Microfluidic Manufacturing of Liposomes: Development and Optimization by Design of Experiment and Machine Learning.

Liposomes constitute the most exploited drug-nanocarrier with several liposomal drugs on the market. Microfluidic-based preparation methods stand up as a promising approach with high reproducibility and the ability to scale up. In this study, liposomes composed of DOPC, cholesterol, and DSPE-PEG 2000 with different molar ratios were fabricated using a microfluidic system. Process and conditions were optimized by applying design of experiments (DoE) principles. Furthermore, data were used to build an artificial neural network (ANN) model, to predict size and polydispersity index (PDI). Sets of runs were designed by DoE and performed on a micromixer microfluidic chip. Lipids' molar ratio and the process parameters, i.e. total flow rate (TFR) and flow rate ratio (FRR), were found to be the most influential factors on the formation of vesicles with target size and PDI under 100 nm and lower than 0.2, respectively. Size and PDI were predicted by the ANN model for 3 preparations with defined experimental conditions. The results showed no significant difference in size and PDI between the preparations and their values calculated with the ANN. In conclusion, production of optimized liposomes with high reproducibility was achieved by the application of microfluidic manufacturing processes, DoE, and Artificial Intelligence (AI). Microfluidic-based preparation methods assisted by computational tools would enable a faster development and clinical transfer of nanobased medications.

Potential use for chronic pain: Poly(Ethylene Glycol)-Poly(Lactic-Co-Glycolic Acid) nanoparticles enhance the effects of Cannabis-Based terpenes on calcium influx in TRPV1-Expressing cells.

The objective of these in vitro studies was to investigate the impact of the encapsulation of three cannabis-based terpenes, namely ß-myrcene (MC), ß-caryophyllene (CPh), and nerolidol (NL), on their potential efficacy in pain management. Terpene-encapsulated poly(ethylene glycol)-poly(lactic-co-glycolic acid) nanoparticles (PEG-PLGA NPs) were prepared by an emulsion-solvent evaporation method. The terpene-loaded NPs were examined in HEK293 cells that express the nociceptive transient receptor potential vanilloid-1 (TRPV1), an ion channel involved in pain perception. TRPV1 activation was assessed by monitoring calcium influx kinetics over 1 h in cells pre-treated with the fluorescent indicator Fluo-4. In addition, the fluorescence intensity changes induced by the NPs in living cells were also explored by a fluorescence microscope. Furthermore, the cytotoxicity of the terpene-loaded NPs was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-3,5-diphenyl tetrazolium bromide (MTT) proliferation assay. The terpene-loaded NPs had a diameter in the range of 250-350 nm and a zeta potential of approximately -20 mV. The encapsulation efficiency was 18.5%, 51.3%, and 60.3% for MC, NL, and CPh NPs, respectively. The nano-formulations significantly increased the fluorescence intensity in comparison with free terpenes. Furthermore, combinations of terpene-loaded NPs produced significantly higher calcium responses when compared to combinations of free terpenes. Similar findings were shown by the fluorescence images. In conclusion, the terpene-PLGA NPs can be promising therapeutics for more effective pain management.

Recent Advances in the Surface Functionalization of PLGA-Based Nanomedicines.

Therapeutics are habitually characterized by short plasma half-lives and little affinity for targeted cells. To overcome these challenges, nanoparticulate systems have entered into the disease arena. Poly(d,l-lactide-co-glycolide) (PLGA) is one of the most relevant biocompatible materials to construct drug nanocarriers. Understanding the physical chemistry of this copolymer and current knowledge of its biological fate will help in engineering efficient PLGA-based nanomedicines. Surface modification of the nanoparticle structure has been proposed as a required functionalization to optimize the performance in biological systems and to localize the PLGA colloid into the site of action. In this review, a background is provided on the properties and biodegradation of the copolymer. Methods to formulate PLGA nanoparticles, as well as their in vitro performance and in vivo fate, are briefly discussed. In addition, a special focus is placed on the analysis of current research in the use of surface modification strategies to engineer PLGA nanoparticles, i.e., PEGylation and the use of PEG alternatives, surfactants and lipids to improve in vitro and in vivo stability and to create hydrophilic shells or stealth protection for the nanoparticle. Finally, an update on the use of ligands to decorate the surface of PLGA nanomedicines is included in the review.

