Agglomeration Control during Ultrasonic Crystallization of an Active Pharmaceutical Ingredient

Application of ultrasound during crystallization can efficiently inhibit agglomeration. However, the mechanism is unclear and sonication is usually enabled throughout the entire process, which increases the energy demand. Additionally, improper operation results in significant crystal damage. Therefore, the present work addresses these issues by identifying the stage in which sonication impacts agglomeration without eroding the crystals. This study was performed using a commercially available API that showed a high tendency to agglomerate during seeded crystallization. The crystallization progress was monitored using process analytical tools (PAT), including focus beam reflectance measurements (FBRM) to track to crystal size and number and Fourier transform infrared spectroscopy (FTIR) to quantify the supersaturation level. These tools provided insight in the mechanism by which ultrasound inhibits agglomeration. A combination of improved micromixing, fast crystal formation which accelerates depletion of the supersaturation and a higher collision frequency prevent crystal cementation to occur. The use of ultrasound as a post-treatment can break some of the agglomerates, but resulted in fractured crystals. Alternatively, sonication during the initial seeding stage could assist in generating nuclei and prevent agglomeration, provided that ultrasound was enabled until complete desupersaturation at the seeding temperature. FTIR and FBRM can be used to determine this end point

Continuous Crystallization Using Ultrasound Assisted Nucleation, Cubic Cooling Profiles and Oscillatory Flow

Continuous tubular crystallizers have the potential to reduce manufacturing costs and increase product quality. However, designing tubular crystallizers is a complex and challenging task as crystallization is a complex, multiphase process with a propensity for fouling and clogging. While several designs have been proposed to overcome these issues, these designs are either unproven or poorly scalable and complex. In this work a continuous crystallizer is designed and evaluated to mitigate these issues. The tubular crystallizer combines a novel method to obtain a cubic cooling profile to control the supersaturation, ultrasound to induce nucleation and oscillatory flow to improve mixing and minimize fouling and sedimentation. The results show that the crystallizer was able to operate for more than 4 h without clogging, with high yields and a narrow particle size distribution. The design proposed here is therefore considered a viable approach for continuous crystallizers

Reducing the Induction Time Using Ultrasound and High-Shear Mixing in a Continuous Crystallization Process

Continuous crystallization in tubular crystallizers is of particular interest to the pharmaceutical industry to accurately control average particle size, particle size distribution, and (polymorphic) shape. However, these types of crystallizers require fast nucleation, and thus, short induction times at the beginning of the flow process, which is challenging for larger and complex organic molecules. High shear and/or the presence of bubbles were identified to influence the nucleation behavior. This work investigates the effects of both high-shear mixing and ultrasound on the anti-solvent crystallization of paracetamol in acetone–water. Both devices generate intense amounts of shear and gas bubbles. Generally, the results show that increasing input power decreases the induction time significantly for both the rotor–stator mixer and ultrasound probe. However, the induction time is almost independent of the supersaturation for the ultrasound probe, while the induction time significantly increases with decreasing supersaturation for the rotor–stator mixer. In contrast, the particle size distribution for the rotor–stator mixer is independent of the supersaturation, while increasing supersaturation decreases the particle size for the ultrasound probe

The cytotoxic activity of amorphous silica nanoparticles is mainly influenced by surface area and not by aggregation.

The aggregation state of NP has been a significant source of difficulty for assessing their toxic activity and great efforts have been done to reduce aggregation of and/or to disperse NP in experimental systems. The exact impact of aggregation on toxicity has, however, not been adequately assessed. Here we compared in vitro the cytotoxic activity of stable monodisperse and aggregated silicon-based nanoparticles (SNP) without introducing a dispersing agent that may affect NP properties. SNP aggregates (180nm) were produced by controlled electrostatic aggregation through addition of KCl to a Ludox SM sol (25nm) followed by stabilization and extensive dialysis. The size of the preparations was characterized by TEM and DLS; specific surface area and porosity were derived from N(2) sorption measurements. Macrophage (J774) and fibroblast (3T3) cell lines were exposed to monodisperse or aggregate-enriched suspensions of SNP in DMEM in absence of serum. The cytotoxic activity of the different preparations was assessed by the WST1 assay after 24h of exposure. Parameters that determined the cytotoxic activity were traced by comparing the doses of the different preparations that induced half a maximal reduction in WST1 activity (ED(50)) in both cell lines. We found that ED(50) (6-9μg/ml and 15-22μg/ml, in J774 and 3T3, respectively) were hardly affected upon aggregation, which was consistent with the fact that the specific surface area of the SNP, a significant determinant of their cytotoxic activity, was unaffected upon aggregation (283-331m(2)/g). Thus studying small aggregated NP could be as relevant as studying disperse primary NP, when aggregates keep the characteristics of NP, i.e. a high specific surface area and a nanosize dimension. This conclusion does, however, not necessarily hold true for other toxicity endpoints for which the determinants may be different and possibly modified by the aggregation process

The nanosilica hazard : another variable entity.

