Tapani Viitala

Inkjet printing of drug substances and use of porous substrates‐towards individualized dosing

Medicines are most often oral solid dosage forms made into tablets or capsules, and there is little room for individualized doses. The drug substance and additives are processed through multiple production phases, including complex powder handling steps. In drug manufacturing, the control of the solid-state properties of active pharmaceutical ingredient (API) is essential and it offers opportunities for enhancement of drug delivery systems. In this context, inkjet printing technologies have emerged over the last decades in pharmaceutical and biological applications and offer solutions for controlling material and product characteristics with high precision. Here we report the concept of conventional inkjet printing technology to produce printable pharmaceutical dosage forms on porous substrates. Data are shown to demonstrate inkjet printing of APIs into paper substrates, and how the model drug substances (paracetamol, theophylline, and caffeine) are penetrating the porous substrates used. The method enables controlling not only the deposition but also the crystallization of the drug substances. We anticipate that the inkjet printing approach has immense potential in making sophisticated drug delivery systems by use of porous substrates in the future. For example, it may offer new perspectives for solving problems around poorly soluble drugs and dosing low-dose medicines accurately. Furthermore, with the advent of genetic mapping of humans, controlled inkjet dosing can bring solutions to fabricate on-demand individualized medicines for patients.

Microvesicle- and exosome-mediated drug delivery enhances the cytotoxicity of Paclitaxel in autologous prostate cancer cells.

BACKGROUND Extracellular vesicles (EVs) are naturally occurring membrane particles that mediate intercellular communication by delivering molecular information between cells. In this study, we investigated the effectiveness of two different populations of EVs (microvesicle- and exosome-enriched) as carriers of Paclitaxel to autologous prostate cancer cells. METHODS EVs were isolated from LNCaP- and PC-3 prostate cancer cell cultures using differential centrifugation and characterized by electron microscopy, nanoparticle tracking analysis, and Western blot. The uptake of microvesicles and exosomes by the autologous prostate cancer cells was assessed by flow cytometry and confocal microscopy. The EVs were loaded with Paclitaxel and the effectiveness of EV-mediated drug delivery was assessed with viability assays. The distribution of EVs and EV-delivered Paclitaxel in cells was inspected by confocal microscopy. RESULTS Our main finding was that the loading of Paclitaxel to autologous prostate cancer cell-derived EVs increased its cytotoxic effect. This capacity was independent of the EV population and the cell line tested. Although the EVs without the drug increased cancer cell viability, the net effect of enhanced cytotoxicity remained. Both EV populations delivered Paclitaxel to the recipient cells through endocytosis, leading to the release of the drug from within the cells. The removal of EV surface proteins did not affect exosomes, while the drug delivery mediated by microvesicles was partially inhibited. CONCLUSIONS Cancer cell-derived EVs can be used as effective carriers of Paclitaxel to their parental cells, bringing the drug into the cells through an endocytic pathway and increasing its cytotoxicity. However, due to the increased cell viability, the use of cancer cell-derived EVs must be further investigated before any clinical applications can be designed.

Enhanced activity of horseradish peroxidase in Langmuir-Blodgett films of phospholipids.

The immobilization of enzymes in nanostructured films has potential applications, e.g. in biosensing, for which the activity may not only be preserved, but also enhanced if optimized conditions are identified. Optimization is not straightforward because several requirements must be fulfilled, including a suitable matrix and film-forming technique. In this study, we show that horseradish peroxidase (HRP) has its activity enhanced when immobilized in Langmuir-Blodgett (LB) films, in conjunction with dipalmitoylphosphatidylglycerol (DPPG). Incorporation of HRP into a DPPG monolayer at the air-water interface was demonstrated with compression isotherms, and Polarization-Modulation Infrared Reflection Absorption Spectroscopy (PM-IRRAS). From the PM-IRRAS data, we inferred that HRP was not denatured when adsorbed on a pre-formed, low pressure DPPG monolayer. A change in orientation was induced by the phospholipid matrix, with the amide C=O and NH groups from HRP being oriented perpendicular to the surface, parallel to the DPPG acyl chains, i.e. the alpha-helix was inserted into the monolayer. The mixed DPPG-HRP monolayer could be transferred onto solid supports, to which HRP activity was ca. 23% higher than in solution. The control of molecular architecture and choice of a suitable phospholipid matrix allowed HRP-containing LB films to be used in sensing peroxide.

