Lipid Nanoparticles with Side-Chain Polymer Coating for Targeted mRNA Delivery through Nanobody Attachment
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Lipid Nanoparticles with Side-Chain Polymer Coating for Targeted mRNA Delivery through Nanobody Attachment
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  • On Ting Choy
    On Ting Choy
    Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland 4072, Australia
    More by On Ting Choy
    Orcidhttps://orcid.org/0000-0002-1932-7797
  • Nicholas L. Fletcher
    Nicholas L. Fletcher
    Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland 4072, Australia
    ARC Research Hub for Advanced Manufacture of Targeted Radiopharmaceuticals, The University of Queensland, Brisbane, Queensland 4072, Australia
    Centre for Advanced Imaging, The University of Queensland, Brisbane, Queensland 4072, Australia
    More by Nicholas L. Fletcher
    Orcidhttps://orcid.org/0000-0002-2993-833X
  • Pie Huda
    Pie Huda
    Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland 4072, Australia
    ARC Research Hub for Advanced Manufacture of Targeted Radiopharmaceuticals, The University of Queensland, Brisbane, Queensland 4072, Australia
    Centre for Advanced Imaging, The University of Queensland, Brisbane, Queensland 4072, Australia
    More by Pie Huda
    Orcidhttps://orcid.org/0000-0002-2197-4993
  • Craig A. Bell
    Craig A. Bell
    Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland 4072, Australia
    Centre for Advanced Imaging, The University of Queensland, Brisbane, Queensland 4072, Australia
    More by Craig A. Bell
    Orcidhttps://orcid.org/0000-0002-8986-2795
  • David J. Owen
    David J. Owen
    Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland 4072, Australia
    ARC Research Hub for Advanced Manufacture of Targeted Radiopharmaceuticals, The University of Queensland, Brisbane, Queensland 4072, Australia
    More by David J. Owen
    Orcidhttps://orcid.org/0000-0002-9165-1540
  • Andrew K. Whittaker*
    Andrew K. Whittaker
    Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland 4072, Australia
    Australian Research Council Centre of Excellence for Green Electrochemical Transformation of Carbon Dioxide, The University of Queensland, Brisbane, Queensland 4072, Australia
    *Email: [email protected]
    More by Andrew K. Whittaker
    Orcidhttps://orcid.org/0000-0002-1948-8355
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Bioconjugate Chemistry

Cite this: Bioconjugate Chem. 2026, XXXX, XXX, XXX-XXX
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https://doi.org/10.1021/acs.bioconjchem.6c00045
Published March 26, 2026

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Abstract

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The use of mRNA therapies has innovated the clinical progress of cancer immunotherapy. However, current immunotherapeutic approaches are unable to achieve site- or immune-cell-specific delivery, resulting in adverse immune responses in off-target tissues. In addition, the commercial lipid nanoparticle (LNP) formulations with a poly(ethylene glycol) coating generally undergo significant hepatic accumulation during clearance. To promote site- and immune-cell-specific delivery of therapeutic mRNA-LNPs, we investigated several bioconjugation approaches to attach targeting antibodies onto the surface of polymer-functionalized mRNA-LNPs. Building on our previous work, side-chain sulfoxide polymer–lipid conjugate PMSEA-DSPE was used to incorporate a low-fouling polymeric LNP coating. trans-Cyclooctene functionality was incorporated within PMSEA-DSPE end groups to allow conjugation to the tetrazine-functionalized nanobody 9G8 for EGFR targeting. Bioconjugation methods were compared, including direct conjugation and post-insertion. The results showed that 9G8-attached PMSEA mRNA-LNPs prepared via direct conjugation significantly enhanced cell association and in vitro transfection efficiency with an EGFR-positive cell line, demonstrating the potency of active targeting for mRNA-LNP platforms with side-chain polymer coatings.

