Nanoparticles Containing Oxidized Cholesterol Deliver mRNA to the Liver Microenvironment at Clinically Relevant Doses

Using mRNA to produce therapeutic proteins is a promising approach to treat genetic diseases. However, systemically delivering mRNA to cell types besides hepatocytes remains challenging. Fast identification of nanoparticle delivery (FIND) is a DNA barcode‐based system designed to measure how over 100 lipid nanoparticles (LNPs) deliver mRNA that functions in the cytoplasm of target cells in a single mouse. By using FIND to quantify how 75 chemically distinct LNPs delivered mRNA to 28 cell types in vivo, it is found that an LNP formulated with oxidized cholesterol and no targeting ligand delivers Cre mRNA, which edits DNA in hepatic endothelial cells and Kupffer cells at 0.05 mg kg^?1. Notably, the LNP targets liver microenvironmental cells fivefold more potently than hepatocytes. The structure of the oxidized cholesterols added to the LNP is systematically varied to show that the position of the oxidative modification may be important; cholesterols modified on the hydrocarbon tail associated with sterol ring D tend to outperform cholesterols modified on sterol ring B. These data suggest that LNPs formulated with modified cholesterols can deliver gene‐editing mRNA to the liver microenvironment at clinically relevant doses.     

A Combinatorial Library of Lipid Nanoparticles for RNA Delivery to Leukocytes

Lipid nanoparticles (LNPs) are the most advanced nonviral platforms for small interfering RNA (siRNA) delivery that are clinically approved. These LNPs, based on ionizable lipids, are found in the liver and are now gaining much attention in the field of RNA therapeutics. The previous generation of ionizable lipids varies in linker moieties, which greatly influences in vivo gene silencing efficiency. Here novel ionizable amino lipids based on the linker moieties such as hydrazine, hydroxylamine, and ethanolamine are designed and synthesized. These lipids are formulated into LNPs and screened for their efficiency to deliver siRNAs into leukocytes, which are among the hardest to transfect cell types. Two potent lipids based on their in vitro gene silencing efficiencies are also identified. These lipids are further evaluated for their biodistribution profile, efficient gene silencing, liver toxicity, and potential immune activation in mice. A robust gene silencing is also found in primary lymphocytes when one of these lipids is formulated into LNPs with a pan leukocyte selective targeting agent (β₇ integrin). Taken together, these lipids have the potential to open new avenues in delivering RNAs into leukocytes.     

Targeted Delivery of siRNA‐Generating DNA Nanocassettes Using Multifunctional Nanoparticles

Molecular therapy using a small interfering RNA (siRNA) has shown promise in the development of novel therapeutics. Various formulations have been used for in vivo delivery of siRNAs. However, the stability of short double‐stranded RNA molecules in the blood and efficiency of siRNA delivery into target organs or tissues following systemic administration have been the major issues that limit applications of siRNA in human patients. In this study, multifunctional siRNA delivery nanoparticles are developed that combine imaging capability of nanoparticles with urokinase plasminogen activator receptor‐targeted delivery of siRNA expressing DNA nanocassettes. This theranostic nanoparticle platform consists of a nanoparticle conjugated with targeting ligands and double‐stranded DNA nanocassettes containing a U6 promoter and a shRNA gene for in vivo siRNA expression. Targeted delivery and gene silencing efficiency of firefly luciferase siRNA nanogenerators are demonstrated in tumor cells and in animal tumor models. Delivery of survivin siRNA expressing nanocassettes into tumor cells induces apoptotic cell death and sensitizes cells to chemotherapy drugs. The ability of expression of siRNAs from multiple nanocassettes conjugated to a single nanoparticle following receptor‐mediated internalization should enhance the therapeutic effect of the siRNA‐mediated cancer therapy.     

