Lipid Nanoparticle and Liposome Reference Materials: Assessment of Size Homogeneity and Long-Term -70 °C and 4 °C Storage Stability.

With recent advances and anticipated proliferation of lipid nanoparticle (LNP)-delivered vaccines and therapeutics, there is a need for the availability of internationally recognized reference materials of LNP systems. Accordingly, we developed six LNP and liposome (anionic, neutral, and cationic each) candidate reference material formulations and thoroughly characterized by dynamic light scattering their particle hydrodynamic size (Z-avr) and polydispersity. We also evaluated the particle size homogeneity and long-term -70 °C and 4 °C storage stability using multiple large sets of randomly selected vials for each formulation. The formulations stored at -70 °C remained stable and homogeneous for a minimum of 9 months. The Z-avr relative combined uncertainty and the long-term variability were both <1.3% for liposome formulations and anionic LNPs, (3.9% and 1.7%) for neutral LNPs, and (6.7% and 4.4%) for cationic LNPs. An inadvertent few-hour-long storage temperature increase to -35 °C due to a freezer malfunction resulted in a small change of the size and size distribution of anionic liposomes and LNPs but, unexpectedly, a larger size increase of the neutral and cationic liposomes (≤5%) and LNPs (≤25%). The mean Z-avr values of the LNPs stored at 4 °C appeared to slowly increase with t1/3, where t is the storage time, and the Z-avr between-vial heterogeneity and mean polydispersity index values appeared to decrease; no change was observed for liposomes. The size and size distribution evolution of LNPs stored at 4 °C was attributed to an incomplete equilibration of the formulations following the addition of sucrose prior to the initial freezing. Such a process of size increase and size distribution narrowing has not been previously discussed nor observed in the context of LNPs.

Lipid-Nanoparticle-Mediated Delivery of Docetaxel Prodrug for Exploiting Full Potential of Gold Nanoparticles in the Treatment of Pancreatic Cancer.

Current chemoradiation therapy suffers from normal tissue toxicity. Thus, we are proposing incorporating gold nanoparticles (GNPs) and docetaxel (DTX), as they have shown very promising synergetic radiosensitization effects. Here, we explored the effect of a DTX prodrug encapsulated in lipid nanoparticles (LNPDTX-P) on GNP uptake in pancreatic cancer models in vitro and in vivo. For the in vitro experiment, a pancreatic cancer cell line, MIA PaCa-2, was cultured and dosed with 1 nM GNPs and 45 nM free DTX or an equivalent dose of LNPDTX-P. For the in vivo experiment, MIA PaCa-2 cells were implanted subcutaneously in NRG mice, and the mice were dosed with 2 mg/kg of GNPs and 6 mg/kg of DTX or an equivalent dose of LNPDTX-P. The results show that LNPDTX-P-treated tumour samples had double the amount GNPs compared to control samples, both in vitro and in vivo. The results are very promising, as LNPDTX-P have superior targeting of tumour tissues compared to free DTX due to their nanosize and their ability to be functionalized. Because of their minimal toxicity to normal tissues, both GNPs and LNPDTX-P could be ideal radiosensitization candidates in radiotherapy and would produce very promising synergistic therapeutic outcomes.

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.

Lipid nanoparticles to silence androgen receptor variants for prostate cancer therapy.

