Cholesterol-modified sphingomyelin chimeric lipid bilayer for improved therapeutic delivery

Liposomes commonly incorporate cholesterol to enhance bilayer packing, reduce fluidity and permeability, and improve drug retention. However, under physiological conditions cholesterol rapidly exchanges between liposomes, biomembranes, and lipoproteins, destabilizing carriers, accelerating leakage and clearance, and limiting clinical efficacy. Prior strategies such as sterol-modified lysophospholipids (SMLs) covalently tethered cholesterol reduced cholesterol exchange and leakage in serum but did not improve tumor uptake or therapeutic outcomes versus approved formulations. To better mimic biological membranes and stabilize bilayers while enabling controlled release, the authors hypothesized that covalently attaching cholesterol to sphingomyelin (SM)—a natural double-chain membrane phospholipid with an amide linkage and a hydroxyl group—would retain cholesterol’s condensing effect, introduce stabilizing hydrogen bonding, increase compressibility and decrease water permeability versus single-chain SMLs, and resist physiological extraction. They further posited that stimuli-responsive linkers (e.g., disulfide for glutathione, glycine for cathepsin B, thioketal for ROS) could enable selective dissociation in disease microenvironments to trigger release. The objective was to engineer and systematically evaluate SM–Chol constructs and identify compositions that minimize cholesterol exchange and leakage, improve pharmacokinetics and biodistribution, and enhance therapeutic efficacy across diverse payloads and disease models.

The study builds on work showing cholesterol stabilizes membranes by promoting liquid-ordered phases, increasing rigidity and reducing permeability. However, cholesterol shuttles between membranes and lipoproteins in vivo, compromising liposomal stability. Szoka’s group developed sterol-modified lysophospholipids (SMLs; PChcPC, PChemsPC, OChemsPC, DChemsPC), covalently attaching cholesterol via ester or carbonate linkers; these reduced cholesterol exchange and leakage in serum but did not improve doxorubicin tumor uptake or antitumor efficacy versus Doxil. An N-cholesteryl sphingomyelin (cholesterol carbamate replacing the amide-linked acyl chain) increased bilayer order and detergent resistance. Numerous cholesterol derivatives (e.g., DC-Chol and other cationic lipids; cholesterol-drug conjugates like paclitaxel–cholesterol; cholesterol–peptide constructs) use cholesterol as a bilayer anchor to aid gene/drug delivery or targeting, but added moieties can jeopardize bilayer stability and generally do not improve bilayer physicochemical properties per se. Clinically, several liposomal nanotherapeutics (e.g., Doxil, Marqibo, Onivyde) improve pharmacokinetics and reduce side effects but often show limited gains in efficacy and survival, underscoring a need for more stable, effective bilayers. The present work aims to address these gaps by covalently linking cholesterol to a double-chain phospholipid backbone (sphingomyelin) using diverse, potentially cleavable linkers to both stabilize the bilayer and enable controlled release.