Mild Innate Immune Activation Overrides Efficient Nanoparticle-Mediated RNA Delivery

Clinical mRNA delivery remains challenging, in large part because how physiology alters delivery in vivo remains underexplored. For example, mRNA delivered by lipid nanoparticles (LNPs) is being considered to treat inflammation, but whether inflammation itself changes delivery remains understudied. Relationships between immunity, endocytosis, and mRNA translation lead to hypothesize that toll-like receptor 4 (TLR4) activation reduced LNP-mediated mRNA delivery. Therefore, LNP uptake, endosomal escape, and mRNA translation with and without TLR4 activation are quantified. In vivo DNA barcoding is used to discover a novel LNP that delivers mRNA to Kupffer cells at clinical doses; unlike most LNPs, this LNP does not preferentially target hepatocytes. TLR4 activation blocks mRNA translation in all tested cell types, without reducing LNP uptake; inhibiting TLR4 or its downstream effector protein kinase R improved delivery. The discrepant effects of TLR4 on i) LNP uptake and ii) translation suggests TLR4 activation can “override” LNP targeting, even after mRNA is delivered into target cells. Given near-future clinical trials using mRNA to modulate inflammation, this highlights the need to understand inflammatory signaling in on- and off-target cells. More generally, this suggests an LNP which delivers mRNA to one inflammatory disease may not deliver mRNA to another.     

Synthesis and Biological Evaluation of Ionizable Lipid Materials for the In Vivo Delivery of Messenger RNA to B Lymphocytes

B lymphocytes regulate several aspects of immunity including antibody production, cytokine secretion, and T‐cell activation; moreover, B cell misregulation is implicated in autoimmune disorders and cancers such as multiple sclerosis and non‐Hodgkin's lymphomas. The delivery of messenger RNA (mRNA) into B cells can be used to modulate and study these biological functions by means of inducing functional protein expression in a dose‐dependent and time‐controlled manner. However, current in vivo mRNA delivery systems fail to transfect B lymphocytes and instead primarily target hepatocytes and dendritic cells. Here, the design, synthesis, and biological evaluation of a lipid nanoparticle (LNP) system that can encapsulate mRNA, navigate to the spleen, transfect B lymphocytes, and induce more than 60 pg of protein expression per million B cells within the spleen is described. Importantly, this LNP induces more than 85% of total protein production in the spleen, despite LNPs being observed transiently in the liver and other organs. These results demonstrate that LNP composition alone can be used to modulate the site of protein induction in vivo, highlighting the critical importance of designing and synthesizing new nanomaterials for nucleic acid delivery.     

Ionizable Lipid with Supramolecular Chemistry Features for RNA Delivery In Vivo

Lipid nanoparticles (LNPs) and ribonucleic acid (RNA) technology are highly versatile tools that can be deployed for diagnostic, prophylactic, and therapeutic applications. In this report, supramolecular chemistry concepts are incorporated into the rational design of a new ionizable lipid, C3-K2-E14, for systemic administration. This lipid incorporates a cone-shaped structure intended to facilitate cell bilayer disruption, and three tertiary amines to improve RNA binding. Additionally, hydroxyl and amide motifs are incorporated to further enhance RNA binding and improve LNP stability. Optimization of messenger RNA (mRNA) and small interfering RNA (siRNA) formulation conditions and lipid ratios produce LNPs with favorable diameter (<150 nm), polydispersity index (<0.15), and RNA encapsulation efficiency (>90%), all of which are preserved after 2 months at 4 or 37 °C storage in ready-to-use liquid form. The lipid and formulated LNPs are well-tolerated in animals and show no deleterious material-induced effects. Furthermore, 1 week after intravenous LNP administration, fluorescent signal from tagged RNA payloads are not detected. To demonstrate the long-term treatment potential for chronic diseases, repeated dosing of C3-K2-E14 LNPs containing siRNA that silences the colony stimulating factor-1 (CSF-1) gene can modulate leukocyte populations in vivo, further highlighting utility.     

Low‐Density Lipoprotein‐Mimicking Nanoparticles for Tumor‐Targeted Theranostic Applications

This study introduces multifunctional lipid nanoparticles (LNPs), mimicking the structure and compositions of low‐density lipoproteins, for the tumor‐targeted co‐delivery of anti‐cancer drugs and superparamagnetic nanocrystals. Paclitaxel (4.7 wt%) and iron oxide nanocrystals (6.8 wt%, 11 nm in diameter) are co‐encapsulated within folate‐functionalized LNPs, which contain a cluster of nanocrystals with an overall diameter of about 170 nm and a zeta potential of about ‐40 mV. The folate‐functionalized LNPs enable the targeted detection of MCF‐7, human breast adenocarcinoma expressing folate receptors, in T₂‐weighted magnetic resonance images as well as the efficient intracellular delivery of paclitaxel. Paclitaxel‐free LNPs show no significant cytotoxicity up to 0.2 mg mL^?1, indicating the excellent biocompatibility of the LNPs for intracellular drug delivery applications. The targeted anti‐tumor activities of the LNPs in a mouse tumor model suggest that the low‐density lipoprotein‐mimetic LNPs can be an effective theranostic platform with excellent biocompatibility for the tumor‐targeted co‐delivery of various anti‐cancer agents.     

