A lipidic mesophase with tunable release properties for the local delivery of macromolecules: the apoferritin nanocage, a case study.

Lipid mesophases are able to incorporate and release a plethora of molecules, spanning from hydrophobic drugs to small hydrophilic proteins and therefore they have been widely used as drug delivery systems. However, their 3-5 nm water channels do not allow the release of large hydrophilic molecules such as monoclonal antibodies and therapeutic proteins. To overcome this major geometrical constraint, we designed a gel by mixing monoacylglycerol lipids, generally recognized as safe for human and/or animal use by FDA, and phospholipids, to obtain a material with swollen water channels suitable to host and further release macromolecules. Apoferritin, a 12 nm nanocage protein with intrinsic tumor-targeting properties able to incorporate several molecules, was selected here as the hydrophilic model protein to be embedded in the biocompatible gel. When immersed completely in the release media, mesophases with a swollen water channel of 22 nm, composed of monoolein and doped with 5 mole% of DOPS and 10 mole% of Chol allowed us to achieve a protein release of 60%, which is 120 times higher with respect to that obtained by employing non swollen-LMPs composed only of monoolein. Thus, the formulation can be administered locally to the rectal or vaginal mucosa, reducing the drawbacks often associated with the parenteral administration of bio-therapeutics. This approach would pave the way for the local application of other biomacromolecules (including human ferritin, monoclonal antibodies and antibody drug-conjugates) in those diseases easily reachable by a local application such as rectal or vaginal cancer

Mucoadhesive 3D printed vaginal ovules to treat endometriosis and fibrotic uterine diseases.

Gynaecological health is a neglected field of research that includes conditions such as endometriosis, uterine fibroids, infertility, viral and bacterial infections, and cancers. There is a clinical need to develop dosage forms for gynecological diseases that increase efficacy and reduce side effects and explore new materials with properties tailored to the vaginal mucosa and milieu. Here, we developed a 3D printed semisolid vaginal ovule containing pirfenidone, a repurposed drug candidate for endometriosis. Vaginal drug delivery allows direct targeting of the reproductive organs via the first uterine pass effect, but vaginal dosage forms can be challenging to self-administer and retain in situ for periods of more than 1-3 h. Here, we show that a semisoft alginate-based vaginal suppository manufactured using semisolid extrusion additive manufacturing is superior to vaginal ovules made using standard excipients. The 3D-printed ovule showed a controlled release profile of pirfenidone in vitro in standard and biorelevant release tests, as well as better mucoadhesive properties ex vivo. An exposure time of 24 h of pirfenidone to a monolayer culture of an endometriotic epithelial cell line, 12Z, is necessary to reduce the cells' metabolic activity, which demonstrates the need for a sustained release formulation of pirfenidone. 3D printing allowed us to formulate mucoadhesive polymers into a semisolid ovule with controlled release of pirfenidone. This work enables further preclinical and clinical studies into vaginally administered pirfenidone to assess its efficacy as a repurposed endometriosis treatment

3D printing injectable microbeads using a composite liposomal ink for local treatment of peritoneal diseases.

The peritoneal cavity offers an attractive administration route for challenging-to-treat diseases, such as peritoneal carcinomatosis, post-surgical adhesions, and peritoneal fibrosis. Achieving a uniform and prolonged drug distribution throughout the entire peritoneal space, though, is difficult due to high clearance rates, among others. To address such an unmet clinical need, alternative drug delivery approaches providing sustained drug release, reduced clearance rates, and a patient-centric strategy are required. Here, we describe the development of a 3D-printed composite platform for the sustained release of the tyrosine kinase inhibitor gefitinib (GEF), a small molecule drug with therapeutic applications for peritoneal metastasis and post-surgical adhesions. We present a robust method for the production of biodegradable liposome-loaded hydrogel microbeads that can overcome the pharmacokinetic limitations of small molecules with fast clearance rates, a current bottleneck for the intraperitoneal (IP) administration of these therapeutics. By means of an electromagnetic droplet printhead, we 3D printed microbeads employing an alginate-based ink loaded with GEF-containing multilamellar vesicles (MLVs). The sustained release of GEF from microbeads was demonstrated. In vitro studies on an immortalized human hepatic cancer cell line (Huh-7) proved concentration-dependent cell death. These findings demonstrate the potential of 3D-printed alginate microbeads containing liposomes for delivering small drug compounds into the peritoneum, overcoming previous limitations of IP drug delivery

