Leide P. Cavalcanti

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

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

Association between cationic liposomes and low molecular weight hyaluronic acid.

This work presents a study of the association between low molecular weight hyaluronic acid (16 kDa HA) and cationic liposomes composed of egg phosphatidylcholine (EPC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). The cationic liposome/HA complexes were evaluated to determine their mesoscopic structure, average size, zeta potential, and morphology as a function of the amount of HA in the system. Small angle X-ray scattering results revealed that neighboring cationic liposomes either stick together after a partial coating of low concentration HA or disperse completely in excess of HA, but they never assemble as multilamellar vesicles. Cryo-transmission electron microscopy images confirm the existence of unilamellar vesicles and large aggregates of unilamellar vesicles for HA fractions up to 80% (w/w). High concentrations of HA (> 20% w/w) proved to be efficient for coating extruded liposomes, leading to particle complexes with sizes in the nanoscale range and a negative zeta potential.

Microfluidic Assembly of pDNA/Cationic Liposome Lipoplexes with High pDNA Loading for Gene Delivery

Microfluidics offers unique characteristics to control the mixing of liquids under laminar flow. Its use for the assembly of lipoplexes represents an attractive alternative for the translation of gene delivery studies into clinical trials on a sufficient throughput scale. Here, it was shown that the microfluidic assembly of pDNA/cationic liposome (CL) lipoplexes allows the formation of nanocarriers with enhanced transfection efficiencies compared with the conventional bulk-mixing (BM) process under high pDNA loading conditions. Lipoplexes generated by microfluidic devices exhibit smaller and more homogeneous structures at a molar charge ratio (R±) of 1.5, representing the ratio of lipid to pDNA content. Using an optimized model to fit small-angle X-ray scattering (SAXS) curves, it was observed that large amounts of pDNA induces the formation of aggregates with a higher number of stacked bilayers (N ∼ 5) when the BM process was used, whereas microfluidic lipoplexes presented smaller structures with a lower number of stacked bilayers (N ∼ 2.5). In vitro studies further confirmed that microfluidic lipoplexes achieved higher in vitro transfection efficiencies in prostate cancer cells at R ± 1.5, employing a reduced amount of cationic lipid. The correlation of mesoscopic characteristics with in vitro performance provides insights for the elucidation of the colloidal arrangement and biological behavior of pDNA/CL lipoplexes obtained by different processes, highlighting the feasibility of applying microfluidics to gene delivery.

Calcium mediated interaction of calf-thymus DNA with monolayers of distearoylphosphatidylcholine: a neutron and X-ray reflectivity study

X-ray and neutron reflection studies, the latter in conjunction with contrast variation, have been combined to study the interaction of calf thymus DNA (ctDNA) with monolayers of distearoylphosphatidylcholine (DSPC) in the presence of 20 mM Ca2+ ions, at the air–liquid interface as a function of surface pressure (10, 20, 30 and 40 mN m−1). Analysis of the X-ray and neutron reflection data showed that, regardless of the surface pressure of the monolayer, a layer of ctDNA was present below the DSPC lipid head groups and that this ctDNA-containing layer (thickness ∼12.5 to 15 A) was separated from the DSPC head groups by a layer of water of ∼9 A thickness. The thickness of the ctDNA-containing layer was thinner than that reported for monolayers of cationic lipid at the air–water interface (18–25 A) although in these monolayers no water layer separating the lipid head groups from the layer containing ctDNA has been reported. At all surface pressures the amount of ctDNA present in the layer was in the range 30–40% by volume. As no significant re-arrangement of the DSPC film was required to accommodate the presence of the ctDNA, this suggests that the distribution of charges in the lipid film matches well the charge spacing of ctDNA. Brewster angle microscopy measurements of DSPC on water in the absence of Ca2+ showed the presence of a continuous film containing small, regular shaped domains at all four surface pressures examined. When Ca2+ ions were present in the sub-phase, although the film was still continuous, the domains comprising the film were more irregular in appearance while the presence of Ca2+ ions and ctDNA resulted in the domains becoming smaller and more regularly packed on the surface.

Technological Aspects of Scalable Processes for the Production of Functional Liposomes for Gene Therapy

