Angewandte Chemie

G-quadruplex (G4) DNA structures are increasingly recognized for their roles in key cellular processes, including transcriptional regulation and genome stability, making them attractive therapeutic targets. Selective recognition of individual G4s remains challenging due to the high structural similarity among human G4 motifs. The G4 Ligand-conjugated Oligonucleotide strategy addresses this need by combining the G4-binding capabilities of small-molecule G4-ligands with the sequence specificity of an oligonucleotide complementary to the flanking region of the target G4. Here, we systematically explore how the oligonucleotide component governs G4 binding and stabilization by varying its length, backbone composition, and sequence complementarity. This revealed that efficient G4 recognition depends on a strong interdependence between hybridization and G4-ligand binding, such that both elements cooperatively reinforce complex stability and site specificity. Central mismatches disrupt this dual recognition and reduce selectivity. While longer oligonucleotides hybridize more slowly, they form more stable complexes and show stronger G4 stabilization in thermal melting and polymerase stop assays. Replacing the DNA oligonucleotide with peptide nucleic acid enhances binding strength, thermal stability, and metabolic stability, but selective G4 stabilization is achieved only upon ligand conjugation. Together, these results show how rational oligonucleotide design enables selective and potent recognition of G4 structures using GL-Os.

Opioid agonists such as morphine and fentanyl exert analgesic effects by binding and activating the {micro}-opioid receptor ({micro}OR), yet agonism of the {micro}OR causes a slate of serious side effects. {micro}OR-mediated addiction and respiratory depression are the major causes of the current opioid overdose crisis, largely driven by the explosion in illicit use of fentanyl, a potent opioid receptor full agonist. Given these serious side effects (and high resulting societal cost), molecules that act as analgesics with distinct mechanisms of action are of great interest. Positive allosteric modulators (PAMs) of the {micro}OR have the potential to avoid many off-target side effects of conventional opioid orthosteric agonists by enhancing the signaling properties of natural opioid peptide systems. We used a DNA-encoded chemical library screening approach to selectively discover active-state-specific {micro}OR PAMs. Two out of 3 selected prospective PAMs displayed the anticipated enhancement in agonist activity. The most effective of these compounds enhanced the activity of all orthosteric opioid agonists tested, including the native opioid peptide met-enkephalin. Little is known about the underlying dynamic basis of allosteric modulation of Family A GPCRs like the {micro}OR. To that end, we used single-molecule fluorescence resonance energy transfer experiments to detail the impact that our novel {micro}OR PAM has on the dynamic activation behavior of a key region on the intracellular face of the receptor. Our results here provide both a new chemical scaffold that acts as a {micro}OR PAM and detailed pharmacological and dynamic insights into its mechanism of action.

Nanodiscs are nanoscale lipid bilayer membrane mimetics surrounded by two membrane scaffold proteins (MSP). They are widely used as soluble cassettes for membrane proteins and lipids in diverse applications. The original MSP1 was derived directly from human apolipoprotein A-1, and novel constructs have been adapted from this original design, including nanodiscs with larger sizes and covalent circularization. Here, we developed MSPs with a range of different fluorescent C-terminal protein tags, including a versatile HaloTag fusion. These fluorescent MSP were purified following typical MSP purification procedures with similar yield. Then, we demonstrate that fluorescent MSPs form nanodiscs with similar structure and stoichiometry to conventional MSP nanodiscs. These fluorescent MSP constructs enable a range of different applications and provide a versatile template for future design of nanodiscs with unique functions. For Table of Contents Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/716332v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@f85870org.highwire.dtl.DTLVardef@764055org.highwire.dtl.DTLVardef@179b7c5org.highwire.dtl.DTLVardef@ff6a7_HPS_FORMAT_FIGEXP M_FIG C_FIG