Advanced-stage prostate cancer remains an incurable disease with poor patient prognosis. There is an unmet clinical need to target androgen receptor (AR) splice variants, which are key drivers of the disease. Some AR splice variants are insensitive to conventional hormonal or androgen deprivation therapy due to loss of the androgen ligand binding domain at the C-terminus and are constitutively active. Here we explore the use of RNA interference (RNAi) to target a universally conserved region of all AR splice variants for cleavage and degradation, thereby eliminating protein level resistance mechanisms. To this end, we tested five siRNA sequences designed against exon 1 of the AR mRNA and identified several that induced potent knockdown of full-length and truncated variant ARs in the 22Rv1 human prostate cancer cell line. We then demonstrated that 2'O methyl modification of the top candidate siRNA (siARvm) enhanced AR and AR-V7 mRNA silencing potency in both 22Rv1 and LNCaP cells, which represent two different prostate cancer models. For downstream in vivo delivery, we formulated siARvm-LNPs and functionally validated these in vitro by demonstrating knockdown of AR and AR-V7 mRNA in prostate cancer cells and loss of AR-mediated transcriptional activation of the PSA gene in both cell lines following treatment. We also observed that siARvm-LNP induced cell viability inhibition was more potent compared to LNP containing siRNA targeting full-length AR mRNA (siARfl-LNP) in 22Rv1 cells as their proliferation is more dependent on AR splice variants than LNCaP and PC3 cells. The in vivo biodistribution of siARvm-LNPs was determined in 22Rv1 tumor-bearing mice by incorporating 14C-radiolabelled DSPC in LNP formulation, and we observed a 4.4% ID/g tumor accumulation following intravenous administration. Finally, treatment of 22Rv1 tumor bearing mice with siARvm-LNP resulted in significant tumor growth inhibition and survival benefit compared to siARfl-LNP or the siLUC-LNP control. To best of our knowledge, this is the first report demonstrating therapeutic effects of LNP-siRNA targeting AR splice variants in prostate cancer.

Synthesis and Characterization of Hybrid Lipid Nanoparticles Containing Gold Nanoparticles and a Weak Base Drug.

Hybrid lipid nanoparticles containing gold nanoparticles (LNP-GNPs) and drugs have potential for imaging applications as well as triggered release of LNP contents in response to pulsed laser or X-ray radiation mediated by the GNPs. However, methods to synthesize LNP-GNP systems that efficiently entrap GNPs (the potential triggered release and imaging agent) and then load and retain the drug cargo in a manner that may have clinical applications have proven elusive. Here, we develop a straightforward "bottom-up" approach to manufacture drug-loaded LNP-GNP systems. We show that negatively charged GNPs of 5 nm diameter can be stably loaded into LNPs containing 10 mol % ionizable cationic lipid using an ethanol dilution, rapid mixing approach and that these systems also exhibit aqueous compartments. Further, we show that such systems can also entrap ammonium sulfate, enabling pH-dependent loading of the weak base anti-cancer drug doxorubicin into the aqueous compartments. Cryo-transmission electron microscopy (Cryo-TEM) imaging clearly demonstrates the presence of GNPs in the interior of the resulting hybrid nanostructures as well as the formation of electron-dense drug precipitates in the aqueous core of the LNP-GNPs. The approach described here is a robust and straightforward method to generate hybrid LNP-GNP-drug and other LNP-metal nanoparticle-drug systems with potential applications for a variety of triggered release protocols.

Modular Lipid Nanoparticle Platform Technology for siRNA and Lipophilic Prodrug Delivery.

Successfully employing small interfering RNA (siRNA) therapeutics requires the use of nanotechnology for efficient intracellular delivery. Lipid nanoparticles (LNPs) have enabled the approval of various nucleic acid therapeutics. A major advantage of LNPs is the interchangeability of its building blocks and RNA payload, which allow it to be a highly modular system. In addition, drug derivatization approaches can be used to synthesize lipophilic small molecule prodrugs that stably incorporate in LNPs. This provides ample opportunities to develop combination therapies by co-encapsulating multiple therapeutic agents in a single formulation. Here, it is described how the modular LNP platform is applied for combined gene silencing and chemotherapy to induce additive anticancer effects. It is shown that various lipophilic taxane prodrug derivatives and siRNA against the androgen receptor, a prostate cancer driver, can be efficiently and stably co-encapsulated in LNPs without compromising physicochemical properties or gene-silencing ability. Moreover, it is demonstrated that the combination therapy induces additive therapeutic effects in vitro. Using a double-radiolabeling approach, the pharmacokinetic properties and biodistribution of LNPs and prodrugs following systemic administration in tumor-bearing mice are quantitatively determined. These results indicate that co-encapsulating siRNA and lipophilic prodrugs into LNPs is an attractive and straightforward plug-and-play approach for combination therapy development.

