Synthetic polyinosinic:polycytidylic acid (poly(I:C)) offers an attractive cancer therapeutic by operating on two fronts at once, combining direct tumor cell killing with immunostimulatory activity. Yet, these dual functions can only be efficiently harnessed when intracellular delivery is sufficiently effective to enable poly(I:C) to reach and activate its intracellular receptors. We addressed this delivery challenge by developing pH-responsive formulations using lipoamino fatty acid xenopeptide (LAF-XP) carriers, composed of polar cationizable succinoyl tetraethylene pentamine (Stp) and apolar cationizable LAF building blocks in defined architectures. In particular, poly(I:C)-lipid nanoparticles (LNPs) formulated with bundle LAF4-Stp1 XP carriers displayed increased anti-tumoral activity at decreased dosage across multiple cancer cell models, compared to control formulations. In parallel, LAF-XP LNP-delivered poly(I:C) activated immune responses, including CXCL10 production by tumor cells, and activation of peripheral blood mononuclear cells (PBMCs), characterized by increased phenotypic markers (CD69 and LAMP-1/CD107a) and functional molecules (e.g., IFN-γ and granzyme B). Conditioned supernatant of pre-stimulated PBMCs with poly(I:C) reduced cancer cell viability, highlighting the contribution of PBMC-released factors to cancer cell death. Of particular novelty is the combination of poly(I:C) with siRNA-mediated survivin knockdown to increase apoptosis in cancer cells using the bundle LAF-XP LNP. Collectively, our findings establish efficient LAF-XP LNPs as a versatile platform that supports multi-layered therapeutic strategies.

LAF-Stp xenopeptides (XPs) comprising lipoamino fatty acid (LAF) units connected with the tetraethylene pentamine derived polyamino acid Stp were identified as a novel class of amphiphilic mRNA carriers. A 'chameleon-like' polarity switch is observed upon transition from neutral physiological to endosomal acidic pH. Protonation of the lipidic tertiary amine of LAF results in an similar to 2 log unit change in the octanol/water partition coefficient (logD), consistent with all-atom molecular dynamics simulations. mRNA delivery efficacy is tunable by the chemical topology (either a flexible 'U-shape' or a constrained 'Bundle' structure) and specific modifications in the LAF-Stp linker domains. Specifically, replacing lysine with shorter ornithine linkers further increases the structural rigidity of Bundles. For all LAF-XPs, protonation-induced solubilization of carriers triggers endosomal destabilization (evidenced by galectin-8 recruitment) and successful cytosolic mRNA transfection. Carriers with U-shape topology strongly depend on endosomal acidification, as demonstrated by an up to similar to 20-fold drop in efficacy upon treatment with v-ATPase inhibitor bafilomycin, similar as found for the ionizable polymer polyethylenimine. In contrast, Bundles are far less affected (<4-fold reduction) by bafilomycin treatment. Small-angle X-ray scattering (SAXS) reveals that lipid bulk phase structures are largely pH-independent for both topologies. Interestingly, U-shapes form lamellar phases, whereas Bundle topology induces non-lamellar bicontinuous phases with negative membrane curvature. These findings suggest distinct endosomal escape mechanisms for Bundle and U-shapes. Both carriers benefit from endosomal protonation and solubilization, but only Bundles can adopt pH-independent membrane-destabilizing fusogenic phases, reflected by faster kinetics and characteristic cellular morphology in a galectin-8 assay.

Double pH-responsive lipo-xenopeptides combine polar oligoamino acids (OAAs) such as succinoyl tetraethylene pentamine (Stp) with apolar lipoamino fatty acid (LAF) domains. The resulting potent mRNA carriers undergo a profound shift in their polarity upon endosomal protonation. In the current chemical evolution approach, three top-performing xenopeptides based on a U-shaped (LAF(2)Stp(1), LAF(4)Stp(2)) or bundle (LAF(4)Stp(1)) topology were modified by replacing Stp with a variety of hydrophobized OAAs. Octanol/water distribution (log D) was determined by a novel label-free mass spectrometry-based method. At neutral pH, the new xenopeptide analogues displayed an up to 85-fold higher hydrophobicity (log D-7.4 > 2) than Stp-based carriers, with a sharp pH-responsive decrease at endosomal pH (log D-5.5 -1). Transfection efficacies were influenced by the amine count of the OAAs, with tetraethylene pentamine outperforming the OAAs with fewer amines. For both topologies, a strong correlation between mRNA expression and endosomal escape, as quantified by calcein release, was found. For U-shaped carriers (LAF/OAA = 2:1), lipophilic OAAs improved endosomal escape and mRNA transfection efficiency. In contrast, for bundle carriers (LAF/OAA = 4:1), lipophilic OAA incorporation did not further enhance endosomal escape or transfection. Mechanistic studies revealed that LAF-xenopeptide polyplexes can adopt both endosomal pH-dependent and pH-independent cytosolic entry mechanisms, with hydrophobization accelerating expression kinetics and enhancing pH-independent entry. Optimized U-shaped xenopeptides were identified with improved mRNA transfection in tumor cells, a dendritic cell line, and low-passage human colon carcinoma cells. In vivo, enhanced mRNA expression was found upon either intramuscular or intravenous administration in several organs and tumor tissue.