• Design and synthesis: Five sphingomyelin–cholesterol (SM–Chol) conjugates were synthesized with different linkers: carbonate ester (SM-C-Ester-Chol), ester (SM-Ester-Chol), glycine (SM-Glycine-Chol, cathepsin B-cleavable), disulfide with a longer linker (SM-CSS-Chol, GSH-responsive), and thioketal with a longer linker (SM-SCS-Chol, ROS-responsive). Structures were confirmed by 1H/13C NMR and ESI-MS. Commercial SMLs (PChcPC, PChemsPC, OChemsPC, DChemsPC) served as controls.
• Liposome preparation and characterization: Liposomes were formed by thin-film hydration and probe sonication, typically with SM or SM–Chol and 5 mol% DSPE-PEG2K; cholesterol mol% was matched to comparisons (e.g., eq. 35–40 mol% for SM–Chol/SM; eq. 64.4 mol% for PChcPC-based controls). Size, polydispersity (PDI), and zeta potential were measured by DLS; morphology by cryo-EM. Stability over time in 5% dextrose at 4 °C was monitored by DLS.
• Calcein encapsulation and leakage assays: Calcein-loaded liposomes were prepared. Leakage under osmotic stress was assessed by transferring liposomes into buffers of varied osmolarity at 37 °C and measuring fluorescence, calculating fraction retained. Serum-induced leakage was evaluated by incubating in 30% FBS at 37 °C and monitoring calcein release over time.
• Cholesterol exchange: Cholesterol transfer between membranes was quantified at 37 °C for compositions with eq. 40 mol% Chol.
• Thermotropic behavior: Differential scanning calorimetry (DSC) assessed SM phase transitions with increasing mol% Chol or SM–Chol to determine effects on Tm and enthalpy and whether transitions were abolished.
• Bilayer rigidity: Atomic force microscopy (AFM) measured particle height and diameter; rigidity was inferred from height/diameter (H/D) ratio.
• Redox-responsiveness: SM-CSS-Chol stability was tested in PBS (pH 7.4) versus GSH at 37 °C by LC-MS/MS; formation of sulfide-linked sphingomyelin intermediate under GSH was confirmed by HRMS. Calcein release kinetics from SM-CSS-Chol liposomes were evaluated in PBS and GSH.
• VCR formulation and MTD study: Vincristine (VCR) was remotely loaded (citrate pH gradient) into various liposomes (including SM/Chol, SM–Chol variants, PChcPC control). Formulations were characterized (DLC, DLE, size, PDI, zeta). Maximum tolerated dose (single i.v.) was assessed in healthy C57BL/6J mice by body weight loss and survival over 14 days, with hematology and serum chemistry on day 14.
• Pharmacokinetics and biodistribution: In an orthotopic MC38 CRC model, free VCR or VCR/liposomes were administered i.v. (2 mg/kg). Blood kinetics and tissue distribution (including tumors) were measured (n=3 mice). PK parameters (T1/2, Vss, AUC, CL, MRT) were derived.
• In vivo content tracking: Dual-labeled liposomes (MU-P payload in core, DiD in bilayer) were used in orthotopic KPC-Luc PDAC model to assess in vivo stability and site-specific release by AUCMU-P/AUCDID ratios and metabolite conversion.
• Antitumor efficacy (VCR): In SU-DHL-4 DLBCL s.c. xenografts, a single i.v. dose (2 mg/kg VCR) of various VCR/liposomes was given at ~200 mm³ tumor size; tumor growth, images, and Kaplan–Meier survival were recorded. Tumor pharmacodynamics (β-tubulin structure, cleaved caspase-3, TUNEL, Ki67) were examined in an independent cohort.
• Antitumor efficacy (IRI in PDAC): Irinotecan (IRI) was remotely loaded using TEASSOS pH gradient into SM–Chol, SM/Chol, PChcPC liposomes and compared to Onivyde. In a late-stage orthotopic KPC-Luc PDAC model (primary tumor ~400 mg with metastasis by day 11), mice received i.v. injections on days 11, 14, 17 (40 mg/kg IRI). Tumor burden by bioluminescence imaging (BLI), ex vivo organ BLI, and metastasis heatmaps were analyzed. Additional SML controls included custom disulfide-linked SMLs (SML-SS, SML-CSS) and commercial SMLs.
• Antitumor efficacy (DOX in TNBC): Doxorubicin (DOX) was remotely loaded using ammonium sulfate gradient. In orthotopic 4T1-Luc2 TNBC (tumor ~200 mm³ on day 15), a single i.v. dose (15 mg/kg DOX) was given. Tumor growth (calipers), whole-body and lung metastasis BLI, and images were collected through day 35; Doxil served as control.
• Anti-inflammatory efficacy (DEX in lung inflammation): Hydrophobic dexamethasone (DEX) was incorporated in the bilayer during liposome formation. In an LPS-induced lung inflammation model (BALB/c), mice received i.v. free DEX or DEX/liposomes (1 mg/kg) 6 h post-LPS; lungs were harvested 12 h later to quantify IL-6, TNF-α, IL-1β by ELISA and assessed by H&E histology for leukocyte recruitment/peribronchial thickening.
• Gene delivery (siRNA): siRNA against P-gp (Abcb1a) was formulated into LNPs using DLin-MC3-DMA with different helper lipid systems (DSPC/Chol, PChcPC, SM/Chol, SM-CSS-Chol; 49.3/10.2/39.0/1.5 DMA/helper/Chol/PEG2K-C-DMG). Serum stability was assessed by gel retardation up to 24 h. In CT26 CRC s.c. tumors, mice received three i.v. doses (200 µg/kg siRNA, days 10, 12, 14); P-gp mRNA knockdown was measured by qRT-PCR on day 15. Combination therapy studies paired IRI/liposomes (SM-CSS-Chol or PChcPC) with matched siRNA/LNP to assess tumor growth and intratumoral IRI by HPLC.
• Statistics: Mean ± SD; Student’s t-test for two-group comparisons; one-way ANOVA with Tukey’s post hoc for multiple groups; log-rank Mantel–Cox for survival analyses. All animal studies were IACUC-approved.

