Sabrina Bochicchio

Liposomes as siRNA Delivery Vectors

Nucleic Acid Based Drugs (NABDs) constitute a class of promising and powerful therapeutic new agents with limited side effects, potentially useable against a wide range of diseases, including cancer. Among them, the short interfering RNAs (siRNAs), represent very effective molecules. Despite their in vitro efficacy, the major drawback that limits siRNAs usage consists in a difficult delivery due to their very low stability in physiological fluids, and to their limited membrane-permeability through physiological barriers. On the other hand, the liposomes (lipid bilayers closed in vesicles of various sizes) represent interesting drug delivery systems (DDSs) which can be tailored in order to get the best performance in terms of load, vesicle size and transfection yield. In this work, the current state of study in these two fields, and the connections between them, are briefly summarized.

In vitro and ex vivo delivery of tailored siRNA-nanoliposomes for E2F1 silencing as a potential therapy for colorectal cancer

Tailored developed nanoliposomes loaded with a siRNA against the transcription factor E2F1 (siE2F1), were produced and delivered to human colorectal adenocarcinoma cell lines and to intestinal human biopsies. siE2F1 loaded nanoliposomes were produced through a dedicated ultrasound assisted technique producing particles with about 40nm size (Small Unilamellar Vesicles, SUVs) and 100% siRNA encapsulation efficiency. Compared to other production methods, the one proposed here can easily produce particles in the nanometric scale by suitable ultrasonic duty cycle treatments. Furthermore, SUVs have a high degree of size homogeneity, a relevant feature for uniform delivery behaviour. siE2F1-loaded SUVs demonstrated a very low cytotoxicity in cells when compared to a commercial transfection agent. Moreover, SUVs loaded with siE2F1 were effective in the down regulation of the target in cultured colon carcinoma cells and in the consequent reduction of cell growth. Finally, a remarkable uptake and target silencing efficiencies were observed in cultured human biopsy of colonic mucosa. In conclusion, whereas further studies in more complex models are required, the siE2F1-SUVs generated have the potential to contribute to the development of novel effective inflammatory bowel diseases-associated colorectal cancer therapies for a future personalized medicine.

New Preparative Approaches for Micro and Nano Drug Delivery Carriers.

The full success of pharmacological therapies is strongly depending on the use of suitable, efficient and smart drug delivery systems (DDSs). Thus DDSs development is one of the main challenges in pharmaceutical industry both to achieve tailored carrier systems based on drug features and to promote manufacturing innovations to reduce energetic resources, emissions, wastes and risks. Main functions of an ideal DDS are: to protect loaded active molecules from degradation in physiological environments; to deliver them in a controlled manner and towards specific organs or tissues, to allow the maintenance of drug concentration within therapeutic window. Smart features, such as those able to induce active molecule release upon the occurrence of specific physiological stimuli, are also desirable. Under the manufacturing point of view, the current industrial scenery is obliged to respond to the increasing market requirements and to the mandatory rules in sustainable productions such as raw material and energy savings. In this work a general framework on drug delivery systems preparation techniques is presented. In particular two sections on innovation in preparative approaches carried out are detailed. These latter involve the use of microwave and ultrasonic energy applied in the production of polymeric and lipidic delivery systems on micro- and nanometric scale. The novelties of these preparative approaches are emphasized and examples of developed drug delivery carriers, loaded with vitamins and drug mimicking siRNA, are shown.

Nanoliposomes Production by a Protocol Based on a Simil-Microfluidic Approach

In this work a protocol based on the microfluidic principles has been developed and applied to produce nanoliposomes. The protocol basically consists in the realization of a contact between two flows, lipids/ethanol and water solutions, inside a tubular device where interdiffusion phenomena allow the formation of lipid vesicles. Effects of solutions flow rates and lipids concentrations on size and size distribution have been investigated. Moreover, ultrasonic energy was used to enhance homogenization of the hydroalcoholic final solutions and to promote the vesicles size reduction. By this protocol a massive output has been achieved; increasing the ratio between the water volumetric flow rate to the lipids-ethanol volumetric flow rate the liposomes dimension decreases; at equal flow rates, when the lipids concentration increases also the liposomes size has been observed increasing.

