Lipid nanoparticles (LNPs) are effective carriers for messenger ribonucleic acid (mRNA) delivery in vaccines; however, their reliance on extreme cold-chain storage limits global manufacturing and distribution. Conventional LNPs are formed by rapidly mixing four lipids with mRNA through electrostatic interactions between cationic ionizable lipids and negatively charged nucleic acids, facilitating nucleation and precipitation of mRNA-loaded LNPs. However, this binding also accelerates mRNA degradation, requiring stringent cold storage which limits widespread vaccine deployment. To overcome this limitation, we introduce a post-loading strategy in which empty LNPs (eLNPs) are first fabricated and RNA is subsequently loaded at a later stage. Using scalable confined impinging jet (CIJ) mixers, we optimized pH, buffer composition, lipid concentration, and ethanol content to produce colloidally stable eLNPs. Controlled adjustment of ethanol content and pH enabled efficient incorporation of four distinct RNA payloads while maintaining loaded LNP diameters below 100 nm. Post-loaded LNPs demonstrated mRNA delivery efficiencies in HeLa cells comparable to those of conventionally co-precipitated LNPs. Consistent size distributions and zeta potentials further confirmed comparable surface properties. Structural characterization by x-ray and neutron scattering revealed similar internal architectures for post-loaded and co-precipitated LNPs without compromising RNA loading efficiency. Together, these results demonstrate equivalent cellular delivery performance between the two formulations. This post-loading approach enables decentralized assembly of mRNA LNPs at the point of administration, with both eLNPs and mRNA stored under mild refrigeration, thereby improving vaccine accessibility. Moreover, eLNPs function as modular laboratory reagents, facilitating the translation of mRNA research toward clinical applications.

Despite the recent advances and clinical demonstration of lipid nanoparticles (LNPs) for therapeutic and prophylactic applications, the extrahepatic delivery of nucleic acids remains a significant challenge in the field. This limitation arises from the rapid desorption of lipid-PEG in the bloodstream and clearance to the liver, which hinders extrahepatic delivery. In response, we explore the substitution of lipid-PEG with biodegradable block copolymers (BCPs), specifically poly(epsilon-caprolactone)-block-poly(ethylene glycol) (PCL-b-PEG). BCPs offer strong anchoring for large macromolecules, potentially enhancing cell-specific targeting. To develop and optimize BCP-stabilized LNPs (BCP-LNPs), we employed a Design of Experiment (DOE) approach. Through a systematic exploration, we identified optimal formulations for BCP-LNPs, achieving desirable physicochemical properties and encapsulation efficiency. Notably, BCP-LNPs exhibit surprising trends in transfection efficiency, with certain formulations showing up to a 40-fold increase in transfection in Hela cells, while maintaining minimal cytotoxicity. The lipid compositions that optimized PCL-b-PEG LNP transfection were different from the compositions that optimized PEG-lipid LNP transfection. Furthermore, our study confirms the versatility of BCP-LNPs in encapsulating and delivering both mRNA and pDNA, demonstrating their cargo-agnostic nature. Lastly, we showcased the targeted BCP-LNPs using a Cetuximab-conjugated formulation. These targeted LNPs show significant promise in delivering cargo specific to EGFR-overexpressing cells (A549 cells), with up to 2.4 times higher transfection compared to nontargeted LNPs. This finding underscores the potential of BCP-LNPs in targeted gene therapy, especially in challenging scenarios such as tumor targeting. Overall, our study establishes the viability of BCP-LNPs as a versatile, efficient, and targeted delivery platform for nucleic acids, opening avenues for advanced therapeutic applications.

AbstractNew generations of vaccines have been developed by encapsulating messenger ribonucleic acid (mRNA) in lipid nanoparticle (LNP) carriers. In addition to the physicochemical properties of LNPs, the encapsulation efficiency (EE) of mRNA in LNPs is a key factor to screen vaccine assembly assays. Fluorescent dyes with amplified signals upon binding with mRNA are at the core of developing assays to quantify EE. However, disregarding the temporal effects during the assay impacts the accuracy of the assay. Here, the kinetics of temporal decay in fluorescence intensity of dye‐RNA complex—in Ribogreen assay—are reported and shown how this dynamic process can be impeded in the presence of a nonionic surfactant. Further, the impact of this dynamic process on the calculated EE is studied. The corrections needed to accurately assay dynamic mRNA loading processes are presented.