• SM–Chol preserves cholesterol’s membrane-condensing capacity: DSC showed SM phase transition was abolished at ~30 mol% free Chol; SM–Chol similarly eliminated SM transitions at equivalent ~30 mol% Chol (40 mol% for SM-SCS-Chol) and reduced Tm and ΔH in an SM–Chol–dependent manner, indicating retained condensing ability.
• Reduced leakage and cholesterol exchange: Under high osmotic stress and in 30% FBS at 37 °C, SM–Chol with longer linkers and cleavable bonds (SM-CSS-Chol and SM-SCS-Chol) exhibited the lowest payload leakage versus other SM–Chol variants, SMLs, and conventional phospholipid/Chol bilayers. Cholesterol transfer rates were markedly lower for SM-CSS-Chol and SM-SCS-Chol than for SMLs and phospholipid/Chol mixtures.
• Increased bilayer rigidity: AFM showed higher H/D ratios for SM–Chol membranes versus SM/Chol. Example: H/D ratio for Lipo-SM-CSS-Chol 0.154 ± 0.054 vs Lipo-SM/Chol 0.056 ± 0.001; increased height (e.g., 17.68 nm vs 8.83 nm) indicated greater stiffness.
• Redox-responsive stability: SM-CSS-Chol liposomes were stable in PBS (pH 7.4) with minimal calcein release but showed rapid SM-CSS-Chol degradation and accelerated release in the presence of GSH; sulfide-linked sphingomyelin intermediate formation confirmed disulfide cleavage.
• Safety and MTD with VCR: In healthy mice, free VCR MTD was 2 mg/kg; VCR/PChcPC increased to 3 mg/kg; VCR/SM–Chol (SM/Chol) and VCR/SM-CSS-Chol both reached 4 mg/kg. VCR/SM-CSS-Chol avoided abnormal serum chemistry and hematologic changes observed in VCR/SM/Chol and VCR/PChcPC groups, supporting an improved safety profile.
• Pharmacokinetics and biodistribution (VCR, orthotopic MC38 CRC): Compared to free VCR (rapid ~90% clearance within 5 min), VCR/SM–Chol formulations substantially prolonged circulation and increased tumor delivery (6.5–13.9-fold). PK parameters (mean ± SD) included: VCR/Lipo-PChcPC T1/2 0.64 h, AUC 38.37 µg·h/mL, CL 35.74 mL/min/kg; VCR/Lipo-SM/Chol T1/2 1.16 h, AUC 47.61, CL 27.04; VCR/Lipo-SM-CSS-Chol T1/2 7.14 h, AUC 326.45, CL 9.10, MRT 10.31 h; VCR/Lipo-SM-SCS-Chol T1/2 6.76 h, AUC 401.71, CL 7.57, MRT 10.67 h. SM-CSS-Chol also showed reduced distribution to heart, lung, and kidney versus SM/Chol.
• In vivo content retention and tumor-triggered release: In KPC-Luc PDAC, dual-labeled SM-CSS-Chol liposomes had higher AUCMU-P/AUCDID (0.86) than SM–Chol variants (0.43–0.46), PChcPC (0.69), and SM/Chol (0.29), indicating superior systemic stability. SM-CSS-Chol released payload more in tumors (consistent with higher GSH) while retaining contents longer in liver, spleen, and kidney versus comparators.
• Antitumor efficacy (SU-DHL-4 DLBCL): A single 2 mg/kg VCR dose showed significant tumor burden reduction with SM–Chol carriers; SM-CSS-Chol delivered the strongest inhibition, eradicating 2/5 tumors and yielding the longest survival. Pharmacodynamic markers (β-tubulin disruption, increased cleaved caspase-3 and TUNEL, decreased Ki67) were most pronounced with SM-CSS-Chol.
• Antitumor efficacy (IRI in KPC-Luc PDAC): With 40 mg/kg IRI on days 11, 14, 17, IRI/SM-CSS-Chol outperformed Onivyde, SM/Chol, PChcPC, and other SM–Chol variants in suppressing primary tumor growth and markedly reducing metastases. Disulfide-linked SML controls (SML-SS, SML-CSS) benefited from disulfide linkages; SML-CSS > SML-SS for delivery; nonetheless, SM-CSS-Chol surpassed all SMLs in efficacy, consistent with improved PK/tumor delivery and enhanced γ-H2AX/CC-3 signals and reduced Ki67.
• Antitumor efficacy (DOX in 4T1-Luc2 TNBC): At 15 mg/kg (single dose), DOX/SM-CSS-Chol outperformed Doxil, PChcPC, SM/Chol, and other SM–Chol variants, shrinking tumors to ~50% of baseline and completely preventing lung metastasis by day 35.
• Anti-inflammatory efficacy (DEX in LPS lung): Incorporation of DEX into SM–Chol bilayers increased DLE 4–5.2-fold versus SM/Chol or PChcPC (e.g., DEX DLE: SM-CSS-Chol 12.67%, SM-SCS-Chol 12.61% vs SM/Chol 2.05%, PChcPC 1.79%). In vivo, DEX/SM-CSS-Chol most strongly reduced IL-6, TNF-α, and IL-1β, and histology showed marked reductions in leukocyte recruitment and peribronchial thickening versus free DEX and other liposomal controls.
• Gene delivery (siRNA, CT26): siRNA in LNPs with SM-CSS-Chol helper lipids maintained serum stability up to 24 h and achieved greater P-gp mRNA knockdown than LNPs with DSPC/Chol, SM/Chol, or PChcPC. Combining IRI/SM-CSS-Chol with siRNA/LNP-SM-CSS-Chol enhanced tumor suppression beyond IRI/SM-CSS-Chol alone and increased intratumoral IRI levels compared with matched PChcPC systems.
• Cellular lipid homeostasis and cytotoxicity: Lipo-SM–Chol did not alter SREBP1, NPC1/2 expression, lipid raft levels, or cellular SM levels versus vehicle controls, and showed no significant cytotoxicity up to 1 mM across tested lipids.

