In Vitro Characterization and Real-Time Label-Free Assessment of the Interaction of Chitosan-Coated Niosomes with Intestinal Cellular Monolayers.

In vitro cell-based characterization methods of nanoparticles are generally static and require the use of secondary analysis techniques and labeling agents. In this study, bare niosomes and chitosan-coated niosomes (chitosomes) and their interactions with intestinal cells are studied under dynamic conditions and without fluorescent probes, using surface plasmon resonance (SPR)-based cell sensing. Niosomes and chitosomes were synthesized by using Tween 20 and cholesterol in a 15 mM:15 mM ratio and then characterized by dynamic light scattering (DLS). DLS analysis demonstrated that bare niosomes had average sizes of ∼125 nm, polydispersity index (PDI) below 0.2, and a negative zeta (ζ)-potential of -35.6 mV. In turn, chitosomes had increased sizes up to ∼180 nm, with a PDI of 0.2-0.3 and a highly positive ζ-potential of +57.9 mV. The viability of HT29-MTX, Caco-2, and Caco-2/HT29-MTX cocultured cells showed that both niosomes and chitosomes are cytocompatible up to concentrations of 31.6 µg/mL for at least 240 min. SPR analysis demonstrated that chitosomes interact more efficiently with HT29-MTX, Caco-2, and Caco-2/HT29-MTX cocultures compared to bare niosomes. The resulting SPR measurements were further supported by confocal microscopy and flow cytometry studies, which demonstrated that this method is a useful complementary or even alternative tool to directly characterize the interactions between niosomes and in vitro cell models in label-free and real-time conditions.

Assembly of Bleomycin Saccharide-Decorated Spherical Nucleic Acids.

Glyco-decorated spherical nucleic acids (SNAs) may be attractive delivery vehicles, emphasizing the sugar-specific effect on the outer sphere of the construct and at the same time hiding unfavorable distribution properties of the loaded oligonucleotides. As examples of such nanoparticles, tripodal sugar constituents of bleomycin were synthesized and conjugated with a fluorescence-labeled antisense oligonucleotide (AONARV7). Successive copper(I)-catalyzed azide-alkyne and strain-promoted alkyne-nitrone cycloadditions (SPANC) were utilized for the synthesis. Then, the glyco-AONARV7 conjugates were hybridized with complementary strands of a C60-based molecular spherical nucleic acid (i.e., a hybridization-mediated carrier). The formation and stability of these assembled glyco-decorated SNAs were evaluated by polyacrylamide gel electrophoresis (PAGE), UV melting profile analysis, and time-resolved fluorescence spectroscopy. Association constants were extracted from time-resolved fluorescence data. Preliminary cellular uptake experiments of the glyco-AONARV7 conjugates (120 nM solutions) and of the corresponding glyco-decorated SNAs (10 nM solutions) with human prostate cancer cells (PC3) showed an efficient uptake in each case. A marked variation in intracellular distribution was observed.

Controlled Monofunctionalization of Molecular Spherical Nucleic Acids on a Buckminster Fullerene Core.

An azide-functionalized 12-armed Buckminster fullerene has been monosubstituted in organic media with a substoichiometric amount of cyclooctyne-modified oligonucleotides. Exposing the intermediate products then to the same reaction (i.e., strain-promoted alkyne-azide cycloaddition, SPAAC) with an excess of slightly different oligonucleotide constituents in an aqueous medium yields molecularly defined monofunctionalized spherical nucleic acids (SNAs). This procedure offers a controlled synthesis scheme in which one oligonucleotide arm can be functionalized with labels or other conjugate groups (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid, DOTA, and Alexa-488 demonstrated), whereas the rest of the 11 arms can be left unmodified or modified by other conjugate groups in order to decorate the SNAs' outer sphere. Extra attention has been paid to the homogeneity and authenticity of the C60-azide scaffold used for the assembly of full-armed SNAs.

Diffusion and Protein Corona Formation of Lipid-Based Nanoparticles in the Vitreous Humor: Profiling and Pharmacokinetic Considerations.

