Use Of Lipid Nanoparticles Research Articles

SLN- and NLC-encapsulating antifungal agents: skin drug delivery and their unexplored potential for treating onychomycosis.

Lipid nanoparticles have been extensively studied for drug delivery of antifungal drugs, especially for dermatophytosis treatments. They can accumulate in skin appendages and release drugs in a controlled manner and also increase skin moisture, due to the formation of an occlusive film. Since moisture heavily influences nail and skin permeability, these systems seem to pose great potential for antifungal drug delivery. We therefore compare skin and nail physiopathological structure and discuss the potential use of lipid nanoparticles in managing skin and nail mycoses, highlighting their unexplored use in onychomycosis. Structural features become particularly relevant when treating local skin/nail disorders. Nail plate represents the most resistant barrier to the penetration of molecules. In recent years, at least 55 researches have been reported about lipid nanoparticles and, antifungal drugs. They have focused on production methods and nanoparticle ingredients influence on entrapment efficiency, fungal activity in vitro, stability, and drug release. Lipid nanoparticles such as SLN and NLC have shown great results in permeating the skin. Currently, however, there is just one study published using NLC applied directly to the nail plate. NLC containing voriconazole had a noteworthy impact on the penetration depth of a nanoencapsulated drug, which allowed its deeper penetration into porcine hooves than the unloaded drug. Evidence of the success of SLN and NLC in achieving high encapsulation efficiencies of antifungals and promoting cutaneous delivery indicates the potential of the systems in enhancing nail hydration and drug penetration into the nail plate.

Photosensitizer in lipid nanoparticle: a nano-scaled approach to antibacterial function

Photosensitization-based antimicrobial therapy (PAT) is an alternative therapy aimed at achieving bacterial inactivation. Researchers use various photosensitizers to achieve bacterial inactivation. However, the most widely used approach involves the use of photosensitizers dispersed in aqueous solution, which could limit the effectiveness of photodynamic inactivation. Therefore, the approaches to encapsulate the photosensitizer in appropriate vehicles can enhance the delivery of the photosensitizer. Herein, Toluidine Blue O (TBO) was the photosensitizer, and lipid nanoparticles were used for its encapsulation. The lipid nanoparticle-based delivery system has been tailor-made for decreasing the average size and viscosity and increasing the formulation stability as well as the wettability of skin. Usage of an appropriate vehicle will also increase the cellular uptake of the photosensitizer into the bacterial cells, leading to the damage on cell membrane and genomic DNA. Evidence of effectiveness of the developed PAT on planktonic bacteria and biofilms was examined by fluorescence microscopy and scanning electron microscopy. Lipid nanoparticles protected the photosensitizer from aggregation and made the application easy on the skin as indicated in data of size distribution and contact angle. The use of lipid nanoparticles for encapsulating TBO could enhance photosensitization-based antimicrobial therapy as compared to the aqueous media for delivering photosensitizers.

Effectiveness of nanoencapsulated methotrexate against osteosarcoma cells: in vitro cytotoxicity under dynamic conditions.

Cancer is a leading cause of mortality in the world, with osteosarcoma being one of the most common types among children between 1 and 14years old. Current treatments including preoperative chemotherapy, surgery and postoperative chemotherapy produce several side effects with limited effectiveness. The use of lipid nanoparticles as biodegradable shells for controlled drug delivery shows promise as a more effective and targeted tumor treatment. However, in vitro validation of these vehicles is limited due to fluid stagnation in current techniques, in which nanoparticles sediment onto the bottom of the wells killing the cells by asphyxiation. In the current series of experiments, results obtained with methotrexate-lipid nanoparticles under dynamic assay conditions are presented as a promising alternative to current free drug based therapies. Effects on the viability of the U-2 OS osteosarcoma cell line of recirculation of cell media, free methotrexate and blank and methotrexate containing lipid nanoparticles in a 11μM concentration were successfully assessed. In addition, several designs for the microfluidic platform used were simulated using COMSOL-Multiphysics, optimized devices were fabricated using soft-lithography and simulated parameters were experimentally validated. Nanoparticles did not sediment to the bottom of the platform, demonstrating the effectiveness of the proposed system. Moreover, encapsulated methotrexate was the most effective treatment, as after 72h the cell population was reduced nearly 40% while under free methotrexate circulation the cell population doubled. Overall, these results indicate that methotrexate-lipid nanoparticles are a promising targeted therapy for osteosarcoma treatment.