The study addresses the central barrier of cholesterol exchange from liposomal membranes, which promotes leakage and reduces therapeutic performance. By covalently tethering cholesterol to sphingomyelin, the SM–Chol bilayer traps cholesterol within the membrane while preserving its condensing effects, as evidenced by DSC and AFM. Systematic linker screening revealed that disulfide-bonded SM-CSS-Chol with a longer spacer optimally balances systemic stability and triggered release: in circulation it minimizes payload leakage and cholesterol transfer (reduced exchange/leakage in osmotic stress and serum assays, high AUCMU-P/AUCDID), prolongs exposure (high AUC, long T1/2, low CL), and reduces off-target tissue distribution; within tumors or inflamed tissues with elevated GSH, the disulfide is cleaved to accelerate content release and enhance on-target pharmacodynamics. These properties translated into improved therapeutic indices across modalities: increased VCR MTD without detectable systemic chemistry abnormalities; superior tumor growth control and survival in DLBCL; enhanced efficacy over FDA-approved comparators (Onivyde, Doxil) in aggressive PDAC and TNBC models with reduced metastasis; greater anti-inflammatory efficacy delivering DEX to lungs; and potentiated gene knockdown and chemo-sensitization via P-gp siRNA LNPs. Importantly, SM–Chol carriers did not perturb cellular cholesterol/SM trafficking or lipid raft status and were non-cytotoxic in vitro, supporting biocompatibility. Overall, the data substantiate that bilayer engineering via SM–Chol, particularly SM-CSS-Chol, directly resolves cholesterol exchange-mediated instability and offers a broadly applicable platform for improved in vivo delivery and efficacy.

Covalently conjugating cholesterol to sphingomyelin produces a chimeric bilayer that confines cholesterol, retains membrane-condensing effects, and markedly reduces payload leakage and cholesterol exchange. Among tested constructs, the disulfide-linked, longer-spacer SM-CSS-Chol optimally enhances in vivo stability, pharmacokinetics, tumor delivery, and GSH-triggered release, thereby improving the therapeutic index across multiple payload classes (VCR, IRI, DOX, DEX) and nucleic acids (P-gp siRNA) in several disease models. Compared with conventional phospholipid/Chol and SML systems, SM-CSS-Chol increased VCR MTD, achieved orders-of-magnitude higher AUC and prolonged half-life, reduced off-target organ exposure, improved antitumor efficacy and survival, mitigated metastasis, and enhanced anti-inflammatory outcomes. These findings position SM–Chol, particularly SM-CSS-Chol, as a universal platform for drug and gene delivery. Future work could include detailed mechanistic and structure–function studies to further optimize linker chemistry, expanded safety and immunogenicity profiling, scale-up and manufacturing robustness assessments, and translational studies across additional therapeutic modalities and disease indications.

• The studies are preclinical and performed in murine models; translation to humans remains to be established.
• Most in vivo efficacy experiments were conducted in female mice; sex-specific responses were not comprehensively evaluated.
• Long-term safety, immunogenicity, and repeat-dosing effects were not fully characterized beyond acute MTD and short-term chemistry/hematology.
• While improved biodistribution and triggered release were demonstrated, detailed mechanistic quantification of cholesterol exchange kinetics in vivo and the precise contribution of each linker’s length/chemistry to performance warrant further investigation.
• Comparative head-to-head studies with additional clinically used lipid compositions or alternative ionizable/helper lipids for siRNA were limited.