Alexander E. Marras

High Resolution Image Download MS PowerPoint Slide Messenger RNA (mRNA) lipid nanoparticles (mRNA-LNPs) are central to emerging vaccines and therapeutics, but their wide implementation is constrained by limited endosomal escape and instability during long-term storage and freezing. While buffers are routinely optimized to prevent instability, the impact of buffer on the internal structural organization of LNPs and, consequently, their delivery efficiency remain unresolved. Here, we study the impact of storage in Tris, histidine, and citrate buffers for mRNA-LNPs formulated with LP-01, MC3, and SM-102 ionizable lipids. We demonstrate that storage buffer identity and concentration govern mRNA-LNP internal ordering before and after freeze–thaw and are thus critical parameters for engineering high-performance formulations. Deconvoluting ordered phases into an mRNA-lipid region and excess lipid region reveals the importance of excess ionizable lipid behavior in enhancing endosomal escape. Prior to freezing, citrate buffer enhances transfection efficiency by promoting a transition to the fusogenic inverse hexagonal (H II ) phase earlier during acidification, facilitated by a greater amount of ordered excess ionizable lipid. In contrast, Tris buffer provides the highest transfection efficiency after freeze–thaw by preventing aggregation and cargo loss while promoting favorable internal structure. Increasing Tris concentration from 10 to 50–150 mM leads to mRNA-rich bleb formation in freeze–thawed mRNA-LNPs, which improves freeze–thaw stability and thus transfection efficiency by mitigating mRNA-lipid adduct formation and accommodating a larger excess ionizable lipid region to facilitate H II phase formation. These findings establish a direct structural link between buffer conditions, particle size, internal morphology, and transfection efficiency, highlighting the importance of buffer composition in modulating mRNA-LNP performance.

Polyelectrolyte complex micelles (PCMs) are promising nucleic acid nanocarriers, however, nucleic acids are usually treated as generic polyanions, leaving the impact of nucleotide sequence and partially hybridized structures unclear. Here we systematically tune DNA stiffness in PCMs using (i) single-stranded sequences with varied purine content and (ii) hybridization structure in the form of hairpins, single-tailed duplexes, and double-tailed duplexes. Small-angle X-ray scattering, electron microscopy, and dynamic light scattering reveal a strong bending penalty imposed by increasing DNA stiffness that drives a transition from spherical to less-curved cylindrical or worm-like PCMs with ordered cores. Increasing purine content produces gradual morphological changes, whereas hybridized constructs show sharper transitions. We further link the availability of base-stacking sites to cylindrical PCM length and find that longer block copolymers lower the DNA-stiffness threshold for cylinder formation consistent with larger PCMs. The results establish design rules connecting DNA sequence, structure, and polymer length to PCM morphology.

Lipid nanoparticles are the leading platform for the delivery of nucleic acid therapeutics, yet their structural complexity remains a significant barrier to achieve rational design and predictable function. Part of this complexity arises from the non-equilibrium assemblies that are difficult to identify using ensemble average techniques given the substantial heterogeneity in all properties. Aiming to overcome the limitations of traditional characterization methods, we combined asymmetric flow field-flow fractionation with in-line small-angle X-ray scattering and spectroscopic analyses, nanoflow cytometry, and cryo-EM to construct detailed structural models of mRNA-loaded nanoparticles formulated with different amounts of mRNA loading (N/P ratios of 3 and 6). This combination of techniques revealed that microfluidic formulation produces structurally diverse nanoparticle subpopulations differing in size, anisotropy, and cargo loading. Notably, these variations extend to the particle internal organization: spheroidal geometries display densely loaded mRNA cores, whereas bleb-like morphologies exhibit reduced mRNA content relative to the lipid amount within segregated domains at the core. NanoFCM further shows that the N/P ratio modulates cargo distribution across individual nanoparticles, with N/P=6 yielding a more uniform mRNA copy number per particle across subpopulations than N/P=3. These differences resulted in higher transfection efficacies for the N/P=6 formulation, highlighting core organization and loading homogeneity as key parameters for efficacious delivery. Together, these results establish a direct link between LNP architecture, internal organization, cargo distribution, and transfection efficiency, underscoring the importance of accounting for heterogeneity in the rational design of nucleic acid delivery systems.

