Lipid Nanovesicles Research Articles

Multifunctional AIE Nanosphere-Based "Nanobomb" for Trimodal Imaging-Guided Photothermal/Photodynamic/Pharmacological Therapy of Drug-Resistant Bacterial Infections.

Injudicious or inappropriate use of antibiotics has led to the prevalence of drug-resistant bacteria, posing a huge menace to global health. Here, a self-assembled aggregation-induced emission (AIE) nanosphere (AIE-PEG1000 NPs) that simultaneously possesses near-infrared region II (NIR-II) fluorescence emissive, photothermal, and photodynamic properties is prepared using a multifunctional AIE luminogen (AIE-4COOH). The AIE-PEG1000 NPs were encapsulated with teicoplanin (Tei) and ammonium bicarbonate (AB) into lipid nanovesicles to form a laser-activated "nanobomb" (AIE-Tei@AB NVs) for the multimodal theranostics of drug-resistant bacterial infections. In vivo experiments validate that the "nanobomb" enables high-performance NIR-II fluorescence, infrared thermal, and ultrasound (AB decomposition during the photothermal process to produce numerous CO2/NH3 bubbles, which is an efficient ultrasound contrast agent) imaging of multidrug-resistant bacteria-infected foci after intravenous administration of AIE-Tei@AB NVs followed by 660 nm laser stimulation. The highly efficient photothermal and photodynamic features of AIE-Tei@AB NVs, combined with the excellent pharmacological property of rapidly released Tei during bubble generation and NV disintegration, collectively promote broad-spectrum eradication of three clinically isolated multidrug-resistant bacteria strains and rapid healing of infected wounds. This multimodal imaging-guided synergistic therapeutic strategy can be extended for the theranostics of superbugs.

Quantification of Anti-Osteoporotic Anabolic Peptide in Stealth Lipid Nanovesicles Through Validated RP-HPLC Method.

Teriparatide is a recombinant fragment of human parathyroid hormone, a potent osteoanabolic agent used for osteoporosis. The present study endeavored to develop a simple, rapid, and reliable reverse phase-high performance liquid chromatography (RP-HPLC) method for the determination of teriparatide in pegylated lipid nanovesicles for rapid formulation development/optimization. A rapid RP-HPLC-based analytical method was developed for the quantification of teriparatide in pegylated lipid nanovesicles. The method was optimized on a Waters XBridge C18 (4.6 × 150 mm, 10 μm) column with a mobile phase consisting of 0.1% formic acid in water and acetonitrile both in a linear gradient program. In the method, a short run time of 9 min was achieved at a flow rate of 1.0 mL/min with an injection volume of 50 µL at a detection wavelength of 210 nm. The developed method was validated according to the ICH Q2 (R2) guideline. The method was applied for the quantification of teriparatide in prepared pegylated lipid nanovesicles. Teriparatide encapsulated pegylated lipid nanovesicles were prepared by the ethanol injection method. Further, these vesicles were characterized for % entrapment efficiency (%EE), particle size, zeta potential, and morphology by Cryo-SEM. The teriparatide was eluting at 4.8 min in the run. Further, for the method validation, the linear relationship between concentration and response was established over the concentration range of 50-250 µg/mL with the R2 > 0.999. The method sensitivity was shown with LOD and LOQ with the value of 100 ng/mL and 500 ng/mL, respectively. The method was found to be accurate and precise with the recovery ranging in 100 ± 2% and RSD <2%, respectively. Minor deliberate changes proved the robustness of the developed method. These results indicated that the developed and validated method is accurate, precise, rapid, reliable, and fit for the quantification of teriparatide in different formulations. The RP-HPLC method was developed and validated for the quantification of teriparatide from novel pegylated lipid nanovesicles.

Skin Penetration Enhancer-Incorporated Lipid Nanovesicles (SPE-LNV) for Skin Brightening and Wrinkle Treatment.

