Lyophilization of a triply unsaturated phospholipid: effects of trace metal contaminants.

As liquid liposomal formulations are prone to chemical degradation and aggregation, these formulations often require freeze drying (e.g., lyophilization) to achieve sufficient shelf-life. However, liposomal formulations may undergo oxidation during lyophilization and/or during prolonged storage. The goal of the current study was to characterize the degradation of 1,2-dilinolenoyl-sn-glycero-3-phosphocholine (DLPC) during lyophilization and to also probe the influence of metal contaminants in promoting the observed degradation. Aqueous sugar formulations containing DLPC (0.01 mg/ml) were lyophilized, and DLPC degradation was monitored using HPLC/UV and GC/MS methods. The effect of ferrous ion and sucrose concentration, as well as lyophilization stage promoting lipid degradation, was investigated. DLPC degradation increased with higher levels of ferrous ion. After lyophilization, 103.1 ± 1.1%, 66.9 ± 0.8%, and 28.7 ± 0.7% DLPC remained in the sucrose samples spiked with 0.0 ppm, 0.2 ppm, and 1.0 ppm ferrous ion, respectively. Lipid degradation predominantly occurs during the freezing stage of lyophilization. Sugar concentration and buffer ionic strength also influence the extent of lipid degradation, and DLPC loss correlated with degradation product formation. We conclude that DLPC oxidation during the freezing stage of lyophilization dramatically compromises the stability of lipid-based formulations. In addition, we demonstrate that metal contaminants in sugars can become highly active when lyophilized in the presence of a reducing agent.

A radiation target method for size determination of supercoiled plasmid DNA.

Supercoiled DNA plasmids were exposed in the frozen state to high-energy electrons. Surviving supercoiled molecules were separated from their degradation products (e.g., open circle and linear forms) by agarose gel electrophoresis and subsequently quantified by staining and image analysis. Complex survival curves were analyzed using radiation target theory, yielding the radiation-sensitive mass of each form. One of the irradiated plasmids was transfected into cells, permitting radiation analysis of gene expression. Loss of this function was associated with a mass much smaller than the entire plasmid molecule, indicating a lack of energy transfer in amounts sufficient to cause structural damage along the DNA polynucleotide. The method of radiation target analysis can be applied to study both structure and function of DNA.

DNA acts as a nucleation site for transient cavitation in the ultrasonic nebulizer.

Several new technologies based upon ultrasound technology have been proposed as a method to enhance the delivery of genetic therapeutics. Using ultrasonic nebulization and a well-established method to quantitatively monitor transient cavitation, this study investigates the extent and factors influencing the degradation of DNA. Results from our studies show that the presence of DNA greatly enhances cavitation, and that the number of transient cavitation events also increases with the hydrodynamic diameter and number of DNA molecules in solution. More importantly, removing saturated gases from the plasmid DNA solutions resulted in a decrease in transient cavitation events but not degradation rate, suggesting that the cavitation event responsible for degradation occurs locally at the DNA molecule. Finally, complexing plasmid DNA with the cationic polymer polyethylenimine protected the native structure by reducing the molecule's potential to act as a heterogeneous nucleation site for transient cavitation.

Shear-induced degradation of plasmid DNA.

The majority of gene therapy clinical trials use plasmid DNA that is susceptible to shear-induced degradation. Many processing steps in the extraction, purification, and preparation of plasmid-based therapeutics can impart significant shear stress that can fracture the phosphodiester backbone of polynucleotides, and reduce biological activity. Much of the mechanistic work on shear degradation of DNA was conducted over 30 years ago, and we rely heavily on this early work in an attempt to explain the empirical observations of more recent investigations concerning the aerosolization of plasmids. Unfortunately, the sporadic reports of shear degradation in the literature use different experimental systems, making it difficult to quantitatively compare results and reach definitive mechanistic conclusions. In this review, we describe the forces imparted to DNA during shear stress, and use published data to quantitatively evaluate their relative effects. In addition, we discuss the effects of molecular weight, strain rate, particle size, flexibility, ionic strength, gas-liquid interfaces, and turbulence on the fluid flow degradation of supercoiled plasmid DNA. Finally, we speculate on computational methods that might allow degradation rates in different experimental systems to be predicted.

Maintenance of nonviral vector particle size during the freezing step of the lyophilization process is insufficient for preservation of activity: insight from other structural indicators.