Ionizable Amino‐Polyesters Synthesized via Ring Opening Polymerization of Tertiary Amino‐Alcohols for Tissue Selective mRNA Delivery

The utility of messenger RNA (mRNA) as a therapy is gaining a broad interest due to its potential for addressing a wide range of diseases, while effective delivery of mRNA molecules to various tissues still poses a challenge. This study reports on the design and characterization of new ionizable amino‐polyesters (APEs), synthesized via ring opening polymerization (ROP) of lactones with tertiary amino‐alcohols that enable tissue and cell type selective delivery of mRNA. With a diverse library of APEs formulated into lipid nanoparticles (LNP), structure‐activity parameters crucial for efficient transfection are established and APE‐LNPs are identified that can preferentially home to and elicit effective mRNA expression with low in vivo toxicity in lung endothelium, liver hepatocytes, and splenic antigen presenting cells, including APE‐LNP demonstrating nearly tenfold more potent systemic mRNA delivery to the lungs than vivo‐jetPEI. Adopting tertiary amino‐alcohols to initiate ROP of lactones allows to control polymer molecular weight and obtain amino‐polyesters with narrow molecular weight distribution, exhibiting batch‐to‐batch consistency. All of which highlight the potential for clinical translation of APEs for systemic mRNA delivery and demonstrate the importance of employing controlled polymerization in the design of new polymeric nanomaterials to improve in vivo nucleic acid delivery.     

Gold Nanoparticles Capped with Polyethyleneimine for Enhanced siRNA Delivery

An efficient and safe delivery system for small interfering RNA (siRNA) is required for clinical application of RNA interfering therapeutics. Polyethyleneimine (PEI)‐capped gold nanoparticles (AuNPs) are successfully manufactured using PEI as the reductant and stabilizer, which bind siRNA at an appropriate weight ratio by electrostatic interaction and result in well‐dispersed nanoparticles with uniform structure and narrow size distribution. With siRNA binding, PEI‐capped AuNPs induce more significant and enhanced reduction in targeted green fluorescent protein expression in MDA‐MB‐435s cells, though more internalized PEI/siRNA complexes in cells are evidenced by confocal laser scanning microscopy observation and fluorescence‐activated cell sorting analyses. PEI‐capped AuNPs/siRNA targeting endogenous cell‐cycle kinase, an oncogene polo‐like kinase 1 (PLK1), display significant gene expression knockdown and induce enhanced cell apoptosis, whereas it is not obvious when the cells are treated with PLK1 siRNA using PEI as the carrier. Without exhibiting cellular toxicity, PEI‐capped AuNPs appear to be suitable as a potential carrier for intracellular siRNA delivery.     

Preparation and Characterization of Dentin Phosphophoryn‐Derived Peptide‐Functionalized Lignin Nanoparticles for Enhanced Cellular Uptake

The surface modification of nanoparticles (NPs) using different ligands is a common strategy to increase NP?cell interactions. Here, dentin phosphophoryn‐derived peptide (DSS) lignin nanoparticles (LNPs) are prepared and characterized, the cellular internalization of the DSS‐functionalized LNPs (LNPs‐DSS) into three different cancer cell lines is evaluated, and their efficacy with the widely used iRGD peptide is compared. It is shown that controlled extent of carboxylation of lignin improves the stability at physiological conditions of LNPs formed upon solvent exchange. Functionalization with DSS and iRGD peptides maintains the spherical morphology and moderate polydispersity of LNPs. The LNPs exhibit good cytocompatibility when cultured with PC3‐MM2, MDA‐MB‐231, and A549 in the conventional 2D model and in the 3D cell spheroid morphology. Importantly, the 3D cell models reveal augmented internalization of peptide‐functionalized LNPs and improve antiproliferative effects when the LNPs are loaded with a cytotoxic compound. Overall, LNPs‐DSS show equal or even superior cellular internalization than the LNPs‐iRGD, suggesting that DSS can also be used to enhance the cellular uptake of NPs into different types of cells, and release different cargos intracellularly.     