In this work, we utilize skin penetration enhancers (SPEs) such as ceramide and fatty acids in lipid nanovesicles to promote the transdermal delivery of active ingredients. These SPE-incorporated lipid nanovesicles (SPE-LNV) interact with the constituents of skin's outermost stratum corneum (SC) layer, enabling even niacinamide and adenosine with high water solubility to effectively permeate through, leading to enhanced skin efficacy. We demonstrate by both in vitro and in vivo skin permeation studies that the SPE-LNV formulation containing both ceramide and fatty acids (LNV-CF) exhibits deeper penetration depth and faster permeation rate compared to conventional lipid nanovesicles (LNV) without SPE as well as LNV-C with only ceramide. Moreover, in vivo clinical trials were also performed to confirm that LNV-CF most effectively mediates the delivery of niacinamide and adenosine, resulting in a substantial decrease in melanin index as well as skin wrinkle compared to the control groups. We envision that the strategy of incorporating both ceramide and fatty acids in lipid nanovesicles offers a simple and convenient route for the rapid and effective delivery of water-soluble active ingredients across the skin barrier layer.

Lipid‐Head‐Polymer‐Tail Chimeric Vesicles

Lipid nanovesicles (LNVs) and polymer nanovesicles (PNVs), also known as liposomes and polymersomes, are becoming increasingly vital in global health. However, the two major classes of nanovesicles both exhibit their own issues that significantly limit potential applications. Here, by covalently attaching a naturally occurring phosphate "lipid head" and a synthetic polylactide "polymer tail" via facile ring-opening polymerization on a 500 g scale, a type of "chimeric" nanovesicles (CNVs) can be easily produced. Compared to LNVs, the reported CNVs exhibit reduced permeability for small and large molecules; on the other hand, the CNVs are less hydrophobic and exhibit enhanced tolerance toward proteins in buffer solutions without the need for hydrophilic polymeric corona such as poly(ethylene glycol)(PEG), in contrast to conventional PNVs. The proof-of-concept in vitro delivery experiments using hydrophilic solutions of fluorescein-PEG, rhodamine-PEG, and anti-cancer drug doxorubicin demonstrate that these CNVs, as a structurally diverse class of nano-materials, are highly promising as alternative carriers for therapeutic molecules in translational nanomedicine.

Lipid nano-vesicles for thyroid hormone encapsulation: A comparison between different fabrication technologies, drug loading, and an in vitro delivery to human tendon stem/progenitor cells in 2D and 3D culture

Phosphatidylcholine (PC) vesicles loaded with Triiodothyronine (T3) were fabricated using different manufacturing methods: thin layer hydration plus sonication (TF-UF), supercritical liposome formation (SC), and microfluidic technology (MF). Vesicles obtained by MF had the lowest mean diameter (88.61 ± 44.48 nm) with a Zeta Potential of −20.1 ± 5.90 mV and loading of 10 mg/g (encapsulation efficiency: 57%). In contrast, SC vesicles showed extremely low encapsulation efficiency (<10%) probably due to T3 solubility in ethanol/carbon dioxide mixture; despite TF-UF vesicles exhibiting good size (167.7 ± 90 nm; Zp −8.50 ± 0.60 mV) and loading (10 mg/g), poor mass recovery was obtained (50% loss). MF vesicles had low cytotoxicity, and they were well enough internalized by both HeLa and human tendon stem/progenitor cells (hTSPCs). Their biological activity was also monitored in both 2D and 3D cultures of hTSPCs supplemented with therapeutical concentrations of PC/T3 nano-liposomes. 2D culture showed almost similar constitutive gene expression compared to control culture supplemented with free-T3. On the contrary, when hTPSCs 3D culture was assembled, it showed a more evident homogeneous distribution of FITC labeled vesicles within the high-density structure and a significant upregulation of cell constitutive genes, such as type I Collagen (4.8-fold; p < 0.0001) at day 7, compared to the control, suggesting that T3/PC formulation has increased T3 cytosolic concentration, thus improving cells metabolic activity. The study supported MF technology for nano-carriers fabrication and opens perspectives on the activity of PC/T3 nano-vesicles as innovative formulations for TPSCs stimulation in ECM secretion.

Encapsulation of Anabolic Peptide in Lipid Nano Vesicles for Osteoporosis

Screening of critical variables, including formulation and process variables, in the development of various dosage forms facilitates the identification of the most influencing parameters, which modulate the responses, thereby helping in building the strong quality target product profile. The objective of the present work was to screen out the most influential and critical variables for the development of an anabolic peptide encapsulated lipid nanovesicles (PTH-LNVs). PTH-LNVs were prepared by the ethanol injection method. Taguchi standard orthogonal array L8 design was employed to assess the effect of formulation and processing variables on different response variables. Independent variables considered were drug concentration, lipid concentration, cholesterol concentration, stirring rate, and rate of injection, whereas dependent variables studied were particle size, PDI, zeta potential, % entrapment efficiency, and % drug loading. Particle size, PDI, and zeta potential were evaluated by a zeta sizer. Drug loading efficiency and % entrapment efficiency were determined by HPLC analysis. The ethanol injection method was employed to formulate PTH-LNVs using Taguchi standard orthogonal array L8 design. From the half-normal plot and Pareto ranking analysis, it was found that drug, lipid, and cholesterol concentration have a significant effect on responses of formulation and are hence considered critical variables during the formulation development. The presented work demonstrates the feasibility of Taguchi orthogonal array design in the screening of potential independent factors in the development of peptide encapsulated nanoformulations.