Achieving high folding yield remains a challenge in DNA origami, particularly as structures increase in complexity and scale. Here, how DNA origami design influences folding is investigated using a combination of real-time fluorometry, gel electrophoresis, electron microscopy, and theoretical analysis. Results reveal a balance of free energy changes from loop formation and hybridization that govern nucleation of nanostructure assembly, while the extent of cooperativity determines the overall assembly. The effect of structural complexity, staple design, and scaffold design on each energetic parameter, folding yield, kinetics, and cooperativity is measured. The results show that the scaffold pattern determines the extent of cooperativity, where fewer scaffold crossovers result in more cooperative folding. These findings use a tool developed in this work to estimate the extent of cooperativity in any structure. It is also found that limiting the number of crossovers per staple should be prioritized over extending staple binding domains, as the entropic penalty dominates the favorable binding. Finally, a 1-2 h focused annealing ramp strategy is demonstrated, that can increase yield up to 17% relative to traditional multi-day ramps. Optimizing energy changes and cooperativity through design can significantly enhance assembly yield and reduce time, particularly for complex structures, aiding large-scale DNA materials.

Achieving high folding yield remains a major challenge in DNA origami, particularly as structures increase in complexity and scale. Here, we investigate how DNA origami design influences folding yield and kinetics using a combination of real-time fluorometry, gel electrophoresis, electron microscopy, and theoretical analysis. Results reveal a balance of the free energy changes from loop formation and hybridization that govern nucleation of nanostructure assembly, while the extent of cooperativity determines the overall assembly behavior. We measure the effect of structural complexity, staple design, and scaffold design on each energetic parameter, folding yield, and kinetics. We show that the scaffold crossover pattern determines the extent of cooperativity and subsequent folding kinetics, where fewer scaffold crossovers result in more cooperative folding. We also demonstrate that limiting the number of crossovers per staple should be prioritized over extending staple binding domains. The entropic penalty dominates the lower energy binding, disrupting folding. Finally, we demonstrate a 1-2 hour focused annealing ramp strategy that can increase yield up to 17% relative to traditional multi-day ramps. Optimizing energy changes and the contribution of cooperativity through design can significantly enhance folding yield and assembly time, particularly for complex structures, aiding the design and assembly of large-scale materials.

In concentrated protein solutions, short-range attractions (SRAs) contribute to liquid–liquid phase separation (LLPS) as a function of temperature and salinity, particularly when the charge and thus long-range repulsions are low near the isoelectric point pI. Herein, we study how SRA and solution morphology vary with the approach to LLPS from increased SRA for two monoclonal antibodies (mAbs) as salt concentration is reduced near the pI. These properties are quantified using small-angle X-ray scattering (SAXS) interpreted via coarse-grained (CG) molecular dynamics (MD) simulations and compared with less descriptive properties from static and dynamic light scattering. Experimental structure factors are fit with a library of MD simulations for a CG 12-bead mAb model to determine the SRA strength ( K ) and cluster size distributions. Proximity to LLPS and clustering characteristics in mAb solutions are impacted by both net charge, which are modified by pH, and the strength of anisotropic electrostatic SRA (charge–charge, charge–dipole, hydrogen bonding, etc.), which are screened and weakened by added salts. The trends in LLPS are consistent with the reduced diffusion interaction parameter kD /B 22 ex for dilute solutions. However, greater insight is provided with SAXS along with CG-MD simulations; in particular, the growth of clusters is observed with the approach to LLPS with decreasing salinity over a wide range of concentrations.

