Acyl Research Articles

PH-Responsive Peptide Nanoparticles Deliver Macromolecules to Cells via Endosomal Membrane Nanoporation.

The synthetically evolved pHD family of peptides is known to self-assemble into macromolecule-sized nanopores of 2-10 nm diameter in synthetic lipid bilayers, but only when the pH is below ∼6. Here, we show that a representative family member, pHD108, has the same pH-responsive nanopore-forming activity in the endosomal membranes of living human cells, which is triggered by endosomal acidification. This enables the cytosolic delivery of endocytosed proteins and other macromolecules. Acylation of either peptide terminus significantly decreases the concentration of peptide required for macromolecule delivery to the cell cytosol while not causing any measurable cytotoxicity. Longer acyl chains are more effective. The N-terminal palmitoylated C16-pHD108 is the most potent of all of the acyl-pHD108 variants and readily delivers a cytotoxic enzyme, fluorescent proteins, and a dye-labeled dextran to the cell cytosol. C16-pHD108 forms stable monodisperse micellar nanoparticles in a buffer at pH 7 with an average diameter of around 120 nm. These nanoparticles are not cytolytic or cytotoxic because the acylated pHD peptide does not partition from the nanoparticles into cell membranes at pH 7. At pH 5, the nanoparticles are unstable, driving acylated pHD108 to bind strongly to membranes. We hypothesize that passive endocytosis of macromolecular cargo and stable peptide nanoparticles, followed by endosomal acidification-dependent destabilization of the nanoparticles, triggers the nanopore-forming activity of acylated pHD peptides in the endosomal membrane, enabling internalized macromolecules to be delivered to the cytosol.

Diet-Induced Obesity Modulates Close-Packing of Triacylglycerols in Lipid Droplets of Adipose Tissue.

Adipose-derived lipid droplets (LDs) are rich in triacylglycerols (TAGs), which regulate essential cellular processes, such as energy storage. Although TAG accumulation and LD expansion in adipocytes occur during obesity, how LDs dynamically package TAGs in response to excessive nutrients remains elusive. Here, we found that LD lipidomes display a remarkable increase in TAG acyl chain saturation under calorie-dense diets, turning them conducive to close-packing. Using high-resolution X-ray diffraction, solid-state NMR, and imaging, we show that beyond size expansion LDs from mice under varied obesogenic diets govern fat accumulation by packing TAGs in different crystalline polymorphs. Consistently, LDs and tissue stiffen for high-calorie-fed mice with more than a 2-fold increase in elastic moduli compared to normal diet. Our data suggest that in addition to expanding, adipocyte LDs undergo structural remodeling by close-packing rigid and highly saturated TAGs in response to caloric overload, as opposed to liquid TAGs in a low-calorie diet. This work provides insights into how lipid packing within LDs can allow for the rapid and optimal expansion of fat during the initial stages of obesity.

Highly selective cytokine induction of nitrated lipid-modified α-GalCer derivatives demonstrating high binding affinity to the lipid antigen presenting molecule CD1d.

Glycolipid antigens are presented by CD1d on antigen-presenting cells to T cell receptors (TCRs) of natural killar (NK)T cells, leading to immune responses via cytokine induction. Although various lipid antigens have been found, there are only limited numbers of glycolipid antigens having selective cytokine induction. In this study, we identified the glycolipids (α-GalCer nitro-type) that exhibits highly selective induction of Th2 and Th17 type cytokine, with very high binding affinity to CD1d, by introducing natural nitro-modified fatty acyl groups. The natural nitroalkene moiety of fatty acyl groups in the glycolipids effectively enhance the affinity to CD1d with presumed hydrogen-bonding and cation-πinteraction, leading to the distinctive function in cytokine induction and selectivity.

Die Kinetik der Kohlenstoff‐Kohlenstoff‐Bindungsbildung in der metazoischen Fettsäure Synthase und ihr Einfluss auf die Produktgenauigkeit

Fatty acid synthase (FAS) multienzymes are responsible for de novo fatty acid biosynthesis and crucial in primary metabolism. Despite extensive research, the molecular details of the FAS catalytic mechanisms are still poorly understood. For example, the b‐ketoacyl synthase (KS) catalyzes the fatty acid elongating carbon‐carbon‐bond formation, which is the key catalytic step in biosynthesis, but factors that determine the speed and accuracy of his reaction are still unclear. Here, we report enzyme kinetics of the KS‐mediated carbon‐carbon bond formation, enabled by a continuous fluorometric activity assay. We observe that the KS is likely rate‐limiting to the fatty acid biosynthesis, its kinetics are adapted to the length of the bound fatty acyl chain, and that the KS is also responsible for the fidelity of biosynthesis by preventing intermediates from undergoing KS‐mediated elongation. To provide mechanistic insight into KS selectivity, we performed computational molecular dynamics (MD) simulations. We identify positive cooperativity of the KS dimer, which we suggest to affect the conformational variability of the multienzyme. Advancing our knowledge about the KS molecular mechanism will pave the ground for engineering FAS for biotechnology applications and the design of new therapeutics targeting the fatty acid metabolism.