Evaluation of poly (lactic-co-glycolic acid) nanoparticles to improve the therapeutic efficacy of paclitaxel in breast cancer.

Introduction: Paclitaxel (PTX) is a cornerstone in the treatment of breast cancer, the most common type of cancer in women. However, this drug has serious limitations, including lack of tissue-specificity, poor water solubility, and the development of drug resistance. The transport of PTX in a polymeric nanoformulation could overcome these limitations. Methods: In this study, PLGA-PTX nanoparticles (NPs) were assayed in breast cancer cell lines, breast cancer stem cells (CSCs) and multicellular tumor spheroids (MTSs) analyzing cell cycle, cell uptake (Nile Red-NR-) and α-tubulin expression. In addition, PLGA-PTX NPs were tested in vivo using C57BL/6 mice, including a biodistribution assay. Results: PTX-PLGA NPs induced a significant decrease in the PTX IC50 of cancer cell lines (1.31 and 3.03-fold reduction in MDA-MB-231 and E0771 cells, respectively) and CSCs. In addition, MTSs treated with PTX-PLGA exhibited a more disorganized surface and significantly higher cell death rates compared to free PTX (27.9% and 16.3% less in MTSs from MCF-7 and E0771, respectively). PTX-PLGA nanoformulation preserved PTX's mechanism of action and increased its cell internalization. Interestingly, PTX-PLGA NPs not only reduced the tumor volume of treated mice but also increased the antineoplastic drug accumulation in their lungs, liver, and spleen. In addition, mice treated with PTX-loaded NPs showed blood parameters similar to the control mice, in contrast with free PTX. Conclusion: These results suggest that our PTX-PLGA NPs could be a suitable strategy for breast cancer therapy, improving antitumor drug efficiency and reducing systemic toxicity without altering its mechanism of action.

An update on liposomes in drug delivery: a patent review (2014-2018).

Introduction: Pharmacotherapy is limited by the inefficient drug targeting of non-healthy cells/tissues. In this pharmacological landscape, liposomes are contributing to the impulse given by Nanotechnology to optimize drug therapy. Areas covered: The analysis of the state-of-the-art in liposomal formulations for drug delivery purposes have underlined that lately published patents (since 2014) are exploring alternative compositions and ways to optimize the stability and drug loading content/release profile. These improvements are complemented by improved long-circulating structures and further liposome functionalizations, which have definitively opened the road for the (co-)delivery of therapeutics to the site of action. Liposomes are also contributing to new drug delivery approaches involving the generation of extracellular vesicles by targeted cells, while opening new ways to combine disease diagnosis and therapy (theranosis). Expert opinion: Patent publications on liposomal formulations have expanded new ways in drug delivery. New lipid compositions and strategies to optimize stability and drug vehiculization capabilities have settle solid pillars in liposome fabrication. Despite, their architecture has been satisfactorily adapted for combining passive and active drug targeting concepts, new inputs of liposomes into the disease arena should answer for: a simple/scalable/cost-effective formulation; a safe/stable/controllable formulation meeting quality control regulations; and, a confirmed therapeutic efficiency in clinical investigations.

A novel nanoformulation of PLGA with high non-ionic surfactant content improves in vitro and in vivo PTX activity against lung cancer.

Paclitaxel (PTX), a chemotherapy agent widely used to treat lung cancer, is characterised by high toxicity, low bioavailability and the need to use of excipients with serious side effects that limit its use. Paclitaxel encapsulation into nanoparticles (NPs) generates drug pharmacokinetic and pharmacodynamic advantages compared to free PTX. In this context, a NP carrier formed from a copolymer of lactic acid and glycolic acid (PLGA) has demonstrated high biocompatibility and low toxicity and therefore being approved by FDA to be used in humans. We synthesised a new PLGA NP and loaded it with PTX to improve drug efficacy and reduce side effects. This nanoformulation showed biocompatibility and no toxicity to human immune system. These NPs favor the intracellular uptake of PTX and enhance its antitumor effect in human and murine lung cancer cells, with up to 3.6-fold reductions in the PTX's IC50. Although PLGA NPs did not show any inhibitory capacity against P-glycoprotein, they increased the antitumor activity of PTX in cancer stem cells. Treatment with PLGA-PTX NPs increased apoptosis and significantly reduced the volume of the tumorspheres derived from A549 and LL2 cells by up to 36% and 46.5%, respectively. Biodistribution studies with PLGA-PTX NPs revealed an increase in drug circulation time, as well as a greater accumulation in lung and brain tissues compared to free PTX. Low levels of PTX were detected in the dorsal root ganglion with PLGA-PTX NPs, which could exert a protective effect against peripheral neuropathy. In vivo treatment with PLGA-PTX NPs showed a greater decrease in tumor volume (44.6%) in immunocompetent mice compared to free PTX (24.4%) and without increasing the toxicity of the drug. These promising results suggest that developed nanosystem provide a potential strategy for improving the chemotherapeutic effect and reducing the side effects of PTX.