Efficient siRNA loading into Argonaute2 (AGO2) requires a 5'-phosphate (5'-P) on the guide strand, yet this group is vulnerable to metabolic degradation in vivo. Although numerous chemical mimics of 5'-P have been reported, structural principles governing AGO2 interactions with organyl substituents on the 5'-P remain unclear. Moreover, structural determinants of 5'-P mimic recognition by known degradative enzymes (principally phosphatases and 5'-exonucleases) are also poorly understood. The 5'-P binding site of the AGO2 MID domain contains a stack of aromatic residues (Y527/F811/Y815), presenting a structural basis for augmenting canonical anchoring interactions. Herein, we systematically synthesized and characterized a diverse panel of organyl 5'-phosphates (5'-POR; R = 35 variable substituents) as guide strand 5'-P mimics designed to engage this unique hydrophobic pocket. Among the compounds evaluated, 5'-POR guide strands bearing methyl (Me) or phenylpropargyl (PhPrp) substituents are well-tolerated by AGO2 in cells. Previously uncharacterized 5'-P mimics, including 5'-phosphorothioate (5'-PS), phenylpropargyl 5'-phosphorothioate (5'-PS-PhPrp), and 5'-mesylphosphoramidate (5'-MsPA), maintain comparable AGO2 compatibility. All examined 5'-P mimics are markedly resistant to phosphatase, while 5'-POR variants and 5'-PS-PhPrp are also resistant to 5'-exonuclease degradation due to masking a negative charge of 5'-P. A crystal structure of a 5'-PO-PhPrp guide strand loaded into AGO2 reveals an unexpected network of {pi}-{pi} interactions between the rigid phenylpropargyl group and the targeted hydrophobic pocket of the MID domain. Collectively, these findings expand the functional chemical space of 5'-P mimics and define new modes for metabolically stabilizing the guide strand 5'-end while augmenting AGO2 MID anchoring. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=85 SRC="FIGDIR/small/705631v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@e6f88forg.highwire.dtl.DTLVardef@1c87dfaorg.highwire.dtl.DTLVardef@1c6e68corg.highwire.dtl.DTLVardef@14a1f60_HPS_FORMAT_FIGEXP M_FIG C_FIG

A programmable 4-input cascade DNA logic gate utilizing toehold mediated strand displacement (TMSD) was implemented on a 3D printed hybrid paper-polymer vertical flow device (3D HPVF) for on/off sensitive and specific fluorescence detection of platelet derived growth factor BB (PDGF BB). Polypropylene was 3D printed directly on paper and thermally cured to create micro paper analytical devices ({micro}PADs). The 3D HPVF comprised of three layers of {micro}PADs enclosed in a casing that clamped each {micro}PAD securely to ensure seamless and efficient wicking between layers. In the presence of PDGF BB, a partially complementary strand to a PDGF B aptamer (PDGF B Apt), cApt, was liberated from a PDGF B Apt/cApt duplex in solution. The solution was then deposited on the 3D HPVF with a dimeric g-quadruplex hairpin. The 4-nucleotide toehold region on the cApt started the hybridization reaction with the dimeric g-quadruplex hairpin (dGH) opening it up allowing formation of a dimeric g-quadruplex structure that binds with thioflavin T (ThT) with enhanced fluorescence intensity at room temperature. The 3D HPVF exhibits a pico molar range of detection from 10pM to 100pM with a 10pM limit of detection (LOD) for PDGF BB concentrations relevant for pregnant women predisposed to early-onset preeclampsia with clear differentiation when compared to similarly competing analytes PDGF AA and AB.

Chemically modified nucleic acids have become a powerful platform for basic research and applied technologies. Universal nucleobases are used in PCR,sequencing, and the design of nanodevices and aptamers. Fluorescent universal nucleobases have an even wider range of applications, including the development of nucleic acid-based sensors, switches, and relay logic gates. However, few such nucleobases have been proposed to date, and most of them have suboptimal optical properties. Here, we propose an adenine-based molecular rotor, 7,8-dihydro-8-oxo-6-(3-methylbenzo[d]thiazol-2(3H)-ylidene)adenine (oxo-Ade BZT), as a new, remarkably bright and potent fluorescent universal nucleobase. Its brightness in both oligodeoxyribonucleotides (ODNs) and DNA duplexes (4200 - 10000 M-1 x cm-1) originates from a high molar extinction coefficient (averaged{varepsilon} 368 37000 M-1 x cm-1), provided by the appended 3-methylbenzo[d]thiazolyl moiety, and a relatively high quantum yield (0.11 - 0.27). Melting temperature variations observed upon the incorporation of oxo-Ade BZT opposite native nucleobases in a duplex context did not exceed 10%. The basis of these universal hybridizing properties was unveiled using computational methods. According to molecular dynamics simulations, oxo-Ade BZT pushes the opposite nucleobase out of the DNA double helix and forms multiple hydrophobic contacts with the flanking base pairs. At the same time, the rotational mobility of the bonds between the oxo-Ade BZT-constituting heterobicycles decreases, and oxo-Ade BZT adopts a planar conformation in both ODNs and their duplexes, resulting in the light-up effect. These properties make oxo-Ade BZT a promising molecular tool for analytical, biophysical and biochemical studies.