Silica nanoparticles (SNPs) are produced on an industrial scale and are an addition to a growing number of commercial products. SNPs also have great potential for a variety of diagnostic and therapeutic applications in medicine. Contrary to the well-studied crystalline micron-sized silica, relatively little information exists on the toxicity of its amorphous and nano-size forms. Because nanoparticles possess novel properties, kinetics and unusual bioactivity, their potential biological effects may differ greatly from those of micron-size bulk materials. In this review, we summarize the physico-chemical properties of the different nano-sized silica materials that can affect their interaction with biological systems, with a specific emphasis on inhalation exposure. We discuss recent in vitro and in vivo investigations into the toxicity of nanosilica, both crystalline and amorphous. Most of the in vitro studies of SNPs report results of cellular uptake, size- and dose-dependent cytotoxicity, increased reactive oxygen species levels and pro-inflammatory stimulation. Evidence from a limited number of in vivo studies demonstrates largely reversible lung inflammation, granuloma formation and focal emphysema, with no progressive lung fibrosis. Clearly, more research with standardized materials is needed to enable comparison of experimental data for the different forms of nanosilicas and to establish which physico-chemical properties are responsible for the observed toxicity of SNPs

Methodological approaches influencing cellular uptake and cyto-(geno) toxic effects of nanoparticles.

Methods are needed to assess cytotoxicity and genotoxicity of nanoparticles (NPs). The influence of serum and the use of cytochalasin-B were assessed on the cellular uptake of amorphous silica NPs (SNPs) and their biological effects. Our observations indicate that some methodological approaches may modulate the outcome of the assay. Therefore the experimental design and choice of the assays are of great importance in nanotoxicology

Size-dependent cytotoxicity of monodisperse silica nanoparticles in human endothelial cells.

The effect that monodisperse amorphous spherical silica particles of different sizes have on the viability of endothelial cells (EAHY926 cell line) is investigated. The results indicate that exposure to silica nanoparticles causes cytotoxic damage (as indicated by lactate dehydrogenase (LDH) release) and a decrease in cell survival (as determined by the tetrazolium reduction, MTT, assay) in the EAHY926 cell line in a dose-related manner. Concentrations leading to a 50% reduction in cell viability (TC(50)) for the smallest particles tested (14-, 15-, and 16-nm diameter) ranging from 33 to 47 microg cm(-2) of cell culture differ significantly from values assessed for the bigger nanoparticles: 89 and 254 microg cm(-2) (diameter of 19 and 60 nm, respectively). Two fine silica particles with diameters of 104 and 335 nm show very low cytotoxic response compared to nanometer-sized particles with TC(50) values of 1095 and 1087 microg cm(-2), respectively. The smaller particles also appear to affect the exposed cells faster with cell death (by necrosis) being observed within just a few hours. The surface area of the tested particles is an important parameter in determining the toxicity of monodisperse amorphous silica nanoparticles

Cytokine production by co-cultures exposed to monodisperse amorphous silica nanoparticles: the role of size and surface area.

The aim of this study was to test the influence of nanoparticle size and surface area (SA) on cytokine secretion by co-cultures of pulmonary epithelial cells (A549), macrophages (differentiated THP-1 cells) and endothelium cells (EA.hy926) in a two-compartment system. We used monodisperse amorphous silica nanoparticles (2, 16, 60 and 104 nm) at concentrations of 5 μg/cm² cell culture SA or 10 cm² particle SA/cm². A549 and THP-1 cells were exposed to nanoparticles for 24h, in the presence of EA.hy926 cells cultured in an insert introduced above the bi-culture after 12h. Supernatants from both compartments were recovered and TNF-α, IL-6, IL-8 and MIP-1α were measured. Significant secretion of all cytokines was observed for the 2 nm particles at both concentrations and in both compartments. Larger particles of 60 nm induced significant cytokine secretion at the dose of 10 cm² particle SA/cm². The use of multiple cellular types showed that cytokine secretion in single cell cultures is amplified or mitigated in co-cultures. The release of pro-inflammatory mediators by endothelial cells not directly exposed to nanoparticles indicates a possible endothelium activation after inhalation of silica particles. This work shows the role of size and SA in cellular response to amorphous nanosilica

Investigation of the cytotoxicity of nanozeolites A and Y.

Nanosized zeolite particles are important materials for many applications in the field of nanotechnology. The possible adverse effects of these nanomaterials on human health have been scarcely investigated and remain largely unknown. This study reports the synthesis of nanozeolites Y and A with particle sizes of 25-100 nm and adequate colloidal stability for in vitro cytotoxicity experiments. The cytotoxic response of macrophages, epithelial and endothelial cells to these nanocrystals was assessed by determining mitochondrial activity (MTT assay) and cell membrane integrity (LDH leakage assay). After 24 h of exposure, no significant cytotoxic activity was detected for nanozeolite doses up to 500 μg/ml. The addition of fetal calf serum to the cell culture medium during exposure did not significantly change this low response. The nanozeolites showed low toxicity compared with monodisperse amorphous silica nanoparticles of similar size (60 nm). These results may contribute to the application of safe nanozeolites for purposes such as medical imaging, sensing materials, low-k films and molecular separation processes