Efficient and Targeted siRNA Delivery to M2 Macrophages by Smart Polymer Blends for M1 Macrophage Repolarization as a Promising Strategy for Future Cancer Treatment.

Cancer remains an issue on a global scale. It is estimated that nearly 10 million people succumbed to cancer worldwide in 2020. New treatment options are urgently needed. A promising approach is a conversion of tumor-promoting M2 tumor-associated macrophages (TAMs) as part of the tumor microenvironment to tumor-suppressive M1 TAMs by small interfering RNA (siRNA). In this work, we present a well-characterized polymeric nanocarrier system capable of targeting M2 TAMs by a ligand-receptor interaction. Therefore, we developed a blended PEI-based polymeric nanoparticle system conjugated with mannose, which is internalized after interaction with macrophage mannose receptors (MMRs), showing low cytotoxicity and negligible IL-6 activation. The PEI-PCL-PEI (5 kDa-5 kDa-5 kDa) and Man-PEG-PCL (2 kDa-2 kDa) blended siRNA delivery system was optimized for maximum targeting capability and efficient endosomal escape by evaluation of different polymer and N/P ratios. The nanoparticles were formulated by surface acoustic wave-assisted microfluidics, achieving a size of ∼80 nm and a zeta potential of approximately +10 mV. Special attention was given to the endosomal escape as the so-called bottleneck of RNA drug delivery. To estimate the endosomal escape capability of the nanocarrier system, we developed a prediction method by evaluating the particle stability via the inflection temperature. Our predictions were then verified in an in vitro setting by applying confocal microscopy. For cellular experiments, however, human THP-1 cells were polarized to M2 macrophages by cytokine treatment and validated through MMR expression. To show the efficiency of the nanoparticle system, GAPDH and IκBα knockdown was performed in the presence or absence of an MMR blocking excess of mannan. Cellular uptake, GAPDH knockdown, and NF-κB western blot confirmed efficient mannose targeting. Herein, we presented a well-characterized nanoparticle delivery system and a promising approach for targeting M2 macrophages by a mannose-MMR interaction.

Scale-up analysis of geometrically dissimilar single-use bioreactors.

Cell culture scale-up is a challenging task due to the simultaneous change of multiple hydrodynamic process characteristics and their different dependencies on the bioreactor size as well as variation in the requirements of individual cell lines. Conventionally, the volumetric power input is the most common parameter to select the impeller speed for scale-up, however, it is well reported that this approach fails when there are huge differences in bioreactor scales. In this study, different scale-up criteria are evaluated. At first, different hydrodynamic characteristics are assessed using computational fluid dynamics data for four single-use bioreactors, the Mobius® CellReady 3 L, the Xcellerex™ XDR-10, the Xcellerex™ XDR-200, and the Xcellerex™ XDR-2000. On the basis of this numerical data, several potential scale-up criteria such as volumetric power input, impeller tip speed, mixing time, maximum hydrodynamic stress, and average strain rate in the impeller zone are evaluated. Out of all these criteria, the latter is found to be most appropriate, and the successful scale-up from 3 to 10 L bioreactor and to 200 L bioreactor is confirmed with cell culture experiments using Chinese Hamster Ovary cell cultivation.

Quantifying the hydrodynamic stress for bioprocesses.

Hydrodynamic stress is an influential physical parameter for various bioprocesses, affecting the performance and viability of the living organisms. However, different approaches are in use in various computational and experimental studies to calculate this parameter (including its normal and shear subcomponents) from velocity fields without a consensus on which one is the most representative of its effect on living cells. In this letter, we investigate these different methods with clear definitions and provide our suggested approach which relies on the principal stress values providing a maximal distinction between the shear and normal components. Furthermore, a numerical comparison is presented using the computational fluid dynamics simulation of a stirred and sparged bioreactor. It is demonstrated that for this specific bioreactor, some of these methods exhibit quite similar patterns throughout the bioreactor-therefore can be considered equivalent-whereas some of them differ significantly.

Impact of post-freeze annealing on shrinkage of sucrose and trehalose lyophilisates.