Scalable Production of Lipid Nanoparticles Containing Amphotericin B.

Lipid-based formulations have been developed to improve stability profiles, tolerability, and toxicity profiles of small molecule drugs. However, manufacture of such formulations involving lipophilic compounds can be labor-intensive and difficult to scale because of solubility and solvent compatibility issues. We have developed a rapid and scalable approach using rapid-mixing techniques to generate homogeneous lipid nanoparticle (LNP) formulations of siRNA, triglycerides, and hydrophilic weak-base drugs. Here, we used this approach to entrap a hydrophobic small molecule, Amphotericin B (AmpB), a hydrophobic drug not soluble in ethanol. The three prototypes presented in this study were derived from LNP-siRNA systems, triglyceride nanoparticles, and liposomal systems. Cryogenic transmission electron microscopy (cryo-TEM) revealed that all three LNP-AmpB formulations retain structural characteristics of the parent (AmpB-free) LNPs, with particles remaining stable for at least 1 month. All formulations showed similar in vitro toxicity profiles in comparison to AmBisome. Importantly, the formulations have a 2.5-fold improved IC50 for fungal growth inhibition as compared to AmBisome in in vitro efficacy studies. These results demonstrate that the rapid-mixing technology combined with dimethyl sulfoxide (DMSO) for drugs insoluble in other organic solvents can be a powerful manufacturing method for the generation of stable LNP drug formulations.

Characterization of Lipid Nanoparticles Containing Ionizable Cationic Lipids Using Design-of-Experiments Approach.

Lipid nanoparticles (LNPs) containing short-interfering RNA (LNP-siRNA systems) are a promising approach for silencing disease-causing genes in hepatocytes following intravenous administration. LNP-siRNA systems are generated by rapid mixing of lipids in ethanol with siRNA in aqueous buffer (pH 4.0) where the ionizable lipid is positively charged, followed by dialysis to remove ethanol and to raise the pH to 7.4. Ionizable cationic lipids are the critical excipient in LNP systems as they drive entrapment and intracellular delivery. A recent study on the formation of LNP-siRNA systems suggested that ionizable cationic lipids segregate from other lipid components upon charge neutralization to form an amorphous oil droplet in the core of LNPs. This leads to a decrease in intervesicle electrostatic repulsion, thereby engendering fusion of small vesicles to form final LNPs of increased size. In this study, we prepared LNP-siRNA systems containing four lipid components (hydrogenated soy phosphatidylcholine, cholesterol, PEG-lipid, and 1,2-dioleoyl-3-dimethylammonium propane) by microfluidic mixing. The effects of preparation parameters [lipid concentration, flow rate ratio (FRR), and total flow rate], dialysis process, and complex formation between siRNA and ionizable cationic lipids on the physicochemical properties [siRNA entrapment on the particle size and polydispersity index (PDI)] were investigated using a design of experiments approach. The results for the preparation parameters showed no impact on siRNA encapsulation, but lipid concentration and FRR significantly affected the particle size and PDI. In addition, the effect of FRR on the particle size was suppressed in the presence of anionic polymers such as siRNA as compared to the case of LNPs alone. More intriguingly, unlike empty LNPs, a decrease in the PDI and an increase in the particle size occurred after dialysis in the LNP-siRNA systems. Such changes by dialysis were suppressed at FRR = 1. These findings provide useful information to guide the development and manufacturing conditions for LNP-siRNA systems.

Protective Effect of Edaravone against Cationic Lipid-Mediated Oxidative Stress and Apoptosis.