The success of gene delivery systems in in vivo or in vitro applications depends on efficient transfection. Cationic liposomes remain a promising alternative for nonviral DNA carriers, mainly because they protect DNA from interstitial fluids and easily interact with cells (Gregoriadis, 1993; Lasic, 1997). However, in order to be effective in the immunological response, cationic liposomes must be functional and reach their specific target. Stability, reduced toxicity, efficiency in delivering genes to cells, and specific targeting to the nucleus are essential requirements for prophylactic and/or therapeutic performance. In order to achieve these standards, important physico-chemical parameters in liposomes must be controlled, such as the functionality of the lipids, the concentration of the cationic lipid, DNA loading (reflected by the R+/molar charge ratio), the zeta potential, size, and polydispersity. Several laboratory experiments have already explored DNA vaccines using cationic lipids. A classical investigation of lipid functionality and composition, as well as the efficiency of cationic liposomes as DNA carriers, was performed by Perrie and colleagues (Perrie & Gregoriadis, 2000; Perrie et al., 2001). The plasmid pRc/CMV HBS encoding the S (small) region of hepatitis B surface antigen was encapsulated in dehydrated-hydrated liposomes composed of egg phosphatidylcholine (EPC, bilayer-forming lipid), 1,2-dioleoyl-3trimethylammonium-propane (DOTAP, cationic lipid), 1,2-dioleoyl-sn-glycero-3phosphoethanolamine (DOPE, helper lipid) in a 50:25:25 percent molar ratio. The authors demonstrated that the encapsulation process protects the DNA vaccine against incubation with sodium dodecyl sulfate (SDS) due to DNA incorporation inside the liposome lamella (Perrie & Gregoriadis, 2000), and a better immunological response was obtained with cationic liposomes compared to naked DNA. Concerning size and polydispersity, different authors have reported that nanoparticle size is an important parameter for transfection success (Ma et al., 2007; Ogris et al., 1998; Rejman et al., 2004; Ross & Hui, 1999; Wiewrodt et al., 2002). Indeed, most of the variability in transfection procedures is a consequence of non-viral gene delivery systems with high polydispersity index values. The polydispersity index is related to the width of the particle

Refractive index and thickness determination in Langmuir monolayers of myelin lipids

Langmuir monolayers at the air/water interface are widely used as biomembrane models and for amphiphilic molecules studies in general. Under controlled intermolecular organization (lateral molecular area), surface pressure, surface potential, reflectivity (R) and other magnitudes can be precisely determined on these planar monomolecular films. However, some physical parameters such as the refractive index of the monolayer (n) still remain elusive. The refractive index is very relevant because (in combination with R) it allows for the determination of the thickness of the film. The uncertainties of n determine important errors that propagate non-linearly into the calculation of monolayers thickness. Here we present an analytical method for the determination of n in monolayers based on refractive index matching. By using a Brewster angle microscopy (BAM) setup and monolayers spread over subphases with variable refractive index (n2), a minimum in R is search as a function of n2. In these conditions, n equals n2. The results shown correspond to monolayers of myelin lipids. The n values remain constant at 1.46 upon compression and equals the obtained value for myelin lipid bilayers in suspension. The values for n and R allow for the determination of thickness. We establish comparisons between these thicknesses for the monolayer and those obtained from two X-ray scattering techniques: 1) GIXOS for monolayers at the air/water interface and 2) SAXS for bilayers in bulk suspension. This allows us to conclude that the thickness that we measure by BAM includes the apolar and polar headgroup regions of the monolayer.

Characterization of a Pt mirror to be used to deflect synchrotron radiation beam onto Langmuir monolayers

A homemade mirror for X-rays has been built to prepare a diffraction beamline for liquid surface diffraction and scattering measurements. This simple approach is in operation at the XRD2 bending-magnet beamline at the Brazilian Synchrotron Light Laboratory.

X‐Ray Scattering Techniques Applied in the Development of Drug Delivery Systems

The advances in nanotechnology have found application in different fields, such as food, agriculture, materials, chemistry, and medicine. However, one of the most important approaches is the development of nanocarriers and, in order to understand their structural organization, different physicochemical techniques have been used. In particular, small angle X‐ray scattering (SAXS) and X‐ray diffraction (XRD) have given important contribution to the study of organization phase of nanocarriers such as organic/inorganic nanoparticles, micelles, liposomes, cyclodextrins, polymers, and their interaction with drugs and other bioactive molecules. In this chapter, we will present theoretical aspects, experimental design, and the applications of both techniques for the development of delivery systems for bioactive molecules.

Modelling the release of biological molecules from ordered mesoporous silica

This work reports preliminary results of the application of a theoretical model [1] in the study of incorporation and release of biological molecules from the porous structure of the SBA-15 [2] ordered mesoporous silica. A theoretical model taking into account the shape and spatial coordination of the pores in the amorphous silica structure is fitted through a non-linear least-squares method and the behavior of the parameters obtained from curves acquired in-situ during incorporation and release experiments are interpreted in the context of different media. Preliminary studies included experiments regarding the coating of the SBA-15 silica with the Eudragit® polymer and the stability of SBA-15 in experimental media (water and PBS solution) and in simulated body fluids. Small angle X-ray scattering experiments were performed mainly with bovine serum albumin (BSA) and insulin, and showed the silica’s capacity of sheltering those molecules inside its structure, as well as the influence of Eudragit® on their release dynamics. In-situ experiments made during the incorporation and release of insulin helped elucidate the dynamics of those phenomena, through the reinterpretation of the theoretical model, which was originally designed to study the synthesis process of SBA-15. In this model, fit parameters were monitored during the experiment and, from their behaviors, some conclusions are drawn, such as the delay in BSA release for the SBA-15 plus Eudragit® in gastric fluid. The in-situ studies of insulin loading showed that this molecule’s uptake takes place in the course of a few minutes and that it remains inside the pores. Also the in-situ studies of insulin release showed that this molecule is protected inside the silica walls, and the use of Eudragit® is, in a way, optional.