Multifunctional membranes for lipidic nanovesicle capture

Tangential flow filtration membrane systems are employed for the isolation and concentration of extracellular vesicles. However, interfacial interactions between the membrane surface and species influence the flux and membrane performance. Here we propose a strategy aimed at introducing functional ligands over the membrane surface to improve the separation process through combined size-exclusion and affinity-based mechanisms, avoiding the binding of contaminants and other non-target molecules. Polysulfone membranes were modified by a nanometric coating of differently functionalized copolymers with the dual purpose of limiting non-specific interactions while promoting the chemoselective conjugation of a membrane-sensing peptide ligand (BPt) for lipid nanovesicles capture. Copoly azide polymer coating positively affects the physico-chemical properties of the membrane, improving filtration performance and antifouling capacity. A decrease of the flux decline ratio from 38.7 ± 3.9% to 21.2 ± 2.4% and an increase of the ratio of protein permeate concentration (Cp) to the respective feed concentration (Cf) to values of 0.97 was measured after coating the membrane with c-(DMA-N3-BP-MAPS) highlighting its capability to reduce protein adsorption. In addition, the BPt-functionalized membrane displayed a high capturing efficiency towards synthetic liposomes which, notably, can be promptly released upon mild treatment with a divalent cation solution. Overall, our work integrates conventional TFF principles with affinity-based isolation, broadening TFF perspective applications.

Potentiality of raloxifene loaded melittin functionalized lipidic nanovesicles against pancreatic cancer cells

Pancreatic cancer (PC) frequency and incidence have grown rapidly in recent years. One of the most serious problems with PC is the existence of asymptotic manifestations, which frequently delays early detection, and until the diagnosis is established, tumor cells progress to the metastatic stage. Another significant concern with PC is the scarcity of well-defined pharmacotherapeutic drugs. The aim of this study was to develop an efficient nanocarrier system to augment the efficacy of raloxifene (RLX) against PC cells. As a result, the current investigation was carried out in order to give an effective treatment method, in which an optimum RLX loaded phospholipid-based vesicles with melittin (PL-MEL) was chosen using experimental design software, with particle size, zeta potential and entrapment efficiency % as dependent variables. Furthermore, anticancer activity against PANC1 cells was assessed. The optimized nanovesicle parameters were 172.5 nm for the measured size, zeta potential of –0.69 mV, and entrapment efficiency of 76.91% that were in good agreement with the expected ones. RLX-raw, plain formula, and optimized RLX-PL-MEL showed IC50 concentrations of 26.07 ± 0.98, 9.166 ± 0.34, and 1.24 ± 0.05 µg/mL, respectively. Furthermore, cell cycle analysis revealed that the nanovesicle was most effective in the G2-M phase, whereas Bax, and Bcl-2 estimates revealed that optimized RLX formula had the highest apoptotic activity among treatments investigated. However, as compared to RLX alone or plain formula alone, the optimized formula demonstrated higher expression of TNFα and Bax while a significant reduction of Bcl-2 and NF-κB expression was observed. mitochondrial membrane potential (MMP) analysis confirmed the apoptosis as well as the anticancer effect of the optimized formula. Thus, the present study results showed an improvement in the anti-PC effects of the RLX with phospholipid conjugated melittin, making it a novel treatment approach against PC.

Biological and Immune Responses to Current Anti-SARS-CoV-2 mRNA Vaccines beyond Anti-Spike Antibody Production.