Development and Characterization of Magnetite/Poly(butylcyanoacrylate) Nanoparticles for Magnetic Targeted Delivery of Cancer Drugs.

A great attention is presently paid to the design of drug delivery vehicles based on surface-modified magnetic nanoparticles. They can, in principle, be directed to a desired target area for releasing their drug payload, a process triggered by pH, temperature, radiation, or even magnetic field. To this, the possibility of forming part of diagnostic tools by enhanced magnetic resonance imaging or that of further treatment by magnetic hyperthermia can be added. Bare particles are rapidly eliminated from the bloodstream by the phagocyte mononuclear system, leading to short biological half-life. It is hence required to coat them in order to increase their biocompatibility and facilitate the drug incorporation. In this work, magnetite nanoparticles were coated with poly(butylcyanoacrylate) (PBCA) manufactured and characterized with regard to their physical properties and their suitability as a platform for magnetically controlled drug delivery. The average diameter of magnetite and core-shell nanoparticles was 97 ± 19 and 140 ± 20 nm, respectively. Infrared analysis, electrophoretic mobility, surface thermodynamics analysis, and X-ray diffraction all confirmed that the magnetic particles were sufficiently covered by the polymer in the composite nanoparticles. In addition, assays using normal (CCD-18 and MCF-10A) and tumoral (T-84 and MCF-7) cell lines derived from colon and breast tissue, respectively, demonstrated that nanocomposites have low or negligible cytotoxicity. It is concluded that PBCA-coated magnetite core-shell nanoparticles represent a remarkable promise as a platform for magnetically controlled drug delivery.

Folic acid-decorated and PEGylated PLGA nanoparticles for improving the antitumour activity of 5-fluorouracil.

5-Fluorouracil (5-FU) is a broad spectrum cytotoxic agent being used in chemotherapy of malignancies. However, 5-FU shows a number of limitations like short half-life, non-selective biodistribution, and the development of drug resistances by tumour cells. It was investigated the potential use of folic acid-decorated and PEGylated poly(D,L-lactide-co-glycolide) nanoparticles (FOL-PEG-PLGA NPs) for the targeted delivery of 5-FU to colon and breast cancers. PEG-PLGA and FOL-PEG-PLGA conjugates were synthesized and characterized. NPs of PLGA, PEG-PLGA, and FOL-PEG-PLGA were prepared by nanoprecipitation under optimal formulation conditions. They were found to be haemocompatible, and exhibited negligible cytotoxicity in normal (CCD-18 and MCF-10A) and tumour (HT-29 and MCF-7) human cell lines. 5-FU loading capabilities were also defined, and the NPs exhibited an initial burst drug release followed by a sustained 5-FU release. In vitro cytotoxicity studies in folate-overexpressed HT-29 colon cancer cells and MCF-7 breast cancer cells demonstrated that the half maximal inhibitory concentration (IC50) of 5-FU-loaded FOL-PEG-PLGA NPs was approximately 4-fold less than that of the 5-FU-loaded PLGA NPs (p<0.05). Consequently, FOL-PEG-PLGA NPs could have great potential as a targeted 5-FU delivery system for colon and breast cancer treatment.

Melatonin reduces hepatic mitochondrial dysfunction in diabetic obese rats.