HDL‐Mimicking Peptide–Lipid Nanoparticles with Improved Tumor Targeting

Targeted delivery of intracellularly active diagnostics and therapeutics in vivo is a major challenge in cancer nanomedicine. A nanocarrier should possess long circulation time yet be small and stable enough to freely navigate through interstitial space to deliver its cargo to targeted cells. Herein, it is shown that by adding targeting ligands to nanoparticles that mimic high‐density lipoprotein (HDL), tumor‐targeted sub‐30‐nm peptide–lipid nanocarriers are created with controllable size, cargo loading, and shielding properties. The size of the nanocarrier is tunable between 10 and 30 nm, which correlates with a payload of 15–100 molecules of fluorescent dye. Ligand‐directed nanocarriers targeting epidermal growth factor receptor (EGFR) are confirmed both in vitro and in vivo. The nanocarriers show favorable circulation time, tumor accumulation, and biodistribution with or without the targeting ligand. The EGFR targeting ligand is proved to be essential for the EGFR‐mediated tumor cell uptake of the nanocarriers, a prerequisite of intracellular delivery. The results demonstrate that targeted HDL‐mimetic nanocarriers are useful delivery vehicles that could open new avenues for the development of clinically viable targeted nanomedicine.     

Nanoparticle‐Enabled,Image‐Guided Treatment Planning of Target Specific RNAi Therapeutics in an Orthotopic Prostate Cancer Model

The abilities to deliver siRNA to its intended action site and assess the delivery efficiency are challenges for current RNAi therapy, where effective siRNA delivery will join force with patient genetic profiling to achieve optimal treatment outcome. Imaging could become a critical enabler to maximize RNAi efficacy in the context of tracking siRNA delivery, rational dosimetry and treatment planning. Several imaging modalities have been used to visualize nanoparticle‐based siRNA delivery but rarely did they guide treatment planning. We report a multimodal theranostic lipid‐nanoparticle, HPPS(NIR)‐chol‐siRNA, which has a near‐infrared (NIR) fluorescent core, enveloped by phospholipid monolayer, intercalated with siRNA payloads, and constrained by apoA‐I mimetic peptides to give ultra‐small particle size (<30 nm). Using fluorescence imaging, we demonstrated its cytosolic delivery capability for both NIR‐core and dye‐labeled siRNAs and its structural integrity in mice through intravenous administration, validating the usefulness of NIR‐core as imaging surrogate for non‐labeled therapeutic siRNAs. Next, we validated the targeting specificity of HPPS(NIR)‐chol‐siRNA to orthotopic tumor using sequential four‐steps (in vivo, in situ, ex vivo and frozen‐tissue) fluorescence imaging. The image co‐registration of computed tomography and fluorescence molecular tomography enabled non‐invasive assessment and treatment planning of siRNA delivery into the orthotopic tumor, achieving efficacious RNAi therapy.     

Rekindling RNAi Therapy: Materials Design Requirements for In Vivo siRNA Delivery

With the recent FDA approval of the first siRNA‐derived therapeutic, RNA interference (RNAi)‐mediated gene therapy is undergoing a transition from research to the clinical space. The primary obstacle to realization of RNAi therapy has been the delivery of oligonucleotide payloads. Therefore, the main aims is to identify and describe key design features needed for nanoscale vehicles to achieve effective delivery of siRNA‐mediated gene silencing agents in vivo. The problem is broken into three elements: 1) protection of siRNA from degradation and clearance; 2) selective homing to target cell types; and 3) cytoplasmic release of the siRNA payload by escaping or bypassing endocytic uptake. The in vitro and in vivo gene silencing efficiency values that have been reported in publications over the past decade are quantitatively summarized by material type (lipid, polymer, metal, mesoporous silica, and porous silicon), and the overall trends in research publication and in clinical translation are discussed to reflect on the direction of the RNAi therapeutics field.     

pH‐Responsive PEG‐Shedding and Targeting Peptide‐Modified Nanoparticles for Dual‐Delivery of Irinotecan and microRNA to Enhance Tumor‐Specific Therapy