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Introduction

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The successful therapeutic application of mRNA (mRNA) has opened the potential of its use in cancer immunotherapy in clinical settings. The ease of manufacture, the effectiveness of mRNA in target inducing protein translation, and its capability to encode multiple proteins has resulted in several mRNA cancer immunotherapies to enter clinical trials. (1,2) These immunotherapeutic approaches can be generally categorized into antigen presentation, immunomodulatory protein production, and adoptive cell therapy. (3) Currently, personalized neoantigen vaccines exploiting antigen presentation are the most promising and commonly adopted immunotherapeutic approaches. mRNA-4157 is an example of a personalized cancer vaccine that can encode up to 34 neoantigens and is delivered via a lipid nanoparticle (LNP) formulation. This formulation has shown promising antitumor response in Phase I and II trials (NCT03313778 and NCT03897881) and has subsequently entered a Phase III trial (NCT05933577). (4−6)
mRNA immunotherapies can elicit robust immune responses; however, in vivo delivery of mRNA remains the biggest obstacle to successful clinical application. LNPs are the most widely adopted delivery platforms and generally incorporate PEG-lipid conjugates to produce a steric low-fouling layer and control payload delivery. (7) However, these PEG-LNP formulations endogenously target hepatocytes, resulting in significant hepatic accumulation and clearance. (8,9) This nonspecific delivery of mRNA-LNPs increases the risk of adverse off-target events such as cytokine release syndrome and liver damage. (10,11) This poses additional challenges for delivery of immunotherapies that produce immunomodulatory proteins to achieve an optimal therapeutic effect. (2) To promote immune response at the target cells while reducing off-target immune responses, precise delivery to the target site or to specific immune cell populations is key for the next generation of immunotherapies. (12)
A major concern for immunotherapies using PEG-coated mRNA-LNPs is the potential for enhanced clearance upon repeated administration due to the induction of higher antibody titers and the interaction of PEG-lipids with pre-existing anti-PEG antibodies. (13) This has driven recent interest in engineering alternate polymer coatings for LNPs with side-chain polymers in place of linear PEG. A series of alternate coating polymers have been explored, including PEGMA (brush polymer with ethylene glycol side chains), poly(2-oxazoline), poly(N-isopropylacrylamide) (PNIPAM), poly(N,N-dimethylacrylamide) (PDMA), and poly(N-(2-hydroxypropyl)methacrylamide) (PHPMA). (14−16) Notably, Xiao et al. recently demonstrated that polymer coatings with comparable physiochemical properties but varied antigenicity were able to diminish polymer binding to antibodies and ascribed this to the structural differences between linear and side-chain polymer chain conformations. (14) In agreement with this, we recently reported the development a mRNA-LNP delivery platform comprising poly(2-methylsulfinyl)ethyl acrylate) (PMSEA) coatings and demonstrated superior in vitro transfection efficiency while maintaining hydrophilic properties and stability similar to PEG-coated analogues. (17)
In order to enhance delivery efficiency, researchers have focused on functionalizing linear PEG-lipids with antibodies on the surface of mRNA-LNPs via direct conjugation or post-insertion approaches. (18−21) In contrast, the antibody conjugation of mRNA-LNPs with side-chain polymer coatings has not been explored. Such studies are important, as in addition to impacts due to changes in antigenicity from the polymer itself, the structural differences between polymer coatings may affect the feasibility of conjugation methods. In this current study, we investigate and compare conjugation methods for the attachment of antibodies to mRNA-LNPs with side-chain polymer coatings. While our previous work has compared the physical and biological properties of PMSEA and PEG mRNA-LNPs, this study focuses on the development of targeted PMSEA mRNA-LNP delivery systems. EGFR-specific nanobody 9G8 was chosen as the targeting ligand, and PMSEA was chosen as the side-chain polymer coating. To facilitate surface nanobody attachment, PMSEA-lipids were synthesized with a trans-cyclooctene (tCO) end group, and tetrazine (Tz) functionality was introduced through unnatural amino acids (UAAs) in the protein backbone of the 9G8 nanobody, enabling tCO-Tz coupling via biorthogonal click chemistry (Scheme 1). (22) The impact of nanobody-functionalization on PMSEA-LNP cellular association and resulting mRNA transfection was then assessed to demonstrate the feasibility of this approach to produce targeted PMSEA-LNP constructs.