Several vaccine strategies are now available to fight the current SARS-CoV-2 pandemic. Those based on the administration of lipid-complexed messenger(m)RNA molecules represent the last frontiers in terms of technology innovation. mRNA molecules coding for the SARS-CoV-2 Spike protein are intramuscularly injected, thereby entering cells by virtue of their encapsulation into synthetic lipid nanovesicles. mRNA-targeted cells express the Spike protein on their plasma membrane in a way that it can be sensed by the immune system, which reacts generating anti-Spike antibodies. Although this class of vaccines appears as the most effective against SARS-CoV-2 infection and disease, their safety and efficiency are challenged by several factors included, but not limited to the following: emergence of viral variants, lack of adequate pharmacokinetics/pharmacodynamics studies, inability to protect oral mucosa from infection, and antibody waning. Emergence of viral variants can be a consequence of mass vaccination carried out in a pandemic time using suboptimal vaccines against an RNA virus. On the other hand, understanding the remainder flaws could be of some help in designing next generation anti-SARS-CoV-2 vaccines. In this commentary, issues regarding the fate of injected mRNA, the tissue distribution of the induced antiviral antibodies, and the generation of memory B cells are discussed. Careful evaluation of both experimental and clinical observations on these key aspects should be taken into account before planning booster administration, vaccination to non-at-risk population, and social restrictions.

Application of lipid nanovesicle drug delivery system in cancer immunotherapy.

Immunotherapy has gradually emerged as the most promising anticancer therapy. In addition to conventional anti-PD-1/PD-L1 therapy, anti-CTLA-4 therapy, CAR-T therapy, etc., immunotherapy can also be induced by stimulating the maturation of immune cells or inhibiting negative immune cells, regulating the tumor immune microenvironment and cancer vaccines. Lipid nanovesicle drug delivery system includes liposomes, cell membrane vesicles, bacterial outer membrane vesicles, extracellular vesicles and hybrid vesicles. Lipid nanovesicles can be used as functional vesicles for cancer immunotherapy, and can also be used as drug carriers to deliver immunotherapy drugs to the tumor site for cancer immunotherapy. Here, we review recent advances in five kinds of lipid nanovesicles in cancer immunotherapy and assess the clinical application prospects of various lipid nanovesicles, hoping to provide valuable information for clinical translation in the future.

Synthesis and Assessment of Lipid Nanovesicles for Efficient Transdermal Delivery of Hydrophilic Molecules

The transdermal delivery of hydrophilic molecules through the dermis is a challenging task because of the various skin barriers. Thus, lipid nanovesicles, including liposomes, ethosomes and transethosomes, are advantageous to transdermal drug delivery. This study was designed to develop nanovesicles like liposomes (LF1), ethosomes (EF1) and transethosomes (TF1) containing hydrophilic agents such as Rhodamine B (RB) as a model compound. Nanovesicles loaded with RB were synthesized using an ethanol injection method followed by probe sonication (hot method). The nonsonicated nanovesicles had vesicle sizes ranging from [Formula: see text] to [Formula: see text][Formula: see text]nm, while sonicated nanovesicles sizes ranged from [Formula: see text] to [Formula: see text][Formula: see text]nm. In addition, the nonsonicated nanovesicles had polydispersity index (PdI) ranging from [Formula: see text] to [Formula: see text], whereas the PdI of the sonicated nanovesicles ranged from [Formula: see text] to [Formula: see text]. Transmission electron microscopy revealed the distinguishable features of the sonicated nanovesicles depending on the compositions. The entrapment efficiency of RB was observed in the order of [Formula: see text]. At the end of 10[Formula: see text]h, RB permeated through the Strat-M[Formula: see text] membrane at [Formula: see text], [Formula: see text] and [Formula: see text]g/cm2 from the LF1, EF1 and TF1 containing RB, respectively. The flux ([Formula: see text]) of TF1 was obtained 5.53 times higher than RB itself. Therefore, the TF1 nanovesicle allowed the highly efficient permeation and entrapment of hydrophilic RB compared to LF1 and EF1 nanovesicles. Hence, it can be concluded that transethosomes would be a better choice for hydrophilic compounds to cross the skin barriers via the transdermal route.

Aerosolizable Lipid-Nanovesicles Encapsulating Voriconazole Effectively Permeate Pulmonary Barriers and Target Lung Cells.