Benzoxaboroles offer unusual reactivity and protein recognition for the development of small molecule drugs. Despite this potential, they are uncommon in drug discovery or in large fragment screening libraries. We synthesized a small series of structurally related benzoxaboroles containing a diazirine/alkyne tag to enable in-cell photoaffinity labeling (PAL) experiments. A subset of this library was found to have high selectivity for eukaryotic translation initiation factor 4E (eIF4E). The benzoxaborole-eIF4E interaction was found to be stereoselective in nature and competitive with the 7-methylguanosine cap of mRNA. Site of labeling experiments revealed that the benzoxaborole fragment interacts with the cap binding pocket of eIF4E. In silico modeling of the modified protein suggests that H-bonding interactions between the main chain of Trp102 and the side chain of Asn155 to the amide carbonyl and anionic boronate of the benzoxaborole, respectively, drive affinity for this challenging to drug pocket.

DNA methylation is a critical epigenetic modification that regulates gene expression, maintains genome stability, and influences cellular function during development and disease. Accurate analysis of DNA methylation often requires amplification to generate sufficient material, yet preserving the original epigenetic information during this process is challenging because standard amplification methods can disrupt methylation patterns. To address this, we developed a one-pot strategy that combines helicase-dependent amplification (HDA) with DNA methyltransferase 1 (DNMT1)-mediated methylation, enabling simultaneous DNA amplification and preservation of native methylation marks. A key challenge is that HDA is optimized at 65 {degrees}C, whereas DNMT1 is unstable at elevated temperatures. We overcame this by establishing a unified buffer and isothermal reaction at 42 {degrees}C that supports both enzymatic activities. Under these conditions, HDA achieved robust amplification ([~]5 Ct), while DNMT1 faithfully methylated the newly synthesized DNA, as confirmed by methylation-sensitive restriction enzyme quantitative PCR (MSRE-qPCR), with methylation levels proportional to the input template. This one-pot workflow demonstrates the feasibility of concurrent amplification and methylation, providing a foundation for scalable, accurate, and methylation-preserving DNA analyses for epigenetic and clinical applications. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=178 SRC="FIGDIR/small/709983v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@fb8d3aorg.highwire.dtl.DTLVardef@f50c94org.highwire.dtl.DTLVardef@cda7aorg.highwire.dtl.DTLVardef@1db82b0_HPS_FORMAT_FIGEXP M_FIG C_FIG

Cell-free gene expression in micro-compartments constitutes a chassis for biotechnology and synthetic biology. Protein synthesis from low concentrations of DNA, a single copy per compartment, is essential for in vitro evolution of biomolecules and synthetic cells. However, insufficient yield of protein synthesized from typically sub-picomolar DNA results in undetectable signals or inadequate activity of desired protein functions. Here we identify and largely mitigate yield-limiting bottlenecks of reconstituted in vitro transcription and translation (IVTT) at low DNA input. Systematic comparison of commercial reconstituted IVTT kits revealed that gene expression starts becoming limited by mRNA scarcity around 20-200 pM DNA input. We further uncovered that the standard ribosome concentration is excessive at low-DNA input and shortens the lifetime of translation. These findings led to a simple optimization recipe that combines supplementation with a highly active T7 RNA polymerase and a reduction in ribosome concentration, which synergistically amplified gene expression by [~]10-fold across diverse fluorescent proteins and enzymes. This low-DNA-optimized formulation in picoliter droplets achieved [~]94 nM protein expression from a single copy of DNA ([~]0.12 pM). The user-friendly boosted IVTT protocol paves the way for straightforward functional screening and in vitro reconstitution of cellular functions in DNA-scarce environments. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/707295v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@89a051org.highwire.dtl.DTLVardef@17c2e52org.highwire.dtl.DTLVardef@1c52800org.highwire.dtl.DTLVardef@c543fe_HPS_FORMAT_FIGEXP M_FIG C_FIG