The instability of nonviral vectors as liquid formulations has stimulated considerable interest in developing dehydrated formulations that would be resistant to shipping stresses and could be stored at room temperature. Recently, we reported that high sucrose/DNA ratios are capable of maintaining particle size during the freezing step of the lyophilization process and we suggested that the separation of individual particles within sugar matrices is responsible for the reported protection of nonviral vectors during the freezing step of a typical lyophilization protocol. The purpose of this study was to extend these observations to other nonviral vectors that incorporate different cationic components. Cationic lipid-based complexes composed of 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), with helper lipid cholesterol (Chol) or dioleoylphosphatidyl-ethanolamine (DOPE), showed similar protection by sucrose. Formulations of a polyethylenimine (PEI)-based vector required much higher excipient/DNA ratios for size protection compared with protamine- and lipid-based vectors. At low sucrose/DNA ratios, zeta potentials for all complexes were significantly lowered during freezing. Similar results were obtained at high sucrose/DNA ratios, except for DOTAP-DOPE-containing vectors which maintained zeta potential values comparable to unfrozen controls. The changes in zeta potential values indicate that complexes are altered during freezing despite the maintenance of particle size as determined by light scattering. Furthermore, these changes might explain the observed reduction in transfection activity and provide new information about the effects of physicochemical changes of nonviral vectors during the freezing step of lyophilization.

Physical stabilization of DNA-based therapeutics.

The development of non-viral vectors for gene delivery has primarily focused on improving the efficiency of gene transfer in vivo. Although there is clearly a need to improve delivery efficiency, studies also indicate that the physical stability of non-viral vectors is not nearly adequate for a marketable pharmaceutical product. Here, we describe the different strategies that have been used to enhance stability and discuss the mechanisms by which prolonged stabilization (>2 years) might be achieved.

Maintenance of quaternary structure in the frozen state stabilizes lactate dehydrogenase during freeze-drying.

Sugars inhibit protein unfolding during the drying step of lyophilization by replacing hydrogen bonds to the protein lost upon removal of water. In many cases, polymers fail to inhibit dehydration-induced damage to proteins because steric hindrance prevents effective hydrogen bonding of the polymer to the protein's surface. However, in certain cases, polymers have been shown to stabilize multimeric enzymes during lyophilization. Here we test the hypothesis that this protection is due to inhibition of dissociation into subunits during freezing. To test this hypothesis, as a model system we used mixtures of lactate dehydrogenase isozymes that form electrophoretically distinguishable hybrid tetramers during reversible dissociation. We examined hybridization and recovery of catalytic activity during freeze-thawing and freeze-drying in the presence of polymers (dextran, Ficoll, and polyethylene glycol), sugars (sucrose, trehalose, glucose), and surfactants (Tween 80, Brij 35, hydroxy-propyl beta-cyclodextrin). The surfactants did not protect LDH during freeze-thawing or freeze-drying. Rather, in the presence of Brij 35, enhanced damage was seen during both freeze-thawing and freeze-drying, and the presence of Tween 80 exacerbated loss of active protein during freeze-drying. Polymers and sugars prevented dissociation of LDH during the freezing step of lyophilization, resulting in greater recovery of enzyme activity after lyophilization and rehydration. This beneficial effect was observed even in systems that do not form glassy solids during freezing and drying. We suggest that stabilization during drying results in part from greater inherent stability of the assembled holoenzyme relative to that of the dissociated monomers. Polymers inhibit freezing-induced dissociation thermodynamically because they are preferentially excluded from the surface of proteins, which increases the free energy of dissociation and denaturation.

Analysis of self-assembled cationic lipid-DNA gene carrier complexes using flow field-flow fractionation and light scattering.

Self-assembled cationic lipid-DNA complexes have shown an ability to facilitate the delivery of heterologous DNA across outer cell membranes and nuclear membranes (transfection) for gene therapy applications. While the size of the complex and the surface charge (which is a function of the lipid-to-DNA mass ratio) are important factors that determine transfection efficiency, lipid-DNA complex preparations are heterogeneous with respect to particle size and net charge. This heterogeneity contributes to the low transfection efficiency and instability of cationic lipid-DNA vectors. Efforts to define structure-activity relations and stable vector populations have been hampered by the lack of analytical techniques that can separate this type of particle and analyze both the physical characteristics and biological activity of the resulting fractions. In this study, we investigated the feasibility of flow field-flow fractionation (flow FFF) to separate cationic lipid-DNA complexes prepared at various lipid-DNA ratios. The compatibility of the lipid-DNA particles with several combinations of FFF carrier liquids and channel membranes was assessed. In addition, changes in elution profiles (or size distributions) were monitored as a function of time using on-line ultraviolet, multiangle light scattering, and refractive index detectors. Multiangle light scattering detected the formation of particle aggregates during storage, which were not observed with the other detectors. In comparison to population-averaged techniques, such as photon correlation spectroscopy, flow FFF allows a detailed examination of subtle changes in the physical properties of nonviral vectors and provides a basis for the definition of structure-activity relations for this novel class of pharmaceutical agents.

