siRNA-loaded Lipid Nanoparticles Research Articles

Molecular-Level Structural Analysis of siRNA-Loaded Lipid Nanoparticles by 1H NMR Relaxometry: Impact of Lipid Composition on Their Structural Properties.

1H NMR relaxometry was applied for molecular-level structural analysis of siRNA-loaded lipid nanoparticles (LNPs) to clarify the impact of the neutral lipids, 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC) and cholesterol, on the physicochemical properties of LNP. Incorporating DSPC and cholesterol in ionizable lipid-based LNP decreased the molecular mobility of ionizable lipids. DSPC reduced the overall molecular mobility of ionizable lipids, while cholesterol specifically decreased the mobility of the hydrophobic tails of ionizable lipids, suggesting that cholesterol filled the gap between the hydrophobic tails of ionizable lipids. The decrease in molecular mobility and change in orientation of lipid mixtures contributed to the maintenance of the stacked bilayer structure of siRNA and ionizable lipids, thereby increasing the siRNA encapsulation efficiency. Furthermore, NMR relaxometry revealed that incorporating those neutral lipids enhanced PEG chain flexibility at the LNP interface. Notably, a small amount of DSPC effectively increased PEG chain flexibility, possibly contributing to the improved dispersion stability and narrower size distribution of LNPs. However, cryogenic transmission electron microscopy represented that adding excess amounts of DSPC and cholesterol into LNP resulted in the formation of deformed particles and demixing cholesterol within the LNP, respectively. The optimal lipid composition of ionizable lipid-based LNPs in terms of siRNA encapsulation efficiency and PEG chain flexibility was rationalized based on the molecular-level characterization of LNPs. Moreover, the NMR relaxation rate of tertiary amine protons of ionizable lipids, which are the interaction site with siRNA, can be a valuable indicator of the encapsulated amount of siRNA within LNPs. Thus, NMR-based analysis can be a powerful tool for efficiently designing LNP formulations and their quality control based on the molecular-level elucidation of the physicochemical properties of LNPs.

Reprogramming activated hepatic stellate cells by siRNA-loaded nanocarriers reverses liver fibrosis in mice

We report on a novel strategy for treating liver fibrosis through reprogramming activated Hepatic Stellate Cells (aHSCs) into quiescent Hepatic Stellate Cells (qHSCs) using siRNA-loaded lipid nanoparticles (LNPs). The in vivo screening of an array of molecularly-diverse ionizable lipids identified two candidates, CL15A6 and CL15H6, with a high siRNA delivery efficiency to aHSCs. Optimization of the composition and physico-chemical properties of the LNPs enabled the ligand-free, selective, and potent siRNA delivery to aHSCs post intravenous administration, with a median effective siRNA dose (ED50) as low as 0.08 mg/Kg. The biosafety of the LNPs was confirmed by escalating the dose to 50-fold higher than the ED50 or by chronic administration. The recruitment of the novel LNPs for the simultaneous knockdown of Hedgehog (Hh) and Transforming Growth Factor Beta 1 (TGFβ1) signaling pathways using an siRNA cocktail enabled the reversal of liver fibrosis and the restoration of the normal liver function in mice. Analysis of the key transcription factors in aHSCs suggested that the reprogramming of aHSCs into qHSCs mediated the therapeutic outcomes. The scalable ligand-free platform developed in this study as well as the novel therapeutic strategy reported herein are promising for clinical translation.

Biophysical characterization of siRNA-loaded lipid nanoparticles with different PEG content in an aqueous system

Although lipid nanoparticles (LNP) are potential carriers of various pharmaceutical ingredients, further investigation for maintaining their stability under various environmental stressors must be performed. This study evaluated the influence of PEGylation and stress conditions on the stability of siRNA-loaded LNPs with different concentrations of PEG (0.5 mol%; 0.5 % PEG-LNP and 1.0 mol%; 1.0 % PEG-LNP) anchored to their surface. We applied end-over-end agitation, elevated temperature, and repeated freeze and thaw (F/T) cycles as physicochemical stressors of pH and ionic strength. Dynamic light scattering (DLS), flow imaging microscopy (FIM), and ionic-exchange chromatography (IEX) were to determine the degree of aggregation and change in siRNA content. The results indicate that 0.5 % PEG-LNP resisted aggregation only at low pH levels or with salt, whereas 1.0 % PEG-LNP had increased colloidal stability except at pH 4. 0.5 % PEG-LNP withstood aggregation until 71 °C and three cycles of F/T. In contrast, 1.0 % PEG-LNP maintained colloidal stability at 90 °C and seven F/T cycles. Moreover, 1.0 % PEG-LNP had higher siRNA stability under all stress conditions. Therefore, to ensure the stability of LNP and encapsulated siRNA, the PEG concentration must be carefully controlled while considering LNPs’ colloidal instability mechanisms under various stress conditions.

