Liposomes Research Articles

Development of Liposomes That Target Axon Terminals Encapsulating Berberine in Cultured Primary Neurons.

Most of the energy in neurons is produced in mitochondria. Mitochondria generate the ATP that is essential for neuronal growth, function, and regeneration. Mitochondrial axonal transport plays a crucial role in maintaining neuronal homeostasis and biological activity. Decreased mitochondrial axonal transport at axon terminals, where the metabolism of substances is likely to be delayed, may contribute to neurological dysfunction. Therefore, regulation of mitochondrial dynamics at axon terminals has attracted considerable interest as a strategy to modulate neuronal function. Nanoparticles may be useful in controlling local mitochondrial dynamics. Nevertheless, there are few reports on the influence of drug delivery that nanoparticles impart on the mitochondrial dynamics in neurons. This paper reports the results of a study using liposomes (LPs) to examine local drug delivery and pharmacological actions on neurons. We tested berberine (BBR), which is an activator of AMP-activated protein kinase (AMPK), to examine the utility of this drug as a cellular energy sensor. Axon terminals targeting LPs were prepared. The amount of axon terminals targeting LPs was increased compared with treatment using cationic LPs. Moreover, axon terminal-targeting LPs increased anterograde transport by about 40% compared with that of either naked BBR or cationic LPs and suppressed axonal retraction. Our findings suggest that local drug delivery to neurons is important for enhancing pharmacological activity in axon terminals.

CD4+ T cell-targeting immunoliposomes for treatment of latent HIV reservoir

Active targeting nano-delivery is a promising approach to enhance therapeutic efficacy and specificity to the target cells. Liposomes (LPs) have been widely studied for the active targeting delivery due to their low toxicity, biodegradability, biocompatibility, and feasibility of surface medication to provide the interactions with cell receptors. One of the strategies is to functionalize the surface of LPs with monoclonal antibodies (mAbs) to obtain immunoliposomes (imLPs) that recognize specific receptors on target cells. Among several target cells, CD4+ T cells are known for playing a pivotal role in controlling the immune system in several diseases, including cancers, inflammatory diseases, and viral infections, particularly HIV-1. Here, we demonstrate two methods for conjugating αCD4 mAb with imLPs for specific targeting of CD4+ T cells that can harbor viral genome and serve as a predominant latent HIV reservoir. LPs conjugated with αCD4 mAb via neutravidin-biotin linkage were used for selectively targeting CD4+ J-Lat 10.6 cells. We demonstrate, via flow cytometry, the importance of the conjugation step, mAb density, and the presence of polyethylene glycol (PEG) for effective drug delivery to CD4+ T cells. The cellular uptake of imLPs is substantially higher if the imLPs are functionalized with the pre-conjugated αCD4 mAb-neutravidin complex. Furthermore, imLPs loaded with HIV-1 latency reversing agent, suberoylanilide hydroxamic acid (SAHA), could reactivate the J-Lat 10.6 cells, suggesting that the αCD4-imLPs could be potentially used as a targeted drug delivery system for HIV-1 latency reactivation or other CD4-targeted immunotherapies.

Catechol chitosan coated dual-loaded liposomes based on oxidation and saccharification mechanisms for enhancing skin anti-aging effects

Skin aging has become a major urgent problem to be solved. Evidence reveals that oxidation and glycosylation are two dominant inducements of aging. Resveratrol (RES) with outstanding anti-oxidant effect and carnosine (CAR) with superb anti-glycation property were selected as two model drugs to evaluate the feasibility of their synergistic anti-aging effect. RES and CAR at the most desired mass ratio, supplying the most superior synergistic anti-aging effects were further encapsulated in liposomes (LP), which were separately coated with chitosan (CS) and catechol chitosan (Cat-CS) to increase the transdermal penetration. Their anti-aging efficacy was explored in human skin fibroblast (HSF) and human immortalized keratinocytes (HaCaT) cells, as well as the back skin of guinea pigs. Herein, RES and CAR at the mass ratio of 2:1 exhibited the most ideal synergistic anti-aging effect. The constructed liposomes have been shown to possess excellent fundamental properties and sustained-release properties. The aging-related indicator levels in the two cells and guinea pigs were obviously improved for the RES + CAR@Cat-CS-LP group. Additionally, skin appearance, tissue morphology, and collagen content were visibly improved, indicating its perfect anti-aging effect. In conclusion, RES + CAR@Cat-CS-LP is expected to be exploited as a potential anti-aging drug delivery system.