Therapeutic peptides combine high target specificity with potent biological activity.1 However, treatment success is often limited by rapid clearance and the need for frequent injections.2, 3 This challenge is particularly acute for therapeutic peptides used in obesity, where clinical benefit must be balanced against dose-dependent adverse effects. In nature, these constraints are overcome by storing hormones as reversible fibrils,4 but pharmacokinetic control is essential for widespread adoption of bio-inspired self-assembled depots for therapeutic peptides. Here, we show that tuneable pharmacokinetics can be achieved and modelled by mapping the fundamental chemical parameters of reversibly self-assembly in vitro. We demonstrate this approach for the amylin analogue pramlintide. Amylin analogues are under development for the next generation of diabetes and obesity treatments, with improved mechanism of action e.g. preserving lean body mass.5-8 Pramlintide is an approved drug with a well-established safety profile, however, it has a comparable half-life to native amylin.8-12 In a pilot study, we achieve in vitro-in vivo correlation, increasing the half-life of pramlintide 20-82-fold in rats, while controlling burst release. These findings demonstrate that the optimisation of pharmacokinetics can be decoupled from peptide engineering, establishing a generalisable framework for generating long-acting peptide formulations by emulating native storage mechanisms.

There has been a surge in antibiotic resistance in recent years, making traditional antibiotics less effective against key pathogens. RNA has recently emerged as a potential target for antibiotics due to its involvement in crucial microbial functions. It is possible to expand the range of therapeutic targets by using RNA-based therapies, but it remains necessary to improve the molecular-level understanding of interactions between RNA and known and potential binders. The SAM-I riboswitch, which controls the transcriptional termination of gene expression involved in sulfur metabolism in most bacteria, is an excellent ligand target. Thus, understanding its behavior with and without ligand complexes would be very helpful for drug design applications. In this manuscript, we studied the interactions between the SAM-I riboswitch and its natural ligand, SAM, which controls riboswitch function, and compared those interactions to its interactions with the very similar small molecular SAH, which does not control riboswitch function, and to its interactions with a potential binder JS4, identified via virtual screening. From our simulations, we gain a deeper understanding of small molecule interactions with the SAM-I riboswitch. The results reveal how differently the small molecules (SAM, SAH and JS4) bind to and potentially induce conformational changes in the riboswitch. Our findings offer valuable insight into the molecular mechanisms underlying riboswitch RNA-ligand interactions for the design of more effective RNA-targeting therapeutics.

Advanced oral squamous cell carcinoma (OSCC) patients with cisplatin-refractory tumors face a poor prognosis and limited therapeutic options. Cisplatin-based systemic chemotherapy has long been the gold standard despite producing unmanageable adverse side effects and toxicity. Compared to Pt(II)-based analogs, octahedral Pt(IV)-based compounds have demonstrated remarkable potential as antitumor prodrugs. Pt(VI) derivates are chemically inert remaining intact until internalized within cells, demonstrating higher tolerability and selectivity towards cancer cells. In this study we compare antitumoral response implementing primary patient-derived 2D and 3D OSCC in vitro models treated with both cisplatin and a novel Pt(IV) compound. We also test the delivery and efficacy of these anticancer drugs via novel encapsulation of this Pt(IV) prodrug in the framework of mesoporous silica nanoparticles (Pt(IV)-cov@MSN). Our results show a significant improvement of delivery and cytotoxicity of Pt(IV)-cov@MSN in both cisplatin-responsive and cisplatin-resistant primary patient-derived in vitro models. We also show how Pt(IV)-cov@MSN elicits p53-dependent apoptotic cell death superior to that obtained with cisplatin treatment in OSCCs. These findings highlight 3D-primary models as key tools for drug and nanocarrier testing, as well as potential targeted and selective delivery strategies for novel chemotherapeutic agents.

Hyperuricemia, a major risk factor for gout and kidney disease, arises from the evolutionary loss of human uricase and remains a significant medical challenge due to its high prevalence. However, limited therapeutic options are available for refractory hyperuricemia that typically require long-term treatment. Here we developed a circRNA-based uricase replacement strategy and evaluated its efficacy in uricase-knockout mice as a model for severe hyperuricemia. Lipid nanoparticle-mediated delivery of circRNA enabled efficient in vivo expression of an engineered human-like uricase, which rapidly reduced serum urate levels after a single injection and maintained the urate-lowering effect for up to 10 days. Repeated administration led to sustained urate reduction for 10 weeks, mitigated renal injury, and exhibited favorable biosafety. These findings highlight the therapeutic potential of circRNA-based uricase replacement for the long-term treatment of hyperuricemia and its associated complications.