Nanoparticle-mediated targeting of the fusion gene RUNX1/ETO in t(8;21)-positive acute myeloid leukaemia

A hallmark of acute myeloid leukaemias (AMLs) are chromosomal rearrangements that give rise to novel leukaemia-specific fusion genes. Most of these fusion genes are both initiating and driving events in AML and therefore constitute ideal therapeutic targets but are challenging to target by conventional drug development. siRNAs are frequently used for the specific suppression of fusion gene expression but require special formulations for efficient in vivo delivery. Here we describe the use of siRNA-loaded lipid nanoparticles for the specific therapeutic targeting of the leukaemic fusion gene RUNX1/ETO. Transient knockdown of RUNX1/ETO reduces its binding to its target genes and alters the binding of RUNX1 and its co-factor CBFβ. Transcriptomic changes in vivo were associated with substantially increased median survival of a t(8;21)-AML mouse model. Importantly, transient knockdown in vivo causes long-lasting inhibition of leukaemic proliferation and clonogenicity, induction of myeloid differentiation and a markedly impaired re-engraftment potential in vivo. These data strongly suggest that temporary inhibition of RUNX1/ETO results in long-term restriction of leukaemic self-renewal. Our results provide proof for the feasibility of targeting RUNX1/ETO in a pre-clinical setting and support the further development of siRNA-LNPs for the treatment of fusion gene-driven malignancies.

Monitoring the in vivo siRNA release from lipid nanoparticles based on the fluorescence resonance energy transfer principle

The siRNA-loaded lipid nanoparticles have attracted much attention due to its significant gene silencing effect and successful marketization. However, the in vivo distribution and release of siRNA still cannot be effectively monitored. In this study, based on the fluorescence resonance energy transfer (FRET) principle, a fluorescence dye Cy5-modified survivin siRNA was conjugated to nanogolds (Au-DR-siRNA), which were then wrapped with lipid nanoparticles (LNPs) for monitoring the release behaviour of siRNA in vivo. The results showed that once Au-DR-siRNA was released from the LNPs and cleaved by the Dicer enzyme to produce free siRNA in cells, the fluorescence of Cy5 would change from quenched state to activated state, showing the location and time of siRNA release. Besides, the LNPs showed a significant antitumor effect by silencing the survivin gene and a CT imaging function superior to iohexol by nanogolds. Therefore, this work provided not only an effective method for monitoring the pharmacokinetic behaviour of LNP-based siRNA, but also a siRNA delivery system for treating and diagnosing tumors.

Development of lipid nanoparticles and liposomes reference materials (II): cytotoxic profiles

Lipid based nanocarriers are one of the most effective drug delivery systems that is evident from the recent COVID-19 mRNA vaccines. The main objective of this study was to evaluate toxicity of six lipid based formulations with three surface charges—anionic, neutral or cationic, to establish certified reference materials (CRMs) for liposomes and siRNA loaded lipid nanoparticles (LNP-siRNA). Cytotoxicity was assessed by a proliferation assay in adherent and non-adherent cell lines. High concentration of three LNP-siRNAs did not affect viability of suspension cells and LNP-siRNAs were non-toxic to adherent cells at conventionally used concentration. Systematic evaluation using multiple vials and repeated test runs of three liposomes and three LNP-siRNA formulations showed no toxicity in HL60 and A549 cells up to 128 and 16 µg/mL, respectively. Extended treatment and low concentration of LNPs did not affect the viability of suspension cells and adherent cells at 96 h. Interestingly, 80% of A549 and HL60 cells in 3D conditions were viable when treated with cationic LNP-siRNA for 48 h. Taken together, anionic, cationic and neutral lipid formulations were non-toxic to cells and may be explored further in order to develop them as drug carriers.

