Preparation of high concentration protein powder suspensions by milling of lyophilizates.

Pharmaceutical formulations utilizing protein drugs as powders can be used as drug delivery systems in various ways. Besides powders for inhalation, another promising approach is their use as suspensions in non-aqueous liquids for subcutaneous administration providing high protein stability and good injectability. In this study protein powder suspensions were prepared using a swing-mill. Milling of lyophilizates containing a model monoclonal antibody in presence of the suspension vehicle was compared to cryogenic dry milling. Wet media milling led to injectable suspensions, but resulted in monomer loss and increase in protein aggregation. When the lyophilizates were cryogenic dry ball milled less aggregation and monomer loss were detected. Differences related to protein integrity were found for different process parameters, which were successfully optimized. If not cooled with liquid nitrogen, dry milling resulted in increased damage to the mAb. The type of polyol stabilizer, as well as the protein to stabilizer ratio, did not affect the preservation of protein integrity. As finding the right milling duration is time and resource intensive, a correlation between lyophilizate cake hardness and milling duration was established. Based on this approach high concentration lyophilizates were successfully micronized. Suspensions of cryogenic milled powders lead to clogging of 25G needles, which could be prevented by an additional sieving step. Depending on the suspension vehicle, low viscosity formulations (<10 mPa·s) even at high concentrations (≥100 mg/ml protein concentration) were obtained featuring good injectability.

Nanofiber Formation and Polymerization of Bolalipids with Diacetylene-Modified Single Alkyl Chains.

The nanofiber formation in aqueous suspension of two classes of symmetric single-chain bolaamphiphiles with different polar headgroups and a diacetylene-modified alkyl chain with a length of 32, 34, and 36 C atoms was investigated by differential scanning calorimetry, transmission electron microscopy, and small-angle neutron scattering. As observed before for other bolalipids with phosphocholine (PC) and dimethyl-phosphoethanolamine (Me2PE) headgroups, the molecules form fibers when suspended in water at low temperatures but disassemble into micellar-like aggregates upon heating. The introduction of a diacetylene group in the middle of the long chain leads to a perturbation of chain packing so that this fiber-micelle transition occurs at lower temperature compared to the other bolalipids having unmodified alkyl chains. The aim of our project was the introduction of diacetylene groups into alkyl chains to be able to polymerize the fibers at low temperature. This should enhance the fiber stability and prevent the disassembly into micellar aggregates at higher temperature. Polymerization of aggregates containing diacetylene-modified bolaamphiphiles can be easily traced by UV/vis spectroscopy as colored products are formed. We found that polymerization of bolaamphiphiles with PC headgroups leads to a breakdown of most fibers into micellelike aggregates, and only some longer fibers segments are still detectable. In contrast, the use of Me2PE headgroups improves polymerizability and length of the polymerized fibers. The compound with 36 C atoms in the chain could be polymerized at low temperatures, and the fibers remained stable at least up to a temperature of 60 °C. This shows that the perturbation of the chain packing due to the diacetylene groups in the chains can be overcome by elongation of the chains, so that thermostable fibers with a diameter of the length of the bolalipid molecule can be successfully formed.

Self-assembly of different single-chain bolaphospholipids and their miscibility with phospholipids or classical amphiphiles.

A variety of bolalipids with a single long alkyl chain and two identical headgroups self-assemble in aqueous solutions into helical entangled nanofibers leading to the formation of a hydrogel. An increase in temperature usually leads to the break-up of the fiber structure into micellar aggregates. In this paper the question is addressed whether bolalipids of different lengths or different headgroup structures can form mixed fibers. Also, the stability of the fiber aggregation of bolalipids in mixtures with phospholipids forming lamellar bilayers is discussed. Here, the question whether single-chain bolalipids can be incorporated into phospholipid bilayers to stabilize bilayer membranes is important, as possibly lipid vesicles used for drug delivery can be improved. Finally, the stability of the fiber aggregate against solubilisation by common surfactants was studied. The paper addresses the question which type of aggregate structure dominates the self-assembly of bipolar and monopolar amphiphiles in aqueous suspension.