Total Synthesis of Euphorbialoid A.

Euphorbialoid A (1) belongs to the rare diterpenoid family of premyrsinanes and exhibits potent anti-inflammatory effects. The 5/7/6/3-membered carbocycle (ABCD-ring) of 1 contains 11 contiguous stereocenters and seven oxygen-containing functional groups. Moreover, four of the six hydroxy groups of 1 are concentrated in the southern sector and flanked by four structurally different acyl groups. The dense array of various functional groups with disparate reactivities on the tetracyclic ABCD-ring presents a daunting challenge for the chemical synthesis of 1. As a reflection of its formidable complexity, synthesis of 1 or any other premyrsinane diterpenoids has not yet been reported. Here, we devised a novel strategy comprising two stages and achieved the first total synthesis of 1 (35 steps as the longest linear sequence). In the first stage, the ABCD-ring was expeditiously assembled by integrating three powerful transformations: (1) Pt-doped TiO2-catalyzed radical coupling to attach a northern chain to a 6/3-membered CD-ring, (2) Pd-catalyzed decarboxylative asymmetric allylation to construct a quaternary carbon with a southern chain, and (3) a Co-mediated Pauson-Khand reaction to cyclize the two chains into the 5/7-membered AB-ring. In the second stage, three-dimensional structures of the ABCD-ring intermediates were utilized to stereoselectively fabricate the A-ring and site-selectively append the four different acyl groups. In the present total synthesis, we revealed the significance of orchestrating the multistep reaction sequence and incorporating cyclic protective groups. The overall strategy and tactics provide new insights into designing synthetic routes to premyrsinanes and densely oxygenated terpenoids decorated with diverse acyl groups.

Hemisynthesis, Anti-Inflammatory, and In-silico Alpha-Amylase Inhibition of Novel Carlina Oxide Analogs

Background: Numerous natural products have been successfully developed for clinical use in the treatment of human diseases in almost every therapeutic area. Objective: This work aimed to synthesize some new analogs of Carlina oxide by functionalizing the fifth position of the furan by different acyl groups using the Friedel-Crafts acylation approach. The synthetic analogs and carlina oxide were then assessed for their in-vitro anti-inflammatory activity and in-silico alpha-amylase inhibition effect. Methods: The new analogs were synthesized at room temperature using different anhydrides with the presence of boron trifluoride diethyl etherate (BF3-Et2O) as an acid catalyst. A protein denaturation assay was performed to evaluate the anti-inflammatory activity, while the in-silico study was conducted using the Molecular Operating Environment (MOE) with different types of alphaamylase sources, such as human salivary pancreatic alpha-amylase and Aspergillus oryzae alphaamylase (PDB: 1Q4N, 5EMY, 7P4W respectively). Results: A total of four analogs of carlina oxide were obtained in yields of 60-7% and then identified with 1H and 13C NMR analysis. Additionally, analog 1 exhibited a better anti-inflammatory effect with an IC50 of 0.280 mg/mL. However, the in-silico study showed that all the synthetic analogs have different interactions with human salivary alpha-amylase (1Q4N) and other interactions with 5EMY and 7P4W. Conclusion: The new analogs of Carlina oxide have the potential to serve as an alternative agent for alpha-amylase inhibition, contributing to the reduction of postprandial hyperglycemia.

Diacylglycerol metabolism and homeostasis in fungal physiology.

Diacylglycerol (DAG) is a relatively simple and primitive form of lipid, which does not possess a phospholipid headgroup. Being a central metabolite of the lipid metabolism network, DAGs are omnipresent in all life forms. While the role of DAG has been established in membrane and storage lipid biogenesis, it can impart crucial physiological functions including membrane shapeshifting, regulation of membrane protein activity and transduction of cellular signalling as a lipid-based secondary messenger. Besides, the chemical diversity of DAGs, due to fatty acyl chain composition, has been proposed to be the basis of its functional diversity. Therefore, cells must regulate DAG level at a spatio-temporal scale for homeostasis and adaptation. The vast network of eukaryotic lipid metabolism has been unravelled majorly by studying yeast models. Here, we review the current understanding and the emerging concepts in metabolic and functional aspects of DAG regulation in yeast. The implications can be extended to understand pathogenic fungi and mammalian counterparts as well as disease aetiology.