The vitreous humor is the first barrier encountered by intravitreally injected nanoparticles. Lipid-based nanoparticles in the vitreous are studied by evaluating their diffusion with single-particle tracking technology and by characterizing their protein coronae with surface plasmon resonance and high-resolution proteomics. Single-particle tracking results indicate that the vitreal mobility of the formulations is dependent on their charge. Anionic and neutral formulations are mobile, whereas larger (>200 nm) neutral particles have restricted diffusion, and cationic particles are immobilized in the vitreous. PEGylation increases the mobility of cationic and larger neutral formulations but does not affect anionic and smaller neutral particles. Convection has a significant role in the pharmacokinetics of nanoparticles, whereas diffusion drives the transport of antibodies. Surface plasmon resonance studies determine that the vitreal corona of anionic formulations is sparse. Proteomics data reveals 76 differentially abundant proteins, whose enrichment is specific to either the hard or the soft corona. PEGylation does not affect protein enrichment. This suggests that protein-specific rather than formulation-specific factors are drivers of protein adsorption on nanoparticles in the vitreous. In summary, our findings contribute to understanding the pharmacokinetics of nanoparticles in the vitreous and help advance the development of nanoparticle-based treatments for eye diseases.

Label-Free Analysis with Multiple Parameters Separates G Protein-Coupled Receptor Signaling Pathways.

Real-time label-free techniques are used to profile G protein-coupled receptor (GPCR) signaling pathways in living cells. However, interpreting the label-free signal responses is challenging, and previously reported methods do not reliably separate pathways from each other. In this study, a continuous angular-scanning surface plasmon resonance (SPR) technique is utilized for measuring label-free GPCR signal profiles. We show how the continuous angular-scanning ability, measuring up to nine real-time label-free parameters simultaneously, results in more information-rich label-free signal profiles for different GPCR pathways, providing a more accurate pathway separation. For this, we measured real-time full-angular SPR response curves for Gs, Gq, and Gi signaling pathways in living cells. By selecting two of the most prominent label-free parameters: the full SPR curve angular and intensity shifts, we present how this analysis approach can separate each of the three signaling pathways in a straightforward single-step analysis setup, without concurrent use of signal inhibitors or other response modulating compounds.

Personalized Cancer Vaccine Platform for Clinically Relevant Oncolytic Enveloped Viruses.

The approval of the first oncolytic virus for the treatment of metastatic melanoma and the compiling evidence that the use of oncolytic viruses can enhance cancer immunotherapies targeted against various immune checkpoint proteins has attracted great interest in the field of cancer virotherapy. We have developed a novel platform for clinically relevant enveloped viruses that can direct the virus-induced immune response against tumor antigens. By physically attaching tumor-specific peptides onto the viral envelope of vaccinia virus and herpes simplex virus 1 (HSV-1), we were able to induce a strong T cell-specific immune response toward these tumor antigens. These therapeutic peptides could be attached onto the viral envelope by using a cell-penetrating peptide sequence derived from human immunodeficiency virus Tat N-terminally fused to the tumor-specific peptides or, alternatively, therapeutic peptides could be conjugated with cholesterol for the attachment of the peptides onto the viral envelope. We used two mouse models of melanoma termed B16.OVA and B16-F10 for testing the efficacy of OVA SIINFEKL-peptide-coated viruses and gp100-Trp2-peptide-coated viruses, respectively, and show that by coating the viral envelope with therapeutic peptides, the anti-tumor immunity and the number of tumor-specific CD8+ T cells in the tumor microenvironment can be significantly enhanced.

Interaction of lecithin:cholesterol acyltransferase with lipid surfaces and apolipoprotein A-I-derived peptides.

LCAT is an enzyme responsible for the formation of cholesteryl esters from unesterified cholesterol (UC) and phospholipid (PL) molecules in HDL particles. However, it is poorly understood how LCAT interacts with lipoproteins and how apoA-I activates it. Here we have studied the interactions between LCAT and lipids through molecular simulations. In addition, we studied the binding of LCAT to apoA-I-derived peptides, and their effect on LCAT lipid association-utilizing experiments. Results show that LCAT anchors itself to lipoprotein surfaces by utilizing nonpolar amino acids located in the membrane-binding domain and the active site tunnel opening. Meanwhile, the membrane-anchoring hydrophobic amino acids attract cholesterol molecules next to them. The results also highlight the role of the lid-loop in the lipid binding and conformation of LCAT with respect to the lipid surface. The apoA-I-derived peptides from the LCAT-activating region bind to LCAT and promote its lipid surface interactions, although some of these peptides do not bind lipids individually. The transfer free-energy of PL from the lipid bilayer into the active site is consistent with the activation energy of LCAT. Furthermore, the entry of UC molecules into the active site becomes highly favorable by the acylation of SER181.