Aerosolized lipid nanoparticles (LNPs) delivering mRNA are an attractive strategy for use in local, inhalable therapy to treat patients with lung diseases. However, a major barrier to delivering aerosolized mRNA LNPs is the shear forces encountered during aerosolization. These forces lead to significant morphology changes and subsequent decrease in efficacy of mRNA delivery. To best retain the physicochemical properties of mRNA LNPs during aerosolization, we took a formulation-based strategy to stabilize LNPs. We used a design-of-experiment (DOE) approach to comprehensively screen rationally chosen excipients at multiple concentrations. Excipients were carefully selected based on their use in clinically approved inhaled products or their ability to support lipid membrane properties. These excipients were added to the same mRNA LNP composition after formulation, were subsequently characterized, and used to transfect human lung cells at air-liquid interface. From this systematic screen, we identified that the addition of our lead candidate, poloxamer 188, best stabilizes LNP size throughout aerosolization and enhances mRNA expression after aerosolization. Additional morphological studies of the inclusion of poloxamer 188 in LNPs suggests that the excipient lowers aerosolization induced fusion or aggregation of particles without altering the internal structure. Our results indicate that poloxamer 188 can support aerosolized mRNA LNP delivery by maintaining LNP size and significantly enhancing therapeutic nucleic acid delivery to lung cells.

Imaging plays a critical role in all stages of cancer care from early detection to diagnosis, prognosis, and therapy monitoring. Recently, photoacoustic imaging (PAI) has started to emerge into the clinical realm due to its high sensitivity and ability to penetrate tissues up to several centimeters deep. Herein, we encapsulated indocyanine green J (ICGJ) aggregate, one of the only FDA-approved organic exogenous contrast agents that absorbs in the near-infrared range, at high loadings up to ∼40% w/w within biodegradable polymersomes (ICGJ-Ps) composed of poly(lactide- co -glycolide- b -polyethylene glycol) (PLGA- b -PEG). The small Ps hydrodynamic diameter of 80 nm is advantageous for in vivo applications, while directional conjugation with epidermal growth factor receptor (EGFR) targeting cetuximab antibodies renders molecular specificity. Even when exposed to serum, the ∼11 nm-thick membrane of the Ps prevents dissociation of the encapsulated ICGJ for at least 48 h with a high ratio of ICGJ to monomeric ICG absorbances (i.e., I 895 / I 780 ratio) of approximately 5.0 that enables generation of a strong NIR photoacoustic (PA) signal. The PA signal of polymersome-labeled breast cancer cells is proportional to the level of cellular EGFR expression, indicating the feasibility of molecular PAI with antibody-conjugated ICGJ-Ps. Furthermore, the labeled cells were successfully detected with PAI in highly turbid tissue-mimicking phantoms up to a depth of 5 mm with the PA signal proportional to the amount of cells. These data show the potential of molecular PAI with ICGJ-Ps for clinical applications such as tumor margin detection, evaluation of lymph nodes for the presence of micrometastasis, and laparoscopic imaging procedures.

Polyelectrolyte complex micelles are hydrophilic nanoparticles that self-assemble in aqueous environments due to associative microphase separation between oppositely charged blocky polyelectrolytes. In this work, we employ a suite of physical characterization tools to examine the effect of charged block length mismatch on the equilibrium structure of double diblock polyelectrolyte complex micelles (D-PCMs) by mixing a diverse library of peptide and synthetic charged-neutral block polyelectrolytes with a wide range of charged block lengths (25-200 units) and chemistries. Early work on D-PCMs suggested that this class of micelles can only be formed from blocky polyelectrolytes with identical charged block lengths, a phenomenon referred to as chain length recognition. Here, we use salt annealing to create PCMs at equilibrium, which shows that chain length recognition, a longstanding hurdle to repeatable self-assembly from mismatched polyelectrolytes, can be overcome. Interestingly, D-PCM structure-property relationships display a range of values that vary systematically with the charged block lengths and chemical identity of constituent polyelectrolyte pairings and cannot be described by generalizable scaling laws. We discuss the interdependent growth behavior of the radius, ionic pair aggregation number, and density in the micelle core for three chemically distinct diblock pairings and suggest a potential physical mechanism that leads to this unique behavior. By comparing the results of these D-PCMs to the scaling laws recently developed for single diblock polyelectrolyte complex micelles (S-PCMs: diblock + homopolymer), we observe that D-PCM design schemes reduce the size and aggregation number and restrict their growth to a function of charged block length relative to S-PCMs. Understanding these favorable attributes enables more predictive use of a wider array of charged molecular building blocks to anticipate and control macroscopic properties of micelles spanning countless storage and delivery applications.