Significance of the Double Bond in the Acyl Chain of Cardiolipin Revealed by the Partial Unfolding Dynamics of Cytochrome c Using Femtosecond Transient Absorption Spectroscopy.

Cytochrome c (Cyt c) released from the mitochondrion acts as a trigger for the onset of apoptosis in which a double bond of cardiolipin (CL) is oxidized upon interaction with Cyt c. To understand the interaction dynamics of Cyt c with the double bond of CL, CLs having acyl chains with a systematic increase in the number of double bonds, 0 (18:0 CL), 1 (18:1 CL), and 2 (18,2 CL), were complexed with Cyt c, and their excited-state dynamics were studied using femtosecond time-resolved pump-probe spectroscopy. Steady-state and femtosecond transient absorption spectra revealed a systematic increase in the partial unfolding of Cyt c with an increase in double bonds in CL, as observed by the enhanced fluorescence intensity and lifetime of tryptophan due to variations in the resonance energy transfer and extended global conformational relaxation time constants. These studies reflect the significance of occurrence of global conformational changes of Cyt c by structural modification near the double bond of CL in the Cyt c-CL complex, which could be prerequisites for the apoptosis.

Convenient synthesis of O-alkylated/N-acylated polyhydroxyazepane based compounds for modulating MD-2-TLR4 complex formation

Toll-like receptor 4 (TLR4) is a critical component of the innate immune system, recognizing lipopolysaccharide (LPS) from Gram-negative bacteria and triggering immune responses. The activation of TLR4 involves several key steps, including interactions with LPS-binding protein (LBP), CD14, and myeloid differentiation protein 2 (MD-2), culminating in the formation of the (LPS.MD-2 TLR4)2 complex. Structural insights show that LPS acyl chains insert into the hydrophobic pocket of MD-2, driving TLR4 activation. Inspired by this understanding, numerous natural and synthetic compounds have been developed to inhibit TLR4 by targeting the MD-2/TLR4 complex. Eritoran 1, is one such illustration. The conformational flexibility of azepane architecture inspired us to visualize O-alkylated/N-acylated polyhydroxyazepane-based compounds toward this objective. The docking studies and molecular simulation studies supported the rationale. Synthesis of O-alkylated/N-acylated polyhydroxyazepane-based compounds 2-4 (a-h) through a key building block is described herein.

New fluorogenic triacylglycerols as sensors for dynamic measurement of lipid oxidation.

Lipids are major constituents of food but are also highly relevant substructures of drugs and are increasingly applied for the development of lipid-based drug delivery systems. Lipids are prone to oxidative degradation, thus affecting the quality of food or medicines. Therefore, analytical methods or tools that enable the degree of lipid oxidation to be assessedare of utmost importance to guarantee food and drug safety. Herein, we report the design, synthesis and application of the first-in-class fluorogenic triacylglycerols that enable dynamic monitoring of lipid oxidation via straightforward fluorescence readout. Our fluorogenic triacylglycerols can be used in both aqueous and lipid-based environments. Furthermore, we showed that the sensitivity of our fluorescent tracers towards oxidation could be tuned by incorporating either saturated or unsaturated acyl chains in their triacylglycerol core structure. With this, we provide a first proofofprinciple for the applicability of fluorescently labelled triacylglycerols as tracers to monitor the dynamics of lipid oxidation, thus paving the way for novel discoveries in the area of lipid analytics.

The application of monoglycerides to improve the quality of fresh noodles: Discerning the roles of acyl chain length and dispersity

Monoglycerides are widely used in flour-based products, but the roles of their dispersibility and acyl chain length remain unclear. This study investigated the effects of monoglycerides with different chain lengths (C12, C16, C18) dispersed in deionized water (DW) or 95 % ethanol (EE) on fresh noodle quality. Ethanol (2 mL per 200 g flour) had no significant effect on noodle properties, but monoglycerides in EE significantly altered gluten structure through covalent and non-covalent interactions, forming a denser gluten network, as observed by CLSM. Starch-lipid complex formation was confirmed by FT-IR, Raman, and XRD, enhancing cooking and immersion performance. Monoglycerides in EE were more effective than in DW, with impact orders: DW (C12 > C16 ≈ C18) and EE (C12 < C16 < C18), indicating solvent selection was more critical than chain length. This study refined the application method of monoglycerides, enhancing their functional performance and contributing to elevated noodle performance.