Abstract 3295: The Nanoprimer: A nanoparticle designed to the efficacy of nucleic acid-based therapeutics in oncology

Abstract Nucleic acid drugs have shown promise in oncology, however their use is limited by hepatic clearance. Due to the mononuclear phagocytic system (MPS), a large part of the intravenously-administered dose does not reach the therapeutic target. Our approach aims to address this issue using biocompatible nanoparticles—the Nanoprimer—that is administered prior to the therapeutic. Due to its physico-chemical properties, the Nanoprimer is transiently internalized by Kupffer cells (KC), and endothelial cells of the MPS, enabling temporary reduction of hepatic clearance. These data further explore the Nanoprimer’s mechanism of action and demonstrate that scavenger receptor-A (SR) is the main receptor involved in Nanoprimer binding and uptake by KC. Efficacy studies in mouse model show that the Nanoprimer dramatically increases the anti-tumor efficacy of nucleic acid-based treatment without introducing additional toxicities. An in vitro toxicity evaluation was performed investigating the Nanoprimer’s hemolytic properties and its potential ability to activate the complement system, platelet aggregation and pro-inflammatory cytokines*. SR-mediated binding and uptake of Nanoprimer by KC were assessed using a competitive binding assay with SR inhibitor polyinosinic acid. The impact of the Nanoprimer on anti-tumor efficacy was evaluated on orthotopic triple negative breast cancer (TNBC) model in combination with siRNA-loaded lipid nanoparticles (LNPs) targeting a novel cancer-specific gene transcript of a master regulator of invadopodia formation and Wnt, Hedgehog and Notch oncogenic pathways. Results showed that the Nanoprimer is safe at the effective dose. The Nanoprimer does not activate the complement components, induce hemolysis, platelet aggregation, or cytokine storm syndrome elements. The competitive inhibition of SR-A decreases Nanoprimer binding and KC-uptake by 70%, indicating SR-A selective uptake of the Nanoprimer by macrophages. The efficacy study shows that combining the Nanoprimer with siRNA LNP therapy significantly increased treatment efficiency, with a mean tumor growth inhibition ratio of 36.3% for LNPs alone versus 84.6% for the combination. Moreover, the combination therapy more efficiently prevents the development of pulmonary metastatic tumors, with a metastasis-inhibition rate of 98%. These data demonstrate the safety of the Nanoprimer, along with its ability to maximize the inhibition of primary tumor growth and pulmonary metastasis by systemic siRNA LNP therapy in a TNBC mouse model. They also highlight the capacity of Nanoprimer technology to unlock the potential of nucleic acid-based treatments. The ubiquitous nature of its mode of action allows its application to a broad spectrum of anti-cancer therapeutics. *we acknowledge NCL, Frederick National Laboratory for Cancer Research sponsored by the NCI for the help with conducting these experiments Citation Format: Julie Devallière, Laurence Poul, Maxime Bergère, Audrey Darmon, Océane Jibault, Francis Mpambani, Kelvin Tsai, Matthieu Germain. The Nanoprimer: A nanoparticle designed to the efficacy of nucleic acid-based therapeutics in oncology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3295.

Abstract P143: Development of siRNA-loaded lipid nanoparticles targeting long non-coding RNA LINC01257 as a novel and safe therapeutic approach for paediatric acute myeloid leukaemia

