{ "format": "listen-step-protocol@1", "name": "Preparation of targeted LNP for precision nucleic acid delivery", "tags": [ "LNP", "Preparation protocol" ], "source": { "filename": "Preparation of targeted lipid nanopa Source Nat Protoc SO 2026.pdf", "citation": "Hannah C. Geisler et al. \"Preparation of targeted lipid nanoparticles for precision nucleic acid delivery\" Nature Protocols (2026) https://doi.org/10.1038/s41596-025-01330-w", "paper": { "doi": "10.1038/s41596-025-01330-w", "url": "https://doi.org/10.1038/s41596-025-01330-w", "year": 2026, "title": "Preparation of targeted lipid nanoparticles for precision nucleic acid delivery", "authors": [ "Hannah C. Geisler", "Elisa Battistini", "Ajay S. Thatte", "Marshall S. Padilla", "Michael J. Mitchell" ], "journal": "Nature Protocols" } }, "steps": [ { "order": 1, "action": "Prepare antibody at concentration of 1-2 mg/ml in water or azide/amine-free aqueous buffer", "timing": "0.5-1 d", "materials": [] }, { "order": 2, "action": "Perform buffer exchange to remove sodium azide using desalting columns (option A), dialysis (option B), or concentrator spin columns (option C)", "timing": "", "materials": [] }, { "order": 3, "action": "Buffer exchange via desalting", "timing": "", "materials": [] }, { "order": 4, "action": "Use a desalting column according to the manufacturer’s instructions. Briefly,centrifuge the desalting column at 1,500g for 1 min to remove the storage solution.Take note of where the desalting resin is slanted upward and place the resin peakfacing away from the rotor in subsequent steps", "timing": "", "materials": [] }, { "order": 5, "action": "Add water to the column and spin at 1,500g for 1 min to rinse the column. Repeatthis three times", "timing": "", "materials": [] }, { "order": 6, "action": "Add antibody sample to the column and centrifuge at 1,500g for 2 min at roomtemperature to collect your sample.", "timing": "", "materials": [] }, { "order": 7, "action": "To generate two types of tLNPs—LNPs functionalized to whole antibodies and LNPsfunctionalized with F(ab′)2 fragments—divide your antibody solution in half. Store half ofthe solution at 4 °C for later use.", "timing": "", "materials": [] }, { "order": 8, "action": "Digest antibodies with enzyme (ficin for IgG1, pepsin or IdeZ for IgG2/IgG3/IgG4) to generate F(ab')2 fragments if desired", "timing": "1-2 h", "materials": [] }, { "order": 9, "action": "Equilibrate the TFP ester-PEG(4)-DBCO vial to room temperature. Using a scale, weigh out1.25 mg of TFP ester-PEG(4)-DBCO into a microcentrifuge tube. Add 500 μl of anhydrous DMSOto the tube to create a 3.5 mM solution. Vortex vigorously until the TFP ester-PEG(4)-DBCO isdissolved in solution.", "timing": "", "materials": [] }, { "order": 10, "action": "Label antibodies with DBCO using TFP ester-PEG(4)-DBCO at 10:1-20:1 DBCO:antibody molar ratio", "timing": "30 min", "materials": [] }, { "order": 11, "action": "Incubate antibodies on the thermomixer for 2 h at room temperature with shaking(400 r.p.m.).", "timing": "", "materials": [] }, { "order": 12, "action": "Purify DBCO-labeled antibodies to remove unreacted DBCO using Zeba spin columns", "timing": "", "materials": [] }, { "order": 13, "action": "Calculate DBCO degree of labeling (DOL) by measuring absorbance at 280 nm and 309 nm", "timing": "", "materials": [] }, { "order": 14, "action": "Quantify antibody concentration using Qubit fluorometric protein assay kit and Qubit flex fluorometer", "timing": "", "materials": [] }, { "order": 15, "action": "Clean the workspace with RNase AWAY to prevent contamination. Use RNase-free tubes,pipette tips and glassware in this