Quercetin‐encapsulated magnetoliposomes: Fabrication, optimization, characterization, and antioxidant studies

AbstractQuercetin (QU) faces challenges in its therapeutic efficacy due to its hydrophobic nature and limited oral bioavailability. Using a Box–Behnken design (BBD) approach, we developed QU‐loaded magnetoliposomes (QMLs) to address these limitations. By encapsulating QU within iron oxide nanoparticles (IONPs) and liposomes (LPs), we enhanced its hydrophilicity and improved its potential for drug delivery. Through systematic adjustments of phosal, polyvinyl alcohol, and magnetic/IONPs, we optimized the particle size, zeta potential, and iron content of the QMLs. The formulations underwent comprehensive structural characterization using techniques, such as Fourier transform infrared spectroscopy, X‐Ray diffraction, differential scanning calorimetry–thermogravimetric analysis, and energy‐dispersive X‐ray analysis, whereas their morphology was examined through field emission scanning electron microscopy. Furthermore, we evaluated the in vitro drug release of the QMLs and antioxidant activity of QU, QU‐loaded LPs, and QMLs using DPPH, 2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulfonic acid), and H2O2 scavenging assays, enabling us to compare their antioxidant potential and the efficiency of QU encapsulation within the magneto LPs.Practical Applications: This research holds significant practical implications, particularly in targeted drug delivery using magnetic liposomes. The developed system shows promise in enhancing cancer therapy, providing localized treatment for inflammation‐related conditions, delivering drugs to the brain to address neurological disorders, promoting wound healing, and incorporating quercetin into skincare products for its antioxidant and antiaging benefits.

Liposome Vaccine for Active Regulation of Cellular and Humoral Immunity.

Despite significant progress in vaccine development, especially in the fight against viral infections, many unexplored areas remain including innovative adjuvants, diversification of vaccine formulations, and research into the coordination of humoral and cellular immune mechanisms induced by vaccines. Effective coordination of humoral and cellular immunity is crucial in vaccine design. In this study, we used the spike protein (S) of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) or ovalbumin (OVA) as antigen models and CpG DNA (an activator of toll-like receptor 9, TLR9) as an adjuvant to prepare a multitargeted liposome (LIPO) vaccine. Once equipped with the ability to target lymph nodes (LN) and the endoplasmic reticulum (ER), the LIPO vaccine significantly enhances the cross-presentation ability of antigen-presenting cells (APCs) for exogenous antigens through the ER-associated protein degradation (ERSD) mechanism. Additionally, the vaccine could fine-tune the efficiency of ER-targeted antigen delivery, actively regulating the presentation of exogenous antigen proteins via the major histocompatibility complex (MHC-I) or MHC-II pathways. Immune data from in vivo mouse experiments indicated that the LIPO vaccine effectively stimulated both humoral and cellular immune responses. Furthermore, it triggers immune protection by establishing a robust and persistent germinal center. Moreover, the multifunctionality of this LIPO vaccine extends to the fields of cancer, viruses, and bacteria, providing insights for skilled vaccine design and improvement.

Enhancing stability of curcumin-loaded casein nanoparticles by adding liposomal nanoparticles

Curcumin (Cur) is a natural polyphenol found in turmeric. Its application as a functional ingredient in foods, supplements, and pharmaceuticals is dramatically limited due to its poor stability, water solubility, and bioavailability. The purpose of this research was to enhance the performance of curcumin using advanced encapsulation technologies. Curcumin-loaded casein nanoparticles (CCNP) were prepared using the pH-driven method, and then different concentrations of liposomes (LP) were added to assess their impact on the stability of this delivery system. Then, CCNP, curcumin liposome nanoparticles (CLP), and the optimal ratio of CCNP/LP complexes were selected for thermal and storage stability studies. Their formation mechanism was studied by atomic force microscopy (AFM), isothermal titration calorimetry (ITC), and Fourier transform infrared (FTIR) methods. Furthermore, the impact of pH and salt (NaCl) on their stability was also investigated. The results show that LP can adsorb onto the surfaces of CCNP through hydrogen bonding and electrostatic attractive interactions at certain concentrations, which reduces the surface hydrophobicity of the nanoparticles, thereby improving their resistance to environmental stresses. This study provides information that can be used to improve the bioavailability of curcumin.