Freeze-drying of pharmaceuticals produces lyophilisates with properties that depend on both the formulation and the process. Characterisation of the lyophilisate in terms of appearance is necessary not only to produce a visually appealing product, but also to gain insight into the freeze-drying process. The present study investigates the impact of post-freeze annealing on the volume of lyophilisates. For this purpose, sucrose and trehalose solutions were freeze-dried with different annealing conditions and the resulting lyophilisates were analysed with a 3D structured light scanner. The external structure of the lyophilisates was found to be dependent on the bulk materials as well as the choice of vials, while the volume was influenced by the annealing time and temperature. Additionally, differential scanning calorimetry was used to determine glass transition temperatures of frozen samples. As a novelty, the volumes of the lyophilisates and their corresponding glass transition temperatures were compared. This resulted in a correlation supporting the theory that the shrinkage of lyophilisates depends on the amount of residual water in the freeze-concentrated amorphous phase before drying. Understanding the volume change of lyophilisates, in combination with material properties such as glass transition temperature, forms the basis for relating physicochemical properties to process parameters in lyophilisation.

Quantitative Analysis of Glassy State Relaxation and Ostwald Ripening during Annealing Using Freeze-Drying Microscopy.

Supercooling during the freezing of pharmaceutical solutions often leads to suboptimal freeze-drying results, such as long primary drying times or a collapse in the cake structure. Thermal treatment of the frozen solution, known as annealing, can improve those issues by influencing properties such as the pore size and collapse temperature of the lyophilisate. In this study we aimed to show that annealing causes a rearrangement of water molecules between ice crystals, as well as between the freeze-concentrated amorphous matrix and the crystalline ice phase in a frozen binary aqueous solution. Ice crystal sizes, as well as volume fractions of the crystalline and amorphous phases of 10% (w/w) sucrose and trehalose solutions, were quantified after annealing using freeze-drying microscopy and image labelling. Depending on the annealing time and temperature, the amorphous phase was shown to decrease its volume due to the crystallisation of vitreous water (i.e., glassy state relaxation) while the crystalline phase was undergoing coarsening (i.e., Ostwald ripening). These results allow, for the first time, a quantitative comparison of the two phenomena. It was demonstrated that glassy state relaxation and Ostwald ripening, although occurring simultaneously, are distinct processes that follow different kinetics.

Numerical and Experimental Investigation of the Hydrodynamics in the Single-Use Bioreactor Mobius® CellReady 3 L.

Two-way Euler-Lagrange simulations are performed to characterize the hydrodynamics in the single-use bioreactor Mobius® CellReady 3 L. The hydrodynamics in stirred tank bioreactors are frequently modeled with the Euler-Euler approach, which cannot capture the trajectories of single bubbles. The present study employs the two-way coupled Euler-Lagrange approach, which accounts for the individual bubble trajectories through Langrangian equations and considers their impact on the Eulerian liquid phase equations. Hydrodynamic process characteristics that are relevant for cell cultivation including the oxygen mass transfer coefficient, the mixing time, and the hydrodynamic stress are evaluated for different working volumes, sparger types, impeller speeds, and sparging rates. A microporous sparger and an open pipe sparger are considered where bubbles of different sizes are generated, which has a pronounced impact on the bubble dispersion and the volumetric oxygen mass transfer coefficient. It is found that only the microporous sparger provides sufficiently high oxygen transfer to support typical suspended mammalian cell lines. The simulated mixing time and the volumetric oxygen mass transfer coefficient are successfully validated with experimental results. Due to the small reactor size, mixing times are below 25 s across all tested conditions. For the highest sparging rate of 100 mL min-1, the mixing time is found to be two seconds shorter than for a sparging rate of 50 mL min-1, which again, is 0.1 s longer than for a sparging rate of 10 mL min-1 at the same impeller speed of 100 rpm and the working volume of 1.7 L. The hydrodynamic stress in this bioreactor is found to be below critical levels for all investigated impeller speeds of up to 150 rpm, where the maximum levels are found in the region where the bubbles pass behind the impeller blades.

CFD-Based and Experimental Hydrodynamic Characterization of the Single-Use Bioreactor XcellerexTM XDR-10.

Understanding the hydrodynamic conditions in bioreactors is of utmost importance for the selection of operating conditions during cell culture process development. In the present study, the two-phase flow in the lab-scale single-use bioreactor XcellerexTM XDR-10 is characterized for working volumes from 4.5 L to 10 L, impeller speeds from 40 rpm to 360 rpm, and sparging with two different microporous spargers at rates from 0.02 L min-1 to 0.5 L min-1. The numerical simulations are performed with the one-way coupled Euler-Lagrange and the Euler-Euler models. The results of the agitated liquid height, the mixing time, and the volumetric oxygen mass transfer coefficient are compared to experiments. For the unbaffled XDR-10, strong surface vortex formation is found for the maximum impeller speed. To support the selection of suitable impeller speeds for cell cultivation, the surface vortex formation, the average turbulence energy dissipation rate, the hydrodynamic stress, and the mixing time are analyzed and discussed. Surface vortex formation is observed for the maximum impeller speed. Mixing times are below 30 s across all conditions, and volumetric oxygen mass transfer coefficients of up to 22.1 h-1 are found. The XDR-10 provides hydrodynamic conditions which are well suited for the cultivation of animal cells, despite the unusual design of a single bottom-mounted impeller and an unbaffled cultivation bioreactor.