Liposomes containing ionizable cationic lipids have been widely used for the delivery of nucleic acids such as small-interfering RNA and mRNA. The utility of cationic lipids with a permanent positive charge, however, is limited to in vitro transfection of cultured cells due to its dose-limiting toxic side effects observed in animals. Several reports have suggested that the permanently charged cationic lipids induce reactive oxygen species (ROS) and ROS-mediated toxicity in cells. We therefore hypothesized that the concomitant use of ROS inhibitor could reduce toxicity and improve drug efficacy. In this study, suppression of the cationic toxicity was evaluated using an ROS scavenger, edaravone, which is a low-molecular-weight antioxidant drug clinically approved for acute-phase cerebral infarction and amyotrophic lateral sclerosis. Cell viability assay in the mouse macrophage-like cell line RAW264 indicated that the concomitant use of edaravone were not able to suppress the cytotoxicity induced by cationic liposomes comprised of monovalent cationic lipid N-(1-[2,3-dioleyloxy]propyl)-N,N,N-trimethylammonium chloride (DOTMA) over a short period of time. Cationic lipids-induced necrosis was assumed to be involved in the cytotoxicity upon short-term exposure to cationic liposomes. On the other hand, the significant improvement of cell viability was observed when the short treatment with cationic liposomes was followed by exposure to edaravone for 24 h. It was also confirmed that apoptosis inhibition by ROS elimination might have contributed to this effect. These results suggest the utility of continuous administration with edaravone as concomitant drug for suppression of adverse reactions in therapeutic treatment using cationic liposomes.

Spontaneous, solvent-free entrapment of siRNA within lipid nanoparticles.

Lipid nanoparticle (LNP) formulations of nucleic acid are leading vaccine candidates for COVID-19, and enabled the first approved RNAi therapeutic, Onpattro. LNPs are composed of ionizable cationic lipids, phosphatidylcholine, cholesterol, and polyethylene glycol (PEG)-lipids, and are produced using rapid-mixing techniques. These procedures involve dissolution of the lipid components in an organic phase and the nucleic acid in an acidic aqueous buffer (pH 4). These solutions are then combined using a continuous mixing device such as a T-mixer or microfluidic device. In this mixing step, particle formation and nucleic acid entrapment occur. Previous work from our group has shown that, in the absence of nucleic acid, the particles formed at pH 4 are vesicular in structure, a portion of these particles are converted to electron-dense structures in the presence of nucleic acid, and the proportion of electron-dense structures increases with nucleic acid content. What remained unclear from previous work was the mechanism by which vesicles form electron-dense structures. In this study, we use cryogenic transmission electron microscopy and dynamic light scattering to show that efficient siRNA entrapment occurs in the absence of ethanol (contrary to the established paradigm), and suggest that nucleic acid entrapment occurs through inversion of preformed vesicles. We also leverage this phenomenon to show that specialized mixers are not required for siRNA entrapment, and that preformed particles at pH 4 can be used for in vitro transfection.

On the role of helper lipids in lipid nanoparticle formulations of siRNA.

Onpattro, the first RNAi-based therapeutic to receive FDA approval, is enabled by a lipid nanoparticle (LNP) system that facilitates siRNA delivery into the cytoplasm of target cells (hepatocytes) following intravenous (i.v.) administration. These LNP-siRNA systems consist of four lipid components (ionizable cationic lipid, distearolyphosphatidycholine or DSPC, cholesterol, and PEG-lipid) and siRNA. The ionizable cationic lipid has been optimised for RNA encapsulation and intracellular delivery, and the PEG-lipids have been engineered to regulate LNP size and transfection potency. The roles of the other "helper" lipids, DSPC and cholesterol, remain less clear. Here we show that in empty LNP systems that do not contain siRNA, DSPC-cholesterol resides in outer layers, whereas in loaded systems a portion of the DSPC-cholesterol is internalised together with siRNA. It is concluded that the presence of internalised helper lipid is vital to the stable encapsulation of siRNA in the LNP and thus to LNP-siRNA function.

Fusion-dependent formation of lipid nanoparticles containing macromolecular payloads.