Beyond Trial and Error: A Systematic Development of Liposomes Targeting Primary Macrophages

Monocytes/macrophages are phagocytic innate immune cells playing a pivotal role in tissue homeostasis, inflammation, and antitumor immunity in a microenvironment-dependent manner. By expressing pattern recognition and scavenger receptors on their surface, macrophages selectively take up pathogens, cellular debris, and often—undesirably—drug delivery systems. On the other hand, the propensity of phagocytic cells to internalize particulate drug carriers is used to load them with a cargo of choice, turning the monocytes/macrophages into a diagnostic or therapeutic Trojan horse. Identifying the ideal physicochemical properties of particulate carriers such as liposomes to achieve the most efficient macrophage-mediated drug delivery has been object of extensive research in the past, but the studies reported so far rely solely on trial-and-error approaches. Herein, a design of experiment (DoE) strategy to identify the optimal liposomal formulation is proposed, fully characterized in terms of size, surface charge, and membrane fluidity, to maximize macrophage targeting. The findings are validated using mouse bone marrow-derived macrophages, a primary preparation modeling in vivo monocyte-derived macrophages, thus confirming the robustness and versatility of the systematic and iterative approach and suggesting the promising potential of the DoE approach for the design of cell-targeting delivery systems

Rapid liposomal formulation for nucleolin targeting to rhabdomyosarcoma cells.

Rhabdomyosarcoma (RMS) is the most common pediatric soft tissue sarcoma. More effective and less toxic therapies are urgently needed for high-risk patients. Peptide-guided targeted drug delivery can increase the therapeutic index of encapsulated drugs and improve patients' well-being. To apply this strategy to RMS, we identified the peptide F3 in a screening for peptides binding to RMS cells surface. F3 binds to nucleolin, which is present on the surface of RMS cells and is abundantly expressed at the mRNA level in RMS patients' biopsies compared to healthy tissues. We developed a rapid microfluidic formulation of F3-decorated PEGylated liposomes and remote loading of the chemotherapeutic drug vincristine. Size, surface charge, drug loading and retention of targeted and control liposomes were studied. Enhanced cellular binding and uptake were observed in three different nucleolin-positive RMS cell lines. Importantly, F3-functionalized liposomes loaded with vincristine were up to 11 times more cytotoxic than non-targeted liposomes for RMS cell lines. These results demonstrate that F3-functionalized liposomes are promising for targeted drug delivery to RMS and warrant further in vivo investigations

Temperature-triggered in situ forming lipid mesophase gel for local treatment of ulcerative colitis

Ulcerative colitis is a chronic inflammatory bowel disease that strongly affects patient quality of life. Side effects of current therapies necessitate new treatment strategies that maximise the drug concentration at the site of inflammation, while minimizing systemic exposure. Capitalizing on the biocompatible and biodegradable structure of lipid mesophases, we present a temperature-triggered in situ forming lipid gel for topical treatment of colitis. We show that the gel is versatile and can host and release drugs of different polarities, including tofacitinib and tacrolimus, in a sustained manner. Further, we demonstrate its adherence to the colonic wall for at least 6 h, thus preventing leakage and improving drug bioavailability. Importantly, we find that loading known colitis treatment drugs into the temperature-triggered gel improves animal health in two mouse models of acute colitis. Overall, our temperature-triggered gel may prove beneficial in ameliorating colitis and decreasing adverse effects associated with systemic application of immunosuppressive treatments

Development and in vivo validation of phospholipid-based depots for the sustained release of bupivacaine.

By direct deposition of the drug at the local site of action, injectable depot formulations - intended for treatment of a local disease or for local intervention - are designed to limit the immediate exposure of the active principle at a systemic level and to reduce the frequency of administration. To overcome known drawbacks in the production of some marketed phospholipid-based depots, here we propose to manufacture drug-loaded negatively charged liposomes through conventional technologies and to control their aggregation mixing a solution of divalent cations prior to administration. We identified phosphatidylglycerol (PG) as the most suitable phospholipid for controlled aggregation of the liposomes and to modulate the release of the anesthetic bupivacaine (BUP) from liposomal depots. In vivo imaging of the fluorescently-labelled liposomes showed a significantly higher retention of the PG liposomes at the injection site with respect to neutral ones. In situ mixing of PG liposomes with calcium salts significantly extended the area under the curve of BUP in plasma compared to the non-depot system. Overall, controlling the aggregation of negatively charged liposomes with divalent cations not only modulated the particle clearance from the injection site but also the release in vivo of a small amphipathic drug such as BUP