Biophysical Characterization of Supported Lipid Bilayers Using Parallel Dual-Wavelength Surface Plasmon Resonance and Quartz Crystal Microbalance Measurements.

Supported lipid bilayers (SLBs) have been used extensively as an effective model of biological membranes, in the context of in vitro biophysics research, and the membranes of liposomes, in the context of the development of nanoscale drug delivery devices. Despite numerous surface-sensitive techniques having been applied to their study, the comprehensive optical characterization of SLBs using surface plasmon resonance (SPR) has not been conducted. In this study, Fresnel multilayer analysis is utilized to effectively calculate layer parameters (thickness and refractive indices) with the aid of dual-wavelength and dispersion coefficient analysis, in which the linear change in the refractive index as a function of wavelength is assumed. Using complementary information from impedance-based quartz crystal microbalance experiments, biophysical properties, for example, area-per-lipid-molecule and the quantity of lipid-associated water molecules, are calculated for different lipid types and mixtures, one of which is representative of a raft-forming lipid mixture. It is proposed that the hydration layer beneath the bilayer is, in fact, an integral part of the measured optical signal. Also, the traditional Jung model analysis and the ratio of SPR responses are investigated in terms of assessing the structure of the lipid layer that is formed.

High-Generation Amphiphilic Janus-Dendrimers as Stabilizing Agents for Drug Suspensions.

Pharmaceutical nanosuspensions are formed when drug crystals are suspended in aqueous media in the presence of stabilizers. This technology offers a convenient way to enhance the dissolution of poorly water-soluble drug compounds. The stabilizers exert their action through electrostatic or steric interactions, however, the molecular requirements of stabilizing agents have not been studied extensively. Here, four structurally related amphiphilic Janus-dendrimers were synthesized and screened to determine the roles of different macromolecular domains on the stabilization of drug crystals. Physical interaction and nanomilling experiments have substantiated that Janus-dendrimers with fourth generation hydrophilic dendrons were superior to third generation analogues and Poloxamer 188 in stabilizing indomethacin suspensions. Contact angle and surface plasmon resonance measurements support the hypothesis that Janus-dendrimers bind to indomethacin surfaces via hydrophobic interactions and that the number of hydrophobic alkyl tails determines the adsorption kinetics of the Janus-dendrimers. The results showed that amphiphilic Janus-dendrimers adsorb onto drug particles and thus can be used to provide steric stabilization against aggregation and recrystallization. The modular synthetic route for new amphiphilic Janus-dendrimers offers, thus, for the first time a versatile platform for stable general-use stabilizing agents of drug suspensions.

Rational design of liposomal drug delivery systems, a review: Combined experimental and computational studies of lipid membranes, liposomes and their PEGylation.

Combined experimental and computational studies of lipid membranes and liposomes, with the aim to attain mechanistic understanding, result in a synergy that makes possible the rational design of liposomal drug delivery system (LDS) based therapies. The LDS is the leading form of nanoscale drug delivery platform, an avenue in drug research, known as "nanomedicine", that holds the promise to transcend the current paradigm of drug development that has led to diminishing returns. Unfortunately this field of research has, so far, been far more successful in generating publications than new drug therapies. This partly results from the trial and error based methodologies used. We discuss experimental techniques capable of obtaining mechanistic insight into LDS structure and behavior. Insight obtained purely experimentally is, however, limited; computational modeling using molecular dynamics simulation can provide insight not otherwise available. We review computational research, that makes use of the multiscale modeling paradigm, simulating the phospholipid membrane with all atom resolution and the entire liposome with coarse grained models. We discuss in greater detail the computational modeling of liposome PEGylation. Overall, we wish to convey the power that lies in the combined use of experimental and computational methodologies; we hope to provide a roadmap for the rational design of LDS based therapies. Computational modeling is able to provide mechanistic insight that explains the context of experimental results and can also take the lead and inspire new directions for experimental research into LDS development. This article is part of a Special Issue entitled: Biosimulations edited by Ilpo Vattulainen and Tomasz Róg.

Real-Time Label-Free Monitoring of Nanoparticle Cell Uptake.

