Detecting polymeric nanoparticles with coherent anti-stokes Raman scattering microscopy in tissues exhibiting fixative-induced autofluorescence

© 2015 SPIE.Recent advances in pharmaceutical nanotechnology have enabled the development of nano-particulate medicines with enhanced drug performance. Although the fate of these nano-particles can be macroscopically tracked in the body (e.g. using radio-labeling techniques), there is little information about the sub-cellular scale mechanistic processes underlying the particle-tissue interactions, or how these interactions may correlate with pharmaceutical efficacy. To rationally engineer these nano-particles and thus optimize their performance, these mechanistic interactions must be fully understood. Coherent Anti-Stokes Raman scattering (CARS) microscopy provides a label-free means for visualizing biological samples, but can suffer from a strong non-resonant background in samples that are prepared using aldehyde-based fixatives. We demonstrate how formalin fixative affects the detection of polymeric nanoparticles within kidneys following oral administration using CARS microscopy, compared with samples that were snap-frozen. These findings have implications for clinical applications of CARS for probing nanoparticle distribution in tissue biopsies

Nanoparticulate peptide delivery exclusively to the brain produces tolerance free analgesia

This is the final version. Available on open access from Elsevier via the DOI in this recordThe delivery of peptide drugs to the brain is challenging, principally due to the blood brain barrier and the low metabolic stability of peptides. Exclusive delivery to the brain with no peripheral exposure has hitherto not been demonstrated with brain quantification data. Here we show that polymer nanoparticles encapsulating leucine5-enkephalin hydrochloride (LENK) are able to transport LENK exclusively to the brain via the intranasal route, with no peripheral exposure and nanoparticle localisation is observed within the brain parenchyma. Animals dosed with LENK nanoparticles (NM0127) showed a strong anti-nociceptive response in multiple assays of evoked and on going pain whereas animals dosed intranasally with LENK alone were unresponsive. Animals did not develop tolerance to the anti-hyperalgesic activity of NM0127 and NM0127 was active in morphine tolerant animals. A microparticulate formulation of clustered nanoparticles was prepared to satisfy regulatory requirements for nasal dosage forms and the polymer nanoparticles alone were found to be biocompatible, via the nasal route, on chronic dosing.The UK Engineering and Physical Sciences Research Council(EP/K502340/1), Nanomerics Ltd.(NM12TSB-NPP) and Innovate UK(16939-124181) are acknowledged for funding

¿Qué modelo de supervision es más útil para guiar a los estudiantes de doctorado? La perspectiva española

Educating our early career researchers is becoming far more difficult. The model of supervision should be flexible and adjusted along the way. There is not a universal model that is beneficial for all the student-supervisor relationships. It is important to find which model is more suitable depending on the stage of the PhD (1-4 year) but also the characteristics of both PhD student and supervisor. Becoming an effective supervisor requires training. Higher research institutions should implement strategies and plans of action to enhance the supervision skills of their staff. This work analyses the PhD student perspective about which model of supervision is considered as the most suitable in Spain

Mimicking bone microenvironment : 2D and 3D in vitro models of human osteoblasts

Understanding the in vitro biology and behavior of human osteoblasts is crucial for developing research models that reproduce closely the bone structure, its functions, and the cell-cell and cell-matrix interactions that occurs in vivo. Mimicking bone microenvironment is challenging, but necessary, to ensure the clinical translation of novel medicines to treat more reliable different bone pathologies. Currently, bone tissue engineering is moving from 2D cell culture models such as traditional culture, sandwich culture, micro-patterning, and altered substrate stiffness, towards more complex 3D models including spheroids, scaffolds, cell sheets, hydrogels, bioreactors, and microfluidics chips. There are many different factors, such cell line type, cell culture media, substrate roughness and stiffness that need consideration when developing in vitro models as they affect significantly the microenvironment and hence, the final outcome of the in vitro assay. Advanced technologies, such as 3D bioprinting and microfluidics, have allowed the development of more complex structures, bridging the gap between in vitro and in vivo models. In this review, past and current 2D and 3D in vitro models for human osteoblasts will be described in detail, highlighting the culture conditions and outcomes achieved, as well as the challenges and limitations of each model, offering a widen perspective on how these models can closely mimic the bone microenvironment and for which applications have shown more successful results

Lomustine Nanoparticles Enable Both Bone Marrow Sparing and High Brain Drug Levels – A Strategy for Brain Cancer Treatments