The success of Onpattro™ (patisiran) clearly demonstrates the utility of lipid nanoparticle (LNP) systems for enabling gene therapies. These systems are composed of ionizable cationic lipids, phospholipid, cholesterol, and polyethylene glycol (PEG)-lipids, and are produced through rapid-mixing of an ethanolic-lipid solution with an acidic aqueous solution followed by dialysis into neutralizing buffer. A detailed understanding of the mechanism of LNP formation is crucial to improving LNP design. Here we use cryogenic transmission electron microscopy and fluorescence techniques to further demonstrate that LNP are formed through the fusion of precursor, pH-sensitive liposomes into large electron-dense core structures as the pH is neutralized. Next, we show that the fusion process is limited by the accumulation of PEG-lipid on the emerging particle. Finally, we show that the fusion-dependent mechanism of formation also applies to LNP containing macromolecular payloads including mRNA, DNA vectors, and gold nanoparticles.

Dexamethasone prodrugs as potent suppressors of the immunostimulatory effects of lipid nanoparticle formulations of nucleic acids.

Lipid nanoparticles (LNPs) are playing a leading role in enabling clinical applications of gene therapies based on DNA or RNA polymers. One factor impeding clinical acceptance of LNP therapeutics is that LNP formulations of nucleic acid polymers can be immunostimulatory, necessitating co-administration of potent corticosteroid immunosuppressive agents. Here, we describe the development of hydrophobic prodrugs of a potent corticosteroid, dexamethasone, that can be readily incorporated into LNP systems. We show that the presence of the dexamethasone prodrug LD003 effectively suppresses production of cytokines such as KC-GRO, TNFα, IL-1ß and IL-6 following intravenous administration of LNP loaded with immune stimulatory oligodeoxynucleotides containing cytosine-guanine dinucleotide motifs. Remarkably, LD003 dose levels corresponding to 0.5 mg/kg dexamethasone achieve a greater immunosuppressive effect than doses of 20 mg/kg of free dexamethasone. Similar immunosuppressive effects are observed for subcutaneously administered LNP-siRNA. Further, the incorporation of low levels of LD003 in LNP containing unmodified mRNA or plasmid DNA significantly reduced pro-inflammatory cytokine levels following intravenous administration. Our results suggest that incorporation of hydrophobic prodrugs such as LD003 into LNP systems could provide a convenient method for avoiding the immunostimulatory consequences of systemic administration of genetic drug formulations.

On the Formation and Morphology of Lipid Nanoparticles Containing Ionizable Cationic Lipids and siRNA.

Lipid nanoparticles (LNPs) containing short interfering RNA (LNP-siRNA) and optimized ionizable cationic lipids are now clinically validated systems for silencing disease-causing genes in hepatocytes following intravenous administration. However, the mechanism of formation and certain structural features of LNP-siRNA remain obscure. These systems are formed from lipid mixtures (cationic lipid, distearoylphosphatidylcholine, cholesterol, and PEG-lipid) dissolved in ethanol that is rapidly mixed with siRNA in aqueous buffer at a pH (pH 4) where the ionizable lipid is positively charged. The resulting dispersion is then dialyzed against a normal saline buffer to remove residual ethanol and raise the pH to 7.4 (above the p Ka of the cationic lipid) to produce the finished LNP-siRNA systems. Here we provide cryogenic transmission electron microscopy (cryo-TEM) and X-ray evidence that the complexes formed between siRNA and ionizable lipid at pH 4 correspond to tightly packed bilayer structures with siRNA sandwiched between closely apposed monolayers. Further, it is shown that ionizable lipid not complexed to siRNA promotes formation of very small vesicular structures at pH 4 that coalesce to form larger LNP structures with amorphous electron dense cores at pH 7.4. A mechanism of formation of LNP-siRNA systems is proposed whereby siRNA is first sandwiched between closely apposed lipid monolayers at pH 4 and subsequently trapped in these structures as the pH is raised to 7.4, whereas ionizable lipid not interacting with siRNA moves from bilayer structure to adopt an amorphous oil phase located in the center of the LNP as the pH is raised. This model is discussed in terms of previous hypotheses and potential relevance to the design of LNP-siRNA systems.

Lipid nanoparticle delivery of glucagon receptor siRNA improves glucose homeostasis in mouse models of diabetes.