section.", "timing": "", "materials": [] }, { "order": 16, "action": "Fill a 4-l beaker with 2 l of 1× PBS and place it on a stir plate. Add a magnetic stir bar and setthe speed to 300 r.p.m. Cover the entire beaker, including the sides and top, with aluminumfoil", "timing": "", "materials": [] }, { "order": 17, "action": "Pre-wet a dialysis cassette (20-kDa MWCO) by inserting the cassette into a Styrofoam buoyand placing it into the beaker. Let it soak while you formulate the LNPs.", "timing": "", "materials": [] }, { "order": 18, "action": "Prepare organic lipid phase by combining ionizable lipid, DOPE, cholesterol, C14-PEG2000, and DSPE-PEG2000-azide in ethanol", "timing": "", "materials": [] }, { "order": 19, "action": "Prepare aqueous RNA phase by dissolving mRNA (luciferase or mCherry) in 10 mM citrate buffer, pH 3", "timing": "", "materials": [] }, { "order": 20, "action": "Aspirate the organic phase into a 1-ml glass syringe with a 26-gauge needle and aspirate theaqueous phase into a 2.5-ml glass syringe with a 26-gauge needle. Ensure that no air bubblesare present in the syringes.", "timing": "", "materials": [] }, { "order": 21, "action": "Formulate LNPs via microfluidic mixing using syringe pump at 12 ml/min total flow rate with 1:3 organic:aqueous ratio", "timing": "", "materials": [] }, { "order": 22, "action": "Add DiR fluorescent dye (1-2 mg/ml in DMSO) to formulated LNPs at 0.5-1% vol/vol", "timing": "", "materials": [] }, { "order": 23, "action": "Dialyze LNPs against 1× PBS for 2 hours to remove ethanol and excess dye", "timing": "", "materials": [] }, { "order": 24, "action": "Add DBCO-labeled antibody to azide-LNPs at calculated stoichiometric ratio", "timing": "", "materials": [ "DBCO-labeled antibody", "Azide-LNPs" ] }, { "order": 25, "action": "Incubate antibody-LNP mixture overnight (18-24 h) at room temperature with shaking at 350 rpm", "timing": "18-24 h", "materials": [] }, { "order": 26, "action": "Prepare SEC column by packing Sepharose CL-4B resin into 20-ml chromatography column", "timing": "", "materials": [] }, { "order": 27, "action": "When the EtOH storage liquid drains through and the resin becomes visible to the eye,gently pour 20% (vol/vol) EtOH into the column, filling up the entire reservoir of the column.Let the EtOH drain through. Repeat one time", "timing": "", "materials": [] }, { "order": 28, "action": "Wash SEC column extensively with ultrapure water to remove ethanol storage solution", "timing": "", "materials": [] }, { "order": 29, "action": "Equilibrate SEC column with 1× PBS buffer", "timing": "", "materials": [] }, { "order": 30, "action": "Store SEC column overnight in 1× PBS until tLNP purification", "timing": "Overnight", "materials": [] }, { "order": 31, "action": "After the tLNPs have incubated for 18–24 h, uncap the top and bottom closures from theSEC column. Let the remaining 1× PBS drip out of the column", "timing": "", "materials": [] }, { "order": 32, "action": "Load tLNP-antibody reaction mixture onto SEC column", "timing": "", "materials": [] }, { "order": 33, "action": "Elute tLNPs with 1× PBS and collect fractions into 96-well plate", "timing": "", "materials": [] }, { "order": 34, "action": "Perform Triton X-100 assay on fractions to identify tLNP-containing fractions using RiboGreen reagent", "timing": "", "materials": [] }, { "order": 35, "action": "Pool tLNP-containing fractions identified by RiboGreen fluorescence", "timing": "", "materials": [] }, { "order": 36, "action": "Concentrate pooled tLNPs using 100-kDa MWCO ultracentrifugal spin columns", "timing": "", "materials": [] }, { "order": 37, "action": "Measure hydrodynamic radius using DLS plate