose gel electrophoresis. The resulting SLN-DNA complexes were further characterised by AFM and zeta potential measurements. Only the SLN batch SII-13, composed of 4% Compritol, 4% Tween/Span and 1% EQ1, was able to form stable complexes with DNA. Typical complexes were 300 to 800 nm in size. Cytotoxicity and transfection efficiency was tested in vitro on Cos-1 cells. Cationic SLN produced by modification with EQ1 were well tolerated, with LD50 values >3 mg/ml in the LDH release assay and >0.6 mg/ml in the WST-1 assay. Further, SLN-DNA complexes containing between 10 and 200 weight equivalents of SII-13 (matrix lipid) efficiently transfected the galactosidase expression plasmid pCMVbeta in the absence and presence of the endosomolytic agent chloroquine.
11550803
Drug retention and stability of solid lipid nanoparticles containing azidothymidine palmitate after autoclaving, storage and lyophilization.
Solid lipid nanoparticles (SLNs) were prepared using trilaurine (TL) as the SLN core and phospholipid (PL) as coating. Neutral and negatively charged PLs were used to produce neutral and negatively charged SLNs. An ester prodrug of 3'-azido-3'-deoxythymidine (Zidovudine, AZT), AZT palmitate (AZT-P), was synthesized and incorporated in the SLNs. The stability of SLN formulations containing AZT-P was studied at different temperatures. Drug retention and mean particle diameter of SLNs were determined after autoclaving, during temperature stability testing, and after lyophilization (with or without cryoprotective sugars) and reconstitution. There were no significant changes in the mean diameter and the zeta potential (zeta) of SLNs after autoclaving (121 degrees C for 20 min). The amount of incorporated AZT-P was, however, slightly reduced due to the formation of hydrosoluble AZT. Autoclaved SLNs were stable for a period of 10 weeks at 20 degrees C but an increase in particle size and loss of AZT-P were observed at 4 and 37 degrees C. Trehalose was an effective cryoprotectant for preventing SLN aggregation during lyophilization and subsequent reconstitution. Thermal gravimetric analysis showed that lyophilized preparations contained approximately 1% water. Using appropriate trehalose to lipid ratios, AZT-P retention in the SLNs was 100% after reconstitution. Our results demonstrate that SLNs containing AZT-P can be autoclaved, lyophilized and reconstituted without significant changes in SLN diameter and zeta potential or in the quantity of incorporated prodrug.
2808641
Application of Novel Solid Lipid Nanoparticle (SLN)-Gene Vector Formulations Based on a Dimeric HIV-1 TAT-Peptide in Vitro and in Vivo
AbstractPurpose. To optimize gene delivery of SLN-based gene vectors by incorporation of a dimeric HIV-1 TAT peptide (TAT2) into SLN gene vectors.
Methods. Plasmid DNA was complexed with two SLN preparations either with or without pre-compaction of DNA by TAT2, poly-l-arginine, or the mutant TAT2-M1. DNA complexed with polyethylenimine (PEI) served as a standard. Gene expression was analyzed upon transfection of bronchial epithelial cells in vitro and after intratracheal instillation or aerosol application to the lungs of mice in vivo. Stability of DNA was analyzed by agarose gel electrophoresis.
Results. Incorporation of TAT2 into SLN gene vectors induced an up to 100-fold sequence-dependent increase of gene expression as compared with the mutant TAT2-M1 and was 4- to 8-times higher as compared with PEI in vitro. In vivo application of TAT2-SLN gene vectors via jet nebulization increased SLN-based gene expression but was accompanied with DNA degradation. DNA degradation was not observed when an innovative device operating on the principle of a perforated vibrating membrane was used.
Conclusions. Incorporation of TAT2 into SLN gene vectors is suitable to optimize gene transfer in vitro. The use of a mild nebulization technology avoids DNA degradation and offers the opportunity for further studies in large animal models.
46298283
Re-dispersible cationic solid lipid nanoparticles (SLNs) freeze-dried without cryoprotectors: characterization and ability to bind the pEGFP-plasmid.
Cationic solid lipid nanoparticles (SLNs) have recently been suggested for non-viral gene delivery as a promising alternative to the liposomes. The aim of this study was to investigate the possibility to obtain re-dispersible cationic SLNs after a freeze-drying process in the absence of lyo- and/or cryoprotectors. The physical-chemical characteristics of cationic SLNs and their ability to bind gene material were investigated before and after the freeze-drying. To perform this study three samples of cationic SLNs, based on stearic acid, Compritol or cetylpalmitate, were prepared and characterized by PCS (photon correlation spectroscopy) and AFM (atomic force microscopy). The results indicated that solely the re-dispersed sample of stearic acid (SLN-SA) became very similar in terms of size and morphology to the fresh prepared sample, although it displayed a sensible reduction of the zeta potential (from 39.2 to 23.3 mV). By both the DSC (differential scanning calorimetry) and the ESCA (electron spectroscopy for chemical analysis) determinations, the reduction of the zeta potential was ascribed to the loss of the cationic lipids from the particle surface due to the rearrangement of the stearic acid lattice after the freeze-drying. Finally, the gel electrophoresis analysis demonstrated that SLN-SA re-suspended in PBS are unable to complex the DNA, while the SLN-SA re-dispersed in water displayed the same ability to bind DNA as the fresh prepared sample. We can conclude that cationic SLNs, based on stearic acid, retain the ability to complex DNA even after the freeze-drying in the absence of lyo- or cryoprotectors; thus, the powder form of this sample represents an attractive candidate to be investigated as in vivo DNA vector formulation.
40453329
Evaluation of polyplexes as gene transfer agents.
Non-viral transfection systems based on the complexes of DNA and polycations ('polyplexes') were evaluated with respect to their effectiveness, toxicity and cell type dependence in a variety of in vitro models. The panel of polycations examined included branched and linear polyethyleneimines, poly[N-ethyl-4-vinyl pyridinium bromide], polyamidoamine dendrimer (Superfect), poly(propyleneimine) dendrimer (Astramol) and a conjugate of Pluronic P123 and polyethyleneimine (P123-g-PEI(2K)), having a graft-block copolymer architecture. Using a panel of cell lines the linear polyethyleneimine ExGen 500, Superfect, branched polyethyleneimine 25 kDa, and P123-g-PEI(2K) were determined as systems displaying highest transfection activity while exhibiting relatively low cytotoxicity. These systems had activity higher than or comparable to lipid transfection reagents (Lipofectin, LipofectAMINE, CeLLFECTIN and DMRIE-C) but did not reveal serum dependence and were less toxic than the lipids. Overall, this study demonstrates good potential of structurally diverse polyplex systems as transfection reagents with relatively low cytotoxicity.