Chemotherapy has been widely used in cancer treatment, but most of the chemotherapeutic drugs rely mainly on passive accumulation due to lack of target specificity, which may lead to systemic toxicity and limited clinical utility. Recent advances in artificial intelligence-based protein design provide new opportunities for developing precision therapeutics. Here we modify the albumin-bound paclitaxel (Nab-PTX), one of the most widely used drugs in chemotherapies, by applying the fusion proteins of albumin with the de novo designed protein binders targeting human EGFR or HER2. The resulting particles, EGFRmb-Nab-PTX and HER2mb-Nab-PTX, retain the similar physicochemical properties of Nab-PTX while acquiring the receptor-specific binding capacities. The in vitro assays show that both binder-modified Nab-PTX particles have increased uptake and inhibitory effects significantly in the cancer cell lines with high receptor expression levels. Furthermore, the data from a xenograft model, including tumor growth, excised tumor analysis, organ histology, and fluorescence imaging, show that the binder-modified Nab-PTX enhances tumor accumulation, improves tumor suppression, and reduces off-target toxicity compared with the conventional Nab-PTX, suggesting that they may have promising clinical potential in cancer treatment. Overall, this strategy provides an adaptable modular platform for generating albumin-based chemotherapeutic drugs with target specificities, which can be readily customized with diverse target binders to enable precise cancer therapies.

Viroimmunotherapy leverages oncolytic viruses to induce antitumor immunity and is increasingly explored for solid tumors. Their activity can be enhanced by arming them with immunostimulatory payloads, but most approaches rely on protein-based transgenes that are constrained by viral genome packaging limits. Here, we establish a replication-competent Delta-24-RGD-based platform for localized production of immunotherapeutic RNA aptamers at the tumor site. RNA aptamers provide compact, highly specific ligands that can, in principle, target diverse immune receptors. As a model, we engineered a Delta-24-RGD derivative encoding circular 4-1BB targeting aptamers and show that infected tumor cells sustain aptamer transcription and release, which is associated with a pro-inflammatory remodeling of the tumor microenvironment and measurable antitumor activity in different mouse models with a comparable effect to that achieved with a 4-1BBL-expressing adenovirus used as a benchmark. Overall, this work delivers a proof of concept that replication-competent adenoviruses can serve as in situ factories for extracellularly active RNA aptamers, supporting their development as flexible platforms for localized non-coding cancer immunotherapy.

Efficient intracellular delivery of nucleic acids, proteins, and other biomolecules is critical to advancing therapeutic strategies and genome-editing technologies. Lipid nanoparticles (LNPs) have emerged as highly promising delivery vehicles owing to their self-assembly properties, biocompatibility, and capacity to encapsulate large molecular cargos. Their biological performance is determined largely by lipid composition, which influences particle stability, cellular uptake, membrane fusion, and intracellular trafficking. In this study, we designed and optimized LNP formulations inspired by the lipid architecture of enveloped viruses. Four distinct formulations were generated and systematically evaluated in mammalian cell culture, leading to the identification of two lead candidates with superior delivery characteristics. The biodistribution and translocation properties of these formulations were subsequently assessed using an in vitro brain endothelial barrier model to mimic brain environment. Furthermore, we demonstrated that the selected LNPs enable efficient and functional delivery of CRISPR-Cas ribonucleoprotein complexes to mammalian cells. Together, these findings underscore the potential of rationally engineered LNPs as versatile, safe, and effective non-viral delivery platforms for advanced genome-editing applications.

In vitro reconstitution of protein systems - e.g., metabolic pathways, genetic circuits or biosensors - often requires optimization to enhance their activity. Combinatorial DNA libraries that simultaneously target multiple genes allow for a holistic optimization strategy by studying the interplay between the systems components, which may reveal DNA variants that would be hidden when testing each element in isolation. Here, we screen large populations of synthetic vesicles that express combinatorial DNA variants of a DNA self-replicator or a phospholipid synthesis pathway. We simultaneously vary the strengths of multiple RBSs or synonymously mutate the first codons of multiple genes to explore the effects of the protein translation rates directly on the functionality of the two core synthetic cell modules. We isolated high performers through DNA self-selection or functional screening by fluorescence-activated cell sorting. Long-read sequencing of the fittest variants informed on the optimal RBS strengths and base substitutions in the first codons and indicated which genes were most impactful in regulating the functionality of the protein systems. Single-mutation data were used to predict the fitness of combinatorial variants, which was compared with the experimental fitness observed. The theoretical fitness of combinatorial variants was extremely predictive for the two-gene library of the DNA replicator but less for the larger pathway library. Altogether, our approach exemplifies how combinatorial testing can be expanded from single proteins to multiprotein systems, which can in the future be extended to the evolutionary engineering of even larger genetic and metabolic networks, and eventually an entire artificial cell. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=68 SRC="FIGDIR/small/707944v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@1ac1d47org.highwire.dtl.DTLVardef@b62339org.highwire.dtl.DTLVardef@1c2a7ddorg.highwire.dtl.DTLVardef@9abedf_HPS_FORMAT_FIGEXP M_FIG C_FIG

