Interplay of protein corona and immune cells controls blood residency of liposomes

Purpose: Liposome clinical translation is limited by opsonization and rapid clearance by immune cells. Although the protein corona is recognized as the liposome’s “biological identity,” it was unclear whether pre-adsorbed corona proteins could help liposomes avoid capture by circulating leukocytes in whole blood and thereby prolong blood residency.

Hypothesis: Pre-coating liposomes with an artificial protein corona made of human plasma proteins—especially at high plasma concentration—will reduce sequestration by circulating leukocytes and immune cells, enabling prolonged circulation. The biological identity of liposomes will depend strongly on plasma protein concentration.

Aims: Characterize size, zeta potential, nanostructure, and protein corona composition of cationic DOTAP, neutral DOPC, and anionic DOPG liposomes after exposure to increasing human plasma concentrations. - Determine how plasma protein concentration affects the biological identity of liposome–protein complexes. - Evaluate cellular uptake of uncoated vs pre-coated liposomes by THP1 monocytes, peripheral blood mononuclear cells (PBMCs), and leukocyte subpopulations in whole blood. - Test whether pre-coating can reduce leukocyte capture and identify mechanistic and formulation requirements for prolonged circulation.

Delivery system: Platform: Model liposomes with different surface charges: - Cationic: DOTAP. - Neutral: DOPC. - Anionic: DOPG. - Payload: None; these are model liposomes, not drug- or gene-loaded. - Targeting ligand: None. - Protein corona pre-coating: Liposomes incubated with human plasma (HP) at 1%, 2.5%, 5%, 10%, 20%, and 50%. Key comparative conditions: HP = 5% (low) and HP = 50% (high). - Fluorescent label: DOPE-NBD incorporated for flow cytometry uptake studies. - Preparation: Lipid films hydrated, extruded through 100-nm polycarbonate filters, stored at 4°C.

Approach: Model: In vitro only; no in vivo circulation or biodistribution study. - Cell systems: Human monocytic THP1 cell line; PBMCs from healthy donors; whole blood from healthy donors. - Uptake conditions: THP1 and PBMCs incubated with NBD-labeled liposomes; whole blood incubated with liposomes for 0.5, 1, 5, 15, 30, and 60 min. - Groups: Uncoated vs pre-coated liposomes; DOTAP, DOPC, DOPG; various HP concentrations. - Controls: Bare liposomes, different HP concentrations, distinct leukocyte subpopulations gated by CD markers. - Replication: n = 3 for size, zeta, proteomics; n = 2 for THP1 FACS and SDS-PAGE; n = 3 for PBMC experiments except DOTAP at HP = 5% (n = 1); whole blood samples in duplicate.

Key methods: Dynamic light scattering and zeta potential for size and surface charge. - Synchrotron small-angle X-ray scattering (SAXS) for liposome nanostructure. - BCA assay for protein binding. - NanoLC-MS/MS for protein corona composition. - 1D SDS-PAGE for temporal protein corona profiles. - Transmission electron microscopy for morphology. - Flow cytometry for cellular uptake in THP1 cells, PBMCs, and whole-blood leukocyte subpopulations.

Key results: Zeta potential: At high HP, zeta potential of DOTAP, DOPC, and DOPG converged to a common negative plateau of ~ −23 mV, independent of pristine surface charge. - Size: DOTAP size increased at low HP, peaked near charge inversion (5–10% HP), then decreased at HP > 10%. DOPC showed a weaker trend; DOPG showed none. At high HP, all formulations had similar size. - Nanostructure: DOTAP formed multilamellar structures at low plasma concentration; this disappeared at high HP. DOPC showed a much weaker effect, and DOPG did not form multilamellar complexes. - Protein binding: DOTAP adsorbed much more protein than DOPC and DOPG. Protein binding increased with HP concentration. - Corona composition: Intermediate-MW proteins (30–100 kDa) and proteins with pI < 7 dominated. At high HP, DOTAP corona showed decreased acute-phase proteins, immunoglobulins, and lipoproteins, and increased coagulation proteins. DOPG showed a large increase in lipoproteins (28.5% to 45.3% RPA) and decreases in immunoglobulins, coagulation, complement, and tissue leakage proteins. DOPC corona changed little. - THP1 uptake: Without corona, DOTAP uptake was highest. At HP = 5%, uptake order was DOTAP > DOPG > DOPC; DOTAP uptake peaked at HP = 5% and then decreased. At HP = 50%, uptake was low for all formulations. - PBMC uptake: Pre-coated DOTAP at HP = 5% was avidly internalized by nearly all populations; DOPC and DOPG were lower. At HP = 50%, liposome–protein complexes largely avoided capture, with monocytes (CD14+CD3−) showing the highest residual uptake. - Whole blood: Uncoated DOTAP was avidly internalized by leukocytes, with uptake order CD19+ > CD14+CD3− > granulocytes > several T/NK subsets. Pre-coating DOTAP significantly reduced capture by all circulating leukocyte populations. Uncoated DOPC and DOPG were captured less than DOTAP, with DOPG > DOPC. Capture occurred largely by 0.5 min and changed little over time. - Corona stability: SDS-PAGE showed pre-coated DOTAP corona was not modified by plasma exposure, and coronas were established by 0.5 min and remained stable. The main difference between uncoated and pre-coated DOTAP was in the 46–56 kDa region, corresponding to fibrinogen γ- and β-chains. - Optimal formulation features: Small size, negative charge, enrichment of anti-opsons such as clusterin, and low immunoglobulin/Fetuin content. HP = 50% pre-coated liposomes best met these criteria.

Interpretation: The authors conclude that the protein corona controls liposome interactions with immune cells, and pre-coating liposomes with an artificial human plasma protein corona significantly reduces capture by circulating leukocytes in whole blood. This may enable prolonged circulation in vivo. They also argue that in vitro studies at high plasma concentration may not mimic in vivo clearance, and that pre-coated cationic liposomes at low protein concentration might be useful for targeting macrophages in cancer.

Limitations: In vitro only; no in vivo pharmacokinetics