Determination of sorption isotherm and rheological properties of lysozyme using a high-resolution humidity scanning QCM-D technique.

The high-resolution humidity scanning QCM-D technique enables investigation of hydration of soft matter films using a quartz crystal microbalance with dissipation monitoring (QCM-D) equipped with a humidity module. Based on a continuous increase of relative humidity, properties of soft matter films can be investigated depending on the water content of the surrounding atmosphere. Determination of complete water sorption isotherms is possible via analysis of the overtone dependence of the resonance frequencies. Rheological properties are monitored via measurement of the dissipation. The glass transition can be identified from the change of viscoelastic properties of the film reflected in changes of the dissipation. A high-resolution water sorption isotherm of lysozyme was measured and compared with results from water sorption calorimetry. Analysis of the rheological behavior during hydration of lysozyme films revealed the presence of two separate sharp transitions at the water activities 0.67 and 0.91, which are connected to the glass transition. In previous works, only the existence of a broad glass transition has been reported so far. Combining the QCM-D data with Raman scattering data presented earlier, a new mechanism of isothermal glass transition in lysozyme is proposed.

Tuning the aggregation behaviour of single-chain bolaphospholipids in aqueous suspension: from nanoparticles to nanofibres to lamellar phases.

The aggregation behaviour in aqueous suspensions of symmetrical bipolar phospholipids (bolalipids) composed of one long alkyl chain and two polar headgroups were studied as a function of their chemical structure, i.e. the length of their chain, the modification of the chain by introduction of hetero-atoms, triple bonds, or phenyl rings, and the size of the headgroups. Three types of aggregate structures are formed by these bolalipids, namely helical nanofibres, micelle-like aggregates and lamellar sheets. The type of aggregate formed depends not only on the chemical structure, particularly the ratio of the cross-sectional area of the headgroup and the chain, but also on the presence of attractive interactions via hydrogen bonds or repulsive electrostatic interactions between the headgroups.

Tuning the aggregation behaviour of single-chain bolaamphiphiles in aqueous suspension by changes in headgroup asymmetry.

The self-assembly process in aqueous suspension of two new asymmetrical single-chain bolaamphiphiles, namely 32-{[hydroxy(2-hydroxyethoxy)phosphinyl]oxy}dotriacontane-1-yl-{2-[N-(3-dimethylaminopropyl)-N,N-dimethylammonio]ethylphosphate} (DMAPPC-C32-POH) and 32-hydroxydotriacontane-1-yl-{2-[N-(3-dimethylaminopropyl)-N,N-dimethylammonio]ethylphosphate} (DMAPPC-C32-OH), was studied as a function of temperature using transmission electron microscopy, differential scanning calorimetry, FT-IR-spectroscopy, small angle neutron and small angle X-ray scattering to determine whether the asymmetry of the molecule induces the formation of types of aggregates other than the well characterized helical nanofibres of structurally similar symmetrical single-chain bolaamphiphiles with identical headgroups. DMAPPC-C32-POH in acetate buffer at pH 5 can still form nanofibres, i.e. the asymmetry does not induce the formation of other aggregate structures. However, the fibres display a tendency to break more easily and to form irregular, circular structures. This is also reflected by the rheological properties of the suspension that reveal decreased strain resistance at pH 5. In aqueous suspensions at pH 10, where the headgroups of the molecule are negatively charged, only short fibre segments are formed and no gel formation occurs. At higher temperature these fibres convert into micellar aggregates as observed before for symmetrical bolalipids with large headgroups. In contrast, in aqueous suspensions of DMAPPC-C32-OH, a bolalipid where the size difference of the headgroups is much larger, lamellar structures are formed at pH 10 where the headgroup of the molecule is zwitterionic. At low temperature, the molecules are packed in an orthorhombic lattice with interdigitated chains and a repeat distance between lamellae of 6.2 nm is observed. An increase in temperature leads to a lamellar phase with hexagonal packing of the chains. The chains become liquid-crystalline only at very high temperature above 90 °C. At low pH, when the headgroup of the molecule becomes positively charged, some short elongated micellar aggregates are seen besides sheet-like structures. A temperature increase leads to a similar sequence of transformations of the chain packing until formation of a liquid-crystalline lamellar phase at a temperature close to 90 °C. The results show that the aggregation behaviour of single-chain bolaamphiphiles can not only be tuned by changes in chain length or size of both headgroups but also by the difference in headgroup size and charge in asymmetric bolaamphiphiles.