Macrophage membrane-coated SN-38-encapsulated liposomes for efficient treatment of colorectal cancer

BackgroundSN-38, recognized as the primary active derivative of the pivotal chemotherapeutic agent CPT-11, demonstrates substantially enhanced efficacy in colorectal cancer (CRC) management compared to CPT-11. Nonetheless, challenges such as low stability, inadequate aqueous solubility, limited bioavailability, and non-specific targeting to cancer cells hinder its clinical adoption. In the present research, we synthesized SN-38-loaded liposomes cloaked with macrophage membranes (SN-38@MM-LPs) to assess their therapeutic potential and safety profile in addressing CRC. MethodsSN-38@MM-LPs were synthesized using an incubation extrusion technique, combining a macrophage membrane with liposomes (LPs). It was characterized by size, zeta potential, transmission electron microscopy observations, polydispersity index and coomassie bright blue staining. CCK-8, EdU, and flow cytometry assays were performed to evaluate the viability and apoptosis rates of HCT116 and HCT8 cells after treatment with SN-38@MM-LPs. A cellular uptake assay was conducted to evaluate the internalization of SN-38@MM-LPs in vitro. Moreover, the biodistribution, therapeutic efficacy, and safety of SN-38@MM-LPs were further assessed in orthotopic HCT116 xenograft model mice. ResultsCharacterization results revealed that SN-38@MM-LPs possess a spherical morphology with a consistent size distribution (129 nm) and a drug loading efficiency of 5.54 ± 0.73%. SN-38 curtailed the growth and promoted apoptosis in both HCT8 and HCT116 cells. The impact of SN-38 was accentuated when delivered via SN-38@LPs and SN-38@MM-LPs. Notably, in the orthotopic xenograft model, SN-38@MM-LPs manifested superior tumor-targeting capabilities and therapeutic outcomes. Additionally, SN-38@MM-LPs presented negligible hepatic toxicity. ConclusionsSN-38@MM-LPs showcased potent and targeted antitumor actions in CRC. Consequently, SN-38@MM-LPs emerge as a potential nanoparticle formulation that could amplify the antitumor efficacy of SN-38, simultaneously mitigating liver toxicity concerns.

Development of novel tracers for sentinel node identification in cervical cancer.

Indocyanine green (ICG) with near-infrared (NIR) fluorescence imaging is used for lymphatic mapping. However, binding of ICG to blood proteins like serum albumin can shorten its retention time in sentinel lymph nodes (SLNs). Here, we investigated the efficacy and safety of a new fluorescence tracer comprising phytate and liposome (LP)-encapsulated ICG. Coadministration of phytate with LP containing phosphatidic acid promotes chelation mediated by Ca2+ in bodily fluids to enhance SLN retention. Uniformly sized LPs (100 nm) encapsulating ICG under conditions that minimized fluorescence self-quenching during storage were produced. We analyzed the behavior of the new tracer (ICG-phytate-LP) and control tracers (ICG and ICG-LP) in the lymphatic flow of mice in terms of lymph node retention time. We also tested lymphatic flow and safety in pigs that have a more human-like lymphatic system. LPs encapsulating stabilized ICG were successfully prepared. Mixing LP with phytate in the presence of Ca2+ increased both the particle size and negative surface charge. In mice, ICG-phytate-LP had the best lymph node retention, with a fluorescence intensity ratio that increased over 6 h and then decreased slowly over the next 24 h. In pigs, administration of ICG and ICG-phytate-LP resulted in no death or weight loss. There were no obvious differences between blood test results for the ICG and ICG-phytate-LP groups, and the overall safety was good. ICG-phytate-LP may be a useful new tracer for gynecological cancers that require time for lymph node identification due to a retroperitoneal approach.