Abstract Introduction: Standard of care therapies for children with acute myeloid leukemia (AML) cause potent off-target toxicity to healthy cells, highlighting the need to develop new therapeutic approaches that are safe and specific for leukemia cells. Long non-coding RNAs (lncRNAs) are an emerging and highly attractive therapeutic target in the treatment of cancer due to their oncogenic functions and selective expression in cancer cells. However, lncRNAs have historically been considered ‘undruggable’ targets because they do not encode for a protein product. Therefore, the discovery of novel lncRNA targets and the development of efficient methods to silence them is critical for developing new therapies for AML. In this study, we describe the development of an siRNA-loaded lipid nanoparticle (LNP) for the therapeutic silencing of the novel oncogenic lncRNA LINC01257. Materials and Methods: Transcriptomic analysis of children with AML from both the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative and AAML1031 study was performed to identify AML specific lncRNAs. The subsequently identified lncRNA LINC01257 was knocked down in AML Kasumi-1 cells via electroporation of siRNA targeting LINC01257 (si-LINC01257). Changes in proliferation and cell viability were assessed via cell count and Annexin V/7AAD staining 48 hours post-transfection. LNPs incorporating non-targeting si-SCR or si-LINC01257 were synthesized through NxGen non-turbulent microfluidics. LNPs were formulated reproducing the FDA-approved Onpattro® siRNA delivery vector, D-Lin-MC3-DMA:DSPC:cholesterol:PEG-DMG. LNP-si-LINC01257 uptake was measured by confocal microscopy and flow cytometry. Kasumi-1 cells and healthy peripheral blood mononuclear cells (PBMCs) were treated with 250nM LNP-si-SCR or LNP-si-LINC01257 for 48 hours. Changes in proliferation and cell viability were assessed with cell counts and Annexin V/7AAD staining 48 hours post-treatment. Results: LINC01257 was significantly overexpressed in patients carrying the t(8;21) translocation compared to healthy controls ( P = <0.001). Compared to si-SCR controls, siRNA knockdown of LINC01257 in Kasumi-1 cells resulted in a 62% reduction in the total cell count after 48 hours (P = <0.001) , which was accompanied by a 70% decrease in overall cell viability (P = <0.004). LNP-siRNA production yielded a mean LNP size of ~65 nm along with a >85% siRNA encapsulation rate. LNP-siRNAs were efficiently taken up by Kasumi-1 cells (>95% of cells) and LNP-si-LINC01257 treatment successfully ablated LINC01257 expression, which was accompanied by a 55% reduction in total cell count following 48 hours of treatment (P= 0.02). In contrast, healthy PBMCs, which do not express LINC01257, were unaffected by LNP-si-LINC01257 treatment despite comparable levels of LNP-siRNA uptake. Conclusion: Our data demonstrate the effective use of LNP-based RNA interference modalities for the silencing of cancer-driving lncRNAs and highlight their potential as a viable therapeutic and non-toxic approach in the management of AML. Citation Format: Patrick Connerty, Ernest Moles, Charles deBock, Nisitha Jayatilleke, Jenny L. Smith, Soheil Meshinchi, Chelsea Mayoh, Maria Kavallaris, Richard B. Lock. Development of siRNA-loaded lipid nanoparticles targeting long non-coding RNA LINC01257 as a novel and safe therapeutic approach for paediatric acute myeloid leukaemia [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2021 Oct 7-10. Philadelphia (PA): AACR; Mol Cancer Ther 2021;20(12 Suppl):Abstract nr P143.

Development of siRNA-Loaded Lipid Nanoparticles Targeting Long Non-Coding RNA LINC01257 as a Novel and Safe Therapeutic Approach for t(8;21) Pediatric Acute Myeloid Leukemia

Standard of care therapies for children with acute myeloid leukemia (AML) cause potent off-target toxicity to healthy cells, highlighting the need to develop new therapeutic approaches that are safe and specific for leukemia cells. Long non-coding RNAs (lncRNAs) are an emerging and highly attractive therapeutic target in the treatment of cancer due to their oncogenic functions and selective expression in cancer cells. However, lncRNAs have historically been considered ‘undruggable’ targets because they do not encode for a protein product. Here, we describe the development of a new siRNA-loaded lipid nanoparticle for the therapeutic silencing of the novel oncogenic lncRNA LINC01257. Transcriptomic analysis of children with AML identified LINC01257 as specifically expressed in t(8;21) AML and absent in healthy patients. Using NxGen microfluidic technology, we efficiently and reproducibly packaged anti-LINC01257 siRNA (LNP-si-LINC01257) into lipid nanoparticles based on the FDA-approved Patisiran (Onpattro®) formulation. LNP-si-LINC01257 size and ζ-potential were determined by dynamic light scattering using a Malvern Zetasizer Ultra. LNP-si-LINC01257 internalization and siRNA delivery were verified by fluorescence microscopy and flow cytometry analysis. lncRNA knockdown was determined by RT-qPCR and cell viability was characterized by flow cytometry-based apoptosis assay. LNP-siRNA production yielded a mean LNP size of ~65 nm with PDI ≤ 0.22 along with a >85% siRNA encapsulation rate. LNP-siRNAs were efficiently taken up by Kasumi-1 cells (>95% of cells) and LNP-si-LINC01257 treatment was able to successfully ablate LINC01257 expression which was accompanied by a significant 55% reduction in total cell count following 48 h of treatment. In contrast, healthy peripheral blood mononuclear cells (PBMCs), which do not express LINC01257, were unaffected by LNP-si-LINC01257 treatment despite comparable levels of LNP-siRNA uptake. This is the first report demonstrating the use of LNP-assisted RNA interference modalities for the silencing of cancer-driving lncRNAs as a therapeutically viable and non-toxic approach in the management of AML.

