Development of Nanopackaging for Storage and Transport of Loaded Lipid Nanoparticles.

Easily deploying new vaccines globally to combat disease outbreaks has been highlighted as a major necessity by the World Health Organization. RNA-based vaccines using lipid nanoparticles (LNPs) as a drug delivery system were employed to great effect during the recent COVID-19 pandemic. However, LNPs are still unstable at room temperature and agglomerate over time during storage, rendering them ineffective for intracellular delivery. We demonstrate the suitability of nanohole arrays (nanopackaging) as patterned surfaces to separate and store functionalized LNPs (fLNPs) in individual recesses, which can be expanded to other therapeutics. Encapsulating calcein as a model drug, we show through confocal microscopy the effective loading of fLNPs into our nanopackaging for both wet and dry systems. We prove quantifiably pH-mediated capture and subsequent unloading of over 30% of the fLNPs using QCM-D on alumina surfaces altering the pH from 5.5 to 7, displaying controllable storage at the nanoscale.

Studying the impact of the pre-exponential factor on templated nucleation.

Traditionally, the enhancement of nucleation rates in the presence of heterogeneous surfaces in crystallisation processes has been attributed to the modification of the interfacial energy of the system according to the classical nucleation theory. However, recent developments have shown that heterogeneous surfaces instead alter the pre-exponential factor of nucleation. In this work, the nucleation kinetics of glycine and diglycine in aqueous solutions have been explored in the presence and absence of a heterogeneous surface. Results from induction time experiments show that the presence of a heterogeneous surface increases the pre-exponential factor by 2-fold or more for both glycine and diglycine, while the interfacial energy remains unchanged for both species. This study suggests that the heterogeneous surface enhances the nucleation rate via hydrogen bond formation with both glycine and diglycine. This is verified by hydrogen bond propensity calculations, molecular functionality analysis, and calculation of the time taken for a solute molecule to attach to the growing nucleus, which is an order of magnitude shorter than the estimated lifetime of the hydrogen bond. The effect of the heterosurface is of greater magnitude for diglycine than for glycine, which may be due to the heightened molecular complementarity between the hydrogen bond donor and acceptor sites on diglycine and the heterosurface.

Influences of Crystal Anisotropy in Pharmaceutical Process Development.

Crystalline materials are of crucial importance to the pharmaceutical industry, as a large number of APIs are formulated in crystalline form, occasionally in the presence of crystalline excipients. Owing to their multifaceted character, crystals were found to have strongly anisotropic properties. In fact, anisotropic properties were found to be quite important for a number of processes including milling, granulation and tableting. An understanding of crystal anisotropy and an ability to control and predict crystal anisotropy are mostly subjects of interest for researchers. A number of studies dealing with the aforementioned phenomena are grounded on over-simplistic assumptions, neglecting key attributes of crystalline materials, most importantly the anisotropic nature of a number of their properties. Moreover, concepts such as the influence of interfacial phenomena in the behaviour of crystalline materials during their growth and in vivo, are still poorly understood. The review aims to address concepts from a molecular perspective, focusing on crystal growth and dissolution. It begins with a brief outline of fundamental concepts of intermolecular and interfacial phenomena. The second part discusses their relevance to the field of pharmaceutical crystal growth and dissolution. Particular emphasis is given to works dealing with mechanistic understandings of the influence of solvents and additives on crystal habit. Furthermore, comments and perspectives, highlighting future directions for the implementation of fundamental concepts of interfacial phenomena in the rational understanding of crystal growth and dissolution processes, have been provided.

Noncovalent Surface Modification of Cellulose Nanopapers by Adsorption of Polymers from Aprotic Solvents.

Basic adsorption of hydrophobic polymers from aprotic solvents was introduced as a platform technology to modify exclusively the surfaces of cellulose nanopapers. Dynamic vapor sorption demonstrated that the water vapor uptake ability of the nanopapers remained unperturbed, despite strong repellency to liquid water caused by the adsorbed hydrophobic polymer on the surface. This was enabled by the fact that the aprotic solvents used for adsorption did not swell the nanopaper unlike water that is generally applied as the adsorption medium in such systems. As case examples, the adsorptions of polystyrene (PS) and poly(trifluoroethylene) (PF3E) were followed by X-ray photoelectron spectroscopy and water contact angle measurements, backed up with morphological analysis by atomic force microscopy. The resulting nanopapers are useful in applications like moisture buffers where repellence to liquid water and ability for moisture sorption are desired qualities.

Model for Interpreting Surface Crystallization Using Quartz Crystal Microbalance: Theory and Experiments.