The surface plasmon resonance technique in combination with whole cell sensing is used for the first time for real-time label-free monitoring of nanoparticle cell uptake. The uptake kinetics of several types of nanoparticles relevant to drug delivery applications into HeLa cells is determined. The cell uptake of the nanoparticles is confirmed by confocal microscopy. The cell uptake of silica nanoparticles and polyethylenimine-plasmid DNA polyplexes is studied as a function of temperature, and the uptake energies are determined by Arrhenius plots. The phase transition temperature of the HeLa cell membrane is detected when monitoring cell uptake of silica nanoparticles at different temperatures. The HeLa cell uptake of the mesoporous silica nanoparticles is energy-independent at temperatures slightly higher than the phase transition temperature of the HeLa cell membrane, while the uptake of polyethylenimine-DNA polyplexes is energy-dependent and linear as a function of temperature with an activation energy of Ea = 62 ± 7 kJ mol-1 = 15 ± 2 kcal mol-1 . The HeLa cell uptake of red blood cell derived extracellular vesicles is also studied as a function of the extracellular vesicle concentration. The results show a concentration dependent behavior reaching a saturation level of the extracellular vesicle uptake by HeLa cells.

Novel cationic polyelectrolyte coatings for capillary electrophoresis.

The use of bare fused silica capillary in CE can sometimes be inconvenient due to undesirable effects including adsorption of sample or instability of the EOF. This can often be avoided by coating the inner surface of the capillary. In this work, we present and characterize two novel polyelectrolyte coatings (PECs) poly(2-(methacryloyloxy)ethyl trimethylammonium iodide) (PMOTAI) and poly(3-methyl-1-(4-vinylbenzyl)-imidazolium chloride) (PIL-1) for CE. The coated capillaries were studied using a series of aqueous buffers of varying pH, ionic strength, and composition. Our results show that the investigated polyelectrolytes are usable as semi-permanent (physically adsorbed) coatings with at least five runs stability before a short coating regeneration is necessary. Both PECs showed a considerably decreased stability at pH 11.0. The EOF was higher using Good's buffers than with sodium phosphate buffer at the same pH and ionic strength. The thickness of the PEC layers studied by quartz crystal microbalance was 0.83 and 0.52 nm for PMOTAI and PIL-1, respectively. The hydrophobicity of the PEC layers was determined by analysis of a homologous series of alkyl benzoates and expressed as the distribution constants. Our result demonstrates that both PECs had comparable hydrophobicity, which enabled separation of compounds with log Po/w > 2. The ability to separate cationic drugs was shown with ß-blockers, compounds often misused in doping. Both coatings were also able to separate hydrolysis products of the ionic liquid 1,5-diazabicyclo[4.3.0]non-5-ene acetate at highly acidic conditions, where bare fused silica capillaries failed to accomplish the separation.

Indocyanine Green-Loaded Liposomes for Light-Triggered Drug Release.

Light-triggered drug delivery systems enable site-specific and time-controlled drug release. In previous work, we have achieved this with liposomes containing gold nanoparticles in the aqueous core. Gold nanoparticles absorb near-infrared light and release the energy as heat that increases the permeability of the liposomal bilayer, thus releasing the contents of the liposome. In this work, we replaced the gold nanoparticles with the clinically approved imaging agent indocyanine green (ICG). The ICG liposomes were stable at storage conditions (4-22 °C) and at body temperature, and fast near-infrared (IR) light-triggered drug release was achieved with optimized phospholipid composition and a 1:50 ICG-to-lipid molar ratio. Encapsulated small molecular calcein and FITC-dextran (up to 20 kDa) were completely released from the liposomes after light exposure for 15 s. Location of ICG in the PEG layer of the liposomes was simulated with molecular dynamics. ICG has important benefits as a light-triggering agent in liposomes: fast content release, improved stability, improved possibility of liposomal size control, regulatory approval to use in humans, and the possibility of imaging the in vivo location of the liposomes based on the fluorescence of ICG. Near-infrared light used as a triggering mechanism has good tissue penetration and safety. Thus, ICG liposomes are an attractive option for light-controlled and efficient delivery of small and large drug molecules.

Photothermally Triggered Lipid Bilayer Phase Transition and Drug Release from Gold Nanorod and Indocyanine Green Encapsulated Liposomes.

In light-activated liposomal drug delivery systems (DDSs), the light sensitivity can be obtained by a photothermal agent that converts light energy into heat. Excess heat increases the drug permeability of the lipid bilayer, and drug is released as a result. In this work, two near-IR responsive photothermal agents in a model drug delivery system are studied: either gold nanorods (GNRs) encapsulated inside the liposomes or indocyanine green (ICG) embedded into the lipid bilayer. The liposome system is exposed to light, and the heating effect is studied with fluorescent thermometers: laurdan and CdSe quantum dots (QDs). Both photothermal agents are shown to convert light into heat in an extent to cause a phase transition in the surrounding lipid bilayer. This phase transition is also proven with laurdan generalized polarization (GP). In addition to the heating results, we show that the model drug (calcein) is released from the liposomal cavity with both photothermal agents when the light power is sufficient to cause a phase transition in the lipid bilayer.