SIRT1 siRNA-loaded lipid nanoparticles enhanced doxorubicin-induced cell death in prostate cancer cell lines

Most chemotherapeutics induce apoptosis by creating DNA damage in tumor cells. However, high expression of Sirtuin 1 (SIRT1), which is a histone deacetylase activates DNA repair mechanisms leading to the repair of chemotherapeutic-induced DNA damage and subsequently prevention of apoptosis. Therefore, inhibition of SIRT1 is a useful strategy in cancer therapy to achieve a more efficient eradication of tumor cells.In this study, novel lipoplexes composed of 1,2-di-O-octadecenyl-3-trimethylammonium propane (DOTMA) containing lipid nanoparticles and SIRT1 siRNA have been formulated by the freeze-drying method. SIRT1 silencing effect has been demonstrated by a 2.5- to 4- fold decrease in mRNA and protein expression levels. The efficiency of the lipoplexes to enhance the activity of doxorubicin, a DNA damage-inducing chemotherapeutic agent, has been studied on prostate cancer cell lines with different p53 expression patterns, namely LNCaP, DU145, and PC3. SIRT1 silencing by lipoplexes enhanced DNA damage recognition and increased the activity of doxorubicin on cancer cell death. These results have shown that SIRT1 siRNA-loaded lipid nanoparticles can enhance the efficiency of doxorubicin on prostate cancer cell lines.

Development of EphA2 siRNA-loaded lipid nanoparticles and combination with a small\u2010molecule histone demethylase inhibitor in prostate cancer cells and tumor spheroids

BackgroundsiRNAs hold a great potential for cancer therapy, however, poor stability in body fluids and low cellular uptake limit their use in the clinic. To enhance the bioavailability of siRNAs in tumors, novel, safe, and effective carriers are needed.ResultsHere, we developed cationic solid lipid nanoparticles (cSLNs) to carry siRNAs targeting EphA2 receptor tyrosine kinase (siEphA2), which is overexpressed in many solid tumors including prostate cancer. Using DDAB cationic lipid instead of DOTMA reduced nanoparticle size and enhanced both cellular uptake and gene silencing in prostate cancer cells. DDAB-cSLN showed better cellular uptake efficiency with similar silencing compared to commercial transfection reagent (Dharmafect 2). After verifying the efficacy of siEphA2-loaded nanoparticles, we further evaluated a potential combination with a histone lysine demethylase inhibitor, JIB-04. Silencing EphA2 by siEphA2-loaded DDAB-cSLN did not affect the viability (2D or 3D culture), migration, nor clonogenicity of PC-3 cells alone. However, upon co-administration with JIB-04, there was a decrease in cellular responses. Furthermore, JIB-04 decreased EphA2 expression, and thus, silencing by siEphA2-loaded nanoparticles was further increased with co-treatment.ConclusionsWe have successfully developed a novel siRNA-loaded lipid nanoparticle for targeting EphA2. Moreover, preliminary results of the effects of JIB-04, alone and in combination with siEphA2, on prostate cancer cells and prostate cancer tumor spheroids were presented for the first time. Our delivery system provides high transfection efficiency and shows great promise for targeting other genes and cancer types in further in vitro and in vivo studies.