Cholesterol Mediates Chitosan Activity on Phospholipid Monolayers and Langmuir-Blodgett Films

The polysaccharide chitosan has been largely used in many biological applications as a fat and cholesterol reducer, bactericide agent, and wound healing material. While the efficacy for some of such uses is proven, little is known about the molecular-level interactions involved in these applications. In this study, we employ mixed Langmuir and Langmuir-Blodgett (LB) films of negatively charged dimyristoyl phosphatidic acid (DMPA) and cholesterol as cell membrane models to investigate the role of cholesterol in the molecular-level action of chitosan. Chitosan does not remove cholesterol from the monolayer. The interaction with chitosan tends to expand the DMPA monolayer due to its interpenetration within the film. On the other hand, cholesterol induces condensation of the DMPA monolayer. The competing effects cause the surface pressure isotherms of mixed DMPA-cholesterol films on a chitosan subphase to be unaffected by the cholesterol mole fraction, due to distinct degrees of chitosan penetration into the film in the presence of cholesterol. By combining polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) and sum-frequency generation spectroscopy (SFG), we showed that chitosan induces order into negatively charged phospholipid layers, whereas the opposite occurs for cholesterol. In conclusion, chitosan has its penetration in the film modulated by cholesterol, and electrostatic interactions with negatively charged phospholipids, such as DMPA, are crucial for the action of chitosan.

Light induced cytosolic drug delivery from liposomes with gold nanoparticles

Externally triggered drug release at defined targets allows site- and time-controlled drug treatment regimens. We have developed liposomal drug carriers with encapsulated gold nanoparticles for triggered drug release. Light energy is converted to heat in the gold nanoparticles and released to the lipid bilayers. Localized temperature increase renders liposomal bilayers to be leaky and triggers drug release. The aim of this study was to develop a drug releasing system capable of releasing its cargo to cell cytosol upon triggering with visible and near infrared light signals. The liposomes were formulated using either heat-sensitive or heat- and pH-sensitive lipid compositions with star or rod shaped gold nanoparticles. Encapsulated fluorescent probe, calcein, was released from the liposomes after exposure to the light. In addition, the pH-sensitive formulations showed a faster drug release in acidic conditions than in neutral conditions. The liposomes were internalized into human retinal pigment epithelial cells (ARPE-19) and human umbilical vein endothelial cells (HUVECs) and did not show any cellular toxicity. The light induced cytosolic delivery of calcein from the gold nanoparticle containing liposomes was shown, whereas no cytosolic release was seen without light induction or without gold nanoparticles in the liposomes. The light activated liposome formulations showed a controlled content release to the cellular cytosol at a specific location and time. Triggering with visual and near infrared light allows good tissue penetration and safety, and the pH-sensitive liposomes may enable selective drug release in the intracellular acidic compartments (endosomes, lysosomes). Thus, light activated liposomes with gold nanoparticles are an attractive option for time- and site-specific drug delivery into the target cells.

Printing technologies in fabrication of drug delivery systems.

Introduction: There has been increased activity in the field recently regarding the development and research on various printing techniques in fabrication of dosage forms and drug delivery systems. These technologies may offer benefits and flexibility in manufacturing, potentially paving the way for personalized dosing and tailor-made dosage forms. Areas covered: In this review, the most recent observations and advancements in fabrication of drug delivery systems by utilizing printing technologies are summarized. A general overview of 2D printing techniques is presented including a review of the most recent literature where printing techniques are used in fabrication of drug delivery systems. The future perspectives and possible impacts on formulation strategies, flexible dosing and personalized medication of using printing techniques for fabrication of drug delivery systems are discussed. Expert opinion: It is evident that there is an urgent need to meet the challenges of rapidly growing trend of personalization of medicines through development of flexible drug-manufacturing approaches. In this context, various printing technologies, such as inkjet and flexography, can play an important role. Challenges on different levels exist and include: i) technological development of printers and production lines; ii) printable formulations and carrier substrates; iii) quality control and characterization; and iv) regulatory perspectives.