Hepatic mitochondrial dysfunction is thought to play a role in the development of liver steatosis and insulin resistance, which are both common characteristics of obesity and type 2 diabetes mellitus (T2DM). It was hypothesized that the antioxidant properties of melatonin could potentially improve the impaired functions of hepatic mitochondria in diabetic obese animals. Male Zucker diabetic fatty (ZDF) rats and lean littermates (ZL) were given either melatonin (10 mg/kg BW/day) orally for 6 wk (M-ZDF and M-ZL) or vehicle as control groups (C-ZDF and C-ZL). Hepatic function was evaluated by measurement of serum alanine transaminase and aspartate transaminase levels, liver histopathology and electron microscopy, and hepatic mitochondrial functions. Several impaired functions of hepatic mitochondria were observed in C-ZDF in comparison with C-ZL rats. Melatonin treatment to ZDF rats decreases serum levels of ALT (P < 0.001), alleviates liver steatosis and vacuolation, and also mitigates diabetic-induced mitochondrial abnormalities, glycogen, and lipid accumulation. Melatonin improves mitochondrial dysfunction in M-ZDF rats by increasing activities of mitochondrial citrate synthase (P < 0.001) and complex IV of electron transfer chain (P < 0.05) and enhances state 3 respiration (P < 0.001), respiratory control index (RCR) (P < 0.01), and phosphorylation coefficient (ADP/O ratio) (P < 0.05). Also melatonin augments ATP production (P < 0.05) and diminishes uncoupling protein 2 levels (P < 0.001). These results demonstrate that chronic oral melatonin reduces liver steatosis and mitochondria dysfunction in ZDF rats. Therefore, it may be beneficial in the treatment of diabesity.

Iron oxide-based multifunctional nanoparticulate systems for biomedical applications: a patent review (2008 - present).

INTRODUCTION: Iron oxide nanoparticles (IO NPs) exhibit remarkable properties, including inherent magnetism, biocompatibility, high surface to volume ratio, and versatility of engineering, making them ideal candidates for a variety of clinical applications. AREAS COVERED: The review provides an in-depth discussion on recent patents and developments related to IO NPs in Biomedicine from the last 7 years. It covers innovations in the chemical synthesis, surface coating and functionalization, and biomedical applications, including MRI and multimodal imaging, molecular imaging, cell labeling, drug delivery, hyperthermia, hyperphosphatemia, and antibacterial activity. A brief outline of the important properties of IO NPs is also presented. EXPERT OPINION: The main focus of current research is the development of new approaches to generate high-quality IO NPs with optimal properties in terms of particle geometry, crystal structure, surface functionalities, stability, and magnetization. Among chemical synthesis methods, thermal decomposition and hydrothermal synthetics processes allow fine control of the particle properties. Plenty of coating materials have been successfully used as shells for these NPs to provide colloidal stability, even enabling the formulation of nanotheranostics for simultaneous disease diagnosis and therapy. However, long-term toxicity and pharmacokinetic studies are necessary before magnetic nanosystems can be approved for clinical use.

Nano-sized platforms for vaginal drug delivery.

Nano-sized systems have shown promise for efficient vaginal drug delivery providing sustained drug release and enhanced permeation. In parallel with advancements in drug discovery of new vaginal therapeutic agents, such as peptides, proteins, nucleic material, antigens, hormones, and microbicides, nanoplatforms are gaining momentum as prospective vectors for these agents. Thus far, extensive research in this arena has been focused on local delivery to the mucus vagina. However, an improved understanding of vaginal route, advantages offered by the vaginal route including being non-invasive and bypassing hepatic first-effect metabolism, and recent success achieved by vaginal drug nanocarriers may open the door for extensive nanotechnology- based research to explore the viability of systemic administration via this route. The review analyzes the possibilities given by nanoplatform-based delivery systems in the vaginal delivery of active agents. Special insight is given to the most important aspects to be considered during nanomedicine development and preclinical evaluation, i.e., the anatomy and physiology of the vagina, advantages of vaginal route of drug administration, and barriers to vaginal drug delivery. Finally, an updated analysis of the recent advancements of nanomedicine technologies and their potential progress into the clinic is compiled in this work.

Syrian pharmacy students' intentions and attitudes toward postgraduate education.