Influence of particle size and blender size on blending performance of bi-component granular mixing: A DEM and experimental study.

The effect of particle size enlargement and blender geometry down-scaling on the blend uniformity (BU) was evaluated by Discrete Element Method (DEM) to predict the blending performance of a binary granular mixture. Three 10 kg blending experiments differentiated by the physical properties specifically particle size were performed as reference for DEM simulations. The segregation behavior observed during the diffusion blending was common for all blends, while the sample BU, i.e., standard deviation of active ingredient content % was different among the three blends reflecting segregation due to the particle size differences between the components. Quantitative prediction of the sample BU probability density distribution in reality based on the DEM simulation results was successfully demonstrated. The average root mean square error normalized by the mean of the mean sample BU in the blends was 0.228. Beside the ratio of blender container to particle size, total number of particles in the blender and the number of particles in a sample were confirmed critical for the blending performance. These in-silico experiments through DEM simulations would help in setting a design space with respect to the particle size and in a broader sense with respect to the physical properties in general.

Evaluation and prediction of powder flowability in pharmaceutical tableting.

The focus of this study is to establish a characterization method determining the powder flowability in context of tableting. At first, flowability of different materials is measured using the ring shear tester, and its prediction from particle size is established. Next, the model die-filling system is presented which is a modified version of previous studies. Using this system, flowability of different materials is measured at varying die speeds. A new curve fit to assess die fill ratio vs die speed is suggested improving predictability, and a novel flowability metric, "Die Fill Index" (DFI), is derived. The DFI is appropriate to describe flowability for most of the tested materials, and sensitivity of a material with respect to tableting speed. A correlation is generated predicting DFI from particle size. Additionally, it is shown that model die filling is the preferable method to assess flowability for tableting compared to ring shear tester.

Selection of a round convex tablet shape that mitigates the risk of chipping and capping based on systematic evaluation by utilizing multivariate analysis.

Selecting a tablet shape that minimizes the risk of chipping and capping during manufacture is important in pharmaceutical industry. Here, the selection was performed based on systematic evaluation for the first time. Abrasion and stress relaxation time were utilized as indices of the occurrences of chipping and capping, respectively. Partial least square regression models that used tablet shape parameters to estimate the tablet's abrasion and stress relaxation time were utilized to develop response surface plots of the effect of the tablet shapes on the occurrence of chipping and capping systematically, and to identify an optimum tablet shape that is expected to have a low occurrence of chipping and capping. A verification study using commercial scale facilities proved that the optimum tablet shape had a lower occurrence of chipping and capping compared to suboptimum examples as speculated by their abrasion and stress relaxation time. The observed mathematical relationship between the tablet shapes and the occurrence of chipping and capping were consistent with the previous studies based on the comparison of limited number of tablet shapes using different formulations. Consequently, it is expected to be applicable to other formulations beyond the evaluated formulation in the present study.

High efficiency dry coating of non-subcoated pellets for sustained drug release formulations using amino methacrylate copolymers.

Dry coating utilizing a fluidized bed was evaluated in order to produce films with sustained drug release using amino methacrylate copolymers as film former. In contrast to other dry coating procedures using amino methacrylate copolymers, the described method enables an appropriate polymer adhesion by the selection of a plasticizer additive mixture in combination with the use of a three-way nozzle for simultaneous application. Well spreading fatty acid esters were found to increase the coating efficiency from 73% to approximately 86%, when they were used in conjunction with the plasticizer. Pellets were used as drug cores without previous treatment. After a curing step at 55 °C, the pellets exhibited a prolongation of the drug release over a period of about 6 h. Mainly the three parameters, coating level, composition of the polymers in the coating mixture, and the type of plasticizer, were found to exert distinct influence on the dissolution profile. Despite the differences in the coating procedure, the dissolution profiles of the coated pellets as well as the influencing parameters were similar to those known from conventional coating techniques.

Nano- and Microstructured model carrier surfaces to alter dry powder inhaler performance.