Manipulating the function of tumor-associated macrophages by siRNA-loaded lipid nanoparticles for cancer immunotherapy

The tumor-microenvironment contains large numbers of tumor-associated macrophages (TAMs) which are largely M2 phenotypes and are involved in pro-tumorous functions. Targeting TAMs so as to manipulate them and to modify their functions could be a novel immunotherapy for the treatment of cancer. Such a strategy would involve targeting TAMs with short interfering RNA (siRNA) to modify their functions by silencing certain genes that are responsible for their M2 polarization. In this study, a lipid nanoparticle (LNP) formulation was used to target and deliver siRNA to TAMs. The LNP was mainly composed of a novel, pH-sensitive cationic lipid, referred to as the CL4H6 lipid, which had previously been optimized to target hepatocytes. The optimized siRNA-loaded CL4H6-LNPs were selectively and efficiently taken up and showed strong gene silencing activity in TAMs in a human tumor xenograft model in nude mice. Furthermore, an anti-tumor therapeutic response in the same tumor model was obtained by targeting TAMs using the optimized siRNA-loaded CL4H6-LNPs. The anti-tumor therapeutic response was obtained through the silencing of the signal transducer and activator of transcription 3 (STAT3) and hypoxia inducible factor 1 α (HIF-1α), which resulted in an increase in the level of infiltrated macrophage (CD11b+ cells) into the tumor-microenvironment (TME) as well as a tendency to increase the concentration of M1 macrophages (CD169+ cells). The treatment also resulted in reversing the pro-tumorous functions of TAMs -mainly angiogenesis and tumor cell activation-, as evidenced by a decrease in the related gene expression at the mRNA level. This research has promising clinical and pharmaceutical applications for achieving novel macrophage-based cancer immunotherapy.

Improved Stability of siRNA-Loaded Lipid Nanoparticles Prepared with a PEG-Monoacyl Fatty Acid Facilitates Ligand-Mediated siRNA Delivery.

Peptide modification is a popular strategy for developing an active targeting lipid nanoparticle (LNP). In modifying the surface of an LNP with a peptide, the sequence and structure of the peptide strongly affects the formation of the LNP. Specifically, a peptide with a high hydrophobicity can induce coarsening and aggregation of the LNP. In an attempt to prevent this from occurring, we incorporated monoacyl and diacyl group-conjugated poly(ethylene glycol) (PEG) into a LNP. We previously developed an original LNP, a multifunctional envelope type nanodevice (MEND) modified with an Epi-1 peptide, a ligand with a high affinity for the epithelial cell adhesion molecule (EpCAM). Using this peptide-modified MEND, the efficiency of delivery of a small interfering RNA (siRNA) encapsulated in the MEND was significantly improved. Although increasing the ratio of modification enhanced cellular uptake, the increase also induced aggregation of the LNP, particularly in the case of a large scale preparation. Our results indicate that a monoacyl PEG-lipid can prevent aggregation, even when the LNP is modified with higher molar ratios of peptide, but that this also results in a decrease in delivery efficiency. Moreover, the Epi-1-modified MEND exhibited a strong silencing effect in an ovarian cancer peritoneal dissemination model. Our results suggest that the simple incorporation of a monoacyl derivative into the PEG-lipid resulted in the formation of a peptide-modified LNP with improved characteristics.

The silencing of indoleamine 2,3-dioxygenase 1 (IDO1) in dendritic cells by siRNA-loaded lipid nanoparticles enhances cell-based cancer immunotherapy

Cell-based therapy using dendritic cells (DC) represents a potent cancer immunotherapy. However, activated DC express indoleamine 2,3-dioxygenase 1 (IDO1), a counter-regulatory and tolerogenic molecule, leading to the inhibition of T cell activation and the promotion of T cell differentiation into regulatory T cells. Silencing the IDO1 gene in DC by small interfering RNA (siRNA) represents a potent therapeutic strategy. We report on the successful and efficient introduction of a siRNA targeting IDO1 into mouse DCs by a means of a multifunctional envelope-type nanodevice (MEND) containing a YSK12-C4 (YSK12-MEND). The YSK12-C4 has both fusogenic and cationic properties. The YSK12-MEND induced an effective level of gene silencing of IDO1 at siRNA doses in the range of 1–20 nM, a concentration that commercially available transfection reagents are not able to silence. The YSK12-MEND mediated IDO1 silencing had no effect on the characteristic determinants of DC phenotype such as CD11c, CD80 and MHC class II. The silencing of IDO1 in DC by the YSK12-MEND significantly enhanced the antitumor effect against E.G7-OVA tumor. Moreover, a decrease in the numbers of regulatory T cells in the tumor was observed in mice that were treated with the IDO1-silenced DC. The YSK12-MEND appears to be a potent delivery system for IDO1-silenced DC based cancer immunotherapy.