Carboxymethyl chitosan and octadecylamine-coated liposome-containing WPTS: design, optimization, and evaluation

Liposomes (LPs) are a delivery system for stabilizing pharmaceuticals with limited use due to their propensity to congregate and fuse. A proposed method of addressing these problems is polymer coating. In this study, the potential of octadecylamine (ODA)-coated liposomes and carboxymethyl chitosan (CMCS/ODA-LPs) for enhancing Wacao pentacyclic triterpene saponin (WPTS) transport capacity was investigated. CMCS/ODA-LPs were produced by electrostatic adsorption and thin-film hydration. Response surface methodology (RSM) was employed to enhance the process and encapsulation efficiency (EE) for optimum drug encapsulation efficiency. The synthesized WPTS-CMCS/ODA-LPs were uniformly dispersed in a circular shape, and during 14 days of storage at 4 °C, the particle size and morphology did not significantly change. Vesicle size, zeta potential, polydispersity index (PDI), and entrapment efficiency (%) were 179.1 ± 7.31 nm, −29.6 ± 1.35 mV, 0.188 ± 0.052, and 75.62 ± 0.43, respectively. The hemolysis test revealed that WPTS-CMCS/ODA-LPs were sufficiently biocompatible. Compared to WPTS-LPs, WPTS-CMCS/ODA-LPs consistently showed a much more significant cytotoxic effect on cancer cells. Early and WPTS-CMCS/ODA-LPs-induced apoptosis resulted in almost seven times more cell death than the control. Compared to physiological pH 7.3, the pH-sensitive CMCS coupled LPs increased drug release at acidic pH 6.5. These findings suggest the efficacy of pH-sensitive CMCS/ODA-LPs as a medication delivery method for WPTS.

Combining microfluidics and coaxial 3D-bioprinting for the manufacturing of diabetic wound healing dressings

Diabetic foot ulcers (DFUs) are a crucial complication of diabetes, as in a diabetic wound, each step of the physiological healing process is affected. This entails a more easily infectable wound, and delayed tissue regeneration due to the inflammation that occurs, leading to a drastic decrease in the overall patient's quality of life. As a strategy to manage DFUs, skin alternatives and wound dressings are currently receiving a lot of attention as they keep the wound environment “under control”, while providing bioactive compounds that help to manage infection and inflammation and promote tissue repair. This has been made possible thanks to the advent of emerging technologies such as 3D Bioprinting to produce skin resembling constructs or microfluidics (MFs) that allows the manufacture of nanoparticles (NPs) that act as drug carriers, in a prompt and less expensive way.In the present proof-of-concept study, the possibility of combining two novel and appealing techniques in the manufacturing of wound dressings has been demonstrated for first time. The novelty of this work consists in the combination of liposomes (LPs) encapsulating the active pharmaceutical ingredient (API) into a hydrogel that is further printed into a three-dimensional scaffold for wound dressing; to the knowledge of the authors this has never been done before.A grid-shaped scaffold has been produced through the coaxial 3D bioprinting technique which has allowed to combine, in one single filament, two different bioinks. The inner core of the filament is a nanocomposite hydrogel consisting of hydroxyethyl cellulose (HEC) and PEGylated LPs encapsulated with thyme oil (TO) manufactured via MFs for the first time. The outer shell of the filament, instead, is represented by a hybrid hydrogel composed of sodium alginate/cellulose nanocrystals (SA/CNC) and enriched with free TO. This provides a combination of two different release ratios of the API, a bulk release for the first 24 h thanks to the free TO in the shell of the filament and a sustained release for up to 10 days provided from the API inside the LPs. Confocal Microscopy verified the actual presence of the LPs inside the scaffold after printing and evaluation using the zone of inhibition test proved the antibacterial activity of the manufactured scaffolds against both Gram-positive and Gram-negative bacteria.

CD71-mediated liposomal arsenic-nickel complex combined with all-trans retinoic acid for the efficacy of acute promyelocytic leukemia