reader", "timing": "", "materials": [] }, { "order": 38, "action": "Measure zeta potential using Zetasizer", "timing": "", "materials": [] }, { "order": 39, "action": "Quantify mRNA concentration using Quant-iT RiboGreen assay", "timing": "", "materials": [] }, { "order": 40, "action": "Dilute tLNPs to 7.5 ng/μl for in vivo administration (1.5 μg mRNA per mouse)", "timing": "", "materials": [] }, { "order": 41, "action": "Administer tLNPs intravenously to C57BL/6 mice via retro-orbital injection", "timing": "", "materials": [] }, { "order": 42, "action": "Wait 4 hours after tLNP administration", "timing": "", "materials": [] }, { "order": 43, "action": "Inject D-luciferin substrate intraperitoneally", "timing": "", "materials": [] }, { "order": 44, "action": "Euthanize mice using CO2 system", "timing": "", "materials": [] }, { "order": 45, "action": "Dissect major organs (heart, lungs, liver, kidneys, spleen)", "timing": "", "materials": [] }, { "order": 46, "action": "Image organs using IVIS for DiR fluorescence (biodistribution) and luminescence (mRNA transfection)", "timing": "", "materials": [] }, { "order": 47, "action": "Analyze images using Living Image software with ROI analysis", "timing": "", "materials": [] }, { "order": 48, "action": "Activate primary human T cells overnight using anti-CD3/CD28 Dynabeads at 1:1 cell:bead ratio", "timing": "Overnight (18 h)", "materials": [] }, { "order": 49, "action": "Plate activated T cells in 96-well plates", "timing": "", "materials": [] }, { "order": 50, "action": "Dilute tLNPs and administer to T cells at various doses (25, 50, 100 ng RNA per 100,000 cells)", "timing": "", "materials": [] }, { "order": 51, "action": "Incubate T cells with tLNPs for 1, 4, and 24 hours at 37°C in CO2 incubator", "timing": "", "materials": [] }, { "order": 52, "action": "Remove Dynabeads using magnet (96S super magnet plate or Mojosort)", "timing": "", "materials": [] }, { "order": 53, "action": "Wash cells with 1× PBS", "timing": "", "materials": [] }, { "order": 54, "action": "Analyze DiR fluorescence and mCherry expression by flow cytometry", "timing": "", "materials": [] }, { "order": 55, "action": "Store SEC column after use by exchanging buffer to water, then 20% ethanol", "timing": "", "materials": [] }, { "order": 56, "action": "Between samples, rinse SEC column thoroughly with 1× PBS to prevent carryover", "timing": "", "materials": [] } ], "materials": [], "tables": { "0": { "rows": [ [ "Maleimide-thiol", "Native disulfide reduction (TCEP, DTT)", "Simple, fast", "Risk of over-reduction" ], [ "Uses native structure", "Potential for diverse reduction products" ], [ "High levels of reducing agent can affect structure/function" ], [ "Thiolation (SATA)", "Avoids reduction; gentler on structure", "Random, heterogeneous thiolation" ], [ "Tunable thiol density", "Extra de-protection step" ], [ "Site-directed thiolation", "Site specific", "Protein engineering required" ], [ "Preserves paratope", "Increases cost; complexity" ], [ "Azide-DBCO", "DBCO modification (NHS- or TFP ester-DBCO)", "Tunable DBCO density", "Random, heterogeneous DBCO modification" ], [ "High reaction yield under mild conditions" ], [ "Site-directed DBCO modification", "Site specific", "Protein engineering required" ], [ "Preserves paratope", "Increases cost; complexity" ] ] }, "1": { "rows": [ [ "Antibody", "~150", "Clinically validated", "Large, sterically bulky, poor functionalization orientation", "71-74" ], [ "Broad epitope coverage", "Costly manufacturing" ], [ "Multivalent binding, high affinity", "Fc domain may increase immunogenicity or alter clearance (application specific)" ], [ "Fc domain may increase