Surface crystallization of calcium sulfate was investigated using a dissipation crystal quartz microbalance (QCM-D) together with microscopy to understand the mechanical property changes occurring during the growth process. The use of optical microscopy and SEM revealed that needle-shaped crystals grow as clusters on the QCM sensor's surface, not in uniform layers. As crystallization growth progressed, QCM-D revealed inversions between negative and positive frequency shifts. This behavior, a function of the growth of crystals in clusters, is not adequately predicted by existing models. As such, a new mass-to-frequency conversion model is proposed herein to explain the observed frequency inversions. This model is derived from a lumped element approach with point-contact loading and Mason equivalent circuit theory. Critically, the physical phenomena occurring form the basis of the model, particularly addressing the three sources of impedance. When a crystal nucleates and grows, its inertial impedance is considered along with a Kelvin-Voigt link with a hydration layer. A comparison between the proposed model and experimental data, of both frequency and dissipation data for the first four harmonics, shows good agreement for the supersaturations (S = C/C*) of S = 3.75, S = 3.48, and S = 3.22. Additionally, significant improvements over existing models for the case of surface crystallization are observed. The proposed model was therefore able to explain that frequency inversions are caused by a shift from inertia-dominated to elastic-dominated impedance, occurring as a result of crystal growth. Using the nucleation induction time and nucleation rates, determined with imaging, an additional understanding of the crystals' mechanical properties (stiffness and dampening) was obtained.

Decoupling the contribution of surface energy and surface area on the cohesion of pharmaceutical powders.

PURPOSE: Surface area and surface energy of pharmaceutical powders are affected by milling and may influence formulation, performance and handling. This study aims to decouple the contribution of surface area and surface energy, and to quantify each of these factors, on cohesion. METHODS: Mefenamic acid was processed by cryogenic milling. Surface energy heterogeneity was determined using a Surface Energy Analyser (SEA) and cohesion measured using a uniaxial compression test. To decouple the surface area and surface energy contributions, milled mefenamic acid was "normalised" by silanisation with methyl groups, confirmed using X-ray Photoelectron Spectroscopy. RESULTS: Both dispersive and acid-base surface energies were found to increase with increasing milling time. Cohesion was also found to increase with increasing milling time. Silanised mefenamic acid possessed a homogenous surface with a surface energy of 33.1 ± 1.4 mJ/m(2) , for all milled samples. The cohesion for silanised mefenamic acid was greatly reduced, and the difference in the cohesion can be attributed solely to the increase in surface area. For mefenamic acid, the contribution from surface energy and surface area on cohesion was quantified to be 57% and 43%, respectively. CONCLUSIONS: Here, we report an approach for decoupling and quantifying the contribution from surface area and surface energy on powder cohesion.

A new method to determine dispersive surface energy site distributions by inverse gas chromatography.

A computational model to predict the relative energy site contributions of a heterogeneous material from data collected by finite dilution-inverse gas chromatography (FD-IGC) is presented in this work. The methodology employed a multisolvent system site filling model utilizing Boltzmann statistics, expanding on previous efforts to calculate "experienced energies" at varying coverage, yielding a retention volume distribution allowing calculation of a surface free energy distribution. Surface free energy distributions were experimentally measured for racemic ibuprofen and ß-mannitol powders, the energies of each were found in the ranges 43-52 and 40-55 mJ/m(2), respectively, over a surface coverage range of 0-8%. The computed contributions to surface energy values were found to match closely with data collected on macroscopic crystals by alternative techniques (±<1.5 mJ/m(2)).

Phase behavior of medium and high internal phase water-in-oil emulsions stabilized solely by hydrophobized bacterial cellulose nanofibrils.

Water-in-oil emulsions stabilized solely by bacterial cellulose nanofibers (BCNs), which were hydrophobized by esterification with organic acids of various chain lengths (acetic acid, C2-; hexanoic acid, C6-; dodecanoic acid, C12-), were produced and characterized. When using freeze-dried C6-BCN and C12-BCN, only a maximum water volume fraction (ϕw) of 60% could be stabilized, while no emulsion was obtained for C2-BCN. However, the maximum ϕw increased to 71%, 81%, and 77% for C2-BCN, C6-BCN, and C12-BCN, respectively, 150 h after the initial emulsification, thereby creating high internal phase water-in-toluene emulsions. The observed time-dependent behavior of these emulsions is consistent with the disentanglement and dispersion of freeze-dried modified BCN bundles into individual nanofibers with time. These emulsions exhibited catastrophic phase separation when ϕw was increased, as opposed to catastrophic phase inversion observed for other Pickering emulsions.

Crystal regeneration - a unique growth phenomenon observed in organic crystals post breakage.