Irinotecan is one of the main chemotherapeutic agents for colorectal cancer (CRC). MicroRNA‐200 (miR‐200) has been reported to inhibit metastasis in cancer cells. Herein, pH‐sensitive and peptide‐modified liposomes and solid lipid nanoparticles (SLN) are designed for encapsulation of irinotecan and miR‐200, respectively. These peptides include one cell‐penetrating peptide, one ligand targeted to tumor neovasculature undergoing angiogenesis, and one mitochondria‐targeting peptide. The peptide‐modified nanoparticles are further coated with a pH‐sensitive PEG‐lipid derivative with an imine bond. These specially‐designed nanoparticles exhibit pH‐responsive release, internalization, and intracellular distribution in acidic pH of colon cancer HCT116 cells. These nanoparticles display low toxicity to blood and noncancerous intestinal cells. Delivery of miR‐200 by SLN further increases the cytotoxicity of irinotecan‐loaded liposomes against CRC cells by triggering apoptosis and suppressing RAS/β‐catenin/ZEB/multiple drug resistance (MDR) pathways. Using CRC‐bearing mice, the in vivo results further indicate that irinotecan and miR‐200 in pH‐responsive targeting nanoparticles exhibit positive therapeutic outcomes by inhibiting colorectal tumor growth and reducing systemic toxicity. Overall, successful delivery of miR and chemotherapy by multifunctional nanoparticles may modulate β‐catenin/MDR/apoptosis/metastasis signaling pathways and induce programmed cancer cell death. Thus, these pH‐responsive targeting nanoparticles may provide a potential regimen for effective treatment of colorectal cancer.     

Photosensitizer‐Incorporated Quadruplex DNA‐Gated Nanovechicles for Light‐Triggered,Targeted Dual Drug Delivery to Cancer Cells

A novel light‐operated vehicle for targeted intracellular drug delivery is constructed using photosensitizer‐incorporated G‐quadruplex DNA‐capped mesoporous silica nanoparticles. Upon light irradiation, the photosensitizer generates ROS, causing the DNA capping to be cleaved and allowing cargo to be released. Importantly, this platform makes it possible to develop a drug‐carrier system for the synergistic combination of chemotherapy and PDT for cancer treatment with spatial/temporal control. Furthermore, the introducing of targeting ligands further improves tumor targeting efficiency. The excellent biocompatibility, cell‐specific intracellular drug delivery, and cellular uptake properties set up the basis for future biomedical application that require in vivo controlled, targeted drug delivery.     

Single Chain Epidermal Growth Factor Receptor Antibody Conjugated Nanoparticles for in vivo Tumor Targeting and Imaging

Epidermal growth factor receptor (EGFR) targeted nanoparticle are developed by conjugating a single‐chain anti‐EGFR antibody (ScFvEGFR) to surface functionalized quantum dots (QDs) or magnetic iron oxide (IO) nanoparticles. The results show that ScFvEGFR can be successfully conjugated to the nanoparticles, resulting in compact ScFvEGFR nanoparticles that specifically bind to and are internalized by EGFR‐expressing cancer cells, thereby producing a fluorescent signal or magnetic resonance imaging (MRI) contrast. In vivo tumor targeting and uptake of the nanoparticles in human cancer cells is demonstrated after systemic delivery of ScFvEGFR‐QDs or ScFvEGFR‐IO nanoparticles into an orthotopic pancreatic cancer model. Therefore, ScFvEGFR nanoparticles have potential to be used as a molecular‐targeted in vivo tumor imaging agent. Efficient internalization of ScFvEGFR nanoparticles into tumor cells after systemic delivery suggests that the EGFR‐targeted nanoparticles can also be used for the targeted delivery of therapeutic agents.     