Clinically, arsenic trioxide (ATO) was applied to the treatment of acute promyelocytic leukemia (APL) as a reliable and effective frontline drug. However, the administration regimen of AsⅢ was limited due to its fast clearance, short therapeutic window and toxicity as well. Based on CD71 overexpressed on APL cells, in present study, a transferrin (Tf)-modified liposome (LP) was established firstly to encapsulate AsⅢ in arsenic-nickel complex by nickel acetate gradient method. The AsⅢ-loaded liposomes (AsLP) exhibited the feature of acid-sensitive release in vitro. Tf-modified AsLP (Tf-AsLP) were specifically taken up by APL cells and the acidic intracellular environment triggered liposome to release AsⅢ which stimulated reactive oxygen species level and caspase-3 activity. Tf-AsLP prolonged half-life of AsⅢ in blood circulation, lowered systemic toxicity, and promoted apoptosis and induced cell differentiation at lesion site in vivo. Considering that ATO combined with RA is usually applied as the first choice in clinic for APL treatment to improve the therapeutic effect, accordingly, a Tf-modified RA liposome (Tf-RALP) was designed to reduce the severe side effects of free RA and assist Tf-AsLP for better efficacy. As expected, the tumor inhibition rate of Tf-AsLP was improved significantly with the combination of Tf-RALP on subcutaneous tumor model. Furthermore, APL orthotopic NOD/SCID mice model was established by 60CO irradiation and HL-60 cells intravenously injection. The effect of co-administration (Tf-AsLP + Tf-RALP) was also confirmed to conspicuous decrease the number of leukemia cells in the circulatory system and prolong the survival time of APL mice by promoting the APL cells’ apoptosis and differentiation in peripheral blood and bone marrow. Collectively, Tf-modified acid-sensitive AsLP could greatly reduce the systemic toxicity of free drug. Moreover, Tf-AsLP combined with Tf-RALP could achieve better efficacy. Thus, transferrin-modified AsⅢ liposome would be a novel clinical strategy to improve patient compliance, with promising translation prospects.

Precision Localization of Lipid‐Based Nanoparticles by Dual‐Fluorescent Labeling for Accurate and High‐Resolution Imaging in Living Cells

In nanomedicine, lipid‐based nanoparticles (NPs) such as liposomes (LPs) have established an important position. Precise delineation of NP interaction with cells and detailed characterization of activity are becoming essential, which mainly rely on labeling with lipophilic fluorescent molecules and assuming stable association with NPs. However, because of label separation from NPs in (biological) media, or when processed by cells, fluorescence‐based detection of an NP incorporating a single label may not necessarily indicate the actual presence of an NP but may be from the dissociated label, rendering results unreliable. Herein, flow cytometry and confocal microscopy are employed to demonstrate that to verify the localization of LPs in a cell with perfect accuracy, dual‐labeling, and contemporaneous detection of both fluorescent signals in one pixel are required. This is combined with size exclusion chromatography (SEC) and mass spectrometry measurements to indicate factors involved in label dissociation, which helps to understand the possible conditions of dissociated label and NP. It is shown that determining label colocalization with, and label dissociation from, dual‐labeled NPs are needed to provide accurate spatiotemporal insight into targeting destination (colocalized signals) and disintegration (separated signals) of NPs during intracellular processing and in studying payload delivery with precision in nanomedicine.

PH-sensitive liposomes bearing a chemotherapeutic agent and a natural apoptosis modulator for effective intracellular delivery to the solid tumor.

The intracellular delivery of the drug to the solid tumor is a major challenge in the treatment of solid tumors. This project aims to increase cytosolic drug delivery using the endosomal escape of drugs. Topotecan (TPT) and capsaicin were used for the treatment of solid tumors. The pH-dependent conversion of active lactone form to inactive carboxylic form is a major problem of TPT that limits its therapeutic use. Liposomal encapsulation of TPT improved the stability of active lactone form and increased the therapeutic efficacy of TPT. Endosomal degradation of liposomes may reduce the content in the target cells. To solve these problems, pH-sensitive liposomes (pSLPs) were developed which improved the intracellular drug delivery by the endosomal escape of drugs. The liposomes (LPs) bearing the drug(s) were prepared using the cast film method and optimized for various formulation and process variables using the Design-Expert 7 software by employing the Box-Behnken design (BBD). The developed hyaluronic acid (HA)-conjugated pSLPs (HA-pSLPs) displayed a vesicle size of 166.5 ± 2.31nm, zeta potential - 30.53 ± 0.91, and entrapment efficiency of 44.39 ± 1.78%, and 73.48 ± 2.15% for TPT and CAP, respectively. HA-pSLPs displayed better cytotoxicity in comparison to free drugs either single or in combination on the MCF-7 cell line. The apoptosis and cellular uptake of HA-pSLPs were increased ⁓ 4.45-fold and ⁓ 6.95-fold as compared to unconjugated pSLPs, respectively. The pharmacokinetic studies in Balb/c mice demonstrated that HA-pSLPs increased the half-life, MRT, and AUC in comparison to the free drug solution. The HA-pSLPs formulation has shown remarkable tumor regression as compared to PpSLPs, pSLPs, and free drug combinations. These results demonstrated that TPT- and CAP-loaded HA-pSLPs offer a potential platform for targeted drug delivery to solid tumors.