A library of xenopeptide-based ionizable lipids (XP-ILs) was evaluated in mRNA lipid nanoparticles (LNPs) to investigate the impact of modifications within the polar oligoamine headgroup. Modifications included hydrophobic or bulky side chains or a reduced number of protonatable amines. The resulting XP-ILs were characterized by their key physicochemical properties (logD, logP, HLB, CMC), which were correlated with LNP transfection efficiency and cytotoxicity. The inverse relationship between computational logP and experimental logD5.5 is consistent with the HLB, indicating a lower tendency for water-in-oil micelle formation in carriers with larger hydrophobic headgroups. Four-tailed oleic acid (OleA)4-XP-ILs strongly benefited from a cyclohexyl substituent in the polar ionizable headgroup. Hydrophobization of already highly effective bundle-shaped lipoamino fatty acid (LAF)4-XP-ILs moderately improved efficiency. Notably, intramuscular LNP administration resulted in enhanced in vivo mRNA activity of hydrophobized carriers over previous lead compounds, LAF4-XP (2-fold) and OleA4-XP (12-fold).

Local pulmonary delivery offers a non-invasive application route for mRNA therapeutics with the potential for high bioavailability at the target-site of applications such as mucosal vaccination or the treatment of lung diseases. However, efficient delivery remains challenging due to major lung-specific barriers, particularly mucus. Herein, pH-responsive, amphiphilic xenopeptides comprising lipoamino fatty acids and oligoamino acids (OAAs) connected in distinct branched U-shape or bundle topologies were evaluated as mRNA polyplexes for delivery to A549 and Calu-3 lung cells under standard submerged or air-liquid interface (ALI) transfection conditions, and upon intratracheal application in BALB/c mice. Optionally, polyplexes were coated with negatively charged hyaluronic acid (HA) or colloidally stabilized with poly(ethylene glycol) (PEG). For U-shapes, hydrophobic modification of the OAA domain boosted their efficiency. Interestingly, best-performing formulations varied across transfection conditions. While the bundle topology showed the highest potential in submerged cell culture, U-shaped carriers were more efficient under ALI conditions. Polyplex surface modification with HA or PEG did not strongly alter in vitro transfections, whereas hydrophobized U-shape core polyplexes combined with surface modification enhanced their efficiency in vivo. Thus, the cationizable core and surface properties of mRNA nanoparticles require specific balancing in various lung cell models and lung.

An effective chimeric antigen receptor (CAR)-based immunotherapy depends on both a suitable immune cell platform and a tumor-specific antigen to overcome barriers in solid tumors. Natural killer (NK) cell lines are promising platforms for CAR constructs due to their inherent tumor-killing ability, safety profile, and feasibility for standardized, off-the-shelf therapeutic use. Herein, four human NK cell lines (YT, KHYG1, NKL, and NK92) were retrovirally transduced with an anti-Hsp70 CAR targeting membrane-bound heat shock protein 70 (mHsp70), a tumor-specific antigen with broad expression on many solid tumors, but not normal cells. Computational modeling suggested a strong binding between the CAR and the extracellular domain of mHsp70. Although all NK cell lines exhibited successful CAR integration and surface expression, only NKL and NK92 cells maintained stable CAR expression and long-term viability. The anti-Hsp70 CAR NKL and NK92 cells demonstrated enhanced expression of activation markers and secretion of cytotoxic effector molecules, and robust target-specific killing of mHsp70-positive cancer cells, while sparing mHsp70-negative targets. Our findings validate the therapeutic potential of anti-Hsp70 CAR NK cells and the suitability of NKL and NK92 cells for advancing off-the-shelf CAR NK cell therapies, thereby offering a promising strategy for targeting a broad range of solid tumors expressing mHsp70.

