Liposome Formulation and In Vitro Testing in Non-Physiological Conditions Addressed to Ex Vivo Kidney Perfusion

Pisani, Silvia; Chiesa, Enrica; Dorati, Rossella; Gregorini, Marilena; Grignano, Maria Antonietta; Ramus, Marina; Ceccarelli, Gabriele; Croce, Stefania; Valsecchi, Chiara; Monti, Manuela; Rampino, Teresa; Conti, Bice

doi:10.3390/ijms23147999

Cited by 5 publications

(5 citation statements)

References 31 publications

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“…The production speed was varied from 4 mL/min to 12 mL/min while aqueous-to-ethanol FRRs among 2 and 6 (namely 2:1–6:1 v : v ) were selected. The levels of each operating factor were set based on past reports [ 18 , 19 , 20 ]. The effect of selected manufacturing parameters on the liposomes’ physical attributes (particle size and polydispersity) was evaluated by a two-level full factorial screening Design of Experiment (DoE).…”

Section: Methodsmentioning

confidence: 99%

Effect of Micromixer Design on Lipid Nanocarriers Manufacturing for the Delivery of Proteins and Nucleic Acids

Chiesa,

Caimi,

Bellotti

et al. 2024

Pharmaceutics

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Lipid-based nanocarriers have emerged as helpful tools to deliver sensible biomolecules such as proteins and oligonucleotides. To have a fast and robust microfluidic-based nanoparticle synthesis method, the setup of versatile equipment should allow for the rapid transfer to scale cost-effectively while ensuring tunable, precise and reproducible nanoparticle attributes. The present work aims to assess the effect of different micromixer geometries on the manufacturing of lipid nanocarriers taking into account the influence on the mixing efficiency by changing the fluid–fluid interface and indeed the mass transfer. Since the geometry of the adopted micromixer varies from those already published, a Design of Experiment (DoE) was necessary to identify the operating (total flow, flow rate ratio) and formulation (lipid concentration, lipid molar ratios) parameters affecting the nanocarrier quality. The suitable application of the platform was investigated by producing neutral, stealth and cationic liposomes, using DaunoXome®, Myocet®, Onivyde® and Onpattro® as the benchmark. The effect of condensing lipid (DOTAP, 3–10–20 mol%), coating lipids (DSPE-PEG550 and DSPE-PEG2000), as well as structural lipids (DSPC, eggPC) was pointed out. A very satisfactory encapsulation efficiency, always higher than 70%, was successfully obtained for model biomolecules (myoglobin, short and long nucleic acids).

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Section: Methodsmentioning

confidence: 99%

Effect of Micromixer Design on Lipid Nanocarriers Manufacturing for the Delivery of Proteins and Nucleic Acids

Chiesa,

Caimi,

Bellotti

et al. 2024

Pharmaceutics

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“…The procedure of this assay was carried out using a modified method (Pisani et al, 2022;D'Souza, 2014;Li et al, 2001). The other residue sample obtained from the purification process was transferred into the top part of an ultra-centrifugal filter (Amicon tube, 100,000 MWCO) with a volume (ranging from 600 -700 µL, depending on sample encapsulation efficiency) that is equivalent to 1 mg of BSA.…”

Section: In Vitro Protein Release Assaymentioning

confidence: 99%

Effect of Freeze-Thaw Cycles Method to Transfersome Characteristics for Growth Protein Encapsulation

Khayrani,

Fahmi,

Nurhayati

et al. 2024

IJTech

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Transfersome, a lipid-based nanovesicle, can be a suitable tool to improve the delivery of valuable growth factors. Through transfersome technology, growth factors and active compounds can be transferred transdermally without the need for invasive delivery procedures. In this study, we evaluated the impact of freeze-thaw cycles on transfersome characteristics, particularly particle size, polydispersity, and encapsulation efficiency. Transfersome particles were prepared from dipalmitoylphosphatidylcholine (DPPC) and Tween 80 with a 97.5:2.5 w/w% using thin film hydration at a temperature of 45° -50°C. Then, the transfersome suspension was subjected to repeated freeze-thaw for 1 minute of freezing and 3 minutes of thawing. The protein release from all transfersome samples were evaluated using Bradford assay, while the particle size and polydispersity were determined with a dynamic scattering analyzer. It was found that freeze-thaw increased encapsulation efficiency, particle size, and polydispersity of transfersomes up to 81.63±0.00%, 180.70±0.87 nm, and 0.369±0.02, respectively, from those without freeze-thaw steps (73.35±0.03%, 144.93±0.21 nm and 0.202±0.02). Moreover, freeze-thawed transfersomes exhibited a release of up to 52.80% of loaded protein within 78 hours, in contrast to 37.48% in nonfreeze-thawed transfersomes. This study shows that an additional freeze-thaw step is a promising method to improve the properties of transfersome particles, especially encapsulation efficiency and sustained protein release.