Purpose The blood brain barrier compromises glioblastoma chemotherapy. However high blood concentrations of lipophilic, alkylating drugs result in brain uptake, but cause myelosuppression. We hypothesised that nanoparticles could achieve therapeutic brain concentrations without dose-limiting myelosuppression. Methods Mice were dosed with either intravenous lomustine Molecular Envelope Technology (MET) nanoparticles (13 mg kg-1) or ethanolic lomustine (6.5 mg kg-1) and tissues analysed. Efficacy was assessed in an orthotopic U-87 MG glioblastoma model, following intravenous MET lomustine (daily 13 mg kg-1) or ethanolic lomustine (daily 1.2 mg kg-1 - the highest repeated dose possible). Myelosuppression and MET particle macrophage uptake were also investigated. Results The MET formulation resulted in modest brain targeting (brain/ bone AUC0-4h ratios for MET and ethanolic lomustine = 0.90 and 0.53 respectively and brain/ liver AUC0-4h ratios for MET and ethanolic lomustine = 0.24 and 0.15 respectively). The MET formulation significantly increased mice (U-87 MG tumours) survival times; with MET lomustine, ethanolic lomustine and untreated mean survival times of 33.2, 22.5 and 21.3 days respectively and there were no material treatment-related differences in blood and femoral cell counts. Macrophage uptake is slower for MET nanoparticles than for liposomes. Conclusions Particulate drug formulations improved brain tumour therapy without major bone marrow toxicity

Understanding Direct Powder Extrusion for Fabrication of 3D Printed Personalised Medicines: A Case Study for Nifedipine Minitablets

Fuse deposition modelling (FDM) has emerged as a novel technology for manufacturing 3D printed medicines. However, it is a two-step process requiring the fabrication of filaments using a hot melt extruder with suitable properties prior to printing taking place, which can be a rate-limiting step in its application into clinical practice. Direct powder extrusion can overcome the difficulties encountered with fabrication of pharmaceutical-quality filaments for FDM, allowing the manufacturing, in a single step, of 3D printed solid dosage forms. In this study, we demonstrate the manufacturing of small-weight (<100 mg) solid dosage forms with high drug loading (25%) that can be easily undertaken by healthcare professionals to treat hypertension. 3D printed nifedipine minitablets containing 20 mg were manufactured by direct powder extrusion combining 15% polyethylene glycol 4000 Da, 40% hydroxypropyl cellulose, 19% hydroxy propyl methyl cellulose acetate succinate, and 1% magnesium stearate. The fabricated 3D printed minitablets of small overall weight did not disintegrate during dissolution and allowed for controlled drug release over 24 h, based on erosion. This release profile of the printed minitablets is more suitable for hypertensive patients than immediate-release tablets that can lead to a marked burst effect, triggering hypotension. The small size of the minitablet allows it to fit inside of a 0-size capsule and be combined with other minitablets, of other API, for the treatment of complex diseases requiring polypharmacy within a single dosage form

Oral particle uptake and organ targeting drives the activity of amphotericin B nanoparticles

There are very few drug delivery systems that target key organs via the oral route, as oral delivery advances normally address gastrointestinal drug dissolution, permeation, and stability. Here we introduce a nanomedicine in which nanoparticles, while also protecting the drug from gastric degradation, are taken up by the gastrointestinal epithelia and transported to the lung, liver, and spleen, thus selectively enhancing drug bioavailability in these target organs and diminishing kidney exposure (relevant to nephrotoxic drugs). Our work demonstrates, for the first time, that oral particle uptake and translocation to specific organs may be used to achieve a beneficial therapeutic response. We have illustrated this using amphotericin B, a nephrotoxic drug encapsulated within <i>N</i>-palmitoyl-<i>N</i>-methyl-<i>N</i>,<i>N</i>-dimethyl-<i>N</i>,<i>N</i>,<i>N</i>-trimethyl-6-<i>O</i>-glycol chitosan (GCPQ) nanoparticles, and have evidenced our approach in three separate disease states (visceral leishmaniasis, candidiasis, and aspergillosis) using industry standard models of the disease in small animals. The oral bioavailability of AmB-GCPQ nanoparticles is 24%. In all disease models, AmB-GCPQ nanoparticles show comparable efficacy to parenteral liposomal AmB (AmBisome). Our work thus paves the way for others to use nanoparticles to achieve a specific targeted delivery of drug to key organs via the oral route. This is especially important for drugs with a narrow therapeutic index

Chitosan amphiphile coating of peptide nanofibres reduces liver uptake and delivers the peptide to the brain on intravenous administration

The clinical development of neuropeptides has been limited by a combination of the short plasma half-life of these drugs and their ultimate failure to permeate the blood brain barrier. Peptide nanofibres have been used to deliver peptides across the blood brain barrier and in this work we demonstrate that the polymer coating of peptide nanofibres further enhances peptide delivery to the brain via the intravenous route. Leucine5-enkephalin (LENK) nanofibres formed from the LENK ester prodrug - tyrosinyl1palmitate-leucine5-enkephalin (TPLENK) were coated with the polymer - N-palmitoyl-N-monomethyl-N,N-dimethyl-N,N,N-trimethyl-6-O-glycolchitosan (GCPQ) and injected intravenously. Peptide brain delivery was enhanced because the GCPQ coating on the peptide prodrug nanofibres, specifically enables the peptide prodrug to escape liver uptake, avoid enzymatic degradation to non-active sequences and thus enjoy a longer plasma half life. Plasma half-life is increased 520%, liver AUC0-4 decreased by 54% and brain AUC0-4 increased by 47% as a result of the GCPQ coating. The increased brain levels of the GCPQ coated peptide prodrug nanofibres result in the pharmacological activity of the parent drug (LENK) being significantly increased. LENK itself is inactive on intravenous injection