The present study investigates the effect of different carrier surface modifications on the aerosolisation performance and on the effective carrier payload of interactive blends for inhalation. Two different active pharmaceutical ingredients (APIs) were used: Formoterol fumarate dihydrate (FF) and budesonide (BUD). Blends were prepared with glass beads as model carriers which have been subjected to mechanical surface modifications in order to introduce surface roughness via treatment with hydrofluoric acid (HF) and/or milling with tungsten carbide (TC). As far as effective carrier payload, in this study expressed as true surface coverage (TSC), is concerned, surface modification had varying effects on blends containing BUD or FF. Aerodynamic characterisation in vitro showed a significant decrease in respirable fraction for glass beads treated with HF (40.2-50.1%), due to the presence of clefts and cavities, where drug particles were sheltered during inhalation. In contrast, grinding with TC leads to surface roughness on a nano scale, ultimately increasing aerodynamic performance up to 20.0-38.1%. These findings are true for both APIs, regardless of their chemical properties.

Setting the process parameters for the coating process in order to assure tablet appearance based on multivariate analysis of prior data.

Designing efficient, robust process parameters in drug product manufacturing is important to assure a drug's critical quality attributes. In this research, an efficient, novel procedure for a coating process parameter setting was developed, which establishes a prediction model for setting suitable input process parameters by utilizing prior manufacturing knowledge for partial least squares regression (PLSR). In the proposed procedure, target values or ranges of the output parameters are first determined, including tablet moisture content, spray mist condition, and mechanical stress on tablets. Following the preparation of predictive models relating input process parameters to corresponding output parameters, optimal input process parameters are determined using these models so that the output parameters hold within the target ranges. In predicting the exhaust air temperature output parameter, which reflects the tablets' moisture content, PLSR was employed based on prior measured data (such as batch records of other products rather than design of experiments), leading to minimal new experiments. The PLSR model was revealed to be more accurate at predicting the exhaust air temperature than a conventional semi-empirical thermodynamic model. A commercial scale verification demonstrated that the proposed process parameter setting procedure enabled assurance of the quality of tablet appearance without any trial-and-error experiments.

Solubility parameters of hypromellose acetate succinate and plasticization in dry coating procedures.

Solubility parameters of HPMCAS have not yet been investigated intensively. On this account, total and three-dimensional solubility parameters of HPMCAS were determined by using different experimental as well as computational methods. In addition, solubility properties of HPMCAS in a huge number of solvents were tested and a Teas plot for HPMCAS was created. The total solubility parameter of about 24 MPa(0.5) was confirmed by various procedures and compared with values of plasticizers. Twenty common pharmaceutical plasticizers were evaluated in terms of their suitability for supporting film formation of HPMCAS under dry coating conditions. Therefore, glass transition temperatures of mixtures of polymer and plasticizers were inspected and film formation of potential ones was further investigated in dry coating of pellets. Contact angles of plasticizers on HPMCAS were determined in order to give a hint of achievable coating efficiencies in dry coating, but none was found to spread on HPMCAS. A few common substances, e.g. dimethyl phthalate, glycerol monocaprylate, and polyethylene glycol 400, enabled plasticization of HPMCAS; however, only triethyl citrate and triacetin were found to be suitable for use in dry coating. Addition of acetylated monoglycerides to triacetin increased coating efficiency, which was likewise previously demonstrated for triethyl citrate.

Crystallization speed of salbutamol as a function of relative humidity and temperature.

Spray dried salbutamol sulphate and salbutamol base particles are amorphous as a result of spray drying. As there is always the risk of recrystallization of amorphous material, the aim of this work is the evaluation of the temperature and humidity dependent recrystallization of spray dried salbutamol sulphate and base. Therefore in-situ Powder X-ray Diffraction (PXRD) studies of the crystallization process at various temperature (25 and 35 °C) and humidity (60%, 70%, 80%, 90% relative humidity) conditions were performed. It was shown that the crystallization speed of salbutamol sulphate and base is a non-linear function of both temperature and relative humidity. The higher the relative humidity the higher is the crystallization speed. At 60% relative humidity salbutamol base as well as salbutamol sulphate were found to be amorphous even after 12 h, however samples changed optically. At 70% and 90% RH recrystallization of salbutamol base is completed after 3 h and 30 min and recrystallization of salbutamol sulphate after 4h and 1h, respectively. Higher temperature (35 °C) also leads to increased crystallization speeds at all tested values of relative humidity.

Influence of surface characteristics of modified glass beads as model carriers in dry powder inhalers (DPIs) on the aerosolization performance.