Effects of treatments with ozonated water in the vineyard (cv Vermentino) on microbial population and fruit quality parameters

Ozone (O3) is currently employed in wineries as a sanitizing agent and is used to control microbial growth and infection. This molecule decomposes spontaneously to oxygen upon application and the use of ozonated water can represent an alternative for the control of pathogens in the vineyard. Entire canopies of Vitis vinifera (cv Vermentino) plants have been sprayed throughout the vegetative growth with water saturated with O3 to assess the effects of these treatments in reducing the microorganism population, and to evaluate if and how the oxidative stress, induced in the plant by this strong oxidizing agent, affects fruit development, the activity of the cellular antioxidant system, and the production of aromas by the grape berries at ripening. Ozonated water treatments resulted in a partial control of microorganism population, especially considering fungi. Furthermore, the treatments induced a slight delay in the technological maturity of grapes, a significant increase in antioxidant capacity and changes of aroma profile of the grapes at harvest, with an accumulation of monoterpenes. In general, ozonized water treatments showed promising results and seem to be a feasible protocol to be applied in the vineyard in order to reduce the use of chemicals

Open pathways for cerebrospinal fluid outflow at the cribriform plate along the olfactory nerves.

BACKGROUND Routes along the olfactory nerves crossing the cribriform plate that extend to lymphatic vessels within the nasal cavity have been identified as a critical cerebrospinal fluid (CSF) outflow pathway. However, it is still unclear how the efflux pathways along the nerves connect to lymphatic vessels or if any functional barriers are present at this site. The aim of this study was to anatomically define the connections between the subarachnoid space and the lymphatic system at the cribriform plate in mice. METHODS PEGylated fluorescent microbeads were infused into the CSF space in Prox1-GFP reporter mice and decalcification histology was utilized to investigate the anatomical connections between the subarachnoid space and the lymphatic vessels in the nasal submucosa. A fluorescently-labelled antibody marking vascular endothelium was injected into the cisterna magna to demonstrate the functionality of the lymphatic vessels in the olfactory region. Finally, we performed immunostaining to study the distribution of the arachnoid barrier at the cribriform plate region. FINDINGS We identified that there are open and direct connections from the subarachnoid space to lymphatic vessels enwrapping the olfactory nerves as they cross the cribriform plate towards the nasal submucosa. Furthermore, lymphatic vessels adjacent to the olfactory bulbs form a continuous network that is functionally connected to lymphatics in the nasal submucosa. Immunostainings revealed a discontinuous distribution of the arachnoid barrier at the olfactory region of the mouse. INTERPRETATION Our data supports a direct bulk flow mechanism through the cribriform plate allowing CSF drainage into nasal submucosal lymphatics in mice. FUNDING This study was supported by the Swiss National Science Foundation (310030_189226), Dementia Research Switzerland-Synapsis Foundation, the Heidi Seiler Stiftung and the Fondation Dr. Corinne Schuler

Liposomal aggregates sustain the release of rapamycin and protect cartilage from friction

Liposomes show promise as biolubricants for damaged cartilage, but their small size results in low joint and cartilage retention. We developed a zinc ion-based liposomal drug delivery system for local osteoarthritis therapy, focusing on sustained release and tribological protection from phospholipid lubrication properties. Our strategy involved inducing aggregation of negatively charged liposomes with zinc ions to extend rapamycin (RAPA) release and improve cartilage lubrication. Liposomal aggregation occurred within 10 min and was irreversible, facilitating excess cation removal. The aggregates extended RAPA release beyond free liposomes and displayed irregular morphology influenced by RAPA. At nearly 100 µm, the aggregates were large enough to exceed the previously reported size threshold for increased joint retention. Tribological assessment on silicon surfaces and ex vivo porcine cartilage revealed the system's excellent protective ability against friction at both nano- and macro-scales. Moreover, RAPA was shown to attenuate the fibrotic response in human OA synovial fibroblasts. Our findings suggest the zinc ion-based liposomal drug delivery system has potential to enhance OA therapy through extended release and cartilage tribological protection, while also illustrating the impact of a hydrophobic drug like RAPA on liposome aggregation and morphology