Crystal regeneration has been observed in macroscopic paracetamol crystals post breakage along their cleavage plane. High resolution imaging confirmed regeneration rates to be 3-fold faster than growth prior to breakage. Further analysis of the solute-solvent interactions is required to elucidate the process which currently lacks linearity with traditional growth theories.

Experimental Elucidation of Templated Crystallization and Secondary Processing of Peptides.

The crystallization of peptides offers a sustainable and inexpensive alternative to the purification process. In this study, diglycine was crystallised in porous silica, showing the porous templates' positive yet discriminating effect. The diglycine induction time was reduced by five-fold and three-fold upon crystallising in the presence of silica with pore sizes of 6 nm and 10 nm, respectively. The diglycine induction time had a direct relationship with the silica pore size. The stable form (α-form) of diglycine was crystallised in the presence of porous silica, with the diglycine crystals obtained associated with the silica particles. Further, we studied the mechanical properties of diglycine tablets for their tabletability, compactability, and compressibility. The mechanical properties of the diglycine tablets were similar to those of pure MCC, even with the presence of diglycine crystals in the tablets. The diffusion studies of the tablets using the dialysis membrane presented an extended release of diglycine through the dialysis membrane, confirming that the peptide crystal can be used for oral formulation. Hence, the crystallization of peptides preserved their mechanical and pharmacological properties. More data on different peptides can help us produce oral formulation peptides faster than usual.

The Effect of Chain Length and Conformation on the Nucleation of Glycine Homopeptides during the Crystallization Process.

To explore the effect of chain length and conformation on the nucleation of peptides, the primary nucleation induction time of glycine homopeptides in pure water at different supersaturation levels under various temperatures has been determined. Nucleation data suggest that longer chains will prolong the induction time, especially for chains longer than three, where nucleation will occur over several days. In contrast, the nucleation rate increased with an increase in the supersaturation for all homopeptides. Induction time and nucleation difficulty increase at lower temperatures. However, for triglycine, the dihydrate form was produced with an unfolded peptide conformation (pPII) at low temperature. The interfacial energy and activation Gibbs energy of this dihydrate form are both lower than those at high temperature, while the induction time is longer, indicating the classical nucleation theory is not suitable to explain the nucleation phenomenon of triglycine dihydrate. Moreover, gelation and liquid-liquid separation of longer chain glycine homopeptides were observed, which was normally classified to nonclassical nucleation theory. This work provides insight into how the nucleation process evolves with increasing chain length and variable conformation, thereby offering a fundamental understanding of the critical peptide chain length for the classical nucleation theory and complex nucleation process for peptides.

A workflow for the development of template-assisted membrane crystallization downstream processing for monoclonal antibody purification.

Monoclonal antibodies (mAbs) are commonly used biologic drugs for the treatment of diseases such as rheumatoid arthritis, multiple sclerosis, COVID-19 and various cancers. They are produced in Chinese hamster ovary cell lines and are purified via a number of complex and expensive chromatography-based steps, operated in batch mode, that rely heavily on protein A resin. The major drawback of conventional procedures is the high cost of the adsorption media and the extensive use of chemicals for the regeneration of the chromatographic columns, with an environmental cost. We have shown that conventional protein A chromatography can be replaced with a single crystallization step and gram-scale production can be achieved in continuous flow using the template-assisted membrane crystallization process. The templates are embedded in a membrane (e.g., porous polyvinylidene fluoride with a layer of polymerized polyvinyl alcohol) and serve as nucleants for crystallization. mAbs are flexible proteins that are difficult to crystallize, so it can be challenging to determine the optimal conditions for crystallization. The objective of this protocol is to establish a systematic and flexible approach for the design of a robust, economic and sustainable mAb purification platform to replace at least the protein A affinity stage in traditional chromatography-based purification platforms. The procedure provides details on how to establish the optimal parameters for separation (crystallization conditions, choice of templates, choice of membrane) and advice on analytical and characterization methods.

Effect of milling on particle shape and surface energy heterogeneity of needle-shaped crystals.