Double pH-responsive lipo-xenopeptides (lipo-XPs) with varying lipo amino fatty acid (LAF) to succinoyl tetraethylene pentamine (Stp) ratios and two distinct bundle and U-shape topologies were identified as highly effective carriers for mRNA delivery. Physicochemical properties and transfection efficacies of the carriers are strongly influenced by the lipid tail length and position of the ionizable nitrogen within the LAF domain, defined as the "molecular catwalk". Bundles containing short LAFs (8Oc, 12Bu) and U-shapes with medium-length LAFs (12Oc) and centrally placed tertiary amines exhibited strongest activity, while extreme catwalk variants (2Hd, 16Et) proved ineffective. These trends establish clear structure-activity relationships which were subjected to further mechanistic investigation. Mechanistic studies revealed topology- and LAF-specific differences in requirement for endosomal acidification and corresponding escape, indicating that different molecular arrangements trigger distinct biological mechanisms. All-atom molecular dynamics simulations at the water-octanol interface further contextualized these behaviors by resolving how protonation reshapes carrier polarity, hydration and interfacial localization. Protonation-induced relocation was more pronounced in bundles, consistent with their higher LAF content, whereas 12Oc-based U-shapes displayed more moderate shifts and retained greater octanol affinity at neutral pH. Variations in nitrogen placement modulated interfacial enrichment and hydrogen bonding, providing molecular comprehension for the experimentally observed activity profiles across the LAF-Stp carrier library.

CRISPR-Cas9 genome editing is a versatile platform for studying and treating various diseases. Homology-directed repair (HDR) with DNA donor templates serves as the primary pathway for gene correction in therapeutic applications, but its efficiency remains a significant challenge. This study investigates strategies to enhance gene correction efficiency using a T-shaped lipo-xenopeptide (XP)-based Cas9 RNP/ssDNA delivery system combined with various HDR enhancers. Nu7441, a known DNA-PKcs inhibitor, was found to be most effective in enhancing HDR-mediated gene correction. An over 10-fold increase in HDR efficiency was achieved by Nu7441 in HeLa-eGFPd2 cells, with a peak HDR efficiency of 53% at a 5 nM RNP concentration and up to 61% efficiency confirmed by Sanger sequencing. Surprisingly, the total gene editing efficiency including non-homologous end joining (NHEJ) was also improved. For example, Nu7441 boosted exon skipping via NHEJ-mediated splice site destruction by 30-fold in a DMD reporter cell model. Nu7441 modulated the cell cycle by reducing cells in the G1 phase and extending the S and G2/M phases without compromising cellular uptake or endosomal escape. The enhancement in genome editing by Nu7441 was widely applicable across several cell lines, several Cas9 RNP/ssDNA carriers (LAF-XPs), and also Cas9 mRNA/sgRNA/ssDNA polyplexes. These findings highlight a novel and counterintuitive role for Nu7441 as an enhancer of both HDR and total gene editing efficiency, presenting a promising strategy for Cas9 RNP-based gene therapy.

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR associated (Cas) protein has been proved as a powerful tool for the treatment of genetic diseases. The Cas9 protein, when combined with single-guide RNA (sgRNA), forms a Cas9/sgRNA ribonucleoprotein (RNP) capable of targeting and editing the genome. However, the limited availability of effective carriers has restricted the broader application of CRISPR/Cas9 RNP. In this study, we evaluated dual pH-responsive amphiphilic xenopeptides (XPs) for delivering CRISPR/Cas9 RNP. These artificial lipo-XPs contain apolar cationizable lipoamino fatty acid (LAF) and polar cationizable oligoaminoethylene acid units such as succinoyl-tetraethylenepentamine (Stp) in various ratios and U-shaped topologies. The carriers were screened for functional Cas9/sgRNA RNP delivery in four different reporter cell lines, including a Duchenne muscular dystrophy (DMD) exon skipping reporter cell model. Significantly enhanced cellular uptake into HeLa cells, effective endosomal disruption in HeLa gal8-mRuby3 cells, and potent genome editing by several Cas9/sgRNA RNP complexes was observed in four different cell lines in the 5 nM sgRNA range. Comparing Cas9/sgRNA RNP complexes with Cas9 mRNA/sgRNA polyplexes in the DMD reporter cell model demonstrated similar splice site editing and high exon skipping of the two different molecular Cas9 modalities. Based on these studies, analogues of two potent U1 LAF2-Stp and LAF4-Stp2 structures were deployed, tuning the amphiphilicity of the polar Stp group by replacement with the six oligoamino acids dmGtp, chGtp, dGtp, Htp, Stt, or GEIPA. The most potent LAF2-Stp analogues (containing dGtp, chGtp or GEIPA) demonstrated further enhanced gene editing efficiency with EC50 values of 1 nM in the DMD exon skipping reporter cell line. Notably, the EC50 of LAF2-dGtp reached 0.51 nM even upon serum incubation. Another carrier (LAF4-GEIPA2) complexing Cas9/sgRNA RNP and donor DNA, facilitated up to 43% of homology-directed repair (HDR) in HeLa eGFPd2 cells visualized by the switch from green fluorescent protein (eGFP) to blue fluorescent protein (BFP). This study presents a delivery system tunable for Cas9 RNP complexes or Cas9 RNP/donor DNA polyplexes, offering an effective and easily applicable strategy for gene editing.