Investigating impacts of surface charge on intraocular distribution of intravitreal lipid nanoparticles

In this paper, the effect of surface charge of intravitreal lipid nanoparticles (LNPs) on their intraocular accumulation and distribution was quantitatively and systematically studied. The retinal distribution of LNPs delivering model drug (siRNA) with optimal surface charge was visualized as well. LNPs and siRNA-loaded LNPs (siLNPs) were prepared with ethanol-injection method. C57BL/6 Mice with the age of 6–8 weeks were used for the animal experiments. Quantitative accumulation in the eye and respective intraocular distribution of the intravitreally injected LNPs with different surface charge were examined after predesignated time points. The fluorescence intensity of the fluorescent labeled siRNA was also quantified based on the confocal microscope images. The release of siRNA from siLNPs was studied in mimicked vitreous environment at 37 °C with the presence of 50% serum protein. We successfully prepared six types of LNPs had relatively uniform size around 70 nm and varied in zeta potential ranging from −30 mV to +50 mV. Negative, neutral and slightly positive charged LNPs were cleared much faster than strongly positive charged LNPs from the mice's eyes. After 6 h, LNPs with zeta potential of +35 mV had highest ratio of retinal to vitreous accumulation and penetrated through the entire layer of the retina. The siLNPs could successfully deliver the siRNA to the ganglion cells and inner plexiform layer of the retina. After 24 h, majority of LNPs and siLNPs were cleared, suggesting that the LNPs with pegylated surface in the absence of any targeting ligand were not internalized by retinal cells. The mechanism of how the surface charge of nanoparticles affects their intraocular retention and distribution was intensively discussed. The results suggested that intravitreally injected LNPs with optimal surface charge around +35 mV can distribute and penetrate the retina, which could be further modified as promising nanocarriers for retinal delivery.

Lipid nanoparticles silence tumor necrosis factor α to improve wound healing in diabetic mice.

Diabetes mellitus is a mounting concern in the United States, as are the mortality and morbidity that result from its complications. Of particular concern, diabetes patients frequently suffer from impaired wound healing and resultant nonhealing diabetic foot ulcers. These ulcers overproduce tumor necrosis factor α (TNFα), which reduces wound bed cell migration and proliferation while encouraging apoptosis. Herein, we describe the use of siRNA‐loaded lipid nanoparticles (LNPs) as a potential wound treatment to combat an overzealous immune response and facilitate wound closure. LNPs were formulated with an ionizable, degradable lipidoid and siRNA specific for TNFα. Topical application of nanoparticles reduced TNFα mRNA expression in the wound by 40–55% in diabetic and nondiabetic mice. In diabetic mice, this TNFα knockdown accelerated wound healing compared to untreated controls. Together, these results serve as proof‐of‐concept that RNA interference therapy using LNPs can reduce the severity and duration of chronic diabetic wounds.

PH-labile PEGylation of siRNA-loaded lipid nanoparticle improves active targeting and gene silencing activity in hepatocytes

Lipid nanoparticles (LNPs) are one of the promising technologies for the in vivo delivery of short interfering RNA (siRNA). Modifying LNPs with polyethyleneglycol (PEG) is widely used to inhibit non-specific interactions with serum components in the blood stream, and is a useful strategy for maximizing the efficiency of active targeting. However, it is a widely accepted fact that PEGylation of the LNP surface strongly inhibits fusion between LNPs and endosomal membranes, resulting in poor cytosolic siRNA delivery, a process that is referred to as the ‘PEG-dilemma’. In the present study, in an attempt to overcome this problem, siRNA-loaded LNPs were modified with PEG through maleic anhydride, a pH-labile linkage. The in vitro, suppression of cationic charge, stealth function at physiological pH up to 1h and the rapid desorption of PEG and restoration of fusogenic activity under slightly acidic conditions (within only 2min) were achieved by PEG modification of the LNPs through maleic anhydride. In vivo, PEG modification through maleic anhydride resulted in a dramatic improvement in the targeting capability of the active targeting of ligand (N-acetyl-d-galactosamine)-modified LNPs to hepatocytes, with an approximately 14-fold increase in gene silencing activity in factor 7 model mice. Taken together, the maleic anhydride-mediated pH-labile PEGylation of the active targeting LNPs is a useful strategy for achieving the specific and efficient delivery of siRNAs in vivo.