Lipid nanoparticles (SLN, NLC): Overcoming the anatomical and physiological barriers of the eye – Part II - Ocular drug-loaded lipid nanoparticles

In the recent decades, various controlled delivery systems have been introduced with the aim to improve solubility, stability and bioavailability of poorly absorbed drugs. Among all, lipid nanoparticles gather interesting properties as drug or gene delivery carriers. These systems, composed either of solid lipids (SLN) or of solid and liquid lipids (NLC) stabilized with surfactants, combine the advantages of other colloidal particles such as polymeric nanoparticles, fat emulsions and liposomes avoiding their main disadvantages. Lipid nanoparticles represent an interesting approach for eye drug delivery as they can improve the corneal absorption of drugs enhancing their bioavailability. The Generally Recognized as Safe status of formulation excipients, the scaling-up facilities and the possibility of sterilization, make them suitable for industrial production. In this review, the latest findings, potential applications, and challenges related to the use of lipid nanoparticles for ocular drug delivery are comprehensively discussed.

614. Microfluidic Manufacture of RNA-Lipid Nanoparticles Leads to Highly Efficient Delivery of Potent Nucleic Acid Therapeutics for Controlling Gene Expression

Lipid nanoparticles (LNPs) have demonstrated efficient nucleic acid delivery in vitro and in vivo, as well as in clinical development. They exploit endogenous delivery pathways, by co-opting apolipoprotein E (apoE), to mediate effective delivery of the encapsulated nucleic acids into cells via the low-density lipoprotein receptor (LDLR). However, use of LNPs from the bench to the clinic has been considerably limited by challenges in manufacturing at both small and large scales. Here, we bridge that gap by describing the robust manufacture and use of clinical-grade lipid-based nanoparticles for highly efficient delivery of nucleic acids at scales suitable for both in vitro screening and in vivo applications.RNA-LNPs manufactured using an optimized microfluidic platform enables efficient encapsulation of nucleic acids (e.g. siRNA, mRNA, pDNA) into biocompatible “solid-core” nanoparticles (~50 nm). The resultant nanoparticles can then be applied to cell cultures in vitro or administered in vivo. The following reports a comprehensive set of studies conducted to evaluate the merits of the technology and further provide insights for delivering short interfering RNA (siRNA) and mRNA in difficult-to-transfect cells both in vitro and in vivo.RNA-LNPs were formulated to encapsulate a potent siRNA directed against PTEN - a clinically relevant gene associated with neural regeneration. Exceptional cellular uptake (>98%) with minimal toxicity was observed in both primary rat hippocampal and mixed cortical cell cultures. High transfection efficency (>95%) of the encapsulated material resulted in concomitant high-level (>85%) PTEN knockdown within the first 4 hours of a low dose (100 ng/ml) treatment; that level of knockdown was further sustained for 21 days. Similarly, RNA-LNPs encapsulating mRNA were also found to mediate early ( 75% for 7 days) following a single (500 ng/ml) treatment in primary rat mixed cortical cultures.Strategies for locally administering RNA-LNPs into the brain and spinal cord of adult Sprague Dawley rats were also investigated. Controlled localized injections of PTEN-encaspulated siRNA into the motorcortex resulted in significant and sustained (7 days) knockdown. Similarly, local administration at the site of a cervical spinal cord injury significantly reduced target PTEN expression, 10 days later. Visible uptake of RNA-LNPs characterized by their presence in the soma of neurons found in the red nucleus provides further insights into a regtrograde transport mechanism involving the axons.Collectively, these studies reflect the simplicity and efficacy of this commercially available technology in presenting a cost-effective and advantageous avenue for screening and validating new targeted nucleic acid therapies.

Effective Treatment of Human CML By RNAi In Vivo in a Xenotransplantation Mouse Model