Bolalipid fiber aggregation can be modulated by the introduction of sulfur atoms into the spacer chains.

The aggregation behavior in aqueous suspension of two symmetrical single-chain bolaamphiphiles, namely 12,21-dithiadotriacontane-1,32-diyl-bis [2-(tri-methylammonio)ethylphosphate] (PC-C32SS-PC) and 12,21-dithiadotriacontane-1,32-diyl-bis[2-(dimethylammonio)ethylphosphate] (Me(2)PE-C32SS-Me(2)PE), containing sulfur as heteroatoms in the chains, was studied using differential scanning calorimetry (DSC), Fourier transform infrared spectroscopy (FT-IR), small angle neutron scattering (SANS), and transmission electron microscopy (TEM). The rheological properties of hydrogels formed by the aggregation into nanofibers were studied by oscillatory rheology. Based on the well-characterized behavior of bolalipids with long alkyl chains which at room temperature can form a network of nanofibers leading to the formation of a hydrogel, we investigated whether the incorporation of two heteroatoms of sulfur into the spacer chain of the molecules has an influence on the aggregation properties. Compared to the analogues without sulfur, the fibrous aggregates formed by sulfur containing compounds are less stable and build weaker viscoelastic gels. This is due to a perturbation of the packing of the chains as the sulfur atoms change the bond angle in the chain compared to the molecules with pure alkyl chains leading to kinks in the chain. For the bolaamphiphile with the Me(2)PE headgroups this effect is less pronounced due to the possibility of forming stabilizing hydrogen bonds between the headgroups.

Self-assembled bolaamphiphile fibers have intermediate properties between crystalline nanofibers and wormlike micelles: formation of viscoelastic hydrogels switchable by changes in pH and salinity.

The aggregation behavior and rheological properties of aqueous suspensions of the bolaamphiphile dotriacontane-1,32-diyl-bis[2-(dimethylammonio)ethylphosphate] (Me(2)PE-C32-Me(2)PE) were investigated depending on the pH value and the salinity by means of differential scanning calorimetry (DSC), transmission electron microscopy (TEM), and oscillatory rheology. This bolaamphiphile self-assembles into helical fibers of approximately 5 nm thickness with an all-trans conformation of the alkyl chains. These nanofibers can gel water very effectively by forming a three-dimensional network. The headgroups' protonation depends on the pH value of the solution and influences the ability of the molecules to aggregate into fibers. At low pH values the headgroups are zwitterionic and stable hydrogels are formed, whereas at high pH values the headgroups are negatively charged and the length of the fiber aggregates diminishes as does the stability of the gel structure. We can show that by the addition of cations it is possible to shield the repulsive interaction between the molecules at high pH values so that the formation of the fiber aggregates and the gelation of the system occur. By addition of salt at high pH values the viscous flow behavior of Me(2)PE-C32-Me(2)PE suspensions could be transformed into the viscoelastic behavior of a gel. The gels show characteristics that are common in systems of wormlike micelles. However, there are also significant differences that arise from the unique bolaamphiphile fiber structure with highly ordered alkyl chains that render the properties similar to crystalline nanofibers.

Amino-functionalized single-chain bolalipids: synthesis and aggregation behavior of new basic building blocks.