pH-responsive polyelectrolytes, including methacrylate-based anionic copolymers (MACs), are widely used as enteric coatings and matrices in oral drug delivery. Despite their widespread use in these macroscopic applications, the molecular understanding of their use as stabilizers for nanoparticles (NPs) is lacking. Here, we investigate how MACs can be used to create NPs for therapeutic drug delivery and the role of MAC molecular properties on the assembly of NPs via flash nanoprecipitation. The NP size is tuned from 59 to 454 nm by changing the degree of neutralization, ionic strength, total mass concentration, and the core-to-MAC ratio. The NP size is determined by the volume of hydrophilic domains on the surface relative to the volume of hydrophobic domains in the core. We calculate the dimensions of the hydrophobic NP core relative to the thickness of the polyelectrolyte layer over a range of ionizations. Importantly, the results are shown to apply to both high-molecular-weight polymers as core materials and small-molecule drugs. The pH responsiveness of MAC-stabilized NPs is also demonstrated. Future development of polyelectrolyte copolymer-stabilized nanomedicines will benefit from the guiding principles established in this study.

Triple negative breast cancers (TNBCs) represent a heterogenous disease with high patient mortality relative to other breast cancers. Doxorubicin (Dox), a DNA intercalating drug, is widely used as chemotherapy for TNBC. However, it often accompanies severe side effects that limit the effective therapeutic dose. It is hypothesized that the combination of Dox with a histone deacetylase inhibitor, panobinostat (Pan), may improve therapeutic efficacy by attacking rapid cell proliferation and abnormal histone deacetylation. Herein, pH-responsive, lipid targeting nanoemulsions (pLNEs) are designed to direct both Dox and Pan to TNBC cells that overexpress lysophosphatidic acid (LPA) receptor 1 (LPAR(1)) via the ratio of LPA to lysophosphatidylcholine and trigger Dox release via disassociation from docosahexanoic acid. Dox/Pan-pLNEs result in higher intranuclear Dox compared to free Dox, free Dox/Pan, and Dox-pLNEs. Tumors treated with Dox/Pan-pLNEs exhibit 2.4-fold reduction in tumor burden relative to free Dox alone. Molecular analysis reveals that MDA-MB-231 tumor growth inhibition is mediated by re-expression of thioredoxin-interacting protein and suppression of cytoplasmic p27(kip1) and c-Jun activation domain-binding protein-1. This lipid targeting, Dox/Pan encapsulating pLNE represents a platform for treating TNBC with greater therapeutic efficacy.

BACKGROUND:Adverse drug reactions (ADRs) are one of the leading causes of morbidity and mortality in health care. Understanding which drug targets are linked to ADRs can lead to the development of safer medicines.METHODS:Here, we analyse in vitro secondary pharmacology of common (off) targets for 2134 marketed drugs. To associate these drugs with human ADRs, we utilized FDA Adverse Event Reports and developed random forest models that predict ADR occurrences from in vitro pharmacological profiles.FINDINGS:By evaluating Gini importance scores of model features, we identify 221 target-ADR associations, which co-occur in PubMed abstracts to a greater extent than expected by chance. Amongst these are established relations, such as the association of in vitro hERG binding with cardiac arrhythmias, which further validate our machine learning approach. Evidence on bile acid metabolism supports our identification of associations between the Bile Salt Export Pump and renal, thyroid, lipid metabolism, respiratory tract and central nervous system disorders. Unexpectedly, our model suggests PDE3 is associated with 40 ADRs.INTERPRETATION:These associations provide a comprehensive resource to support drug development and human biology studies.FUNDING:This study was not supported by any formal funding bodies.

We describe inverse-Flash NanoPrecipitation as a new scalable platform for co-encapsulation of insulin (a model therapeutic peptide) with soybean trypsin inhibitor (a peptidase inhibitor) and so-dium caprate (a permeation enhancer) forming 300 nm particles for oral delivery. The co-encapsulation of the protein therapeutic with a protease inhibitor and a permeation enhancer into nanoparticles addresses enzymatic attack on the protein drug and the transport across the gastroin-testinal tract barrier. Inverse-Flash NanoPrecipitation (iFNP) encompasses two sequential precipita-tion processes. First, insulin, soybean trypsin inhibitor (SBTI), and hydroxypropyl methylcellulose acetate-succinate polymer (HPMCAS, a stabilizing block copolymer)(at mass ratios of 1:4:5) are rapidly precipitated into an organic antisolvent forming the inverted nano-core (iNC) with 50 wt% insulin and SBTI. Encapsulation efficiencies are greater than 98%. The second step involves the precipitation of sodium caprate and a stabilizing polymer coating onto the iNCs surface. HPMCAS, chitosan, and PEG polymers are used to coat the iNCs to generate nanoparticles with anionic, cation-ic, and neutral surfaces, respectively. This demonstrates that the iFNP platform can encapsulate complex biologics mixtures at high loadings into particles with customizable surface properties.