The Role of Cardiolipin in Brain Bioenergetics, Neuroinflammation, and Neurodegeneration.

Cardiolipin (CL) is an essential phospholipid that supports the functions of mitochondrial membrane transporters and oxidative phosphorylation complexes. Due to the high level of fatty acyl chain unsaturation, CL is prone to peroxidation during aging, neurodegenerative disease, stroke, and traumatic brain or spinal cord injury. Therefore, effective therapies that stabilize and preserve CL levels or enhance healthy CL fatty acyl chain remodeling are needed. In the last few years, great strides have been made in determining the mechanisms through which precursors for CL biosynthesis, such as phosphatidic acid (PA), are transferred from the ER to the outer mitochondrial membrane (OMM) and then to the inner mitochondrial membrane (IMM) where CL biosynthesis takes place. Many neurodegenerative disorders show dysfunctional mitochondrial ER contact sites that may perturb PA transport and CL biosynthesis. However, little is currently known on how neuronal mitochondria regulate the synthesis, remodeling, and degradation of CL. This review will focus on recent developments on the role of CL in neurological disorders. Importantly, due to CL species in the brain being more unsaturated and diverse than in other tissues, this review will also identify areas where more research is needed to determine a complete picture of brain and spinal cord CL function so that effective therapeutics can be developed to restore the rates of CL synthesis and remodeling in neurological disorders.

Membrane fusion reactions limited by defective SNARE zippering or stiff lipid fatty acyl composition have distinct requirements for Sec17, Sec18, and adenine nucleotide.

Intracellular membrane fusion is catalyzed by SNAREs, Rab GTPases, SM proteins, tethers, Sec18/NSF and Sec17/SNAP. Membrane fusion has been reconstituted with purified vacuolar proteins and lipids to address 3 salient questions: whether ATP hydrolysis by Sec18 affects its promotion of fusion, whether fusion promotion by Sec17 and Sec18 is only seen with mutant SNAREs or can also be seen with wild-type SNAREs, and whether Sec17 and Sec18 only promote fusion when they work together or whether they can each work separately. Fusion is driven by two engines, completion of SNARE zippering (which does not need Sec17/Sec18) and Sec17/Sec18-mediated fusion (needing SNAREs but not the energy from their complete zippering). Sec17 is required to rescue fusion that is blocked by incomplete zippering, though optimal rescue also needs the ATPase Sec18. ATP is an essential Sec18 ligand, but at limiting Sec17 levels Sec18 ATP hydrolysis also drives release of Sec17 without concomitant trans-SNARE complex disassembly. At higher (physiological) Sec17 levels, or without ATP hydrolysis, fusion prevails over Sec17 release. Stiff 16:0, 18:1 fatty acyl chain lipids provide an alternative route to suppressing fusion, with entirely wild-type SNAREs and without impediment to zippering. In this case, Sec17 and Sec18 restore comparable fusion with either ATP or a nonhydrolyzable analog. Fusion blocked by impaired zippering can be restored by concentrated Sec17 alone (but not by Sec18), while fusion inhibited by stiff fatty acyl chains is partially restored by Sec18 alone (but not by Sec17). With distinct fusion impediments, Sec18 and Sec17 have both shared roles and independent roles in promoting fusion.

Chemical Landscape of Adipocytes Derived from 3T3-L1 Cells Investigated by Fourier Transform Infrared and Raman Spectroscopies.

Adipocytes derived from 3T3-L1 cells are a gold standard for analyses of adipogenesis processes and the metabolism of fat cells. A widely used histological and immunohistochemical staining and mass spectrometry lipidomics are mainly aimed for examining lipid droplets (LDs). Visualizing other cellular compartments contributing to the cellular machinery requires additional cell culturing for multiple labeling. Here, we present the localization of the intracellular structure of the 3T3-L1-derived adipocytes utilizing vibrational spectromicroscopy, which simultaneously illustrates the cellular compartments and provides chemical composition without extensive sample preparation and in the naïve state. Both vibrational spectra (FTIR-Fourier transform infrared and RS-Raman scattering spectroscopy) extended the gathered chemical information. We proved that both IR and RS spectra provide distinct chemical information about lipid content and their structure. Despite the expected presence of triacylglycerols and cholesteryl esters in lipid droplets, we also estimated the length and unsaturation degree of the fatty acid acyl chains that were congruent with known MS lipidomics of these cells. In addition, the clustering of spectral images revealed that the direct surroundings around LDs attributed to lipid-associated proteins and a high abundance of mitochondria. Finally, by using quantified markers of biomolecules, we showed that the fixative agents, paraformaldehyde and glutaraldehyde, affected the cellular compartment differently. We concluded that PFA preserves LDs better, while GA fixation is better for cytochromes and unsaturated lipid analysis. The proposed analysis of the spectral images constitutes a complementary tool for investigations into the structural and molecular features of fat cells.