immunogenicity (application specific)", "Moderate stability" ], [ "$F(ab')_2$ fragment", "~110", "Broad epitope coverage", "Relatively large, sterically bulky, poor orientation", "73-75" ], [ "Multivalent binding, high affinity", "Costly manufacturing" ], [ "No Fc effector functions", "Lower stability than IgG" ], [ "Fab fragment", "~50", "Smaller, reduced steric hindrance", "Monovalent binding is weaker", "72-76" ], [ "No Fc effector functions", "Costly manufacturing" ], [ "Lower stability than IgG" ], [ "More prone to denaturation/aggregation" ], [ "Single-chain variable fragment", "25-35", "Small size, low steric hindrance", "Costly manufacturing", "72,73,75-77" ], [ "High affinity", "Lower stability than Fab" ], [ "Flexible engineering", "More prone to denaturation/aggregation" ], [ "Potential immunogenicity" ], [ "Engineered scaffold", "10-20", "Small size, low steric hindrance", "Less clinically validated", "75,78,79" ], [ "High affinity", "Potential immunogenicity" ], [ "Very high stability" ], [ "Flexible engineering" ], [ "Lower-cost manufacturing" ], [ "Nanobody", "12-15", "Very small size, low steric hindrance", "Potential immunogenicity", "72,73,75,77,80" ], [ "High affinity" ], [ "Very high stability" ], [ "Flexible engineering" ], [ "Low-cost manufacturing" ], [ "Peptide", "1-5", "Small size, minimal steric hindrance", "Weaker affinity", "71,106,107" ], [ "Low immunogenicity", "Low stability; can be improved with modifications" ], [ "Flexible engineering" ], [ "Low-cost manufacturing" ] ] }, "2": { "rows": [ [ "Mouse", "IgG1", "Ficin", "Murine IgG1 is resistant to pepsin cleavage", "108,109" ], [ "IgG2a", "IdeZ or pepsin", "Pepsin rate and completeness of digestion: rabbit > human > mouse", "109-111" ], [ "IgG2b", "Pepsin", "Pepsin rate and completeness of digestion: rabbit > human > mouse", "109,110" ], [ "IgG3", "IdeZ or pepsin", "Pepsin rate and completeness of digestion: rabbit > human > mouse", "109,111" ], [ "Human", "IgG1", "IdeZ or $pepsin^a$", "Pepsin rate and completeness of digestion: rabbit > human > mouse", "111-113" ], [ "Human sensitivity to pepsin: IgG3 > IgG4 > IgG1 > IgG2" ], [ "IgG2", "IdeZ or pepsin", "Pepsin rate and completeness of digestion: rabbit > human > mouse", "111,112" ], [ "Human sensitivity to pepsin: IgG3 > IgG4 > IgG1 > IgG2" ], [ "IgG3", "IdeZ or pepsin", "Pepsin rate and completeness of digestion: rabbit > human > mouse", "111,112" ], [ "Human sensitivity to pepsin: IgG3 > IgG4 > IgG1 > IgG2" ], [ "IgG4", "IdeZ or $pepsin^a$", "Pepsin rate and completeness of digestion: rabbit > human > mouse", "111-113" ], [ "Human sensitivity to pepsin: IgG3 > IgG4 > IgG1 > IgG2" ], [ "Rat", "IgG1", "Ficin", "Similar to mouse IgG1", "114,115" ], [ "IgG2a", "Pepsin", "Rat IgG2 similar to mouse IgG2", "115" ], [ "Rat sensitivity to pepsin: IgG2c > IgG2b > IgG2a > IgG1" ], [ "IgG2b", "Pepsin", "Rat IgG2 similar to mouse IgG2", "115" ], [ "Rat sensitivity to pepsin: IgG2c > IgG2b > IgG2a > IgG1" ], [ "IgG2c", "Pepsin", "Rat IgG2 similar to mouse IgG2", "115" ], [ "Rat sensitivity to pepsin: IgG2c > IgG2b > IgG2a > IgG1" ], [ "Rabbit", "IgG", "Pepsin", "Pepsin rate and completeness of digestion: rabbit > human > mouse", "116" ] ] }, "3": { "rows": [ [ "1,500", "150", "10:1", "675", "2,025", "1:3" ] ] }, "4": { "rows": [ [ "A4 LNPs" ], [ "A4 ionizable lipid", "35", "40", "37.8" ], [ "DOPE", "16", "5", "83.3" ], [ "Cholesterol", "46.5", "5", "125.8" ], [ "C14-PEG2000", "2.08", "5", "39.3" ], [ "DSPE-PEG2000-azide", "0.42", "5", "8.2" ], [ "EtOH", "-", "-", "380.6" ], [ "Final volume", "-", "-", "675" ], [ "cKK-E12 LNPs" ], [ "cKK-E12 ionizable lipid", "25", "40", "37.8" ], [ "DOPE", "5", "5", "44.9" ], [ "Cholesterol", "18.5", "5", "86.4" ], [ "DOTAP", "50", "5", "421.8" ], [ "DMG-PEG2000", "0.75", "5", "22.7" ], [ "DSPE-PEG2000-azide", "0.75", "5", "25.5" ], [ "EtOH", "-", "-", "35.9" ], [ "Final volume", "-", "-", "675" ], [ "C14-4 LNPs" ], [ "C14-4 ionizable lipid", "35", "40", "37.8" ], [ "DOPE", "16", "5", "74.7" ], [ "Cholesterol", "46.5", "5", "112.8" ], [ "C14-PEG2000", "2.08", "5", "35.2" ], [ "DSPE-PEG2000-azide", "0.42", "5", "7.4" ], [ "EtOH", "-", "-", "407.1" ], [ "Final volume", "-", "-", "675" ] ] }, "5": { "rows": [ [ "3", "Poor antibody cleavage into F(ab')2 fragments visualized via gel electrophoresis", "Wrong enzyme selected", "Confirm compatibility between the enzyme and antibody isotype" ], [ "8", "Antibody concentration not detectable or far below expected", "Too little antibody labeled", "Label a higher amount of antibody" ], [ "Antibody loss during antibody preparation", "Prepare the antibody by using one of the alternative methods described in Step 1" ], [ "Buffer contaminant not accounted for during Qubit calibration", "See the manufacturer's instructions" ], [ "9", "DOL << 6", "Unsuccessful DBCO labeling", "Increase the molar excess of DBCO; increase the reaction time" ], [ "DOL >> 6", "Too much DBCO added", "Add less DBCO" ], [ "Antibody concentration too low for accurate spectrophotometer readings", "Label a higher amount of antibody" ], [ "33", "mRNA tLNPs span more wells than expected", "Column too old, resin integrity compromised", "Prepare a fresh SEC column" ], [ "36", "Increase in size not observed on DLS", "Antibody not conjugated", "Perform the protocol by using a fluorophore-labeled antibody to confirm whether antibody signal overlaps with LNP signal after SEC separation. If the signals do not overlap, see the Troubleshooting solutions for Step 38" ], [ "LNPs have aggregated", "Mix LNP solutions very well before preparing DLS samples" ], [ "tLNP concentration too high", "Dilute tLNP sample more, re-run DLS" ], [ "37", "Low mRNA concentration", "Poor microfluidic formulation", "Prepare fresh stocks of lipid excipients" ], [ "Increase batch size" ], [ "38", "Fluorescent antibody is eluted much later than mRNA LNPs", "Antibody not conjugated to LNPs", "Optimize the DBCO DOL reaction to achieve a DOL of 6-8 (increase DBCO amount and incubation time)" ], [ "Add a higher DBCO-antibody molar equivalent to the LNP solution in Step 20" ], [ "Allow tLNPs to incubate with antibody for 24-36 h" ], [ "Calculated low antibody conjugation efficiency", "Inefficient reaction between DBCO-antibody and azide-LNP", "Add a higher DBCO-antibody molar equivalent to the LNP solution in Step 20" ], [ "Allow tLNPs to incubate with antibody for 24-36 h" ], [ "Poor separation between tLNPs and residual antibody (overlapping peaks)", "The column is not large enough; the resin bed is not tall enough", "Pour a fresh SEC column, ensuring ~20-ml resin volume" ], [ "Try a larger (taller) SEC column" ], [ "45", "Inconsistent or no luminescence signal detected on the IVIS", "mRNA LNPs did not deliver nucleic acid cargo", "Confirm LNP integrity (radius, polydispersity index, mRNA encapsulation efficiency and mRNA concentration)" ], [ "Missed intravenous injections of LNPs" ], [ "Missed or inconsistent intraperitoneal injections of D-luciferin" ], [ "46 and 56", "tLNPs do not demonstrate targeting compared to NT LNPs", "Antibody not conjugated", "See the Troubleshooting solutions for Step 38" ], [ "Low target receptor abundance or wrong target receptor chosen", "Confirm expression of the desired target receptor in your cell population via flow cytometry" ], [ "55", "All cell populations are >90% DiR+", "DiR fluorescence is too strong", "Add less DiR to LNPs" ], [ "Turn down the laser on the flow cytometer" ] ] }, "6": { "rows": [ [ "Anti-EGFR", "$F(ab')_2$", "7.0" ], [ "Wab", "8.5" ], [ "Anti-PECAM", "$F(ab')_2$", "9.8" ], [ "Wab", "8.7" ], [ "Anti-CD5", "$F(ab')_2$", "5.1" ], [ "Wab", "6.6" ] ] }, "7": { "rows": [ [ "A4 NT", "40.4", "92.3" ], [ "A4 + EGFR (Fab') $_{2}$", "54.8", "94.5" ], [ "A4 + EGFR Wab", "49.7", "93.8" ], [ "cKK NT", "42.2", "92.8" ], [ "cKK + PECAM (Fab') $_{2}$", "10.3", "79.6" ], [ "cKK + PECAM Wab", "11.1", "80.7" ], [ "C14-4 NT", "40.8", "93.6" ], [ "C14-4 + CD5 (Fab') $_{2}$", "10.8", "84.8" ], [ "C14-4 + CD5 Wab", "10.0", "84.5" ] ] }, "8": { "rows": [ [ "Reporting on race, ethnicity, or other socially relevant groupings", "N/A" ], [ "Population characteristics", "N/A" ], [ "Recruitment", "N/A" ], [ "Ethics oversight", "N/A" ] ] }, "9": { "rows": [ [ "Data exclusions", "One animal was excluded from the study due to a failed IP injection of D-luciferin salt. This failed injection resulted in no luminescence signal during imaging of the dissected organs. This animal was excluded and a new animal was injected, dissected, and imaged on a separate day. The IVIS images in Figure 5f are stitched together from the 2 separate days. This is explained in the Figure caption." ], [ "Replication", "Results were replicated across multiple experiments with all replicates generating similar results" ], [ "Randomization", "For animal experiments, treatment groups were partitioned by randomly selecting from the same pool of animals. Groups in all in vitro and in vivo experiments were selected randomly." ], [ "Blinding", "All investigators were blinded to group allocation during data collection and analysis." ] ] }, "10": { "rows": [ [ "", "Antibodies" ], [ "", "Eukaryotic cell lines" ], [ "", "Palaeontology and archaeology" ], [ "", "Animals and other organisms" ], [ "", "Clinical data" ], [ "", "Dual use research of concern" ], [ "", "Plants" ] ] }, "11": { "rows": [ [ "", "ChIP-seq" ], [ "", "Flow cytometry" ], [ "", "MRI-based neuroimaging" ] ] }, "12": { "rows": [ [ "Validation", "Each antibody purchased from vendors had the following statement on their website: \"Each lot of this antibody is quality control tested by immunofluorescent staining with flow cytometric analysis.\"" ] ] }, "13": { "rows": [ [ "Authentication", "T cells were authenticated by staff at the Human Immunology Core at the University of Pennsylvania" ], [ "Mycoplasma contamination", "T cell contamination was verified by staff at the Human Immunology Core at the University of Pennsylvania" ], [ "Commonly misidentified lines (See ICLAC register)", "N/A" ] ] }, "14": { "rows": [ [ "Wild animals", "No wild animal were used in this study" ], [ "Reporting on sex", "Female mice were used in this study" ], [ "Field-collected samples", "The study did not involve samples collected from field" ], [ "Ethics oversight", "All animal experiment protocols were reviewed and approved by the institutional animal care and use committee of the University of Pennsylvania" ] ] }, "15": { "rows": [ [ "Novel plant genotypes", "N/A" ], [ "Authentication", "N/A" ] ] }, "16": { "rows": [ [ "Instrument", "BD LSR II Flow Cytometer" ], [ "Software", "FlowJo software package (Flowjo V10)" ], [ "Cell population abundance", "A minimum of 10,000 events per sample was collected" ], [ "Gating strategy", "Cells were first gated using FSC-A and SSC-A. Singlet cells were gated using FSC-A and FSC-H. Live cells (Sytox negative) were gated from Singlets. mCherry and DiR fluorescence were gated from the Live population (Supplementary Figure 8)." ] ] } } }