Fluorescence-suppressed time-resolved Raman spectroscopy of pharmaceuticals using complementary metal-oxide semiconductor (CMOS) single-photon avalanche diode (SPAD) detector.

In this work, we utilize a short-wavelength, 532-nm picosecond pulsed laser coupled with a time-gated complementary metal-oxide semiconductor (CMOS) single-photon avalanche diode (SPAD) detector to acquire Raman spectra of several drugs of interest. With this approach, we are able to reveal previously unseen Raman features and suppress the fluorescence background of these drugs. Compared to traditional Raman setups, the present time-resolved technique has two major improvements. First, it is possible to overcome the strong fluorescence background that usually interferes with the much weaker Raman spectra. Second, using the high photon energy excitation light source, we are able to generate a stronger Raman signal compared to traditional instruments. In addition, observations in the time domain can be performed, thus enabling new capabilities in the field of Raman and fluorescence spectroscopy. With this system, we demonstrate for the first time the possibility of recording fluorescence-suppressed Raman spectra of solid, amorphous and crystalline, and non-photoluminescent and photoluminescent drugs such as caffeine, ranitidine hydrochloride, and indomethacin (amorphous and crystalline forms). The raw data acquired by utilizing only the picosecond pulsed laser and a CMOS SPAD detector could be used for identifying the compounds directly without any data processing. Moreover, to validate the accuracy of this time-resolved technique, we present density functional theory (DFT) calculations for a widely used gastric acid inhibitor, ranitidine hydrochloride. The obtained time-resolved Raman peaks were identified based on the calculations and existing literature. Raman spectra using non-time-resolved setups with continuous-wave 785- and 532-nm excitation lasers were used as reference data. Overall, this demonstration of time-resolved Raman and fluorescence measurements with a CMOS SPAD detector shows promise in diverse areas, including fundamental chemical research, the pharmaceutical setting, process analytical technology (PAT), and the life sciences.

Enhancement of Bioavailability and Pharmacodynamic Effects of Thymoquinone Via Nanostructured Lipid Carrier (NLC) Formulation.

Thymoquinone (TQ), obtained from black cumin (Nigella sativa), is a natural product with anti-oxidant, anti-inflammatory, and hepatoprotective effects but unfortunately with poor bioavailability. Aiming to improve its poor oral bioavailability, TQ-loaded nanostructured lipid carriers (NLCs) were prepared by high-speed homogenization followed by ultrasonication and evaluated in vitro. Bioavailability and pharmacodynamic studies were also performed. The resultant NLCs showed poor physical homogeneity in Compritol 888 ATO Pluronic F127 system which consequently produced larger particle size and polydispersity index, smaller zeta potential values, and lower short-term (30 days) physical stability than other systems. Encapsulation efficiency percentage (EE%) lied between 84.6 ± 5% and 96.2 ± 1.6%. TQ AUC0-t values were higher in animals treated with NLCs, with a relative bioavailability of 2.03- and 3.97-fold (for F9 and F12, respectively) higher than TQ suspension, indicating bioavailability enhancement by NLC formulation. Hepatoprotective effects of F12 showed significant (P < 0.05) decrease in both serum alanine amino transferase and aspartate amino transferase to reach 305.0 ± 24.88 and 304.7 ± 23.55 U/ml, respectively, when compared with untreated toxic group. Anti-oxidant efficacy of F12 showed significant (P < 0.05) decline of malondialdehyde and elevation of reduced glutatione. This improvement was also confirmed histopathologically.

Interaction studies between indomethacin nanocrystals and PEO/PPO copolymer stabilizers.