Scheme 1

Scheme 1. Schematic Diagram of Nanobody-LNP Conjugation Via trans-Cyclooctene (tCO)-Functionalization of PMSEA-Lipid Conjugate and Attachment of Tz-9G8 Nanobody to the tCO Group on PMSEA mRNA-LNPs
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Results and Discussion

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Preparation and Characterization of tCO-Functionalized PMSEA-Lipid Conjugates

In our previous work, we designed a sulfoxide polymer–lipid conjugate PMSEA-DSPE for a new LNP formulation in place of mPEG2000-DSPE. (23) The conjugate was prepared via reversible addition–fragmentation chain-transfer (RAFT) polymerization, a common approach to graft side-chain polymers from/to lipids. (14,15) As 2-(n-butyltrithiocarbonate) propionic acid-1,2-distearoyl-sn-glycero-3-phosphorylethanolamine (BTPA-DSPE) was used as the chain transfer agent for RAFT polymerization, the PMSEA-DSPE conjugate retained an alkyl end group post-polymerization (Scheme 1). To enable post-polymerization modification, the PMSEA-lipid conjugate was functionalized with a carboxyl (−COOH) group by radical-induced reduction with the use of 4,4’-azobis(4-cyanovaleric acid) (ACVA) as the azoinitiator (Scheme 1). (24) This process involved removal of the trithiocarbonyl group from PMSEA-DSPE, followed by the addition of radicals from ACVA, resulting in attachment of the COOH end group to PMSEA-DSPE. Following purification, the trithiocarbonyl group removal was confirmed by the absence of UV–vis absorbance at 300–310 nm (Figure 1(a)) and removal of a 1H NMR peak at 0.98–0.91 ppm (Figure 1(b)) corresponding to the methyl group. The weak proton peak shown at 9.58 ppm in the 1H spectrum of COOH-PMSEA-DSPE (Figure 1(b) and S1) indicated the attachment of the carboxyl group of COOH-PMSEA-DSPE after the trithiocarbonyl group removal.

Figure 1

Figure 1. (a) UV–vis spectra of PMSEA-DSPE and COOH-PMSEA-DSPE; the trithiocarbonyl peak at 300 – 310 nm was evident in PMSEA-DSPE. (b) 1H NMR (500 MHz, CDCl3) of COOH-PMSEA-DSPE with the peak comparison of PMSEA-DSPE. The pink arrows indicate the presence of the methyl group, while the orange arrow indicates the proton in carboxyl groups.

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To facilitate the bioconjugation of Tz-9G8 and PMSEA mRNA-LNPs, tCO-amine was conjugated to the carboxyl end group in COOH-PMSEA-DSPE by amidation using hexafluorophosphate azabenzotriazole tetramethyl uronium (HATU). Following dialysis, the successful attachment of tCO was confirmed by 1H and two-dimensional diffusion-ordered (2D DOSY) NMR spectra (Figure 2 and S2). The 2D DOSY showed the broad tCO peak (peak a at 5.61–5.44 ppm) had similar translational diffusion properties to the polymer peaks (peak b at 4.7–4.3 ppm; peak c at 3.3–2.9 ppm; peak d at 2,7 ppm) and the peak corresponding to the lipid hydrocarbon tails (peak e at 0.95–0.81 ppm). In addition, the tCO peak was not observed with D > 1 × 10–5 cm2/s in 2D DOSY, indicating the small-molecule tCO residue was successfully removed from tCO-PMSEA-DSPE through dialysis. By comparing the integral of the lipid peak (peak e) and the tCO peak (peak a) in 1H NMR, all PMSEA-DSPE was modified with tCO, thereby resulting in a high-yield functionalization (yield = 83.9%).

Figure 2

Figure 2. 1H NMR 2D DOSY spectrum (500 MHz, CDCl3) of tCO-PMSEA-DSPE, showing successful tCO attachment to the PMSEA-lipid conjugate and the absence of free tCO residues.

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Tz-9G8 Nanobody Attachment of PMSEA mRNA-LNPs Via Different Preparation Methods

Following the preparation of the tCO-functionalized PMSEA-lipid, the two most commonly utilized bioconjugation method approaches in the literature were assessed for Tz-9G8 nanobody attachment to PMSEA mRNA-LNPs. These were either direct conjugation (Nb attachment to assembled LNP) or post-insertion (LNP assembled from Nb-functionalized polymer–lipid micelles) (Figure 3(a)). (18,20,25,26) The molar ratio of tCO-PMSEA-DSPE was maintained at 0.3 mol % of lipids for all the targeted LNP batches to allow comparison between preparation methods. A tCO/Tz ratio of 1.5 was used in all of the nanobody conjugation experiments to ensure Tz-9G8 nanobodies were attached to tCO-PMSEA-DSPE. In the direct conjugation method, tCO-PMSEA-DSPE was mixed with other lipid components for mRNA-LNP production. Following LNP assembly, Tz-9G8 was directly conjugated to the tCO functional groups on the surface of the mRNA-LNPs. For the post-insertion method, tCO-PMSEA-DSPE micelles were formed via sonication, and then Tz-9G8 was subsequently conjugated to the micelles, followed by the post-insertion of micelles into the LNPs.