Polymer-lipid hybrid nanoparticles as enhanced indomethacin delivery systems

&NA; Non‐steroidal anti‐inflammatory drugs (NSAIDs), i.e. indomethacin used for rheumatoid arthritis and non‐rheumatoid inflammatory diseases, are known for their injurious actions on the gastrointestinal (GI) tract. Mucosal damage can be avoided by using nanoscale systems composed by a combination of liposomes and biodegradable natural polymer, i.e. chitosan, for enhancing drug activity. Aim of this study was to prepare chitosan‐lipid hybrid delivery systems for indomethacin dosage through a novel continuous method based on microfluidic principles. The drop‐wise conventional method was also applied in order to investigate the effect of the two polymeric coverage processes on the nanostructures features and their interactions with indomethacin. Thermal‐physical properties, mucoadhesiveness, drug entrapment efficiency, in vitro release behavior in simulated GI fluids and stability in stocking conditions were assayed and compared, respectively, for the uncoated and chitosan‐coated nanoliposomes prepared by the two introduced methods. The prepared chitosan‐lipid hybrid structures, with nanometric size, have shown high indomethacin loading (about 10%) and drug encapsulation efficiency up to 99%. TEM investigation has highlighted that the developed novel simil‐microfluidic method is able to put a polymeric layer, surrounding indomethacin loaded nanoliposomes, thicker and smoother than that achievable by the drop‐wise method, improving their storage stability. Finally, double pH tests have confirmed that the chitosan‐lipid hybrid nanostructures have a gastro retentive behavior in simulated gastric and intestinal fluids thus can be used as delivery systems for the oral‐controlled release of indomethacin. Based on the present results, the simil‐microfluidic method, working with large volumes, in a rapid manner, without the use of drastic conditions and with a precise control over the covering process, seems to be the most promising method for the production of suitable indomethacin delivery system, with a great potential in industrial manufacturing. Graphical abstract Figure. No caption available.

Microfluidic Investigation of the Effect of Liposome Surface Charge on Drug Delivery in Microcirculation

Nano-carrier drug transport in blood microcirculation is one of the hotspots of current research in drug development due to many advantages over traditional therapies, such as reduced sideeffects, target delivery, controlled release, improved pharmacokinetics and therapeutic index. Despite the substantial efforts made in the design of nanotherapeutics, the big majority of the used strategies failed to overcome the biological barriers to drug transport encountered in human microvasculature, such as transport by blood flow via the microcirculatory network and margination, the mechanism according to which particles migrate along vessel radius to the wall. In fact, drug transport efficiency in microvasculature is affected by both the particulate nature of blood and drug carrier properties, such as size, shape and surface charge. In this work, the effect of the surface charge of liposomes on their margination in blood flow in microcapillaries was experimentally evaluated. By high-speed video microscopy and image analysis it was found that the two custom-made liposomes (one neuter and the other positively charged) tend to drift laterally, moving towards the wall and accumulating in the cell-free layer. In particular, neuter and cationic liposomes showed a comparable margination propensity, suggesting that the presence of blood cells governs the flow behavior independently on liposome surface charge.

Design and production of hybrid nanoparticles with polymeric-lipid shell–core structures: conventional and next-generation approaches

Liposomes constitute a class of prominent drug delivery systems due their cell-mimetic behaviour. Despite their high biocompatibility, biodegradability and low intrinsic toxicity, their poor stability in biological fluids as well as in stock conditions (high tendency to degrade or aggregate) have led to new approaches for liposome stabilization (e.g., surface covering with polymers). Here, liposomes were enwrapped by the natural biocompatible polymer chitosan to achieve stable shell–core nanostructures. Covered nanoliposomes were produced using an innovative continuous method based on microfluidic principles. The produced hybrid polymeric-lipid nanoparticles were characterized in terms of structural properties, size and stability. Moreover, phenomenological aspects in formation of nanoliposomal vesicles and chitosan layering, product quality (structure, size) and manufacturing yield related to this novel method were compared with those of the conventional dropwise method and the obtained products. The proposed simil-microfluidic method led to the production of stable and completely chitosan-covered liposomes with a shell–core nanostructure that avoided the disadvantages inherent in the conventional method (which are time-consuming and/or require bulky and more expensive equipment).

On the design of tailored liposomes for KRX29 peptide delivery

The high interest in therapeutic peptides, due to the specificity of their mechanisms of action, has stimulated the research of new delivery strategies to overcome bioavailability problems concerning the use of peptides in their naked form. In particular, in this study, a novel small cyclic peptide, KRX29, with a potential therapeutic effect on Heart Failure (HF) pathology, was encapsulated into large and unilamellar small vesicles (LVs, SUVs) using the thin film-hydration method followed by ultrasound assisted size reduction processes, generating loaded liposomes with nanometric sizes. Loaded and unloaded liposomes were produced exploring three different formulations by changing the charge ratio (−/+) between the anionic phosphatidylglycerol (PG) and the cationic KRX29 peptide. LVs and SUVs were designed using a 1 : 1, 7 : 1 and 13 : 1 (−/+) PG/KRX29 charge ratio and, for each formulation, the charge effect on the liposome morphology, size and zeta potential was analyzed together with peptide encapsulation performance, load, recovery efficiencies and stability through an analytical HPLC protocol purposely developed. Best results in terms of encapsulation in nanoliposomal formulations for KRX29 delivery were achieved using a 13 : 1 (−/+) charge ratio (99% in 35 nm SUVs). The influence of the PG/KRX29 charge ratio on the recovery efficiencies was also investigated obtaining that the maximal peptide recovery from liposomes (81–94%) was achieved by using a 1 : 1 (−/+) charge ratio formulation and pure ethanol as a solvent for the extraction.