RNA therapy has emerged as a transformative approach for treating cancer. However, the clinical application of RNA therapy faces significant challenges due to the biological barriers such as enzymatic degradation, reticuloendothelial system (RES) clearance, and low transfection efficiency. To address these limitations, a huge number of engineered nano/micro-scale vehicles have been thoroughly investigated and extensively used for targeted delivery of RNA. In this work, we summarized a comprehensive review of the nano/micro-scale targeted RNA delivery systems, coupled with critical insights into challenges and opportunities. We firstly reviewed the various nano/micro-scale vectors and technologies for RNA delivery in cancer therapy, including lipid nanoparticles, polymeric micelles, hydrogels, microneedles, exosomes, nanoparticles, metal-organic framework, covalent organic frameworks and living cells. We further summarized the challenges, highlighting the potential to achieve high therapeutic efficacy and minimal side effects. Ultimately, the perspectives of nano/micro-scale targeted RNA delivery systems are thoroughly discussed. Collectively, we believe that RNA therapies based on nano/micro-scale delivery strategies could be potentially applied in clinical fields to promote the improvement of cancer treatments.

Supplementary Figure 1: Radioiodide uptake of GL261 brain tumors after systemic MSC-mediated NIS reporter gene delivery in comparison to thyroid as endogenous NIS-expressing organ assessed by 124I PET/CT imaging. Results are expressed as mean value ± SEM.Supplementary Figure 2: Ex vivo analysis of non-target organs after NIS-MSC gene delivery. NIS (A) and Neomycin (B; selection marker of NIS-MSCs) mRNA expression was detected by qPCR in liver, spleen, kidney and lung after NIS-MSC application and showed no expression above the background level of untreated tumors (which was arbitrarily set to one; NIS mRNA levels of untreated ΔΔCt = 0.0003 and Neomycin mRNA levels of untreated ΔΔCt = 0.002). Data are represented as mean-fold change ± SEM (*p<0.05).

For complexation of mRNA into polyplexes, double-pH-responsive lipo-xenopeptides (XP), comprising tetraethylene pentamino succinic acid (Stp) and lipoamino fatty acids (LAFs), were combined with PEGylated lipids, either DMG-PEG 2 kDa (DMG-PEG) or azido-group-containing DSPE-PEG 2 kDa (DSPE-PEG-N3), to increase colloidal stability and to facilitate ligand-mediated targeted mRNA delivery. LAF-XPs mixed with DMG-PEG at low (1.5% and 3%) molar ratios improved colloidal stability and retained transfection efficiency. PEGylation also enabled the formulation of otherwise unstable carrier complexes and prevented aggregation induced by salt, proteins, and serum. PEGylation of more positively charged Stp-LAF2 mRNA polyplexes decreased fibrinogen adsorption. More neutral, LAF-rich Stp-LAF4 polyplexes exhibited low fibrinogen binding without PEGylation. Intravenous administration of these stabilized mRNA complexes demonstrated enhanced biosafety while preserving transfection efficiency. DSPE-PEG-N3 was selected for cell targeting after strain-promoted azide-alkyne cycloaddition (SPAAC)-mediated click-coupling of DBCO-modified ligands. Higher PEG ratios (10% and 20%) provided effective shielding but reduced transfection efficiency, a drawback known as the “PEG dilemma”. Functionalization with an EGFR-targeting ligand restored transfection in EGFR-positive cell lines in a ligand-specific manner. High transfection efficiency is consistent with a lipophilic-to-hydrophilic polarity switch of LAF-XP carriers upon endosomal protonation, triggering dissociation of the PEG lipids and deshielding of the polyplex.