The entire world has recently been witnessing an unprecedented upsurge in microbial lung infections. The major challenge encountered in treating the same is to ensure the optimum drug availability at the infected site. Aerosolization of antimicrobials, in this regard, has shown immense potential owing to their localized and targeted effect. Efforts, therefore, have been undertaken to systematically develop lung-phosphatidylcholine-based lipid nanovesicles of voriconazole for potential management of the superinfections like aspergillosis. LNVs, prepared by thin-film hydration method, exhibited a globule size of 145.4 ± 19.5 nm, polydispersity index of 0.154 ± 0.104 and entrapment efficiency of 71.4 ± 2.2% with improved in vitro antifungal activity. Aerodynamic studies revealed a microdroplet size of ≤5 μm, thereby unraveling its promise to target the physical barrier of lungs effectively. The surface-active potential of LNVs, demonstrated through Langmuir-Blodgett troughs, indicated their ability to overcome the biochemical pulmonary surfactant monolayer barrier, while the safety and uptake studies on airway-epithelial cells signified their immense potential to permeate the cellular barrier of lungs. The pharmacokinetic studies showed marked improvement in the retention profile of voriconazole in lungs following LNVs nebulization compared to pristine voriconazole. Overall, LNVs proved to be safe and effective delivery systems, delineating their distinct potential to efficiently target the respiratory fungal infections.

Tumour-derived extracellular vesicle membrane hybrid lipid nanovesicles enhance siRNA delivery by tumour-homing and intracellular freeway transportation.

Extracellular vesicles (EVs) have been proved a promising small interfering RNA (siRNA) delivery vehicle to mediate gene‐silencing. Tumour‐derived extracellular vesicles (TDEVs) as genetic exchange vectors in the tumour microenvironment, enable intercellular communication for a wide range of endogenous cargo molecules, such as RNAs and proteins. However, the oncogenic cargo of TDEVs limits their application in siRNA delivery for cancer therapy. Herein, we isolated TDEVs from hepatocellular carcinoma (HCC) cells and derived TDEV membranes by abandoning their content. Innovative TDEV membrane hybrid lipid nanovesicles (LEVs) were then fabricated by fusion of TDEV membranes and phospholipids to realize precise delivery to tumours and highly efficient transfection of siRNA. The TDEV membranes endow LEVs with ‘homing’ targeting ability, facilitating specific internalisation into parent HCC cells primarily through heparan sulfate proteoglycan‐mediated pathways. Unlike conventional lipid‐based nanovesicles, LEVs can bypass the endosomal degradation pathway, boost the delivery of siRNA through the Golgi and endoplasmic reticulum (ER) intracellular ‘freeway’ transportation, achieving a 1.7‐fold improvement in siRNA transfection efficiency compared with liposomes. Additionally, siRNA loaded LEVs were demonstrated to enhance the antitumour efficacy in HCC bearing mice through effective gene silencing in the tumour sites. Our results highlight the potential application of the TDEV membrane‐derived nanovesicles as an advanced siRNA delivery strategy for cancer therapy.

New repurposed rolapitant in nanovesicular systems for lung cancer treatment: Development, in-vitro assessment and in-vivo biodistribution study

Lung cancer is characterized by poor prognosis, and is considered a serious disease that causes a significant mortality. The available conventional chemotherapeutic agents suffer from several limitations; hence, new drug molecules are constantly being sought. In the current study, lipid nanovesicles (LNVs) were selected as a colloidal vehicle for encapsulation of the FDA-approved drug; rolapitant (RP), which is used particularly for the treatment of nausea and vomiting, but is repurposed for the treatment of lung cancer in the current work. RP was loaded into various LNVs (liposomes, ethosomes and transethosomes) using the thin film hydration method, and the LNVs were evaluated for particle size, zeta potential, entrapment efficiency (EE%), storage stability and surface morphology. Besides, the in-vitro drug release, in-vitro cytotoxicity on A549 lung cancer cells, nebulization performance using next generation impactor (NGI), and the in-vivo biodistribution behavior were evaluated. The selected ethosomal and transethosomal vesicles displayed a particle size less than 400 nm, a positive charge, and EE% exceeding 90% for RP, with a sustained release pattern over 15 days. The in-vivo biodistribution results proved the high lung deposition potential of RP-LNVs with a considerable safety. Besides, the developed RP-LNVs were able to reach the metastatic organs of lung cancer, hence they were proven promising as a possible treatment modality for lung cancer.