Figure 3

Figure 3. (a) Demonstration of two preparation methods to conjugate the Tz-9G8 nanobody to PMSEA mRNA-LNPs, including direct conjugation and post-insertion. (b) Gel electrophoresis of 9G8 and LNP samples prepared by either direct conjugation or post-insertion; (i) gel image with Coomassie Blue staining and (ii) gel analysis by ImageJ. (c) Z-average (d.nm) and PDI measured by DLS, statistical data is presented as the mean ± SD (n = 3).

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After 9G8-conjugated mRNA-LNPs were prepared using different conjugation methods, the nanobody conjugation efficiency and the stability of mRNA-LNPs were assessed through physical characterization by SDS-PAGE, DLS, and the RiboGreen assay (Figure 3 and Table S1). From the gel analysis (Figure 3(b)), an intense free nanobody band, referenced by the 9G8 nanobody control (line 1; around 15 kDa), was found in the 9G8-PMSEA-DSPE micelle sample (line 2). From the densitometry analysis in ImageJ, 23.3% of 9G8 (around 30 kDa) was found to be conjugated to the micelle sample. After the post-insertion of micelles into mRNA-LNPs, a free nanobody band was still observable (line 4). In contrast, the free nanobody band was not noticeable when the 9G8-PMSEA mRNA-LNPs were prepared via direct conjugation (line 6). The densitometry analysis from the gel plots revealed that the LNPs prepared by post-insertion had a 4.3-fold larger amount of free nanobody than the LNPs prepared by direct conjugation with the same total amount of nanobody loadings.
Considering the physical formation and stability of the mRNA-LNPs, all of the nanobody-functionalized LNPs had high size uniformity (PDI ∼ 0.1) and mRNA encapsulation efficiencies (93%). However, the DLS results showed a large increase in size (22 nm) after the LNPs were modified by post-insertion, while the size of LNPs prepared by direct conjugation was close to the untargeted LNPs. Together, the physical characterization of nanobody-functionalized LNPs suggested the direct conjugation method may be preferred to the post-insertion method for LNP systems incorporating side-chain polymers, as this method has a better size maintenance and a higher tCO-Tz conjugation efficiency.
The physical characterization results showed that the direct conjugation performed better than the post-insertion method in terms of nanobody conjugation efficiency. This was further supported through in vitro analysis to probe the resulting cellular association (Figure 4(a)), with the use of EGFR-positive cell line MDA-MB-468. The initial rate of cellular association as a function of 9G8 conjugation was assessed by flow cytometry after 1 h incubation of 9G8-PMSEA mRNA-LNPs with MDA-MB-468. The flow cytometry results successfully showed 4.0-fold and 1.3-fold increases in mean fluorescence intensity with 9G8 conjugation via direct conjugation or post-insertion, respectively, indicating the initial uptake rate was significantly enhanced with the 9G8-functionalized mRNA-LNPs via direct conjugation. This difference in association was correlated with trends in the resulting functional mRNA expression (Figure 4(b)). The 9G8-functionalized mRNA-LNPs exhibited 1.7-fold and 1.4-fold enhancement in luciferase expression via direct conjugation or post-insertion, respectively, although there were no statistical differences between PMSEA mRNA-LNPs with and without 9G8. Of note, the 9G8-functionalized mRNA-LNPs prepared by direct conjugation showed significantly higher luciferase expression compared to the commercial PEG LNP formulation with a 2.7-fold enhancement, revealing the potential use of targeted PMSEA formulations. Overall, these initial results supported that the cellular association and transfection efficiency of PMSEA mRNA-LNPs had been promoted with 9G8 functionalization, and nanobody attachment to PMSEA mRNA-LNPs via direct conjugation performed better than post-insertion.