The aim of this work is to investigate the effect of surface characteristics (surface roughness and specific surface area) of surface-modified glass beads as model carriers in dry powder inhalers (DPIs) on the aerosolization, and thus, the in vitro respirable fraction often referred to as fine particle fraction (FPF). By processing glass beads in a ball mill with different grinding materials (quartz and tungsten carbide) and varying grinding time (4 h and 8 h), and by plasma etching for 1 min, glass beads with different shades of surface roughness and increased surface area were prepared. Compared with untreated glass beads, the surface-modified rough glass beads show increased FPFs. The drug detachment from the modified glass beads is also more reproducible than from untreated glass beads indicated by lower standard deviations for the FPFs of the modified glass beads. Moreover, the FPF of the modified glass beads correlates with their surface characteristics. The higher the surface roughness and the higher the specific surface area of the glass beads the higher is the FPF. Thus, surface-modified glass beads make an ideal carrier for tailoring the performance of DPIs in the therapy of asthma and chronically obstructive pulmonary diseases.

Preparation and characterization of physically modified glass beads used as model carriers in dry powder inhalers.

The aim of this work is the physical modification and characterization of the surface topography of glass beads used as model carriers in dry powder inhalers (DPIs). By surface modification the contact area between drug and carrier and thereby interparticle forces may be modified. Thus the performance of DPIs that relies on interparticle interactions may be improved. Glass beads were chosen as model carriers because various prospects of physical surface modification may be applied without affecting other factors also impacting interparticle interactions like particle size and shape. To generate rough surfaces glass beads were processed mechanically by friction and impaction in a ball mill with different grinding materials that were smaller and harder with respect to the glass beads. By varying the grinding time (4 h, 8 h) and by using different grinding media (tungsten carbide, quartz) surfaces with different shades of roughness were generated. Depending on the hardness of the grinding material and the grinding time the surface roughness was more or less pronounced. Surface roughness parameters and specific surface area were determined via several complementary techniques in order to get an enhanced understanding of the impact of the modifying procedure on the surface properties of the glass beads.

Evaluating the process parameters of the dry coating process using a 2(5-1) factorial design.

CONTEXT: A recent development of coating technology is dry coating, where polymer powder and liquid plasticizer are layered on the cores without using organic solvents or water. Several studies evaluating the process were introduced in literature, however, little information about the critical process parameters (CPPs) is given. AIM: Aim of the study was the investigation and optimization of CPPs with respect to one of the critical quality attributes (CQAs), the coating efficiency of the dry coating process in a rotary fluid bed. MATERIALS AND METHODS: Theophylline pellets were coated with hydroxypropyl methylcellulose acetate succinate as enteric film former and triethyl citrate and acetylated monoglyceride as plasticizer. A 2(5-1) design of experiments (DOEs) was created investigating five independent process parameters namely coating temperature, curing temperature, feeding/spraying rate, air flow and rotor speed. The results were evaluated by multilinear regression using the software Modde(®) 7. RESULTS AND DISCUSSION: It is shown, that generally, low feeding/spraying rates and low rotor speeds increase coating efficiency. High coating temperatures enhance coating efficiency, whereas medium curing temperatures have been found to be optimum in terms of coating efficiency. CONCLUSION: This study provides a scientific base for the design of efficient dry coating processes with respect to coating efficiency.

Perfusion calorimetry in the characterization of solvates forming isomorphic desolvates.

In this study, the potential of perfusion calorimetry in the characterization of solvates forming isomorphic desolvates was investigated. Perfusion calorimetry was used to expose different hydrates forming isomorphic desolvates (emodepside hydrates II-IV, erythromycin A dihydrate and spirapril hydrochloride monohydrate) to stepwise increasing relative vapour pressures (RVP) of water and methanol, respectively, while measuring thermal activity. Furthermore, the suitability of perfusion calorimetry to distinguish the transformation of a desolvate into an isomorphic solvate from the adsorption of solvent molecules to crystal surfaces as well as from solvate formation that is accompanied by structural rearrangement was investigated. Changes in the samples were confirmed using FT-Raman and FT-IR spectroscopy. Perfusion calorimetry indicates the transformation of a desolvate into an isomorphic solvate by a substantial exothermic, peak-shaped heat flow curve at low RVP which reflects the rapid incorporation of solvent molecules by the desolvate to fill the structural voids in the lattice. In contrast, adsorption of solvent molecules to crystal surfaces is associated with distinctly smaller heat changes whereas solvate formation accompanied by structural changes is characterized by an elongated heat flow. Hence, perfusion calorimetry is a valuable tool in the characterization of solvates forming isomorphic desolvates which represents a new field of application for the method.