Controlled rate slow freezing with lyoprotective agent to retain the integrity of lipid nanovesicles during lyophilization

We designed a novel lyophilization method using controlled rate slow freezing (CSF) with lyoprotective agent (LPA) to achieve intact lipid nanovesicles after lyophilization. During the freezing step, LPA prevented water supercooling, and the freezing rate was controlled by CSF. Regulating the freezing rate by various liquid media was a crucial determinant of membrane disruption, and isopropanol (freezing rate of 0.933 °C/min) was the optimal medium for the CSF system. Lyophilized lipid nanovesicle using both CSF and LPA retained 92.9% of the core material and had uniform size distributions (Z-average diameter = 133.4 nm, polydispersity index = 0.144), similar to intact vesicles (120.7 nm and 0.159, respectively), after rehydration. Only lyophilized lipid nanovesicle using both CSF and LPA showed no changes in membrane fluidity and polarity. This lyophilization method can be applied to improve storage stability of lipid nanocarriers encapsulating drugs while retaining their original activity.

Engineering pH-Sensitive Stable Nanovesicles for Delivery of MicroRNA Therapeutics.

MicroRNAs (miRNAs) are small non-coding endogenous RNAs, which are attracting a growing interest as therapeutic molecules due to their central role in major diseases. However, the transformation of these biomolecules into drugs is limited due to their unstability in the bloodstream, caused by nucleases abundantly present in the blood, and poor capacity to enter cells. The conjugation of miRNAs to nanoparticles (NPs) could be an effective strategy for their clinical delivery. Herein, the engineering of non-liposomal lipid nanovesicles, named quatsomes (QS), for the delivery of miRNAs and other small RNAs into the cytosol of tumor cells, triggering a tumor-suppressive response is reported. The engineered pH-sensitive nanovesicles have controlled structure (unilamellar), size (<150nm) and composition. These nanovesicles are colloidal stable (>24 weeks), and are prepared by a green, GMP compliant, and scalable one-step procedure, which are all unavoidable requirements for the arrival to the clinical practice of NP based miRNA therapeutics. Furthermore, QS protect miRNAs from RNAses and when injected intravenously, deliver them into liver, lung, and neuroblastoma xenografts tumors. These stable nanovesicles with tunable pH sensitiveness constitute an attractive platform for the efficient delivery of miRNAs and other small RNAs with therapeutic activity and their exploitation in the clinics.

Stiffness of Fluid and Gel Phase Lipid Nanovesicles: Weighting the Contributions of Membrane Bending Modulus and Luminal Pressurization

The mechanical properties of biogenic membranous compartments are thought to be relevant in numerous biological processes; however, their quantitative measurement remains challenging for most of the already available force spectroscopy (FS)-based techniques. In particular, the debate on the mechanics of lipid nanovesicles and on the interpretation of their mechanical response to an applied force is still open. This is mostly due to the current lack of a unified model being able to describe the mechanical response of both gel and fluid phase lipid vesicles and to disentangle the contributions of membrane rigidity and luminal pressure. In this framework, we herein propose a simple model in which the interplay of membrane rigidity and luminal pressure to the overall vesicle stiffness is described as a series of springs; this approach allows estimating these two contributions for both gel and fluid phase liposomes. Atomic force microscopy-based FS, performed on both vesicles and supported lipid bilayers, is exploited for obtaining all the parameters involved in the model. Moreover, the use of coarse-grained full-scale molecular dynamics simulations allowed for better understanding of the differences in the mechanical responses of gel and fluid phase bilayers and supported the experimental findings. The results suggest that the pressure contribution is similar among all the probed vesicle types; however, it plays a dominant role in the mechanical response of lipid nanovesicles presenting a fluid phase membrane, while its contribution becomes comparable to the one of membrane rigidity in nanovesicles with a gel phase lipid membrane. The results presented herein offer a simple way to quantify two of the most important parameters in vesicle nanomechanics (membrane rigidity and internal pressurization), and as such represent a first step toward a currently unavailable, unified model for the mechanical response of gel and fluid phase lipid nanovesicles.

Application of Asymmetrical Flow Field-Flow Fractionation for Characterizing the Size and Drug Release Kinetics of Theranostic Lipid Nanovesicles.

Liposome size and in vitro release of the active substance belong to critical quality attributes of liposomal carriers. Here, we apply asymmetric flow field-flow fractionation (AF4) to characterize theranostic liposomes prepared by thin lipid film hydration/extrusion or microfluidics. The vesicles’ size was derived from multi-angle laser light scattering following fractionation (AF4) and compared to sizes derived from dynamic light scattering measurements. Additionally, we adapted a previously developed AF4 method to study zinc phthalocyanine (ZnPc) release/transfer from theranostic liposomes. To this end, theranostic liposomes were incubated with large acceptor liposomes serving as a sink (mimicking biological sinks) and were subsequently separated by AF4. During incubation, ZnPc was transferred from donor to acceptor fraction until reaching equilibrium. The process followed first-order kinetics with half-lives between 119.5–277.3 min, depending on the formulation. The release mechanism was postulated to represent a combination of Fickian diffusion and liposome relaxation. The rate constant of the transfer was proportional to the liposome size and inversely proportional to the ZnPc/POPC molar ratio. Our results confirm the usefulness of AF4 based method to study in vitro release/transfer of lipophilic payload, which may be useful to estimate the unwanted loss of drug from the liposomal carrier in vivo.