Figure 4

Figure 4. In vitro characterization of 9G8-PMSEA mRNA-LNPs prepared with different conjugation methods. (a) Cellular association of MDA-MB-468 incubated with PMSEA FLuc mRNA-LNPs with or without 9G8 attachment for 1 h and (b) luciferase expression of MDA-MB-468 incubated with PMSEA FLuc mRNA-LNPs with or without 9G8 attachment for 24 h. Statistical data is presented as the mean ± SD (n = 3 for flow cytometry; n = 4 for luciferase assay), ***p < 0.001, **p < 0.01, *p < 0.05, and ns p > 0.05 with 95% of confidence level from Welch’s unpaired t test.

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Effect of 9G8 Nanobody Density on PMSEA mRNA-LNPs

After the direct conjugation method was confirmed as the optimal approach for producing Nb-targeted PMSEA mRNA-LNPs, this preparation method was used for all subsequent analyses. To understand the effect of nanobody density on the surface of the LNPs, various molar ratios of tCO-PMSEA-DSPE (0, 0.1, 0.3, 0.5, 0.75, and 1 mol %) were incorporated into the LNP formulations while the total molar ratio of PMSEA-lipids (1.5 mol %) and tCO-PMSEA-DSPE:Tz-9G8 molar ratio of 1.5:1 was maintained. All 9G8-PMSEA mRNA-LNPs achieved high mRNA encapsulation efficiencies close to 90% (Table S1). The gel images showed free nanobody as an observable band from 0.5 mol % of tCO-PMSEA-DSPE and above (Figure 5(a)). The size of all PMSEA mRNA-LNPs was observed to increase after conjugation by DLS (Figure 5(b)). mRNA-LNPs with 0.1 to 0.5 mol % of tCO-PMSEA-DSPE only resulted in a size increase below 16 nm, while mRNA-LNPs with 0.75 and 1 mol % of tCO-PMSEA-DSPE gained 24 and 33 nm in size, respectively, likely due to the higher abundance of 9G8 on the surface of the functionalized mRNA-LNPs.

Figure 5

Figure 5. Physical and in vitro characterization of 9G8-PMSEA mRNA-LNPs with various molar ratios of tCO-PMSEA-DSPE (0 – 1 mol %) prepared by the direct conjugation method. (a) Gel image of 9G8-PMSEA mRNA-LNPs stained with Coomassie Blue. (b) Z-average and PDI of PMSEA mRNA-LNPs before and after conjugation, measured by DLS. (c) Flow cytometry analysis of 9G8 attachment on mRNA-LNPs via titration with various concentrations of AF488-labeled EGFR; (i) mean fluorescence intensity (MFI) of AF488 and (ii) half-maximum binding at equilibrium (Kd) calculated with the Saturation Binding model based on the titration results (R2 = 0.9459). (d) Cellular association of MDA-MB-468 incubated with PMSEA FLuc mRNA-LNPs with or without 9G8 for 1 h; and (e) luciferase expression of MDA-MB-468 incubated with PMSEA FLuc mRNA-LNPs with or without 9G8 for 24 h. Statistical data is presented as the mean ± SD (n = 3 for DLS and cell association assay; n = 4 for luciferase assay), ***p < 0.001, **p < 0.01, *p < 0.05, and ns p > 0.05 with 95% of confidence level from one-way ANOVA.