Current advances in non‐viral gene delivery systems: Liposomes versus extracellular vesicles

AbstractIn the past decade, nucleic acid‐based drugs have emerged as an extremely promising approach for silencing specific disease‐related genes and targeting undruggable ones. However, most nucleic acid drug therapies have not advanced to clinical practice due to their poor stability against serum enzyme degradation and cytotoxicity. Nanoscale drug delivery vehicles show potential to improve efficacy of nucleic acid drugs via targeted delivery to disease‐causing genes, yet, safe and efficient nanocarriers remain elusive. Lipid‐based nanoparticles such as liposomes (LSs) and extracellular vesicles (EVs) are among the most extensively exploited nanoscale vehicles for therapeutic cargo delivery. LS‐based nucleic acid therapies have been used for several years, with many already adopted to the clinic. More recently, EVs hold considerable promise as nucleic acid delivery vehicles due to their high biocompatibility, low immunogenicity, and the inherent abilities to interact with target cells and cross biological barriers. Moreover, a novel LS/EV hybrid gene delivery system has been engineered to preserve the benefits of both systems and generate an advanced drug delivery system. The current review focuses specifically on LS‐ and EV‐based gene therapies and provides the key advantages and shortcomings of these systems with particular emphasis on their potential use as therapeutic vectors.

Abstract 2711: Smart encapsulation of a MEK inhibitor into M2-9SLP/mApoE-liposomes for specific GBM targeting

Abstract Among the main aberrations occurring in GBM, those in MEK/ERK and PI3K/akt/mTOR pathways predominate and confer GBM Stem-like Cells (GSCs) sustained proliferation and resistance to therapy. A panel of eight patient-derived primary GSCs lines have been screened for their sensitivity to a small kinase MEK inhibitor (MEKi) with AnnexinV/PI staining. Among these, five display a sensitive phenotype with at least 50% reduction on cell viability after 72 hours of treatment. Then, four cell lines, two MEKi-sensitive (ICH001 and ICH003) and two MEKi-resistant (ICH013 and ICH027) were selected for a deeper molecular characterization based on MGMT methylation status, mesenchymal index and main hotspot mutations associated with GBM pathology MEKi incubation on GSC caused a prompt phospho-ERK reduction already after 3 hours. Of note, we report a concomitant activation of AKT and downstream molecules pointing to an ERK-mTOR redundant activity. To this end, we combined MEKi to PI3K/akt/mTOR inhibitor and we observed an increased cell death even in GSCs displaying moderate sensitivity to MEKi as single-agent (MEKi: 90% vs MEKi-PI3K/akt/mTOR inhibitor: 30% cell viability). Then, MEKi ability to cross the Blood Brain Barrier (BBB) and target GBM cells was investigated using a transwell BBB in vitro model. The PI3K/akt/mTOR pathway inhibitor, known from the literature to readily cross the BBB, was included as positive control. Obtained results showed MEKi inability to efficiently cross the BBB, thus limiting its utility as GBM therapy. These results suggest the need for a specific drug delivery strategy in the brain that might be therapeutically effective. Recently our laboratory has provided proof-of-concept of a combination strategy based on radiation and adjuvant drug-loaded liposomes (LPs) conjugated with a modified Apolipoprotein E-derived peptide (mApoE), known to facilitate BBB crossing. To strengthen therapeutic efficacy and to lower off-target effects, we implemented mApoE-LPs with a matrix metalloproteinases 2 and 9 sensitive lipopeptide (M2-9SLP) that allows controlled payload release only in the tumor microenvironment rich in MMPs. To this end, MEKi was encapsulated into the M2-9SLP/mApoE-LPs, and its capacity to promote cell death was evaluated. M2-9SLP/mApoE-MEKi-LPs caused in all the sensitive cell lines GSCs proliferation inhibition and induction of apoptosis upon 72h in vitro treatment indicating that the encapsulation process did not alter drug efficacy. In conclusion, our in vitro results support MEKi encapsulation into M2P/mApoE-LP as nanotherapeutic strategy that could guarantee specific delivery of MEKi in a MMP2-enriched tumor microenvironment without altering its capacity to inhibit GSC proliferation and survival. Funding by FRRB grant NEVERMIND (CP2_16/2018) Citation Format: Milena Mattioli, Elisabetta Stanzani, Valentino Ribecco, Marco Pizzocri, Eliana Lauranzano, Margherita Maria Ravanelli, Simone Olei, Maria Pia Tropeano, Pierfausto Seneci, Francesca Re, Federico Pessina, Michela Matteoli, Lorena Passoni. Smart encapsulation of a MEK inhibitor into M2-9SLP/mApoE-liposomes for specific GBM targeting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2711.