OBJECTIVE: Hyperglucagonemia is present in many forms of diabetes and contributes to hyperglycemia, and glucagon suppression can ameliorate diabetes in mice. Leptin, a glucagon suppressor, can also reverse diabetes in rodents. Lipid nanoparticle (LNP) delivery of small interfering RNA (siRNA) effectively targets the liver and is in clinical trials for the treatment of various diseases. We compared the effectiveness of glucagon receptor (Gcgr)-siRNA delivered via LNPs to leptin in two mouse models of diabetes. METHODS: Gcgr siRNA encapsulated into LNPs or leptin was administered to mice with diabetes due to injection of the ß-cell toxin streptozotocin (STZ) alone or combined with high fat diet (HFD/STZ). RESULTS: In STZ-diabetic mice, a single injection of Gcgr siRNA lowered blood glucose levels for 3 weeks, improved glucose tolerance, and normalized plasma ketones levels, while leptin therapy normalized blood glucose levels, oral glucose tolerance, and plasma ketones, and suppressed lipid metabolism. In contrast, in HFD/STZ-diabetic mice, Gcgr siRNA lowered blood glucose levels for 2 months, improved oral glucose tolerance, and reduced HbA1c, while leptin had no beneficial effects. CONCLUSIONS: While leptin may be more effective than Gcgr siRNA at normalizing both glucose and lipid metabolism in STZ diabetes, Gcgr siRNA is more effective at reducing blood glucose levels in HFD/STZ diabetes.

Rapid synthesis of lipid nanoparticles containing hydrophobic inorganic nanoparticles.

A straightforward "bottom-up" synthesis is described for efficient entrapment of inorganic hydrophobic nanoparticles (HNPs) consisting of iron oxide, gold, or quantum dots within the hydrophobic core of lipid nanoparticles (LNPs). These LNPs consist of hydrophobic "core" lipids such as triolein surrounded by a monolayer of amphipathic "surface" lipids, such as phosphatidylcholine and polyethylene-glycol-lipid. It is shown that rapid, controlled mixing of HNPs, core lipids and surface lipids in an organic solvent with an aqueous phase resulted in stable, monodisperse LNPs containing HNPs (LNP-HNP). This method allows 40-fold more hydrophobic iron oxide nanoparticles (IONPs) to be entrapped within an LNP than previous methods and can be readily extended to encapsulate other HNPs. The LNP-HNP diameter can be modulated over the range of 35-150 nm by varying the flow rate during particle synthesis or by varying the core-to-surface lipid ratio. LNP-IONPs can be generated using a variety of "core" lipids, including other triglycerides as well as cholesteryl-palmitate and tocopherol. Finally, it is shown that LNP-IONPs are accumulated in the liver, resulting in enhanced contrast for in vivo MRI. It is concluded that the bottom-up approach for encapsulating HNPs within LNPs has advantages of homogeneity, reproducibility and stability required for biomedical applications.

Design of lipid nanoparticles for in vitro and in vivo delivery of plasmid DNA.

Lipid nanoparticles (LNPs) containing distearoylphosphatidlycholine (DSPC), and ionizable amino-lipids such as dilinoleylmethyl-4-dimethylaminobutyrate (DLin-MC3-DMA) are potent siRNA delivery vehicles in vivo. Here we explore the utility of similar LNP systems as transfection reagents for plasmid DNA (pDNA). It is shown that replacement of DSPC by unsaturated PCs and DLin-MC3-DMA by the related lipid DLin-KC2-DMA resulted in highly potent transfection reagents for HeLa cells in vitro. Further, these formulations exhibited excellent transfection properties in a variety of mammalian cell lines and transfection efficiencies approaching 90% in primary cell cultures. These transfection levels were equal or greater than achieved by Lipofectamine, with much reduced toxicity. Finally, microinjection of LNP-eGFP into the limb bud of a chick embryo resulted in robust reporter-gene expression. It is concluded that LNP systems containing ionizable amino lipids can be highly effective, non-toxic pDNA delivery systems for gene expression both in vitro and in vivo.