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While the gel and DLS analysis gives primary information about ligand attachment on LNPs, a flow cytometry method was developed to assess the functionality of surface-bound nanobodies before moving toward in vitro evaluation (Figure 5(c)). 9G8 attached on LNPs was used to capture fluorescently labeled EGFR antigen. In this approach, violet laser scatter (405 nm) was used to directly detect individual DiD-labeled LNPs. The DiD-labeled LNP population (>90%) was gated, and the level of AF488-EGFR association was then assessed using measurement of associated fluorescence to provide a direct measure of antigen-capture on Nb-functionalized LNPs. The level of AF488-EGFR associated with LNPs increased as a function of antigen concentration until plateauing in all cases, congruent with saturation of LNP-nanobody binding sites (Figure 5(c)(i)). The level at which saturation was observed was directly related to the degree of nanobody functionalization. Next, the measured association data was fit to a saturation binding model (Figure 5(c)(ii)). The model revealed that the half-maximum binding at equilibrium (Kd) is highly associated with the molar ratio of 9G8 attaching on the LNPs (R2 = 0.9459). This indicated that the nanobody conjugation to PMSEA mRNA-LNPs was constant and effective at all the molar ratios (0.1 – 1 mol % tCO-PMSEA-DSPE), showing the avidity of 9G8 was not impacted by the steric effect of polymer chains or other factors within the level of functionalization assayed.
The in vitro targeting performance of 9G8-PMSEA mRNA-LNPs was evaluated next to determine the effect of nanobody density. The initial association of PMSEA mRNA-LNPs with MDA-MB-468 cells was significantly boosted at 1 h by EGFR targeting at all 9G8 molar ratios, with increasing trends in association (Figure 5(c)). This confirmed the effective specific binding of 9G8-PMSEA mRNA-LNPs to MDA-MB-468. However, the receptor binding tended to reach saturation at around 0.5 mol % of tCO-PMSEA-DSPE, potentially revealing LNPs were already well-attached to the cellular surface, and did not provide any further functional benefit, or they could be self-competing with free 9G8 in the in vitro assays, as observable free 9G8 band was evident from LNPs with 0.5 mol % of tCO-PMSEA-DSPE in a gel analysis. A similar trend was observed for the luciferase assay (Figure 5(d)). All the 9G8-functionalized mRNA-LNPs elicited higher luciferase expression than the untargeted LNPs, ranging from a 1.9- to 4.7-fold enhancement with an increasing trend and reaching a plateau from LNPs with 0.5 mol % of tCO-PMSEA-DSPE. This confirmed the mRNA payload delivery and trafficking was enhanced by the promoted cell association, thereby resulting in higher transfection efficiencies. Overall, all the 9G8-functionalized mRNA-LNPs in various 9G8 molar ratios display higher rates of cellular association and higher transfection efficiencies than the untargeted mRNA-LNPs, with a constant increasing trend up to 0.5 mol % of tCO-PMSEA-DSPE.

In Vitro Transfection Efficiency of PMSEA mRNA-LNPs Via Targeting

To further validate the promotion of transfection efficiency of PMSEA mRNA-LNPs via EGFR targeting, we selected the LNP formulation with 0.3 mol % of tCO-PMSEA-DSPE to accurately quantify and probe the in vitro transfection of LNPs, with eGFP as the reporter gene. This tCO molar ratio was chosen as free nanobodies were not observable in the gel analysis (Figure 5(a)). In addition, this ratio is equivalent to the nanobody molar ratio of 0.2 mol % (1.5:1 tCO:Tz in feed), which has been tested in vivo for promoted site-specific delivery. (25,27)
From the flow cytometry analysis, the 9G8-functionalized LNPs successfully transfected 65.9% of the cells (Figure S3(b)). The targeted LNPs significantly elicited higher eGFP expression than the untargeted PEG and PMSEA LNP systems with a 3.5-fold and a 1.5-fold enhancement of mean eGFP fluorescence intensity (Figure 6(a)). An increased eGFP fluorescence signal of the targeted LNPs was also evident in the confocal images (Figure 6(b)). Combining all the in vitro results, the 9G8-functionalized LNPs had a higher specific delivery and transfection efficiency than their untargeted PEG and PMSEA counterparts.

Figure 6

Figure 6. eGFP expression of MDA-MB-468 incubated with PMSEA eGFP mRNA-LNPs with or without 9G8 attachment for 24 h. (a) Mean fluorescence intensity (MFI) of eGFP quantified by flow cytometry. Statistical data is presented as the mean ± SD (n = 3), ***p < 0.001, **p < 0.01, *p < 0.05, and ns p > 0.05 with 95% of confidence level from Welch’s unpaired t test. (b) Confocal images of eGFP transfection and cellular association with DiD-labeled eGFP mRNA-LNPs for 24 h with Hoechst staining MDA-MB-468 nuclei; scale bars are 20 μm).