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“…Both inlet streams were controlled by syringe pumps connected to a computer that controlled the whole process. The total flow rate (TFR) of aqueous and ethanolic streams was 8 mL/min, and the flow rate ratio (FRR) was 3:1, as set in a previous work of the same authors and reported in Table 3-#PM1 [40]. The liposomes preparation process with Nanoassemblr was performed at room temperature (25 ± 3 • C).…”

Section: Empty Liposome Preparation By Microfluidic Techniquementioning

confidence: 99%

“…BSA was loaded into liposomes using the passive method, contextual to liposome preparation (#PM2), as the control. The microfluidic process conditions were those already reported above in Table 3 [40]. BSA was solubilized in MilliQ water (brought to pH 7.4 by NaOH addition) supplemented with trehalose 20% w/w.…”

Section: Empty Liposome Preparation By Microfluidic Techniquementioning

confidence: 99%

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Investigation and Comparison of Active and Passive Encapsulation Methods for Loading Proteins into Liposomes

Pisani,

Di Martino,

Cerri

et al. 2023

IJMS

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In this work, four different active encapsulation methods, microfluidic (MF), sonication (SC), freeze–thawing (FT), and electroporation (EP), were investigated to load a model protein (bovine serum albumin—BSA) into neutral liposomes made from 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC):cholesterol (Chol) and charged liposomes made from DSPC:Chol:Dioleoyl-3-trimethylammonium propane (DOTAP), DSPC:Chol:1,2-dioleoyl-sn-glycero-3-phospho-L-serine (DOPS), and DSPC:Chol:phosphatidylethanolamine (PE). The aim was to increase the protein encapsulation efficiency (EE%) by keeping the liposome size below 200 nm and the PDI value below 0.7, which warrants a nearly monodisperse preparation. Electroporation (100 V) yielded the best results in terms of EE%, with a dramatic increase in liposome size (>600 nm). The FT active-loading method, either applied to neutral or charged liposomes, allowed for obtaining suitable EE%, keeping the liposome size range below 200 nm with a suitable PDI index. Cationic liposomes (DSPC:Chol:DOTAP) loaded with the FT active method showed the best results in terms of EE% (7.2 ± 0.8%) and size (131.2 ± 11.4 nm, 0.140 PDI). In vitro release of BSA from AM neutral and charged liposomes resulted slower compared to PM liposomes and was affected by incubation temperature (37 °C, 4 °C). The empty charged liposomes tested for cell viability on Human Normal Dermal Fibroblast (HNDF) confirmed their cytocompatibility also at high concentrations (1010 particles/mL) and cellular uptake at 4 °C and 37 °C. It can be concluded that even if both microfluidic passive and active methods are more easily transferable to an industrial scale, the FT active-loading method turned out to be the best in terms of BSA encapsulation efficiencies, keeping liposome size below 200 nm.

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Liposome Formulation and In Vitro Testing in Non-Physiological Conditions Addressed to Ex Vivo Kidney Perfusion

Cited by 5 publications

References 31 publications

Effect of Micromixer Design on Lipid Nanocarriers Manufacturing for the Delivery of Proteins and Nucleic Acids

Effect of Micromixer Design on Lipid Nanocarriers Manufacturing for the Delivery of Proteins and Nucleic Acids

Effect of Freeze-Thaw Cycles Method to Transfersome Characteristics for Growth Protein Encapsulation

Investigation and Comparison of Active and Passive Encapsulation Methods for Loading Proteins into Liposomes

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