PURPOSE: Milling and micronization of particles are routinely employed in the pharmaceutical industry to obtain small particles with desired particle size characteristics. The aim of this study is to demonstrate that particle shape is an important factor affecting the fracture mechanism in milling. METHODS: Needle-shaped crystals of the ß polymorph of D-mannitol were prepared from recrystallization in water. A portion of the recrystallized materials was ball-milled. Unmilled and milled sieved fractions of recrystallized D-mannitol were analyzed by dynamic image analysis (DIA) and inverse gas chromatography (IGC) at finite concentration to explain the breakage/fracture behavior. RESULTS: In the process of ball-milling, D-mannitol preferentially fractured along their shortest axis, exposing (011) plane with increased hydrophilicity and increased bounding rectangular aspect ratio. This is in contrary to attachment energy modeling which predicts a fracture mechanism across the (010) plane with increased hydrophobicity, and small change in particle shape. CONCLUSIONS: Crystal size, and more importantly, crystal shape and facet-specific mechanical properties, can dictate the fracture/cleavage behavior of organic crystalline materials. Thorough understanding of the crystal slip systems, combining attachment energy prediction with particle shape and surface characterization using DIA and IGC, are important in understanding fracture behavior of organic crystalline solids in milling and micronization.

Unraveling the Impact of pH on the Crystallization of Pharmaceutical Proteins: A Case Study of Human Insulin.

One of the most crucial parameters in protein crystallization is pH, as it governs the protein's electrostatic interactions. However, the fundamental role of pH on crystallization still remains unknown. Here, we systematically investigated the crystallization of human insulin (isoelectric point 5.3) at various pHs between 6.0 and 6.7 at different supersaturation ratios, up to 20.9. Our results demonstrate that the pH has an opposing effect on solubility and nucleation rate as a shift in pH toward a more basic milieu increases the solubility by 5-fold while the onset of nucleation was accelerated by a maximum of 8.6-fold. To shed light on this opposing effect, we evaluated the protein-protein interactions as a function of pH by measuring the second virial coefficient and hydrodynamic radius and showed that a change in pH of less than one unit has no significant impact on the protein-protein interactions. As it is widely understood that the increase in protein solubility as a function of pH is due to the increase in the repulsive electrostatic interactions, we have demonstrated that the increase in insulin solubility and decrease in the onset of nucleation are independent of the protein-protein interactions. We hypothesize that it is the electrostatic interactions between both ions and solvent molecules and the protein residues that are governing the crystallization of human insulin. The findings of this study will be of crucial importance for the design of novel crystallization pathways.

Relating Crystal Structure to Surface Properties: A Study on Quercetin Solid Forms.

The surface energy and surface chemistry of a crystal are of great importance when designing particles for a specific application, as these will impact both downstream manufacturing processes as well as final product quality. In this work, the surface properties of two different quercetin solvates (quercetin dihydrate and quercetin DMSO solvate) were studied using molecular (synthonic) modeling and experimental techniques, including inverse gas chromatography (IGC) and contact angle measurements, to establish a relationship between crystal structure and surface properties. The attachment energy model was used to predict morphologies and calculate surface properties through the study of their growth synthons. The modeling results confirmed the surface chemistry anisotropy for the two forms. For quercetin dihydrate, the {010} facets were found to grow mainly by nonpolar offset quercetin-quercetin stacking interactions, thus being hydrophobic, while the {100} facets were expected to be hydrophilic, growing by a polar quercetin-water hydrogen bond. For QDMSO, the dominant facet {002} grows by a strong polar quercetin-quercetin hydrogen bonding interaction, while the second most dominant facet {011} grows by nonpolar π-π stacking interactions. Water contact angle measurements and IGC confirmed a greater overall surface hydrophilicity for QDMSO compared to QDH and demonstrated surface energy heterogeneity for both structures. This work shows how synthonic modeling can help in the prediction of the surface nature of crystalline particles and guide the choice of parameters that will determine the optimal crystal form and final morphology for targeted surface properties, for example, the choice of crystallization conditions, choice of solvent, or presence of additives or impurities, which can direct the crystallization of a specific crystal form or crystal shape.

Template-Assisted Crystallization Behavior in Stirred Solutions of the Monoclonal Antibody Anti-CD20: Probability Distributions of Induction Times.

We present a method to determine the template crystallization behavior of proteins. This method is a statistical approach that accounts for the stochastic nature of nucleation. It makes use of batch-wise experiments under stirring conditions in volumes smaller than 0.3 mL to save material while mimicking larger-scale processes. To validate our method, it was applied to the crystallization of a monoclonal antibody of pharmaceutical interest, Anti-CD20. First, we determined the Anti-CD20 phase diagram in a PEG-400/Na2SO4/water system using the batch method, as, to date, no such data on Anti-CD20 solubility have been reported. Then, the probability distribution of induction times was determined experimentally, in the presence of various mesoporous silica template particles, and crystallization of Anti-CD20 in the absence of templates was compared to template-assisted crystallization. The probability distribution of induction times is shown to be a suitable method to determine the effect of template particles on protein crystallization. The induction time distribution allows for the determination of two key parameters of nucleation, the nucleation rate and the growth time. This study shows that the use of silica particles leads to faster crystallization and a higher nucleation rate. The template particle characteristics are shown to be critical parameters to efficiently promote protein crystallization.