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Conclusions

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In summary, we report a modular mRNA-LNP delivery platform with side-chain polymer coatings targeting specific cells as a next-generation immunotherapeutic delivery approach. The polymer–lipid conjugate with side chains, PMSEA-DSPE, was functionalized with a tCO end group to enable conjugation to Tz-functionalized nanobodies. Conventional direct or post-insertion bioconjugation methods were compared for LNP functionalization with the anti-EGFR 9G8 nanobody. The physical characterization and in vitro results suggested that the direct conjugation method is more suitable for antibody conjugation of mRNA-LNPs with side-chain polymer coatings. 9G8-PMSEA mRNA-LNPs prepared by this method successfully promote specific delivery for EGFR targeting as a function of surface Nb conjugation, thereby enhancing the transfection efficiency to specific cells. This demonstrates the potential of producing targeted mRNA-LNP using this modular approach to effect delivery with side-chain polymer coatings as to enhance cancer immunotherapy in the future.

Supporting Information

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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.bioconjchem.6c00045.

  • Experiment procedure (materials and methods), 1H NMR spectra of COOH-PMSEA-DSPE and tCO-PMSEA-DSPE, flow cytometry data of mRNA-LNPs, physical characterization of mRNA-LNPs (PDF)

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Author Information

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  • Corresponding Author
    • Andrew K. Whittaker - Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland 4072, Australia;  Australian Research Council Centre of Excellence for Green Electrochemical Transformation of Carbon Dioxide, The University of Queensland, Brisbane, Queensland 4072, Australia;  Orcidhttps://orcid.org/0000-0002-1948-8355;  Email: [email protected]
  • Authors
    • On Ting Choy - Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland 4072, Australia;  Orcidhttps://orcid.org/0000-0002-1932-7797
    • Nicholas L. Fletcher - Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland 4072, Australia;  ARC Research Hub for Advanced Manufacture of Targeted Radiopharmaceuticals, The University of Queensland, Brisbane, Queensland 4072, Australia;  Centre for Advanced Imaging, The University of Queensland, Brisbane, Queensland 4072, Australia;  Orcidhttps://orcid.org/0000-0002-2993-833X
    • Pie Huda - Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland 4072, Australia;  ARC Research Hub for Advanced Manufacture of Targeted Radiopharmaceuticals, The University of Queensland, Brisbane, Queensland 4072, Australia;  Centre for Advanced Imaging, The University of Queensland, Brisbane, Queensland 4072, Australia;  Orcidhttps://orcid.org/0000-0002-2197-4993
    • Craig A. Bell - Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland 4072, Australia;  Centre for Advanced Imaging, The University of Queensland, Brisbane, Queensland 4072, Australia;  Orcidhttps://orcid.org/0000-0002-8986-2795
    • David J. Owen - Australian Institute for Bioengineering and Nanotechnology, The University of Queensland, Brisbane, Queensland 4072, Australia;  ARC Research Hub for Advanced Manufacture of Targeted Radiopharmaceuticals, The University of Queensland, Brisbane, Queensland 4072, Australia;  Orcidhttps://orcid.org/0000-0002-9165-1540
  • Author Contributions

    On Ting Choy: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation. Nicholas L. Fletcher: Writing – review and editing, Methodology, Investigation, Formal analysis. Pie Huda: Writing – review and editing, Methodology, Conceptualization, Investigation. Craig A. Bell: Methodology, Conceptualization. David J. Owen: Conceptualization, Resources. Andrew K. Whittaker: Writing – review and editing, Supervision, Resources, Project administration.

  • Notes
    The authors declare no competing financial interest.

Acknowledgments

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The authors acknowledge the financial support from the Australian Research Council Discovery grants (DP210101496) and the Advance Queensland Industry Research Fellowship Scheme (AQIRF094-2023RD6; NLF). This work used the Queensland node of the NCRIS-enabled Australian National Fabrication Facility (ANFF). The Australian National Fabrication Facility is acknowledged for access to some items of equipment. Protein Expression Facility (PEF) and BASE facilities, The University of Queensland were acknowledged for the scientific and technical assistance. BASE is supported by Therapeutic Innovation Australia (TIA). TIA is supported by the Australian Government through the National Collaborative Research Infrastructure Strategy (NCRIS) program. The authors also acknowledge Biorender.com, which was used to create Scheme 1, Figure 3 and Figure 5 in this article.