Synthesis and Characterization of a Novel Intrinsically Fluorescent Analog of Cholesterol with Improved Photophysical Properties.

Live-cell imaging of cholesterol trafficking depends on suitable cholesterol analogs. However, existing fluorescent analogs of cholesterol either show very different physicochemical properties compared to cholesterol or demand excitation in the ultraviolet spectral region. We present a strategy to synthesize two novel intrinsically fluorescent sterol probes with a close resemblance of cholesterol. The analogs contain four conjugated double bonds in the ring system and either a keto group (probe 5) or a hydroxy group (probe 6) in the C3 position. The emission of 5 is in the visible range of the spectrum, i.e., red-shifted by 150 nm compared to the widely used dehydroergosterol. Together with its high multiphoton absorption, this allows for imaging of 5 on conventional microscopes, including multicolor 3D and time-lapse microscopy. Molecular dynamics simulations and nuclear magnetic resonance spectroscopy reveal that 5 can condense the fatty acyl chains of phospholipids in model membranes. In giant unilamellar vesicles, 5 partitions equally into the liquid-ordered and disordered phases. In contrast, 6 emits in the ultraviolet range and is unstable in solution, preventing its use in live-cell imaging applications. The good photophysical properties of 5 make it a suitable analogue for improved live-cell imaging of sterol transport.

Stem cell models of TAFAZZIN deficiency reveal novel tissue-specific pathologies in Barth syndrome.

Barth syndrome (BTHS) is a rare mitochondrial disease caused by pathogenic variants in the gene TAFAZZIN, which leads to abnormal cardiolipin (CL) metabolism on the inner mitochondrial membrane. Although TAFAZZIN is ubiquitously expressed, BTHS involves a complex combination of tissue specific phenotypes including cardiomyopathy, neutropenia, skeletal myopathy, and growth delays, with a relatively minimal neurological burden. To understand both the developmental and functional effects of TAZ-deficiency in different tissues, we generated isogenic TAZ knockout (TAZ-KO) and WT cardiomyocytes (CMs) and neural progenitor cells (NPCs) from CRISPR-edited induced pluripotent stem cells (iPSCs). In TAZ-KO CMs we discovered evidence of dysregulated mitophagy including dysmorphic mitochondria and mitochondrial cristae, differential expression of key autophagy-associated genes, and an inability of TAZ-deficient CMs to properly initiate stress-induced mitophagy. In TAZ-deficient NPCs we identified novel phenotypes including a reduction in CIV abundance and CIV activity in the CIII2&CIV2 intermediate complex. Interestingly, while CL acyl chain manipulation was unable to alter mitophagy defects in TAZ-KO CMs, we found that linoleic acid or oleic acid supplementation was able to partially restore CIV abundance in TAZ-deficient NPCs. Taken together, our results have implications for understanding the tissue-specific pathology of BTHS and potential for tissue-specific therapeutic targeting. Moreover, our results highlight an emerging role for mitophagy in the cardiac pathophysiology of BTHS and reveal a potential neuron-specific bioenergetic phenotype.

Cu-Catalyzed Asymmetric Three-Component Radical Acylarylation of Vinylarenes with Aldehydes and Aryl Boronic Acids.

The direct use of readily available aldehydes as acyl radical precursors has facilitated diverse three-component acylative difunctionalization reactions of alkenes, offering a powerful route to synthesize β-branched ketones. However, asymmetric three-component acylative difunctionalization of alkenes with aldehydes still remains elusive. Here we report a copper-catalyzed asymmetric three-component radical acylarylation of vinylarenes with aldehydes and aryl boronic acids. This method begins with acyl radical formation from an aldehyde via hydrogen atom transfer. The acyl radical adds to the alkene, forming a new benzylic radical that then undergoes copper-catalyzed enantioselective arylation. A chiral binaphthyl-tethered bisoxazoline ligand is essential for achieving high stereocontrol. This strategy enables the direct synthesis of a range of synthetically valuable chiral β,β-diaryl ketones from aldehydes and vinylarenes.