The Niemann-Pick C1 Inhibitor NP3.47 Enhances Gene Silencing Potency of Lipid Nanoparticles Containing siRNA.

The therapeutic applications of lipid nanoparticle (LNP) formulations of small interfering RNA (siRNA), are hampered by inefficient delivery of encapsulated siRNA to the cytoplasm following endocytosis. Recent work has shown that up to 70% of endocytosed LNP-siRNA particles are recycled to the extracellular medium and thus cannot contribute to gene silencing. Niemann-Pick type C1 (NPC1) is a late endosomal/lysosomal membrane protein required for efficient extracellular recycling of endosomal contents. Here we assess the influence of NP3.47, a putative small molecule inhibitor of NPC1, on the gene silencing potency of LNP-siRNA systems in vitro. Intracellular uptake and colocalization studies revealed that the presence of NP3.47 caused threefold or higher increases in accumulation of LNP-siRNA in late endosomes/lysosomes as compared with controls in a variety of cell lines. The gene silencing potency of LNP siRNA was enhanced up to fourfold in the presence of NP3.47. Mechanisms of action studies are consistent with the proposal that NP3.47 acts to inhibit NPC1. Our findings suggest that the pharmacological inhibition of NPC1 is an attractive strategy to enhance the therapeutic efficacy of LNP-siRNA by trapping LNP-siRNA in late endosomes, thereby increasing opportunities for endosomal escape.

Influence of particle size on the in vivo potency of lipid nanoparticle formulations of siRNA.

Lipid nanoparticles (LNP) can provide a clinically effective method for delivering small interfering RNA (siRNA) to silence pathological genes in hepatocytes. The gene silencing potency of these LNP-siRNA systems has been shown to depend on a variety of factors including association with serum factors such as ApoE and the pKa of component ionizable lipids. Here we investigate the influence of LNP size, an important parameter affecting tissue penetration of LNP systems, on the pharmacokinetics, biodistribution, and hepatic gene silencing potency of LNP-siRNA systems following intravenous administration. For LNP systems stabilized by a polyethylene glycol (PEG)-lipid that can dissociate from the LNP following injection, it is shown that small (diameter≤30nm) systems are considerably less potent than their larger counterparts. This is attributed in part to the ability of other lipid components, particularly the ionizable amino-lipid, to dissociate from the LNP following dissociation of the PEG-lipid. Small LNP stabilized by PEG-lipids with slow dissociation rates exhibited much reduced amino-lipid dissociation rates, however such systems are relatively impotent due to the continued presence of the PEG coating. These results demonstrate the delicate balance between the in vivo potency of LNP-siRNA systems and the residence times of component lipids in the LNP particle itself and suggest new directions to optimize the in vivo gene silencing potency of small LNP-siRNA systems.

A Glu-urea-Lys Ligand-conjugated Lipid Nanoparticle/siRNA System Inhibits Androgen Receptor Expression In Vivo.

The androgen receptor plays a critical role in the progression of prostate cancer. Here, we describe targeting the prostate-specific membrane antigen using a lipid nanoparticle formulation containing small interfering RNA designed to silence expression of the messenger RNA encoding the androgen receptor. Specifically, a Glu-urea-Lys PSMA-targeting ligand was incorporated into the lipid nanoparticle system formulated with a long alkyl chain polyethylene glycol-lipid to enhance accumulation at tumor sites and facilitate intracellular uptake into tumor cells following systemic administration. Through these features, and by using a structurally refined cationic lipid and an optimized small interfering RNA payload, a lipid nanoparticle system with improved potency and significant therapeutic potential against prostate cancer and potentially other solid tumors was developed. Decreases in serum prostate-specific antigen, tumor cellular proliferation, and androgen receptor levels were observed in a mouse xenograft model following intravenous injection. These results support the potential clinical utility of a prostate-specific membrane antigen-targeted lipid nanoparticle system to silence the androgen receptor in advanced prostate cancer.