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Bioconjugate Chemistry

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  • Abstract

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    Scheme 1

    Scheme 1. Schematic Diagram of Nanobody-LNP Conjugation Via trans-Cyclooctene (tCO)-Functionalization of PMSEA-Lipid Conjugate and Attachment of Tz-9G8 Nanobody to the tCO Group on PMSEA mRNA-LNPs
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    Figure 1

    Figure 1. (a) UV–vis spectra of PMSEA-DSPE and COOH-PMSEA-DSPE; the trithiocarbonyl peak at 300 – 310 nm was evident in PMSEA-DSPE. (b) 1H NMR (500 MHz, CDCl3) of COOH-PMSEA-DSPE with the peak comparison of PMSEA-DSPE. The pink arrows indicate the presence of the methyl group, while the orange arrow indicates the proton in carboxyl groups.

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    Figure 2

    Figure 2. 1H NMR 2D DOSY spectrum (500 MHz, CDCl3) of tCO-PMSEA-DSPE, showing successful tCO attachment to the PMSEA-lipid conjugate and the absence of free tCO residues.

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    Figure 3

    Figure 3. (a) Demonstration of two preparation methods to conjugate the Tz-9G8 nanobody to PMSEA mRNA-LNPs, including direct conjugation and post-insertion. (b) Gel electrophoresis of 9G8 and LNP samples prepared by either direct conjugation or post-insertion; (i) gel image with Coomassie Blue staining and (ii) gel analysis by ImageJ. (c) Z-average (d.nm) and PDI measured by DLS, statistical data is presented as the mean ± SD (n = 3).

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    Figure 4

    Figure 4. In vitro characterization of 9G8-PMSEA mRNA-LNPs prepared with different conjugation methods. (a) Cellular association of MDA-MB-468 incubated with PMSEA FLuc mRNA-LNPs with or without 9G8 attachment for 1 h and (b) luciferase expression of MDA-MB-468 incubated with PMSEA FLuc mRNA-LNPs with or without 9G8 attachment for 24 h. Statistical data is presented as the mean ± SD (n = 3 for flow cytometry; n = 4 for luciferase assay), ***p < 0.001, **p < 0.01, *p < 0.05, and ns p > 0.05 with 95% of confidence level from Welch’s unpaired t test.

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    Figure 5

    Figure 5. Physical and in vitro characterization of 9G8-PMSEA mRNA-LNPs with various molar ratios of tCO-PMSEA-DSPE (0 – 1 mol %) prepared by the direct conjugation method. (a) Gel image of 9G8-PMSEA mRNA-LNPs stained with Coomassie Blue. (b) Z-average and PDI of PMSEA mRNA-LNPs before and after conjugation, measured by DLS. (c) Flow cytometry analysis of 9G8 attachment on mRNA-LNPs via titration with various concentrations of AF488-labeled EGFR; (i) mean fluorescence intensity (MFI) of AF488 and (ii) half-maximum binding at equilibrium (Kd) calculated with the Saturation Binding model based on the titration results (R2 = 0.9459). (d) Cellular association of MDA-MB-468 incubated with PMSEA FLuc mRNA-LNPs with or without 9G8 for 1 h; and (e) luciferase expression of MDA-MB-468 incubated with PMSEA FLuc mRNA-LNPs with or without 9G8 for 24 h. Statistical data is presented as the mean ± SD (n = 3 for DLS and cell association assay; n = 4 for luciferase assay), ***p < 0.001, **p < 0.01, *p < 0.05, and ns p > 0.05 with 95% of confidence level from one-way ANOVA.

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    Figure 6

    Figure 6. eGFP expression of MDA-MB-468 incubated with PMSEA eGFP mRNA-LNPs with or without 9G8 attachment for 24 h. (a) Mean fluorescence intensity (MFI) of eGFP quantified by flow cytometry. Statistical data is presented as the mean ± SD (n = 3), ***p < 0.001, **p < 0.01, *p < 0.05, and ns p > 0.05 with 95% of confidence level from Welch’s unpaired t test. (b) Confocal images of eGFP transfection and cellular association with DiD-labeled eGFP mRNA-LNPs for 24 h with Hoechst staining MDA-MB-468 nuclei; scale bars are 20 μm).

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  • Supporting Information

    Supporting Information

    The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.bioconjchem.6c00045.

    • Experiment procedure (materials and methods), 1H NMR spectra of COOH-PMSEA-DSPE and tCO-PMSEA-DSPE, flow cytometry data of mRNA-LNPs, physical characterization of mRNA-LNPs (PDF)


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