The Kinetics of Carbon-Carbon-Bond Formation in Metazoan Fatty Acid Synthase and its Impact on Product Fidelity.

Fatty acid synthase (FAS) multienzymes are responsible for de novo fatty acid biosynthesis and crucial in primary metabolism. Despite extensive research, the molecular details of the FAS catalytic mechanisms are still poorly understood. For example, the b-ketoacyl synthase (KS) catalyzes the fatty acid elongating carbon-carbon-bond formation, which is the key catalytic step in biosynthesis, but factors that determine the speed and accuracy of his reaction are still unclear. Here, we report enzyme kinetics of the KS-mediated carbon-carbon bond formation, enabled by a continuous fluorometric activity assay. We observe that the KS is likely rate-limiting to the fatty acid biosynthesis, its kinetics are adapted to the length of the bound fatty acyl chain, and that the KS is also responsible for the fidelity of biosynthesis by preventing intermediates from undergoing KS-mediated elongation. To provide mechanistic insight into KS selectivity, we performed computational molecular dynamics (MD) simulations. We identify positive cooperativity of the KS dimer, which we suggest to affect the conformational variability of the multienzyme. Advancing our knowledge about the KS molecular mechanism will pave the ground for engineering FAS for biotechnology applications and the design of new therapeutics targeting the fatty acid metabolism.

Structural basis of the mechanism and inhibition of a human ceramide synthase.

Ceramides are bioactive sphingolipids crucial for regulating cellular metabolism. Ceramides and dihydroceramides are synthesized by six ceramide synthase (CerS) enzymes, each with specificity for different acyl-CoA substrates. Ceramide with a 16-carbon acyl chain (C16 ceramide) has been implicated in obesity, insulin resistance and liver disease and the C16 ceramide-synthesizing CerS6 is regarded as an attractive drug target for obesity-associated disease. Despite their importance, the molecular mechanism underlying ceramide synthesis by CerS enzymes remains poorly understood. Here we report cryo-electron microscopy structures of human CerS6, capturing covalent intermediate and product-bound states. These structures, along with biochemical characterization, reveal that CerS catalysis proceeds through a ping-pong reaction mechanism involving a covalent acyl-enzyme intermediate. Notably, the product-bound structure was obtained upon reaction with the mycotoxin fumonisin B1, yielding insights into its inhibition of CerS. These results provide a framework for understanding CerS function, selectivity and inhibition and open routes for future drug discovery.

Liposomal Nω-hydroxy-l-norarginine, a proof-of-concept: Arginase inhibitors can be incorporated in liposomes while retaining their therapeutic activity ex vivo

Cancer immunotherapy has evolved significantly over the last decade, with therapeutics targeting the adaptive immune system showing exciting effects in clinics. Yet, the modulation of the innate immune system, particularly the tumor-associated innate immune cells which are an integral part of immune responses in cancer, remains less understood. The arginase 1 (Arg1) pathway is a pivotal metabolic pathway that tumor-associated innate immune cells exploit to create an immunosuppressive tumor microenvironment, leading to the evasion of immune surveillance. The inhibition of Arg1 presents a therapeutic opportunity to reverse this immunosuppression, and Nω‑hydroxy-l-norarginine (nor-NOHA) has emerged as a potent arginase inhibitor with promising in vivo efficacy. However, the rapid systemic clearance of nor-NOHA poses a significant challenge for its therapeutic application. This study pioneers the encapsulation of nor-NOHA in liposomes, aiming to enhance its bioavailability and prolong its inhibitory activity against Arg1. Historically, the extensive interaction between innate immune cells and nanoparticles has been one of the biggest drawbacks in nanomedicine. Here we seek to utilize this effect and deliver liposomal nor-NOHA to the arginase 1 expressing innate immune cells. We systematically investigated the effect of lipid composition, acyl chain length, manufacturing and loading methodology on the encapsulation efficiency (EE%) and release profile of nor-NOHA. Our results indicate that while the manufacturing method and lipid acyl chain length do not significantly impact EE%, they crucially influence the release kinetics of nor-NOHA, with longer acyl chains demonstrating a more sustained release of nor-NOHA from liposomes enabling continuous inhibition of Arg1. Our findings suggest that liposomal nor-NOHA retains its functional inhibitory activity and could offer improved pharmacokinetic properties, making it a compelling base for iterations for further innovative cancer immunotherapeutic strategies in preclinical and clinical evaluations.