OBJECTIVE: To investigate Syrian pharmacy students' intentions and attitudes toward postgraduate study, and to determine and evaluate the factors that influence their preferences. METHODS: A questionnaire was developed and used to collect data from final-year bachelor of pharmacy (BPharm) students at Damascus University. RESULTS: Of the 265 students who responded to the survey, approximately 50% intended to work, 25% intended to pursue further study, and 25% were undecided. Personal fulfillment was the factor that most influenced students' intentions concerning future education. Men were more concerned over their financial future, while women's intentions were more influenced by scientific issues. The 3 most preferred pharmaceutical areas of further study were biochemistry and laboratory diagnosis, pharmaceutics and pharmaceutical industry, and clinical pharmacy. More students favored pursuing graduate school abroad rather than in Syria. The majority of those who intended to enroll in local graduate programs were interested in academic programs while less than a fifth favored residency programs. CONCLUSIONS: The graduate programs in Syria do not appear to satisfy pharmacy students' ambitions or have the capacity to accommodate the growing demand associated with the rapid increase in the number of pharmacy graduates in the country. Consequently, a majority of students prefer to pursue postgraduate study abroad.

Investigating the use of liquisolid compacts technique to minimize the influence of pH variations on loratadine release.

Loratadine is a class II water-insoluble drug and its dissolution rate and, consequently, absorption are dependent on the gastrointestinal pH. The resulting very high variability in bioavailability and related inter- and intra-subject absorption variations present a major challenge that hinders the realization of an effective and uniform therapy. Among the several techniques that have been used to minimize pH dependency of dissolution rate, liquisolid compacts technique can be suggested as a promising solution. In this study, it was hypothesized that the formulation of loratadine using liquisolid compacts technique may reduce the effect of pH variation on the drug dissolution rate. Solubilities of loratadine in propylene glycol, Tween 80, and polyethylene glycol 400 were first measured and propylene glycol was selected as for producing the highest solubility among the tested solvents. Several liquisolid tablet formulations containing various ratios of drug: propylene glycol (5%, 10%, and 20% w/w) were prepared. The ratio of microcrystalline cellulose (carrier) to silica (coating powder material) was kept constant in all formulations. The dissolution behavior of loratadine from liquisolid compacts was investigated in several buffered media with different pH values (pH 1.2, 2.5, and 5). The results showed that the drug release rates produced by liquisolid compacts were significantly higher and less affected by pH variation compared with conventionally made (direct compression) and commercial (Clarityn) tablets. In conclusion, liquisolid compacts technique may be used as a tool to minimize the effects of pH variation on the dissolution rate of drugs with poor water solubility.

Gene transfer with three amphiphilic glycol chitosans--the degree of polymerisation is the main controller of transfection efficiency.

A number of studies have examined the possibility of delivering genes for the treatment of genetic diseases using various polymers and lipids. We have previously demonstrated the gene transfer ability of amphiphilic polymers (a soluble amine polymer covalently bound to lipid pendant groups). In the current communication we explore the gene transfer activity of amphiphilic glycol chitosans. Glycol chitosan was acid depolymerised to give polymers of various molecular weights. Palmitoyl or hexadecyl and in some cases additional N-methyl quaternary ammonium groups were attached to the polymers. DNA binding was studied by measuring the reduced fluorescence of ethidium bromide and the polyplex particle size and zeta potential. Biological characterisation of the polyplexes involved haemolysis, cytotoxicity and gene transfer assays. For the 22 polymers tested, DNA binding was optimum at a nitrogen to phosphate ratio of 2:1 and above. Polyplexes were 200-500 nm in diameter with a neutral or positive zeta potential. The haemolytic activity of the N-methyl polymers was studied and no haemolysis was detected up to a concentration of 10 mg ml-1. Cytotoxicity studies showed that the biocompatibility of glycol chitosan was adversely affected by a combination of a palmitoyl group and depolymerisation and that biocompatibility was subsequently restored with the introduction of N-methyl groups. In vitro transfection efficiency superior to the cationic lipid formulation N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium methyl sulphate (DOTAP) was seen with depolymerised glycol chitosan in the A431 cell line only and with the depolymerised N-methyl quaternary ammonium amphiphilic derivatives in both the A431 and A549 cell lines. Degree of polymerisation (DP) was the most important controller of transfection efficiency and transfection resided within polymers with a DP of 73-171. High DP polymers diminished DNA-cell association, the first step in the cellular gene transfer process, thus apparently diminishing cell uptake. In vivo transfection with the N-methyl quaternary ammonium amphiphile was best at a DP of 86 and this glycol chitosan amphiphile gave superior liver and heart gene expression levels when compared to both Exgen 500 (linear polyethylenimine) and Superfect (a polyamidoamine dendrimer).