The plasticity of cancer epigenetics makes them plausible candidates for therapeutic intervention. We took advantage of elevated expression of lysophosphatidic acid receptor 1 (LPAR1) in triple negative breast cancer (TNBC) tissues to target decitabine (DAC) and panobinostat (PAN) to breast cancer cells. DAC and PAN were shown to reverse abnormal methylation of DNA and altered chromatin structure, respectively, leading to increased expression of tumor suppressor genes and decreased expression of oncogenes. Although DAC and PAN have therapeutic benefits, they are limited by chemical instability and systemic toxicity. Herein, we present LPAR1-targeted, lipid nanoemulsions (LNEs) encapsulating both DAC and PAN. Our results demonstrated that the cell uptake and in vivo biodistribution of LNEs was dependent on LPAR1 expression in TNBCs. DAC/PAN-LNEs were effective in inhibiting the growth of mesenchymal breast cancer cells by restoring CDH1/E-cadherin and suppressing forkhead box M1 (FOXM1) expression. Epithelial breast cancer cells that inherently express low FOXM1 and high CDH1 were unaffected by DAC/PAN-LNEs. Overall, we successfully designed LPAR1-targeted LNEs that selectively act on CDH1(low)/FOXM1(high) TNBC cell lines.

Breast cancer is the second leading cause of cancer-related mortality in women. Successful development of sensitive nanoprobes for breast cancer cell detection is of great importance for breast cancer diagnosis and symptomatic treatment. Herein, inspired by the intrinsic peroxidase property of gold nanoclusters, high loading, and targeting ability of ErbB2/Her2 antibody functionalized liposomes, we report that gold nanoclusters-loaded, target-directed, functionalized liposomes can serve as a robust sensing platform for amplified colorimetric detection of HER2-positive breast cancer cells. This approach allows HER2-positive breast cancer cell identification at high sensitivity with high selectivity. In addition, the colorimetric "readout" offers extra advantages in terms of low-cost, portability, and easy-to-use applications. The practicality of this platform was further proved by successful detection of HER2-positive breast cancer cells in human serum samples and in breast cancer tissue, which indicated our proposed method has potential for application in cancer theranostics.

Tissue-equivalents (TEs), simple model tissues with tunable properties, have been used to explore many features of biological soft tissues. Absent in most formulations however, is the residual stress that arises due to interactions among components with different unloaded levels of stress, which has an important functional role in many biological tissues. To create a pre-stressed model system, co-gels were fabricated from a combination of hyaluronic acid (HA) and reconstituted Type-I collagen (Col). When placed in solutions of varying osmolarity, HA-Col co-gels swell as the HA imbibes water, which in turn stretches (and stresses) the collagen network. In this way, co-gels with residual stress (i.e., collagen fibers in tension and HA in compression) were fabricated. When the three gel types tested here were immersed in hypotonic solutions, pure HA gels swelled the most, followed by HA-Col co-gels; no swelling was observed in pure collagen gels. The greatest swelling rates and swelling ratios occurred in the lowest salt concentration solutions. Tension on the collagen component of HA-Col co-gels was calculated from a stress balance and increased nonlinearly as swelling increased. The swelling experiment results were in good agreement with the stress predicted by a fibril network + non-fibrillar interstitial matrix computational model.

Collagen gel tissue-equivalents (TEs), which are simple model tissues with tunable properties, have been used to explore many properties of soft tissues, such as how structural and compositional properties affect mechanical function [1–4]. One aspect not captured in previous TE formulations is residual stress due to interactions among components, which has an important functional role in many tissues (e.g., blood vessels [5], ligaments [6], annulus fibrosus [7]). Since the in vivo stress state of native tissues is not easily replicated in TE fabrication, a different method for “pre-stressing” collagen networks of TEs was necessary. To this end, co-gel TEs were fabricated by adding hyaluronic acid (HA) to reconstituted Type-I collagen (Col) gels. When placed in solutions of varying osmolarity, HA-Col TEs swell as the HA binds water, which in turn will stretch (and stress) the collagen network. In this way, TEs with residual stress (i.e., pre-stressed collagen fibers) can be fabricated and evaluated in order to elucidate relationships between residual stress and functional properties. Therefore, the goals of the present study were to fabricate HA-Col TEs, make initial measurements of their swelling properties, and quantify the mechanical response and changes in microstructural organization under applied tensile load.