Improving Dermal Delivery of Rose Bengal by Deformable Lipid Nanovesicles for Topical Treatment of Melanoma

Cutaneous melanoma is one of the most aggressive and metastatic forms of skin cancer. However, current therapeutic options present several limitations, and the annual death rate due to melanoma increases every year. Dermal delivery of nanomedicines can effectively eradicate primary melanoma lesions, avoid the metastatic process, and improve survival. Rose Bengal (RB) is a sono-photosensitizer drug with intrinsic cytotoxicity toward melanoma without external stimuli but the biopharmaceutical profile limits its clinical use. Here, we propose deformable lipid nanovesicles, also known as transfersomes (TF), for the targeted dermal delivery of RB to melanoma lesions to eradicate them in the absence of external stimuli. Considering RB’s poor ability to cross the stratum corneum and its photosensitizer nature, transfersomal carriers were selected simultaneously to enhance RB penetration to the deepest skin layers and protect RB from undesired photodegradation. RB-loaded TF dispersion (RB-TF), prepared by a modified reverse-phase evaporation method, were nanosized with a ζ-potential value below −30 mV. The spectrophotometric and fluorimetric analysis revealed that RB efficiently interacted with the lipid phase. The morphological investigations (transmission electron microscopy and small-angle X-ray scattering) proved that RB intercalated within the phospholipid bilayer of TF originating unilamellar and deformable vesicles, in contrast to the rigid multilamellar unloaded ones. Such outcomes agree with the results of the in vitro permeation study, where the lack of a burst RB permeation peak for RB-TF, observed instead for the free drug, suggests that a significant amount of RB interacted with lipid nanovesicles. Also, RB-TF proved to protect RB from undesired photodegradation over 24 h of direct light exposure. The ex vivo epidermis permeation study proved that RB-TF significantly increased RB’s amount permeating the epidermis compared to the free drug (78.31 vs 38.31%). Finally, the antiproliferative assays on melanoma cells suggested that RB-TF effectively reduced cell growth compared to free RB at the concentrations tested (25 and 50 μM). RB-TF could potentially increase selectivity toward cancer cells. Considering the outcomes of the characterization and cytotoxicity studies performed on RB-TF, we conclude that RB-TF represents a valid potential alternative tool to fight against primary melanoma lesions via dermal delivery in the absence of light.

Hydrolysable tannins change physicochemical parameters of lipid nano-vesicles and reduce DPPH radical - Experimental studies and quantum chemical analysis

Tannins belong to plant secondary metabolites exhibiting a wide range of biological activity. One of the important aspects of the realization of the biological effects of tannins is the interaction with lipids of cell membranes. In this work we studied the interaction of two hydrolysable tannins: 1,2,3,4,6-penta-O-galloyl-β-d-glucose (PGG) and 1,2-di-O-galloyl-4,6-valoneoyl-β-d-glucose (T1) which had the same number of both aromatic rings (5) and hydroxyl groups (15) but differing in flexibility due to the presence of valoneoyl group in the T1 molecule with DMPC (dimyristoylphosphatidylcholine) lipid nano-vesicles (liposomes). Tannins-liposomes interactions were investigated using fluorescence spectroscopy, differential scanning calorimetry, laser Doppler velocimetry, dynamic light scattering and Fourier Transform Infra-Red spectroscopy. It was shown that more flexible PGG molecules stronger decreased the microviscosity of the liposomal membranes and increased the values of negative zeta potential in comparison with the more rigid T1. Both compounds diminished the phase transition temperature of DMPC membranes, interacted with liposomes via PO groups of head of phospholipids and their hydrophobic regions. These tannins neutralized DPPH free radicals with the stoichiometry of the reaction equal 1:1.The effects of the studied compounds on liposomes were discussed in relation to tannin quantum chemical parameters calculated by molecular modeling.