Preparation of targeted LNP for precision nucleic acid delivery

LNPPreparation protocol

56 steps · 0 materials & instruments

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Steps

#	Action	Timing
1	Prepare antibody at concentration of 1-2 mg/ml in water or azide/amine-free aqueous buffer	0.5-1 d
2	Perform buffer exchange to remove sodium azide using desalting columns (option A), dialysis (option B), or concentrator spin columns (option C)
3	Buffer exchange via desalting
4	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
5	Add water to the column and spin at 1,500g for 1 min to rinse the column. Repeatthis three times
6	Add antibody sample to the column and centrifuge at 1,500g for 2 min at roomtemperature to collect your sample.
7	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.
8	Digest antibodies with enzyme (ficin for IgG1, pepsin or IdeZ for IgG2/IgG3/IgG4) to generate F(ab')2 fragments if desired	1-2 h
9	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.
10	Label antibodies with DBCO using TFP ester-PEG(4)-DBCO at 10:1-20:1 DBCO:antibody molar ratio	30 min
11	Incubate antibodies on the thermomixer for 2 h at room temperature with shaking(400 r.p.m.).
12	Purify DBCO-labeled antibodies to remove unreacted DBCO using Zeba spin columns
13	Calculate DBCO degree of labeling (DOL) by measuring absorbance at 280 nm and 309 nm
14	Quantify antibody concentration using Qubit fluorometric protein assay kit and Qubit flex fluorometer
15	Clean the workspace with RNase AWAY to prevent contamination. Use RNase-free tubes,pipette tips and glassware in this section.
16	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
17	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.
18	Prepare organic lipid phase by combining ionizable lipid, DOPE, cholesterol, C14-PEG2000, and DSPE-PEG2000-azide in ethanol
19	Prepare aqueous RNA phase by dissolving mRNA (luciferase or mCherry) in 10 mM citrate buffer, pH 3
20	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.
21	Formulate LNPs via microfluidic mixing using syringe pump at 12 ml/min total flow rate with 1:3 organic:aqueous ratio
22	Add DiR fluorescent dye (1-2 mg/ml in DMSO) to formulated LNPs at 0.5-1% vol/vol
23	Dialyze LNPs against 1× PBS for 2 hours to remove ethanol and excess dye
24	Add DBCO-labeled antibody to azide-LNPs at calculated stoichiometric ratio
25	Incubate antibody-LNP mixture overnight (18-24 h) at room temperature with shaking at 350 rpm	18-24 h
26	Prepare SEC column by packing Sepharose CL-4B resin into 20-ml chromatography column
27	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
28	Wash SEC column extensively with ultrapure water to remove ethanol storage solution
29	Equilibrate SEC column with 1× PBS buffer
30	Store SEC column overnight in 1× PBS until tLNP purification	Overnight
31	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
32	Load tLNP-antibody reaction mixture onto SEC column
33	Elute tLNPs with 1× PBS and collect fractions into 96-well plate
34	Perform Triton X-100 assay on fractions to identify tLNP-containing fractions using RiboGreen reagent
35	Pool tLNP-containing fractions identified by RiboGreen fluorescence
36	Concentrate pooled tLNPs using 100-kDa MWCO ultracentrifugal spin columns
37	Measure hydrodynamic radius using DLS plate reader
38	Measure zeta potential using Zetasizer
39	Quantify mRNA concentration using Quant-iT RiboGreen assay
40	Dilute tLNPs to 7.5 ng/μl for in vivo administration (1.5 μg mRNA per mouse)
41	Administer tLNPs intravenously to C57BL/6 mice via retro-orbital injection
42	Wait 4 hours after tLNP administration
43	Inject D-luciferin substrate intraperitoneally
44	Euthanize mice using CO2 system
45	Dissect major organs (heart, lungs, liver, kidneys, spleen)
46	Image organs using IVIS for DiR fluorescence (biodistribution) and luminescence (mRNA transfection)
47	Analyze images using Living Image software with ROI analysis
48	Activate primary human T cells overnight using anti-CD3/CD28 Dynabeads at 1:1 cell:bead ratio	Overnight (18 h)
49	Plate activated T cells in 96-well plates
50	Dilute tLNPs and administer to T cells at various doses (25, 50, 100 ng RNA per 100,000 cells)
51	Incubate T cells with tLNPs for 1, 4, and 24 hours at 37°C in CO2 incubator
52	Remove Dynabeads using magnet (96S super magnet plate or Mojosort)
53	Wash cells with 1× PBS
54	Analyze DiR fluorescence and mCherry expression by flow cytometry
55	Store SEC column after use by exchanging buffer to water, then 20% ethanol
56	Between samples, rinse SEC column thoroughly with 1× PBS to prevent carryover

Materials & instruments

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Tables

Table 1

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

Table 2

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

Table 3

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

Table 4

1,500	150	10:1	675	2,025	1:3

Table 5

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

Table 6

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

Table 7

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

Table 8

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

Table 9

Reporting on race, ethnicity, or other socially relevant groupings	N/A
Population characteristics	N/A
Recruitment	N/A
Ethics oversight	N/A

Table 10

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.

Table 11

	Antibodies
	Eukaryotic cell lines
	Palaeontology and archaeology
	Animals and other organisms
	Clinical data
	Dual use research of concern
	Plants

Table 12

	ChIP-seq
	Flow cytometry
	MRI-based neuroimaging

Table 13

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."

Table 14

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

Table 15

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

Table 16

Novel plant genotypes	N/A
Authentication	N/A

Table 17

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).

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 DOI ↗

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