Microneedle-mediated delivery of Ziconotide-loaded liposomes fused with exosomes for analgesia.

Ziconotide (ZIC) is an N-type calcium channel antagonist for treating severe chronic pain that is intolerable, or responds poorly to the administration of other drugs, such as intrathecal morphine and systemic analgesics. As it can only work in the brain and cerebrospinal fluid, intrathecal injection is the only administration route for ZIC. In this study, borneol (BOR)-modified liposomes (LIPs) were fused with exosomes from mesenchymal stem cells (MSCs) and loaded with ZIC to prepare microneedles (MNs) to improve the efficiency of ZIC across the blood-brain barrier. To evaluate local analgesic effects of MNs, the sensitivity of behavioral pain to thermal and mechanical stimuli was tested in animal models of peripheral nerve injury, diabetes-induced neuropathy pain, chemotherapy-induced pain, and ultraviolet-B (UV-B) radiation-induced neurogenic inflammatory pain. BOR-modified LIPs loaded with ZIC were spherical or nearly spherical, with a particle size of about 95nm and a Zeta potential of -7.8mV. After fusion with MSC exosomes, the particle sizes of LIPs increased to 175nm, and their Zeta potential increased to -3.8mV. The nano-MNs constructed based on BOR-modified LIPs had good mechanical properties and could effectively penetrate the skin to release drugs. The results of analgesic experiments showed that ZIC had a significant analgesic effect in different pain models. In conclusion, the BOR-modified LIP membrane-fused exosome MNs constructed in this study for delivering ZIC provide a safe and effective administration for chronic pain treatment, as well as great potential for clinical application of ZIC.

Functional liposome loaded curcumin for the treatment of Streptococcus mutans biofilm.

Introduction: Plaque biofilms, mainly formed by Streptococcus mutans (S. mutans), play an important role in the occurrence and development of dental caries. Antibiotic treatment is the traditional way to control plaque. However, problems such as poor drug penetration and antibiotic resistance have encouraged the search for alternative strategies. In this paper, we hope to avoid antibiotic resistance through the antibacterial effect of curcumin, a natural plant extract with photodynamic effects, on S. mutans. However, the clinical application of curcumin is limited due to its low water solubility, poor stability, high metabolic rate, fast clearance rate, and limited bioavailability. In recent years, liposomes have become a widely used drug carrier due to their numerous advantages, such as high drug loading efficiency, high stability in the biological environment, controlled release, biocompatibility, non-toxic, and biodegradability. So, we constructed a curcumin-loaded liposome (Cur@LP) to avoid the defect of curcumin. Methods: Cur@LP functioned with NHS can adhere to the surface of the S. mutans biofilm by condensation reaction. Liposome (LP) and Cur@LP was characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). The cytotoxicity of Cur@LP was evaluated by CCK-8 assay and LDH assay. The adhesion of Cur@LP to S. mutans biofilm was observed by confocal laser scanning microscope (CLSM). The antibiofilm efficiency of Cur@LP were evaluated by crystal violet staining, CLSM, and scanning electron microscope (SEM). Results: The mean diameter of LP and Cur@LP were 206.67 ± 8.38nm and 312 ± 18.78nm respectively. The ζ-potential of LP and Cur@LP were ∼-19.3mV and ∼-20.8mV respectively. The encapsulation efficiency of Cur@LP was (42.61 ± 2.19) %, and curcumin was rapidly released up to ±21% at 2h. Cur@LP has negligible cytotoxicity, and can effectively adhered to the S. mutans biofilm and inhibited its growth. Discussion: Curcumin has been widely studied in many fields such as cancer, which can be attributed to its antioxidant and anti-inflammatory effects. At present, there are few studies on the delivery of curcumin to S. mutans biofilm. In this study, we verified the adhesion and antibiofilm of Cur@LP to S. mutans biofilm. This biofilm removal strategy has the potential to be translated into the clinic.