PURPOSE: The lack of effective screening methods and systemic understanding of interaction mechanisms complicates the stabilizer selection process for nanocrystallization. This study focuses on the efficiency of stabilizers with various molecular compositions and structures to stabilize drug nanocrystals. METHODS: Five structurally different polymers were chosen as stabilizers for indomethacin nanocrystals. The affinity of polymers onto drug surfaces was measured using surface plasmon resonance (SPR) and contact angle techniques. Nanosuspensions were prepared using the wet-ball milling technique and their physico-chemical properties were thoroughly characterized. RESULTS: SPR and contact angle measurements correlated very well with each other and showed that the binding efficiency decreased in the order L64 > 17R4 > F68 ≈ T908 ≈ T1107, which is attributed to the reduced PPO/PEO ratio and different polymer structures. The electrostatic interactions between the protonated amine of poloxamines and ionized indomethacin enhanced neither the affinity nor the properties of nanosuspensions, such as particle size and physical stability. CONCLUSIONS: A good stabilizer should have high binding efficiency, full coverage, and optimal hydrophobic/hydrophilic balance. A high affinity combined with short PEO chains (L64, 17R4) caused poor physical stability of nanosuspensions, whereas moderate binding efficiencies (F68, T908, T1107) with longer PEO chains produced physically stable nanosuspensions.

Control of the morphology of lipid layers by substrate surface chemistry.

In this study, surface coatings were used to control the morphology of the deposited lipid layers during vesicle spreading, i.e., to control if liposomes self-assemble on a surface into a supported lipid bilayer or a supported vesicular layer. The influence of the properties of the surface coating on formation of the deposited lipid layer was studied with quartz crystal microbalance and two-wavelength multiparametric surface plasmon resonance techniques. Control of lipid self-assembly on the surface was achieved by two different types of soft substrate materials, i.e., dextran and thiolated polyethylene glycol, functionalized with hydrophobic linkers for capturing the lipid layer. The low-molecular-weight dextran-based surface promoted formation of supported lipid bilayers, while the thiolated polyethylene glycol-based surface promoted supported vesicular layer formation. A silicon dioxide surface was used as a reference surface in both measurement techniques. In addition to promoting supported lipid bilayer formation of known lipid mixtures, the dextran surface also promoted supported lipid bilayer formation of vesicles containing the cell membrane extract of human hepatoblastoma cells. The new dextran-based surface was also capable of protecting the supported lipid bilayer against dehydration when exposed to a constant flow of air. The well-established quartz crystal microbalance technique was effective in determining the morphology of the formed lipid layer, while the two-wavelength surface plasmon resonance analysis enabled further complementary characterization of the adsorbed supported lipid bilayers and supported vesicular layers.

An impedimetric study of DNA hybridization on paper-supported inkjet-printed gold electrodes.

In this study, two different supramolecular recognition architectures for impedimetric detection of DNA hybridization have been formed on disposable paper-supported inkjet-printed gold electrodes. The gold electrodes were fabricated using a gold nanoparticle based ink. The first recognition architecture consists of subsequent layers of biotinylated self-assembly monolayer (SAM), streptavidin and biotinylated DNA probe. The other recognition architecture is constructed by immobilization of thiol-functionalized DNA probe (HS-DNA) and subsequent backfill with 11-mercapto-1-undecanol (MUOH) SAM. The binding capacity and selectivity of the recognition architectures were examined by surface plasmon resonance (SPR) measurements. SPR results showed that the HS-DNA/MUOH system had a higher binding capacity for the complementary DNA target. Electrochemical impedance spectroscopy (EIS) measurements showed that the hybridization can be detected with impedimetric spectroscopy in picomol range for both systems. EIS signal indicated a good selectivity for both recognition architectures, whereas SPR showed very high unspecific binding for the HS-DNA/MUOH system. The factors affecting the impedance signal were interpreted in terms of the complexity of the supramolecular architecture. The more complex architecture acts as a less ideal capacitive sensor and the impedance signal is dominated by the resistive elements.

Rheological insights into 3D printing of drug products: Drug nanocrystal-poloxamer gels for semisolid extrusion.

The importance of ink rheology to the outcome of 3D printing is well recognized. However, rheological properties of printing inks containing drug nanocrystals have not been widely investigated. Therefore, the objective of this study was to establish a correlation between the composition of nanocrystal printing ink, the ink rheology, and the entire printing process. Indomethacin was used as a model poorly soluble drug to produce nanosuspensions with improved solubility properties through particle size reduction. The nanosuspensions were further developed into semisolid extrusion 3D printing inks with varying nanocrystal and poloxamer 407 concentrations. Nanocrystals were found to affect the rheological properties of the printing inks both by being less self-supporting and having higher yielding resistances. During printing, nozzle blockages occurred. Nevertheless, all inks were found to be printable. Finally, the rheological properties of the inks were successfully correlated with various printing and product properties. Overall, these experiments shed new light on the rheological properties of printing inks containing nanocrystals.