Background: Considering the heterogeneity of leukemic cells in patients, current treatment regimens of chemotherapy and bone marrow transplantation lack specificity and are associated with frequent relapses and severe adverse effects. Hence, there is a need to develop novel therapeutics that can target the disease by its molecular fingerprint with minimal side effects.Despite the wide potential of RNA interference (RNAi) for translational therapeutics, systemic application of siRNA is hampered by rapid renal clearance, degradation by serum nucleases or associated immune responses. Lipid nanoparticles (LNPs) containing ionizable cationic lipids, when mixed with siRNA, embody the most advanced delivery platform for systemic administration of siRNA based therapeutics. Here, we established and employed the BCR-ABL dependent K562-CML xenotransplantation model as a proof of principle to validate LNP mediated siRNA functional delivery in vivo.Methods and Results: A microfluidic mixing technology was used to obtain reproducible ionizable cationic LNPs loaded with anti-BCR-ABL or CTRL siRNA. To determine the delivery efficiency of LNP-siRNA formulations, human leukemic K562 cells were incubated with siRNA-containing LNPs at various concentrations. Almost 100% of cells had taken up siRNA containing LNPs even at the lowest concentration of 0.0625µg/ml with stable uptake kinetics. We also observed near 100% uptake of LNP-siRNA in hard to transfect primary patient cells (CML, AML, ALL and MDS). Looking at the on-target functional efficacy of LNP-siRNA formulations, we observed a time and dose dependent increase in apoptosis (annexin V assay) and decrease in cell viability (alamar blue assay) of K562 cells treated with anti-BCR-ABL siRNA but not CTRL siRNA. A robust knockdown in BCR-ABL mRNA levels (65-90%) at 72 hours and protein at 96 hours was observed which confirmed that cell death was an on-target effect. Colony-forming potential of primary human CD34+ CML cells was significantly reduced when treated with anti-BCR-ABL siRNA compared to CTRL siRNA and to CD34+ cells from healthy donors.To translate our findings in vivo, we evaluated the safety profile, delivery potential and functional efficacy of LNP-siRNA in mice. A total dose of 15mg/kg (3 injections of 5mg/kg at day 0, 1 and 2) in healthy NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice resulted in 100% LNP positive cells in peripheral blood up to day 10. The formulations were highly tolerable in vivo with no significant differences in body weight and complete blood counts between treated and control mice. Moreover, serum analysis showed no significant toxicity in mice following LNP-siRNA treatment. With a focus on hematopoietic tissues following systemic administration, NSG mice received transplants of human K562 cells (stably expressing GFP and luciferase) intrafemorally and were injected intravenously for 3 consecutive injections of LNP-siRNA (1 or 5mg/kg body weight) at 8 hours interval. Interestingly, almost 100% LNP uptake was observed in xenograft leukemic cells in bone marrow at 48 hours at both doses. The leukemic burden of luciferase expressing K562 cells in mice was quantified using in vivo imaging before and during treatment. Treatment with anti-BCR-ABL siRNA for 10 days (n=7) resulted in a 0.5 fold decrease, whereas CTRL siRNA (n=7) resulted in a net 5-fold increase of luciferase signal, thus proving the efficacy of our approach in vivo.Conclusion: We show a highly efficient and non-toxic delivery in vitro and in vivo with nearly 100% uptake of LNP-siRNA formulations in bone marrow of leukemic mice. By inhibiting BCR-ABL we show a reduction of leukemic burden in our xenotransplant model, while leukemic cells expanded in CTRL siRNA treated mice. Our study provides a proof-of-principle that the combined use of lipid nanoparticles and RNAi technology can be used to target leukemia cells in vivo with promising therapeutic implications. DisclosuresWalsh:Precision Nanosystems Inc.: Employment. Thomas:Precision Nanosystems Inc.: Employment. Ramsay:Precision Nanosystems Inc.: Employment. Heuser:Karyopharm: Research Funding.

Lipid nanoparticles for the delivery of biopharmaceuticals.

Biopharmaceuticals comprise therapeutic protein-based, nucleic acids and cell-based products. According to their therapeutic success, the clinical use of these products has been growing. Therefore, the development of efficient biopharmaceuticals delivery systems, which overcome their limitations for administration, remains an excellent prospect for pharmaceutical technologists. In this area, lipid nanoparticles have been increasingly recognized as one of the most promising delivery systems, due to their exclusive advantages. However, no clinical biopharmaceutical lipid nanoparticle-based products are yet available. This fact could be explained by the lack or failure of in vivo studies, regarding stability and toxicological concerns, and also by the complex regulatory issues that must be accomplished. The present review article focuses on the different classes of biopharmaceuticals, their characteristics and limitations for administration. A state of the art regarding the use of lipid nanoparticles to improve biopharmaceuticals delivery is presented and a critical prospect of the future directions that should be addressed by pharmaceutical technologists is also discussed.