Raman Spectroscopy Characterization of Multi-Functionalized Liposomes as Drug-Delivery Systems for Neurological Disorders.

The characterization of nanoparticle-based drug-delivery systems represents a crucial step in achieving a comprehensive overview of their physical, chemical, and biological features and evaluating their efficacy and safety in biological systems. We propose Raman Spectroscopy (RS) for the characterization of liposomes (LPs) to be tested for the control of neuroinflammation and microglial dysfunctions in Glioblastoma multiforme and Alzheimer's disease. Drug-loaded LPs were functionalized to cross the blood-brain barrier and to guarantee localized and controlled drug release. The Raman spectra of each LP component were used to evaluate their contribution in the LP Raman fingerprint. Raman data analysis made it possible to statistically discriminate LPs with different functionalization patterns, showing that each molecular component has an influence in the Raman spectrum of the final LP formulation. Moreover, CLS analysis on Raman data revealed a good level of synthetic reproducibility of the formulations and confirmed their stability within one month from their synthesis, demonstrating the ability of the technique to evaluate the efficacy of LP synthesis using small amount of sample. RS represents a valuable tool for a fast, sensitive and label free biochemical characterization of LPs that could be used for quality control of nanoparticle-based therapeutics.

Pre-clinical safety of topically administered sunitinib-loaded lipid and polymeric nanocarriers targeting corneal neovascularization

Three different types of sunitinib-loaded (SUN-loaded) nanocarriers were compared, aiming at the topical treatment of corneal neovascularization (CNV): polymeric nanospheres (NS), liposomes (LIP), and solid lipid nanoparticles (SLN). Three out of eleven formulations prepared for an optimization study – the best SUN-loaded nanocarrier of each assessed type (NS, LIP, and SLN) – were selected, based on their size, polydispersity index (PdI), drug load (DL), and encapsulation efficiency (EE). These three optimal formulations were further characterized by nanoparticle tracking analysis (NTA), electron paramagnetic resonance (EPR) spectroscopy, and zeta potential. In vitro SUN release profiles were obtained for the optimal formulations, along with ex vivo corneal permeability/retention studies, and ocular tolerance assays, namely: the bovine corneal opacity and permeability (BCOP) assay, the HET-CAM test (hen’s egg test – chorioallantoic membrane), and hemolytic potential (HP) assay. None of the optimal formulations exhibited toxicity or potential for ocular irritation. SLN showed higher surface fluidity, drug release more suitable for topical ocular applications, besides greater SUN corneal retention. Our results suggest that SLN are the best CNV-targeting SUN-loaded nanocarriers for clinical translation when compared to their NS and LIP analogues.

Development and Comparative In Vitro and In Vivo Study of BNN27 Mucoadhesive Liposomes and Nanoemulsions for Nose-to-Brain Delivery

Intranasal administration offers an alternative and promising approach for direct nose-to-brain delivery. Herein, we developed two chitosan (CHT)-coated (and uncoated) nanoformulations of BNN27 (a synthetic C-17-spiro-dehydroepiandrosterone analogue), liposomes (LIPs), and nanoemulsions (NEs), and compared their properties and brain disposition (in vitro and in vivo). LIPs were formulated by thin film hydration and coated with CHT by dropwise addition. BNN27-loaded NEs (BNEs) were developed by spontaneous emulsification and optimized for stability and mucoadhesive properties. Mucoadhesive properties were evaluated by mucin adherence. Negatively charged CHT-coated LIPs (with 0.1% CHT/lipid) demonstrated the highest coating efficiency and mucoadhesion. BNEs containing 10% w/w Capmul-MCM and 0.3% w/w CHT demonstrated the optimal properties. Transport of LIP or NE-associated rhodamine-lipid across the blood-brain barrier (in vitro) was significantly higher for NEs compared to LIPs, and the CHT coating demonstrated a negative effect on transport. However, the CHT-coated BNEs demonstrated higher and faster in vivo brain disposition following intranasal administration compared to CHT-LIPs. For both BNEs and LIPs, CHT-coating resulted in the increased (in vivo) brain disposition of BNN27. Current results prove that CHT-coated NEs consisting of compatible nasal administration ingredients succeeded in to delivering more BNN27 to the brain (and faster) compared to the CHT-coated LIPs.