Infrared spectroscopic characterization of the interaction of cationic lipids with plasmid DNA.

Fourier transform infrared spectroscopy was used to characterize the interaction of the cationic lipids 1,2-dioleoyl-3-trimethylammonium-propane and dioctadecyldimethylammonium bromide with plasmid DNA. The effect of incorporating the neutral colipids cholesterol and dioleoylphosphatidylethanolamine on this interaction was also examined. Additionally, dynamic and phase analysis light scattering were used to monitor the size and zeta potential of the resulting complexes under conditions similar to the Fourier transform infrared measurements. Results suggest that upon interaction of cationic lipids with DNA, the DNA remains in the B form. Distinct changes in the frequency of several infrared bands arising from the DNA bases, however, suggest perturbation of their hydration upon interaction with cationic lipids. A direct interaction of the lipid ammonium headgroup with and dehydration of the DNA phosphate is observed when DNA is complexed with these lipids. Changes in the apolar regions of the lipid bilayer are minimal, whereas the interfacial regions of the membrane show changes in hydration or molecular packing. Incorporation of helper lipids into the cationic membranes results in increased conformational disorder of the apolar region and further dehydration of the interfacial region. Changes in the hydration of the DNA bases were also observed as the molar ratio of helper lipid in the membranes was increased.

Lyophilization of nonviral gene delivery systems.

This chapter presents a qualitative description of the freeze-drying process as it pertains to the development of stable, dry polycation-DNA complex formulations. It is not intended to be a comprehensive treatise on freeze-drying. Readers are referred to a series of excellent papers by Pikal (1-5) for more detailed, quantitative explanations of the freeze-drying process.

Stabilization of lipid/DNA complexes during the freezing step of the lyophilization process: the particle isolation hypothesis.

The instability of nonviral vectors in aqueous suspensions has stimulated an interest in developing lyophilized formulations for use in gene therapy. Previous work has demonstrated a strong correlation between the maintenance of particle size and retention of transfection rates. Our earlier work has shown that aggregation of nonviral vectors typically occurs during the freezing step of the lyophilization process, and that high concentrations of sugars are capable of maintaining particle size. This study extends these observations, and demonstrates that glass formation is not the mechanism by which sugars protect lipid/DNA complexes during freezing. We also show that polymers (e.g., hydroxyethyl starch) are not capable of preventing aggregation despite their ability to form glasses at relatively high subzero temperatures. Instead, our data suggest that it is the separation of individual particles within the unfrozen fraction that prevents aggregation during freezing, i.e., the particle isolation hypothesis. Furthermore, we suggest that the relatively low surface tension of mono- and disaccharides, as compared to starch, allows phase-separated particles to remain dispersed within the unfrozen excipient solution, which preserves particle size and transfection rates during freezing.

Mechanisms of protection of cationic lipid-DNA complexes during lyophilization.

Gene therapy using nonviral vectors offers advantages over viral methods. However, the instability of aqueous suspensions of cationic lipid-DNA complexes is a major problem that must be overcome to develop this therapeutic modality on a pharmaceutical scale. Disaccharides have been reported to protect lipid-DNA complexes during lyophilization, and recovery of transfection correlates with the retention of particle size. However, the mechanism by which disaccharides achieve this protection is not known. The purpose of this study was to investigate the protective mechanism by lyophilizing cationic lipid-DNA complexes with a variety of solutes that have different physical behaviors during the lyophilization process. In agreement with previous studies, disaccharides conferred protection to lipid-DNA complexes. By contrast, a large polymeric sugar, hydroxyethyl starch, did not protect as well. The level of protection by additives, such as mannitol, that crystallized during lyophilization was also less than that of the disaccharides, but some protection was nonetheless observed. These data suggest that water replacement plays a significant role in protecting complexes during lyophilization. A second mechanism that prevents aggregation by diluting complexes within freeze-concentrated solutions or dried cakes may also contribute to protection. Sample vitrification did not correlate with maintenance of transfection efficiency. Elucidation of the mechanism(s) by which cationic lipid-DNA complexes are protected during lyophilization will permit a rational approach to the development of stable, lyophilized formulations.

Physical stability of nonviral plasmid-based therapeutics.