Triple negative breast cancers (TNBCs) represent a heterogenous disease with high patient mortality relative to other breast cancers. Conventional chemotherapy, such as Doxorubicin (DOX), is widely used for TNBC treatment. However, it often accompanies severe side effects that limit the effective therapeutic dose for cancer treatment. The plasticity of cancer epigenetics makes them plausible candidates for therapeutic intervention. DNA methyltransferases (DNMTs) and histone deacetyltransferases (HDACs) are two of most well studied enzymes that regulate transcription and chromatin compaction. Aberrant expressions of DNMTs and HDACs have been implicated in a variety of cancers. A few DNMT inhibitors (DNMTi) and HDAC inhibitors (HDACi) have been approved for treating T-cell lymphoma, multiple myeoloma, and myelodysplastic syndromes. For instance, DNMTi Decitabine (DAC) and HDACi Panobinostat (PAN) were shown to reverse abnormal methylation of DNA and altered chromatin structure, respectively, leading to increased expression of tumor suppressor genes (TSGs) and decreased expression of oncogenes (OGs). However, epigenetic agents as a monotherapy did not show a therapeutic benefit against solid tumors. A targeted drug delivery vehicle may improve efficacy of epigenetic drugs. Increased synthesis of lysophosphatidic acid (LPA) and its receptors (LPAR1-3) are implicated in breast cancers. The LPA/LPAR1 axis has been linked to breast cancer metastasisboth in vitro and in vivo. Herein, we took advantage of elevated expression of LPAR1 in TNBC tissues to direct chemotherapeutics to breast cancer cells. DAC and PAN were delivered using LPAR1-targeted, lipid nanoemulsions (LNEs). Our results demonstrated that the cell uptake and in vivo biodistribution of LNEs were dependent on LPAR1 expression in TNBCs. DAC/PAN-LNEs were effective in inhibiting the growth of mesenchymal breast cancer cells by restoring CDH1 and suppressing FOXM1 expression. Epithelial breast cancer cells that inherently express low FOXM1 and high CDH1 were unaffected by DAC/PAN-LNEs. Overall, we successfully designed LPAR1-targeted LNEs that selectively act on CDH1(low)/FOXM1(high) TNBC cell lines. However, in vivo therapeutic efficacy of DAC/PAN-LNEs did not show significant tumor suppression in the time frame of the experiment. We then hypothesized that the combination of DOX with PAN may improve therapeutic efficacy by attacking abnormal DNA replication and dysregulated transcription landscape simultaneously. Thus, DOX was delivered in combination with PAN using previously designed LPAR1-targeted LNEs. DOX/PAN LNEs had a significantly higher uptake of DOX compared to DOX delivered by non-targeted formulations or free drug. DOX and PAN synergistically inhibited tumor cell growth in vitro and in vivo. Lastly, a PCR microarray was used to screen the change of oncogene and tumor suppressor gene expressions induced by LNEs encapsulating DOX and/or PAN. We identified that Dox/Pan LNE treatment upregulated a tumor suppressor gene that sequesters a ROS (reactive oxygen species) scavenging protein, leading to higher ROS accumulation in TNBCs. Invasiveness biomarkers, also found in papillary thyroid carcinoma, were also expressed in TNBC; the expression of these proteins were reversed upon Dox/Pan LNE treatment. Fine-tuning of the hydrophobic core of LNEs to improve the solubility of chemotherapeutics is challenging due to heterogeneity of fats in oil. To maximize the encapsulation of hydrophobic drugs in a lipid nanoparticle, a careful selection of matrix lipids are required. We developed a strategy to fabricate multi-drug delivery nanocarriers to overcome matrix-limited containment of more than one drug. First, solubility of model drugs, 3, 3'-diindolylmethane (DIM) and PAN, were measured in several lipids. Second, a solid lipid and a liquid lipid that have maximal affinity toward model drugs were screened. Third, dual-phase lipid nanocarriers (DPLNs) were fabricated and physiochemical properties were optimized. Lastly, cytotoxicity of DIM and PAN encapsulating DIM/PAN lipid nanoparticles (LNPs) were evaluated in TNBCs. Overall, we demonstrated the therapeutic efficacy and mechanism of LPAR1-targeted LNEs in vitro and in vivo. A strategy to develop DPLNs was suggested to facilitate the construction and optimization of a multi-drug delivery carrier to overcome matrix-limited solubility. (*) This work was supervised by Professor Debra Auguste