Herein, we report the synthesis of two novel, amino-functionalized single-chain bolalipids and, based on those, a general synthetic approach for the insertion of various carboxylic acids into the bolalipid headgroups, e.g. alpha-lipoic acid for one-dimensional fixation of gold nanoparticles, sorbic acid for polymerization experiments, or lysine for the use in gene delivery systems. The temperature- and pH-dependent self-assembly of amino-functionalized bolalipids into nanofibers and micelles was investigated by differential scanning calorimetry (DSC), transmission electron microscopy (TEM) and dynamic light scattering (DLS). Rheological measurements were used to describe the macroscopic behavior of the formed temperature switchable hydrogels that can be fine-tuned for drug delivery applications. We showed that the viscoelastic properties of the hydrogel strongly depend on ionic interactions between bolalipid headgroups as well as on the ability to form hydrogen bonds.

General synthesis and aggregation behaviour of new single-chain bolaphospholipids: variations in chain and headgroup structures.

The chemical structures of polymethylene-1,omega-bis(phosphocholines) that self-assemble into nanofibres was modified on the one hand in the hydrophobic chain region, by introduction of sulfur and oxygen atoms, and on the other hand by variation of the polar headgroup structure with functionalised tertiary amines. The temperature-dependent self-assembly of these novel bolaphospholipids into nanofibres and spherical micelles was investigated by differential scanning calorimetry (DSC) and transmission electron microscopy (TEM). The thermal stabilities of the nanofibres strongly depend on the chemical compositions of the headgroups and of the hydrophobic chains. The insertion of new functionalities in the headgroup region by click chemistry makes these substances interesting for potential applications in bioscience and materials science.

Temperature-dependent self-assembly and mixing behavior of symmetrical single-chain bolaamphiphiles.

The temperature-dependent self-assembly and the mixing behavior of symmetrical single-chain bolaamphiphiles with different polymethylene chain lengths and different headgroup structures were investigated in water by differential scanning calorimetry (DSC), cryo transmission electron microscopy (cryo-TEM), and small angle neutron scattering (SANS). The even-numbered polymethylene-1,omega-bis(phosphocholines) (PC-C n-PC) are known to form nanofibers composed of stretched molecules with an all- trans alkyl chain conformation (Drescher, S.; Meister, A.; Blume, A.; Karlsson, G.; Almgren, M.; Dobner, B. Chem.Eur. J. 2007, 13, 5300-5307). The odd-numbered analogues were synthesized to study a possible even-odd effect of these bolaamphiphiles during their aggregation in water. In addition to these bolaamphiphiles with phosphocholine headgroups, a new series of polymethylene-1,omega-bis(phosphodimethylethanolamines) (Me2PE-Cn-Me2PE) with smaller headgroup sizes was synthesized. These bolaamphiphiles show an additional fiber-fiber transition when the alkyl chain length exceeds 26 carbon atoms. The mixing behavior of both types of bolaamphiphiles indicates that differences in the alkyl chain length up to six carbon atoms are tolerated within the fiber structure. The mixing of two Me2PE-Cn-Me2PE or PC-Cn-PC type bolaamphiphiles with different alkyl chain lengths offers the possibility to adjust the temperature, where the cross-linking of the fibers is disrupted and where the fibers break apart. As a consequence, temperature switchable hydrogels are obtained that can be fine-tuned for drug delivery applications. The comparison with dotriacontane-1,32-diyl-bis[2-(methylammonio)-ethylphosphate] (MePE-C32-MePE), a new bolaamphiphile with even smaller phosphomonomethylammonio headgroups, illustrates the importance of the headgroup size for the aggregation behavior. This bolaamphiphile self-assembles exclusively into lamellar structures, and this aggregate type persists in mixtures with the fiber forming Me2PE-C32-Me2PE.

Helical nanofibers of self-assembled bipolar phospholipids as template for gold nanoparticles.

Bipolar phospholipids (bolalipids) represent an exciting class of amphiphilic molecules as they self-assemble in water to distinct structures of nanoscopic dimensions. Reported here are structural details of helical nanofibers, composed of achiral, symmetrical single-chain bolalipids with phosphocholine headgroups. These nanofibers are used as template for the fixation of gold nanoparticles (AuNPs) without prior functionalization. This realization of a metal array on bolalipid nanofibers is one of the rare examples of one-dimensional AuNP arrangements in solution. The loading and the heat of binding of AuNPs are determined applying transmission electron microscopy and isothermal titration calorimetry.