Understanding flow properties of mannitol powder at a range of temperature and humidity.

Inadequate flowability of powders in industries during handling can cause many problems. For example, lack of flow from hoppers, poor tablet weight consistency, and low production rate in tableting. Many factors are known to commonly affect flow properties of powders, such as temperature, humidity and conditioning duration. In this paper, flow properties of a mannitol powder, which was conditioned between 24 and 72 h at various high relative humidities and temperature, were measured using a shear tester. A statistical model was developed to investigate the relative importance of these variables on the mannitol flow properties. The developed model showed all independent variables are significant in estimating bulk cohesion. Two separate approaches were used to evaluate inter-particle forces in the bulk, and how these changed with environmental conditions. First, inter-particle forces were inferred from the measured bulk properties using the Rumpf model approach. Secondly, inter-particle forces were predicted based on a model of moisture present on the particle surface using a combination of Kelvin model with the Laplace-Young (KLY) equation. The second approach also involved a new method to measure surface energy of mannitol powder based on measurements using Finite Dilution Inverse Gas Chromatography (FD-IGC). The surface energies of the mannitol powder were measured at high temperature (35 °C) and at different range of relative humidities. In spite of the fundamentally different approaches to the two ways of inferring inter-particles forces, these forces came out within less than 1.5:1 in magnitude. The Rumpf approach from bulk behaviour data obviously reflected the measured change in behaviour with humidity in particular, but this was not predicted from the KLY approach, however the likely reasons for this are postulated and recommendations for improvement are made.

Triglycine (GGG) Adopts a Polyproline II (pPII) Conformation in Its Hydrated Crystal Form: Revealing the Role of Water in Peptide Crystallization.

Polyproline II (pPII) is a left-handed 31-helix conformation, which has been observed to be the most abundant secondary structure in unfolded peptides and proteins compared to α-helix and ß-sheet. Although pPII has been reported as the most stable conformation for several unfolded short chain peptides in aqueous solution, it is rarely observed in their solid state. Here, we show for the first time a glycine homopeptide (gly-gly-gly) adopting the pPII conformation in its crystalline dihydrate structure. The single crystal X-ray structure with molecular dynamic simulation suggests that a network of water and the charged carboxylate group is critical in stabilizing the pPII conformation in solid state, offering an insight into the structures of unfolded regions of proteins and the role of water in peptide crystallization.

Influence of interparticle structuring on the surface energetics of a binary powder system.

The structuring of component particles in binary compositions affects the solid-solid interfacial properties. This work reports the effect of interparticle interactions in binary powder compositions of D-Mannitol and glass beads through the heterogeneity data obtained from Finite Dilution Inverse Gas Chromatography (FD-IGC). Three different scenarios viz. structured, random and segregated systems of the binary powder composition were considered for the analysis in the IGC column. Binary mixtures with large size disparity between the components produced structured mixtures exhibiting a guest-host type of interactions and energetic homogeneity irrespective of the energetics of the finer component. Random and segregated systems revealed a heterogeneous trend in the data indicating preferential probing of the active sites of the composition, particularly at the lower probe coverages. The results demonstrate that in the multicomponent binary systems the surface energetics is influenced by the solid-solid interfaces and structuring of the component particles within the mix i.e., the surface energy analysis could reveal a mixing behavior in powders. Furthermore, an adsorption energy distribution model based on Boltzmann statistics and simulation fitting approach was employed to deconvolute the distribution of the changing energy landscape of the binary mixtures.

Surface Energy Mapping of Modified Silica Using IGC Technique at Finite Dilution.

The reinforcing silica filler, which can be more than 40% of an elastomer composite, plays a key role to achieve the desired mechanical properties in elastomer vulcanizates. However, the highly hydrophilic nature of silica surface causes silica particle aggregation. It remained a challenge for many tire manufacturers when using silica-filled elastomer compounds. Here, the silica surface energy changes when the surface is modified with coupling or noncoupling silanes; coupling silanes can covalently bond the silica to the elastomers. The surface energy of silica was determined using inverse gas chromatography (IGC) at finite dilution (FD-IGC) and found to be reduced by up to 50% when the silica surface was silanized. The spatial distribution of silica aggregates within the tire matrix is determined by transmission electron microscopy (TEM) and a direct correlation between aggregate size (silica microdispersion) and work of cohesion from IGC is reported, highlighting surface energy and work of cohesion being excellent indicators of the degree of dispersion of silica aggregates.