Lipid nanoparticles protect from edelfosine toxicity in vivo

Edelfosine, an alkyl-lysophospholipid antitumor drug with severe side-effects, has previously been encapsulated into lipid nanoparticles (LN) with the purpose of improving their toxicity profile. LN are made of lipids recognized as safe by the Food and Drug Administration (FDA) and, therefore, these systems are generally considered as nontoxic vehicles. However, toxicity studies regarding the use of LN as vehicles for drug administration are limited. In the present study, we investigated the in vivo toxicity of free edelfosine, and the protection conferred by LN. The free drug, non-loaded LN and edelfosine-loaded LN were orally administered to mice. Our results show that the oral administration of the free drug at 4 times higher than the therapeutic dose caused the death of the animals within 72h. Moreover, histopathology revealed gastrointestinal toxicity and an immunosuppressive effect. In contrast, LN showed a protective effect against edelfosine toxicity even at the higher dose and were completely safe. LN are, therefore, a safe vehicle for the administration of edelfosine by the oral route. The nanosystems developed could be further used for the administration of other drugs.

Reactivating PP2A by FTY720-Loaded Lipid Nanoparticles, a Novel Therapeutic Strategy in Acute Myeloid Leukemia

Abstract Abstract 1511 Protein phosphatase 2A (PP2A) is a human tumor suppressor that inhibits cellular transformation by regulating the activity of several signaling proteins critical for malignant cell behavior. Our group has shown that PP2A activity is reduced in acute myeloid leukemia (AML), and that restoration of PP2A activity in AML cells blocks proliferation, induces caspase-dependent apoptosis, and affects AKT and ERK1/2 activity. It has been proposed that FTY720 suppress PP2A activity by directly interacting with PP2AA-C dimers, and possibly interfering with the SET-PP2A interplay. Reactivation of PP2A by FTY720 in vivo restricts leukemogenesis in different types of hematological malignancies; however, its antitumor effects in AML remain underexplored. Here, we developed lipid nanoparticles (LN) encapsulating FTY720, and evaluated its pharmacokinetic profile and biodistribution in mice, and its antileukemic effects in AML in vitro and in vivo. FTY720-LN show suitable size (around 210 nm) and encapsulation efficiency (nearly 75%) for the oral and intravenous administration of the compound. Blood concentration of FTY720 decreased rapidly 2 hours after the intravenous administration of FTY720-LN; however, orally administered LN provided both a sustained release of the drug over four days and a high distribution in tissues, significantly increasing the accumulation of FTY720 in spleen, compared to the administration of the same dose of FTY720 solution and intravenous administration of FTY720-LN. In vitro studies revealed that FTY720 and FTY720-LN restore PP2A phosphatase activity in AML cells, leading to cell growth arrest, induction of caspase-dependent apoptosis and decrease in phosphorylation of PP2A targets AKT and ERK. Moreover, in vivo daily intraperitoneal administration of FTY720 in RAG2−/− γc−/− mice bearing an AML xenograft tumor reduced significant tumor growth. Of note, oral administration of FTY720-LN every three days was as efficient in reducing AML xenograft tumor growth as a daily oral administration of FTY720. These results provide the first evidence for the potential use of lipid nanoparticles encapsulating FTY720 as a therapeutic agent in AML. Disclosures: No relevant conflicts of interest to declare.

Lipid-based delivery of CpG oligodeoxynucleotides for cancer immunotherapy

The anti-tumor activity of CpG-containing oligodeoxynucleotides (ODNs) has been well established in numerous animal models and confirmed in a number of early clinical trials. While the use of chemical modifications has effectively reduced the sensitivity of ODNs to nuclease degradation and a number of human trials have yielded promising results, the clinical utility of free CpG ODN still faces several significant challenges that must be addressed to achieve optimal potency and therapeutic activity. These include unfavorable pharmacokinetic/biodistribution characteristics, lack of specificity for target cells and poor intracellular uptake. To overcome these challenges, lipid-based delivery systems have been developed to protect the CpG ODN payload, modify their circulation/distribution characteristics, enhance immune cell targeting and facilitate intracellular uptake. In preclinical cancer models, lipid-mediated delivery has demonstrated the capacity to increase the immunopotency of CpG ODNs and dramatically enhance their anti-tumor efficacy as monotherapies, vaccine adjuvants and combination therapies with monoclonal antibodies or chemotherapy. This review will focus on investigating CpG ODNs as a cancer immunotherapeutic and the promising enhancement in efficacy that can be achieved through the use of lipid nanoparticles as delivery vehicles.