Efficient delivery of siRNA-based polyplexes to tumors remains a major challenge. Nonspecific interactions in the bloodstream, limited circulation time, and nontargeted biodistribution hamper sufficient tumor accumulation. To address these challenges, we developed an ionic hyaluronic acid (HA) coating to shield sequence-defined oligoaminoamide-based polyplexes. This coating should shield the positive polyplex surface charge, thus reducing nonspecific interactions and enhancing serum stability. Additionally, we modified the HA coating with the cyclic RGDfK (cRGD) peptide to specifically target tumor endothelial cells (TECs). Optionally, a polyethylene glycol (PEG) spacer was also introduced to improve ligand presentation on the polyplex surface. The HA-coated polyplexes exhibited favorable physicochemical properties, including a negative zeta potential and effective siRNA retention within the polyplex, which was not adversely affected by PEG or cRGD modification. In vitro analyses revealed that these polyplexes not only enhanced tumor cell association and preserved the high transfection efficiency of plain cationic polyplexes but also exhibited coating-dependent cellular internalization, as evidenced by a competitive inhibition experiment. Even in the presence of serum, the HA-coated polyplexes encapsulated siRNA effectively, exhibited suitable particle sizes, and maintained a high gene silencing efficiency. In vivo studies involving intravenous administration into Neuro2a tumor-bearing mice showed that the HA coating, particularly when modified with PEG and cRGD, significantly increased the tumor accumulation of polyplexes. HA-PEG-cRGD-shielded polyplexes exhibited significantly enhanced in vivo gene silencing in tumors compared with plain polyplexes. Collectively, our results indicate a superior performance of HA-coated polyplexes in terms of stability and cellular uptake, both in vitro and in vivo.

Although small interfering RNA (siRNA) holds immense promise for treating genetic diseases and cancers, its clinical application is constrained by instability, cellular uptake barriers, and inefficient cytosolic delivery, underscoring the need for effective delivery systems. Therefore, this study focuses on screening novel T-shaped lipo-xenopeptide (XP) nanocarriers for siRNA polyplex formulation, integrating two single succinoyl-tetraethylene pentamine (Stp) units for electrostatic interaction and tyrosine tripeptides (Y3) for aromatic stabilization, along with structural modifications such as the addition of histidine (H) with or without terminal cysteines (C), and the incorporation of various fatty acids (FAs). A systematic evaluation of siRNA binding, nanoparticle stability, and gene silencing efficiency in multiple cell lines illustrated that the novel Stp1-HC lipo-XPs carriers outperform their Stp2-HC analogs, despite having fewer cationizable Stp units. This advantage stems from increased fatty acid, Y3, and C density, which compensates for reduced electrostatic interactions. The presence of H in combination with unsaturated FAs significantly improved the functional siRNA delivery. Our findings highlight the complex interplay of electrostatic, hydrophobic, covalent, hydrogen-bonded, and aromatic interactions to achieve efficient siRNA delivery, which is best-balanced in the oleic acid-containing Stp1-HC/OleA lipo-XP, exceeding the previously best standard carrier Stp2-HC/OleA in efficiency.

Abstract Natural killer (NK) cells are central components of the innate immunity system against cancers. Since tumor cells have evolved a series of mechanisms to escape from NK cells, developing methods for increasing the NK cell antitumor activity is of utmost importance. It is previously shown that an ex vivo stimulation of patient‐derived NK cells with interleukin (IL)‐2 and Hsp70‐derived peptide TKD (TKDNNLLGRFELSG, aa450‐461) results in a significant upregulation of activating receptors including CD94 and CD69 which triggers exhausted NK cells to target and kill malignant solid tumors expressing membrane Hsp70 (mHsp70). Considering that TKD binding to an activating receptor is the initial step in the cytolytic signaling cascade of NK cells, herein this interaction is studied by molecular docking and molecular dynamics simulation computational modeling. The in silico results showed a crucial role of the heterodimeric receptor CD94/NKG2A and CD94/NKG2C in the TKD interaction with NK cells. Antibody blocking and CRISPR/Cas9–mediated knockout studies verified the key function of CD94 in the TKD stimulation and activation of NK cells which is characterized by an increased cytotoxic capacity against mHsp70 positive tumor cells via enhanced production and release of lytic granules and pro‐inflammatory cytokines.