Antisense oligonucleotides (ASOs) are short, synthetic nucleic acids that bind to complementary RNA sequences and alter gene expression, making them versatile therapeutic agents. Here, we identify transcription termination windows of protein-coding genes as previously unrecognized targets for ASOs. We show that ASOs can act on nascent RNA synthesised downstream of the annotated genes, leading to a pronounced decrease in the corresponding mRNA levels. These downstream-of-gene ASOs (DG-ASOs) induce RNase H1-dependent cleavage, impairing mRNA 3 end processing, directing the unprocessed mRNAs for exosome-dependent degradation and creating early entry points in the nascent RNA for the XRN2 exonuclease termination factor. Altogether, we show that termination windows are genuine ASO targets which can be exploited to suppress gene expression. Importantly, we also reveal an underappreciated source of off-target effects which may arise from ASOs binding downstream of genes. Our findings indicate the necessity to expand ASO design and off-target assessment guidelines to include termination window sequences, thereby improving therapeutic efficacy and safety.

Fibroblast activation protein (FAP) is an attractive target for the development of cancer theranostics due to its selective expression on cancer-associated fibroblasts (CAFs). While a number of small-molecule FAP inhibitors (FAPIs) have been developed, few biologics have been investigated as FAP targeting vectors. Camelid-derived single-domain antibodies, or variable-heavy-heavy domains (VHHs), offer a compelling alternative, combining high affinity with versatile engineering options. In this study, we first identified a novel anti-FAP VHH, F7, from an affinity-matured camelid phage display library. To investigate how valency and molecular weight affected target engagement and in vivo properties, F7 was engineered into three formats: a monomer (F7), a tethered dimer (F7D), and an Fc-fusion protein (F7-Fc). All three were specific for FAP with the two bivalent constructs demonstrating picomolar affinity. Positron emission tomography imaging in FAP-positive xenograft models revealed distinct pharmacokinetic profiles across constructs with notable differences in tumor uptake and clearance. F7 had rapid uptake and clearance resulting in significantly higher tumor uptake than FAPI-46. Low molecular weight bivalent F7D demonstrated similar kinetics but was retained by the tumor resulting in a high tumor-to-blood ratio with secondary uptake limited to clearance organs. The largest construct, F7-Fc, resulted in the highest tumor uptake and allowed for longitudinal imaging. Absorbed dose calculations confirmed that tumors received significantly higher radiation doses compared to normal tissues. These findings demonstrate that tuning VHH scaffold size and valency can improve biodistribution and retention, establishing F7-based constructs as promising targeting vectors for FAP.

The clinical translation of RNA interference (RNAi) therapeutics remains limited by inefficient delivery and cancer-target accumulation. Here, we report the development of a new cationic liposome (CLP) nanocarrier engineered for delivery and controlled-release of small interfering RNA (siRNA) targeting the epidermal growth factor receptor (EGFR) in human colorectal cancer. CLPs were synthesized from ethylphosphocholine-based lipids and PEGylated components, with folic acid (FA) tissue-specific ligand and fluorophore labelling. These nanocarriers exhibited robust physicochemical stability across a broad pH and temperature range, efficient siRNA complexation, and nuclease-protection of siRNA. Functional studies revealed that CLP-siEGFR achieved effective cytosolic siRNA cargo release and EGFR silencing in vitro, proving to be more effective than conventional lipid-based transfection systems. In human xenograft models, intravenously administered CLP-siEGFR showed enhanced tumor localization, prolonged siRNA retention, and significant tumor growth suppression, accompanied by marked downregulation of EGFR. Importantly, systemic dosing was well-tolerated, with no evidence of hepatotoxicity, nephrotoxicity, or hematological abnormalities. These results position CLP nanocarriers as an effective platform for targeted RNAi therapeutics, offering translational potential for precision oncology applications.