Single exosome study reveals subpopulations distributed among cell lines with variability related to membrane content

Current analysis of exosomes focuses primarily on bulk analysis, where exosome-to-exosome variability cannot be assessed. In this study, we used Raman spectroscopy to study the chemical composition of single exosomes. We measured spectra of individual exosomes from 8 cell lines. Cell-line-averaged spectra varied considerably, reflecting the variation in total exosomal protein, lipid, genetic, and cytosolic content. Unexpectedly, single exosomes isolated from the same cell type also exhibited high spectral variability. Subsequent spectral analysis revealed clustering of single exosomes into 4 distinct groups that were not cell-line specific. Each group contained exosomes from multiple cell lines, and most cell lines had exosomes in multiple groups. The differences between these groups are related to chemical differences primarily due to differing membrane composition. Through a principal components analysis, we identified that the major sources of spectral variation among the exosomes were in cholesterol content, relative expression of phospholipids to cholesterol, and surface protein expression. For example, exosomes derived from cancerous versus non-cancerous cell lines can be largely separated based on their relative expression of cholesterol and phospholipids. We are the first to indicate that exosome subpopulations are shared among cell types, suggesting distributed exosome functionality. The origins of these differences are likely related to the specific role of extracellular vesicle subpopulations in both normal cell function and carcinogenesis, and they may provide diagnostic potential at the single exosome level.

Cholesterol level affects surface charge of lipid membranes in saline solution

Cholesterol is an important component of all biological membranes as well as drug delivery liposomes. We show here that increasing the level of cholesterol in a phospholipid membrane decreases surface charge in the physiological environment. Through molecular dynamics simulation we have shown that increasing the level of cholesterol decreases Na+ ion binding. Complementary experimental ζ – potential measurements have shown a decreased ζ – potential with increasing cholesterol content, indicative of reduced surface charge. Both experiments and simulations have been carried out on both saturated 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and monounsaturated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) membranes. This result is particularly important because membrane surface charge plays an important role in the interactions of biomembranes with peripheral membrane proteins and drug delivery liposomes with the immune system.

Silymarin loaded liposomes for hepatic targeting: In vitro evaluation and HepG2 drug uptake

Silymarin has hepatoprotective properties and is used in treatment of various liver diseases, but its bioavailability from oral products is very poor. In order to overcome its poor oral bioavailability we have prepared silymarin loaded hepatic targeting liposomes suitable for parenteral administration. The liposomal formulations were composed of hydrogenated soy phosphatidylcholine and cholesterol with or without distearoylphosphoethanolamine-(polyethyleneglycol)-2000 and various amounts of β-sitosterol β-D-glucoside (Sito-G) as the hepatic targeting moiety. Increasing the amount of Sito-G in the liposomes gradually decreased drug encapsulation efficiencies from ∼70% to ∼60%; still showing promising drug encapsulation efficiencies. Addition of Sito-G to non-PEGylated liposomes clearly affected their drug release profiles and plasma protein interactions, whereas no effect on these was seen for the PEGylated liposomes. Non-PEGylated liposomes with 0.17 M ratio of Sito-G exhibited the highest cellular drug uptake of 37.5% for all of the studied liposome formulations. The highest cellular drug uptake in the case of PEGylated liposomes was 18%, which was achieved with 0.17 and 0.33 M ratio of added Sito-G. The liposome formulations with the highest drug delivery efficacy in this study showed hemolytic activities around 12.7% and were stable for at least 2 months upon storage in 20 mM HEPES buffer (pH 7.4) containing 1.5% Polysorbate 80 at 4 °C and room temperature. These results suggest that the Sito-G containing liposomes prepared in this work have hepatic targeting capability and that they are promising candidates for delivering silymarin to the liver.

Characterizing ultrathin and thick organic layers by surface plasmon resonance three-wavelength and waveguide mode analysis.

A three-wavelength angular-scanning surface plasmon resonance based analysis has been utilized for characterizing optical properties of organic nanometer-thick layers with a wide range of thicknesses. The thickness and refractive index were determined for sample layers with thicknesses ranging from subnanometer to hundreds of nanometers. The analysis approach allows for simultaneous determination of both the refractive index and thickness without prior knowledge of either the refractive index or the thickness of the sample layers and without the help of other instruments, as opposed to current methods and approaches for characterizing optical properties of organic nanometer-thick layers. The applicability of the three-wavelength angular-scanning surface plasmon resonance approach for characterizing thin and thick organic layers was demonstrated by ex situ deposited mono- and multilayers of stearic acid and hydrogenated soy phosphatidylcholine and in situ layer-by-layer deposition of two different polyelectrolyte multilayer systems. In addition to the three-wavelength angular-scanning surface plasmon resonance approach, another surface plasmon resonance optical phenomenon, i.e., the surface plasmon resonance waveguide mode, was utilized to characterize organic sample layers whose thicknesses border the micrometer scale. This was demonstrated by characterizing both in situ layer-by-layer deposited polyelectrolyte multilayer systems and an ex situ deposited spin-coated polymer layer.