Scalable preparation of poly(ethylene glycol)-grafted siRNA-loaded lipid nanoparticles using a commercially available fluidic device and tangential flow filtration

While a number of siRNA delivery systems have been developed, the methods used in their preparation are not suitable for large-scale production. We herein report on methodology for the large-scale preparation of liposomal siRNA using a fluidic device and tangential flow filtration (TFF). A number of studies have appeared on the use of fluidic devices for preparing and purifying liposomes, but no systematic information regarding appropriate membrane type of commercially available apparatus is available. The findings reported herein indicate that, under optimized conditions, a microfluidic device and TFF can be used to produce siRNA lipid nanoparticles with the same characteristics as traditional ones’. The in vivo silencing efficiency of these lipid nanoparticles in the liver was comparable to laboratory-produced nanoparticles. In addition, con-focal laser scanning microscopy analyses revealed that they accumulated in the liver accumulation at the same levels as particles produced by batch-type and continuous-type procedures. This methodology has the potential to contribute to the advancement of this process from basic research to clinical studies of liposomal DDS.

The intracellular pharmacodynamics of siRNA is responsible for the low gene silencing activity of siRNA-loaded nanoparticles in dendritic cells

The delivery of small interfering RNA (siRNA) to dendritic cells (DCs) is a challenging issue for siRNA-loaded lipid nanoparticles. The cause of this difficulty is unknown. The findings reported herein indicate that the rate-limiting step in gene silencing using siRNA-loaded lipid nanoparticles in DCs, as evidenced by a quantitative analysis of each process in siRNA delivery between mouse bone marrow derived DC (BMDC) and other cell lines, was not associated with the actual delivery of siRNA. A gene silencing of only 50% was observed in BMDC, even when a high dose was used. Contrary to our expectation, the interval between cellular uptake and the delivery of siRNA to the cytosol was not responsible for the low gene silencing. Meanwhile, a drastic difference was found in the relationship between the efficiency of gene silencing and the amount of intracellular intact siRNA. This fact indicates that the processes after cytosolic delivery of siRNA, namely the intracellular pharmacodynamics (PD) of siRNA, appear to be the rate-limiting step in gene silencing in BMDC. The findings reported here demonstrate the importance of the intracellular PD of siRNA delivered to cytosol in the development of siRNA delivery systems for gene silencing in DCs.

Analysis of lipid nanoparticles by Cryo-EM for characterizing siRNA delivery vehicles

Lipid nanoparticles are self-assembling, dynamic structures commonly used as carriers of siRNA, DNA, and small molecular therapeutics. Quantitative analysis of particle characteristics such as morphological features can be very informative as biophysical properties are known to influence biological activity, biodistribution, and toxicity. However, accurate characterization of particle attributes and population distributions is difficult. Cryo-Electron Microscopy (Cryo-EM) is a leading characterization method and can reveal diversity in particle size, shape and lamellarity, however, this approach is traditionally used for qualitative review or low throughput image analysis due to inherent EM micrograph contrast characteristics and artifacts in the images which limit extraction of quantitative feature values. In this paper we describe the development of a semiautomatic image analysis framework to facilitate reliable image enhancement, object segmentation, and quantification of nanoparticle attributes in Cryo-EM micrographs. We apply this approach to characterize two formulations of siRNA-loaded lipid nanoparticles composed of cationic lipid, cholesterol, and poly(ethylene glycol)-lipid, where the formulations differ only by input component ratios. We found Cryo-EM image analysis provided reliable size and morphology information as well as the detection of smaller particle populations that were not detected by standard dynamic light scattering (DLS) analysis.