A Poly(Propyleneimine) Dendrimer‐Based Polyplex‐System for Single‐Chain Antibody‐Mediated Targeted Delivery and Cellular Uptake of SiRNA

Therapeutics based on small interfering RNAs (siRNAs) offer a great potential to treat so far incurable diseases or metastatic cancer. However, the broad application of siRNAs using various nonviral carrier systems is hampered by unspecific toxic side effects, poor pharmacokinetics due to unwanted delivery of siRNA‐loaded nanoparticles into nontarget organs, or rapid renal excretion. In order to overcome these obstacles, several targeting strategies using chemically linked antibodies and ligands have emerged. This study reports a new modular polyplex carrier system for targeted delivery of siRNA, which is based on transfection‐disabled maltose‐modified poly(propyleneimine)‐dendrimers (mal‐PPI) bioconjugated to single chain fragment variables (scFvs). To achieve targeted delivery into tumor cells expressing the epidermal growth factor receptor variant III (EGFRvIII), monobiotinylated anti‐EGFRvIII scFv fused to a Propionibacterium shermanii transcarboxylase‐derived biotinylation acceptor (P‐BAP) is bioconjugated to mal‐PPI through a novel coupling strategy solely based on biotin–neutravidin bridging. In contrast to polyplexes containing an unspecific control scFv‐P‐BAP, the generated EGFRvIII‐specific polyplexes are able to exclusively deliver siRNA to tumor cells and tumors by receptor‐mediated endocytosis. These results suggest that receptor‐mediated uptake of otherwise noninternalized mal‐PPI‐based polyplexes is a promising avenue to improve siRNA therapy of cancer, and introduce a novel strategy for modular bioconjugation of protein ligands to nanoparticles.     

N-Alkyl-PEI-functionalized iron oxide nanoclusters for efficient siRNA delivery

Small-interfering RNA (siRNA) is an emerging class of therapeutics, which works by regulating the expression of a specific gene involved in disease progression. Despite the promises, effective transport of siRNA with minimal side effects remains a challenge. In this study, a nonviral nanoparticle gene carrier is developed and its efficiency for siRNA delivery and transfection is validated at both in vitro and in vivo levels. Such a nanocarrier, abbreviated as Alkyl-PEI2k-IO, was constructed with a core of iron oxide nanoparticles (IOs) and a shell of alkylated polyethyleneimine of 2000 Da [corrected] molecualr weight (Alkyl-PEI2k). It is found to be able to bind with siRNA, resulting in well-dispersed nanoparticles with a controlled clustering structure and narrow size distribution. Electrophoresis studies show that the Alkyl-PEI2k-IOs could retard siRNA completely at N:P ratios (i.e., PEI nitrogen to nucleic acid phosphate) above 10, protect siRNA from enzymatic degradation in serum, and release complexed siRNA efficiently in the presence of polyanionic heparin. The knockdown efficiency of the siRNA-loaded nanocarriers is assessed with 4T1 cells stably expressing luciferase (fluc-4T1) and further, with a fluc-4T1 xenograft model. Significant down-regulation of luciferase is observed, and unlike high-molecular-weight analogues, the Alkyl-PEI2k-coated IOs show good biocompatibility. In conclusion, Alkyl-PEI2k-IOs demonstrate highly efficient delivery of siRNA and an innocuous toxic profile, making it a potential carrier for gene therapy.     

Cationic Bovine Serum Albumin Based Self‐Assembled Nanoparticles as siRNA Delivery Vector for Treating Lung Metastatic Cancer

It is generally believed that intravenous application of cationic vectors is limited by the binding of abundant negatively charged serum components, which may cause rapid clearance of the therapeutic agent from the blood stream. However, previous studies show that systemic delivery of cationic gene vectors mediates specific and efficient transfection within the lung, mainly as a result of interaction of the vectors with serum proteins. Based on these findings, a novel and charge‐density‐controllable siRNA delivery system is developed to treat lung metastatic cancer by using cationic bovine serum albumin (CBSA) as the gene vector. By surface modification of BSA, CBSA with different isoelectric points (pI) is synthesized and the optimal cationization degree of CBSA is determined by considering the siRNA binding and delivery ability, as well as toxicity. The CBSA can form stable nanosized particles with siRNA and protect siRNA from degradation. CBSA also shows excellent abiliies to intracellularly deliver siRNA and mediate significant accumulation in the lung. When Bcl2‐specific siRNA is introduced to this system, CBSA/siRNA nanoparticles exhibit an efficient gene‐silencing effect that induces notable cancer cell apoptosis and subsequently inhibits the tumor growth in a B16 lung metastasis model. These results indicate that CBSA‐based self‐assembled nanoparticles can be a promising strategy for a siRNA delivery system for lung targeting and metastatic cancer therapy.