Nonviral, plasmid-based therapeutics are a new class of pharmaceutical agents that offer the potential to cure many diseases that are currently considered untreatable. While nonviral vectors have shown promise in clinical trials, their physical instability in liquid formulations represents a major barrier to the development of these agents as marketable products. While several different approaches have been used to improve the stability of liquid formulations, it is unclear whether aqueous suspensions can be rendered sufficiently stable to withstand the stresses associated with shipping and storage. Some studies have demonstrated the potential of frozen formulations to be stored for prolonged periods of time, however the potential for phase changes in frozen samples combined with the expense of maintaining the frozen state during shipping has stimulated an interest in developing dehydrated preparations. Although the stresses associated with dehydration are considerable, several studies have reported that sugars are capable of preserving the physical characteristics and transfection activity of nonviral vectors during acute lyophilization stress. This paper discusses the merits and drawbacks of the different approaches to preserving nonviral vectors, and identifies research areas in which more work is needed to develop stable formulations of plasmid-based therapeutics.

Stability of lipid/DNA complexes during agitation and freeze-thawing.

It is well established that cationic liposomes facilitate the delivery of DNA and offer substantial advantages over viral-based delivery systems. However, these synthetic vectors readily aggregate in liquid formulations which in clinical trials requires preparation of lipid/DNA complexes at the bedside immediately before injection. This temporal requirement could be eliminated if complexes were formulated as stable preparations that could be shipped, stored, and administered as needed. To this end, our study investigates the stability of lipid/DNA complexes during physical stresses that might be encountered during shipping and storage, i.e., agitation and freeze-thawing. Our data show that agitation significantly reduces transfection rates in complexes prepared with three different commercially available lipid formulations. Additional experiments indicate that slow freezing is more damaging than rapid freezing, and that sucrose is able to preserve transfection and complex size during freeze-thawing. These results are consistent with previous reports and demonstrate that frozen formulations may be suitable for maintaining transfection rates of lipid/DNA complexes. Under certain conditions, we observe a reproducible 3-fold increase in transfection after freeze-thawing that is prevented by high concentrations of sucrose. Together, these data suggest that physical stresses can alter structural characteristics of lipid/DNA complexes that can markedly affect rates of DNA delivery.

Maintenance of transfection rates and physical characterization of lipid/DNA complexes after freeze-drying and rehydration.

It is well established that cationic liposomes form complexes with DNA and effectively transfect cells in vivo and ex vivo. Lipid/DNA complexes have proven safe and nonimmunogenic in clinical trials; however, they are known to aggregate readily in liquid formulations. This physical instability requires clinicians to prepare lipid/DNA complexes immediately prior to injection. In order to eliminate problems associated with this temporal requirement, we investigated the feasibility of preserving complexes as a dried preparation that could be tested, stored, and rehydrated as needed. To this end, our study evaluated the ability of different stabilizers to preserve transfection rates of complexes during acute freeze-drying stress. Our data show that complexes lyophilized in 0.5 M sucrose or trehalose possessed transfection rates similar to those of fresh preparations. In addition, dried complexes that exhibited full transfection activity upon rehydration had sizes comparable to nonlyophilized controls. Our work demonstrates that lipid/DNA complexes can be stabilized as dried powders that offer significant advantages over current liquid formulations. Furthermore, the correlation of transfection rates with maintenance of complex diameter suggests that size plays a critical role in lipid-based DNA delivery.

Polymers protect lactate dehydrogenase during freeze-drying by inhibiting dissociation in the frozen state.

Enzymes subjected to freeze-thawing are known to be protected by polymers that are preferentially excluded from the hydrated surface of proteins [reviewed in Carpenter et al. (1994) ACS Symp. Ser. 567, 134-147]. Preferentially excluded solutes are also known to stabilize quaternary structure, which enhances the thermostability of multimeric proteins in aqueous systems. Also, it has been suggested that retention of quaternary structure may play a role in the protection of multimeric proteins by polymers during freeze-drying (lyophilization). Although preferential solute exclusion cannot occur in the absence of water, we reasoned that polymers could protect multimeric proteins during freeze-drying by stabilizing quaternary structure in the frozen state. Our results are consistent with this hypothesis and demonstrate that bovine serum albumin and polyvinylpyrrolidone stabilize lactate dehydrogenase by inhibiting dissociation in the frozen solution, during the initial phase of the sublimation step of lyophilization. Dissociation at this critical step correlated directly with decreased recovery of enzyme activity after rehydration. The damage to the protein, under conditions where dissociation was studied, was due to a large decrease in pH in the frozen state (e.g., from pH 7.5 to 4.5), which was attenuated by protective levels of polymers. Thus, inhibition of freezing-induced pH shifts, in addition to stabilization by the preferential exclusion mechanism, plays an important role in the protection conferred by polymers. Furthermore, high concentrations of these polymers were capable of maintaining quaternary structure during subsequent drying and rehydration. We suggest that the proximate cause for increased recovery of active, native protein after lyophilization is that the holoenzyme is more resistant to the stresses of drying/rehydration than unassociated monomers.