Time-of-flight secondary ion mass spectrometry employing an SF5+ polyatomic primary ion source was utilized to obtain a series of in-depth profiles from PLLA/Pluronic-P104 (poly(ethylene oxide-co-propylene oxide) triblock copolymer) blends in attempts to quantify the in-depth surface segregated Pluronic region. The resultant in-depth profiles were consistent with theoretical models describing the surface segregated region in polymeric blends and copolymer systems, with a surface enriched Pluronic-P104 region, followed by a P104 depletion layer, and finally a constant composition bulk region. These results were consistent over a range of concentrations (1-25%). The depth profiles obtained using cluster SIMS were compared to information obtained using X-ray photoelectron spectroscopy. The results demonstrate that, with cluster primary ion bombardment, we are for the first time able to quantify the polymeric composition as a function of depth within certain multicomponent polymer blends. This success can be attributed to the sputter characteristics of polyatomic primary ion bombardment (SF5+) as compared to monatomic primary ion beams.

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The purpose of this study is to investigate the effects of moisture content on the storage stability of freeze-dried lipoplex formulations. DC-Cholesterol: DOPE (dioleoyl phosphatidylethanolamine) / plasmid DNA lipoplexes were prepared at a 3-to-2 DC-Cholesterol + to DNA − molar ratio and lyophilized prior to storing at room temperature, 40 °C, and 60 °C for three months. Different residual moistures (1.93%, 1.10%, 1.06% and 0.36%) were obtained by altering the secondary drying temperatures. In addition to moisture content, lipoplex formulations were evaluated after freezedrying and/ or storage for particle size, transfection efficiency, accumulation of TBARS (thiobarbituric reactive substances), glass transition temperature, DNA supercoil content, and surface area. Lipoplex formulations stored at room temperature for 3 months maintain TBARS concentrations and supercoil contents. At higher storage temperatures, formulations possessing the highest moisture content (1.93%) maintained significantly lower TBARS concentrations and higher supercoil content than those with the lowest (0.36%) moisture content. Curiously, the intermediate moisture contents exhibited marked differences in stability despite virtually identical moisture contents. Subsequent measurements of surface area indicated that the lower stability corresponded to higher surface area in the dried cake, suggesting that there may be an interplay between water content and surface area that contributes to storage stability.

The stability of non-viral vectors during freeze-drying has been well-studied, and it has been established that sugars can protect lipoplexes during freeze-drying. However low levels of damage are often observed after freeze-drying, and this damage is more evident in dilute lipoplex preparations. By investigating the stability of lipoplexes after each step in the freeze-drying cycle (i.e., freezing, primary drying, and secondary drying) we strive to understand the mechanisms responsible for damage and identify improved stabilization strategies. N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP)-cholesterol/plasmid DNA lipoplexes were prepared at an equimolar DOTAP to cholesterol ratio, and a 3-to-1 DOTAP + to DNA − charge ratio. Our experiments indicate that despite sufficient levels of "stabilizing" sugars, significant damage is still evident when dilute lipoplex preparations are subjected to freeze-drying. Analysis of the different stages of freeze-drying suggests that significant damage occurs during freezing, and that sugars have a limited capacity to protect against this freezing-induced damage. Similar effects have been observed in studies with proteins, and surfactants have been employed in protein formulations to protect against surface-induced damage, e.g., at the ice crystal, solid, air or sugar glass surfaces. However, the use of surfactants in a lipid-based formulation is inherently risky due to the potential for altering/solubilizing the lipid delivery vehicle. Our data indicate that judicious use of surfactants can reduce surface-induced damage, and result in better preservation of lipoplex size and transfection activity after freeze-drying.

The surface chemistry and in-depth distribution of the composition of a poly(ethylene oxide) (PEO)-containing biodegradable poly(l-lactic acid) (PLLA) blend matrix system have been investigated using X-ray photoelectron spectroscopy (XPS). This study reports detailed quantitative compositional information using a novel numerical method for determining depth profiles. The PEO system studied is an amphiphilic Pluronic P104 surfactant, PEO–b-poly(propylene oxide) (PPO)–b-PEO. The extent of phase separation is analyzed by determining the surface enrichment of the PEO component via measurement of chemical composition at the polymer–air interface. For this blend system, the combination of the PPO component in the Pluronic surfactants drives the formation of a surface excess of Pluronic in the blends with PLLA. The surface excess profile shows a rapid increase in Pluronic surface composition versus bulk Pluronic mass fractions of 1–5%, but the profile levels off above bulk Pluronic mass fractions of 5%.

Secondary Ion Mass Spectrometry (SIMS) has proven to be a useful tool in the analysis of biomaterials and drug delivery systems. With SIMS, the distribution of components in biomaterials systems can potentially be determined with a high degree of spatial resolution (<1 µm) and sensitivity (as low as ppm (µg / g)) when compared to other analytical methods such as Raman and IR spectroscopies [1]. Unfortunately, the widespread use of SIMS for imaging and depth profiling of organic constituents in drug delivery systems has been severely limited by low secondary ion yields and beam-induced damage effects that result from the use of monatomic primary ion beams, resulting in low sensitivity and precluding compositional depth profiling. One potential solution to this limitation is to use cluster or polyatomic primary ion bombardment. Cluster primary ion sources (such as SF 5 + and C 60 + ) have already generated considerable interest for organic SIMS analysis, where they have resulted in significant improvements (up to 1000 fold) in characteristic molecular secondary ion yields and in some cases have resulted in decreased beam-induced damage [2,3,4]. This decreased beam-induced damage coupled with an increased sputter rate has led to the ability to depth profile through some organic and polymeric materials without the characteristic rapid signal decay observed with monatomic primary ion sources [2,4]. Figure 1 illustrates an example of successful polymeric depth profiling in a model drug delivery system comprised of poly(lactic acid) (PLA) doped with 20 % (w/w) acetaminophen cast on Si. Characteristic secondary ion signals are observed from both PLA and acetaminophen as a function of increasing SF 5 + primary ion dose (increasing depth). These characteristic secondary ion signals remain stable throughout the depth profile until the Si substrate is reached. Intensity variations in the surface and bulk regions are consistent with domain formations within the film and a surface depleted drug region. This capability can be combined with the ability to obtain secondary ion images to obtain information on the 3-D molecular structures within these films. An example of this is shown in Figure 2, which represents the surface and in-depth distribution in three component blend films of poly(L-lactic acid) (PLLA) containing Pluronic surfactant [poly(ethylene oxide) (A) poly(propylene oxide) (B) ABA block copolymer] and insulin. It can be clearly seen from this figure that there is a surface enriched P104 region. In the subsurface region, domains on the order of 40 µm -50 µm are observed. These domains disappear shortly thereafter until after 210 s of sputtering where there is complete removal of the organic material as indicated by the intense Si signal intensity and corresponding loss in organic signals.