Efrosini Kokkoli

Aging negatively impacts skin health, notably through the senescent cell phenotype, which reduces collagen production and leads to thinner, more fragile skin prone to injuries and chronic wounds. We designed a drug delivery system that addresses these age-related issues using a hybrid hydrogel-nanoparticle system that utilizes a poly(δ-valerolactone-co-lactide)-b-poly(ethylene-glycol)-b-poly(δ-valerolactone-co-lactide) (PVLA-PEG-PVLA) hydrogel. This hydrogel allows for the local, extended release of therapeutics targeting both proliferating and senescent cells. The PVLA-PEG-PVLA hydrogel entrapped valsartan, and metformin-loaded liposomes functionalized with a fibronectin-mimetic peptide, PR_b. Metformin acts as a senomorphic, reversing aspects of cellular senescence, and valsartan, an angiotensin receptor blocker, promotes collagen production. This combination treatment partially reversed the senescent phenotype and improved collagen production in senescent dermal fibroblasts from both young and old adults. Our codelivery hydrogel-nanoparticle system offers a promising treatment for improving age-related dermal pathologies.

Size-dependent phagocytosis is a well-characterized phenomenon in monocytes and macrophages. However, this size effect for preferential gene delivery to these important cell targets has not been fully exploited because commonly adopted stabilization methods for electrostatically complexed nucleic acid nanoparticles, such as PEGylation and charge repulsion, typically arrest the vehicle size below 200 nm. Here, we bridge the technical gap in scalable synthesis of larger submicron gene delivery vehicles by electrostatic self-assembly of charged nanoparticles, facilitated by a polymer structurally designed to modulate internanoparticle Coulombic and van der Waals forces. Specifically, our strategy permits controlled assembly of small poly(β-amino ester)/messenger ribonucleic acid (mRNA) nanoparticles into particles with a size that is kinetically tunable between 200 and 1,000 nm with high colloidal stability in physiological media. We found that assembled particles with an average size of 400 nm safely and most efficiently transfect monocytes following intravenous administration and mediate their differentiation into macrophages in the periphery. When a CpG adjuvant is co-loaded into the particles with an antigen mRNA, the monocytes differentiate into inflammatory dendritic cells and prime adaptive anticancer immunity in the tumor-draining lymph node. This platform technology offers a unique ligand-independent, particle-size-mediated strategy for preferential mRNA delivery and enables therapeutic paradigms via monocyte programming.

Effectively utilizing MEK inhibitors in the clinic remains challenging due to off-target toxicity and lack of predictive biomarkers. Recent findings propose E-cadherin, a breast cancer diagnostic indicator, as a predictor of MEK inhibitor success. To address MEK inhibitor toxicity, traditional methodologies have systemically delivered nanoparticles, which require frequent, high-dose injections. Here, we present a different approach, employing a thermosensitive, biodegradable hydrogel with functionalized liposomes for local, sustained release of MEK inhibitor PD0325901 and doxorubicin. The poly(δ-valerolactone-co-lactide)-b-poly(ethylene-glycol)-b-poly(δ-valerolactone-co-lactide) triblock co-polymer gels at physiological temperature and has an optimal degradation time in vivo. Liposomes were functionalized with PR_b, a biomimetic peptide targeting the α₅β₁ integrin receptor, which is overexpressed in E-cadherin-positive triple negative breast cancer (TNBC). In various TNBC models, the hydrogel-liposome system delivered via local injection reduced tumor progression and improved animal survival without toxic side effects. Our work presents the first demonstration of local, sustained delivery of MEK inhibitors to E-cadherin-positive tumors alongside traditional chemotherapeutics, offering a safe and promising therapeutic strategy.

Lipid nanoparticles (LNP) have emerged as pivotal delivery vehicles for RNA therapeutics. Previous research and development usually assumed that LNPs are homogeneous in population, loading density, and composition. Such perspectives are difficult to examine due to the lack of suitable tools to characterize these physicochemical properties at the single-nanoparticle level. Here, we report an integrated spectroscopy-chromatography approach as a generalizable strategy to dissect the complexities of multicomponent LNP assembly. Our platform couples cylindrical illumination confocal spectroscopy (CICS) with single-nanoparticle free solution hydrodynamic separation (SN-FSHS) to simultaneously profile population identity, hydrodynamic size, RNA loading levels, and distributions of helper lipid and PEGylated lipid of LNPs at the single-particle level and in a high-throughput manner. Using a benchmark siRNA LNP formulation, we demonstrate the capability of this platform by distinguishing seven distinct LNP populations, quantitatively characterizing size distribution and RNA loading level in wide ranges, and more importantly, resolving composition-size correlations. This SN-FSHS-CICS analysis provides critical insights into a substantial degree of heterogeneity in the packing density of RNA in LNPs and size-dependent loading-size correlations, explained by kinetics-driven assembly mechanisms of RNA LNPs.

With its lack of commonly targeted receptors, triple negative breast cancer (TNBC) is aggressive and difficult to treat. To address this problem, nanotubes self-assembled from single stranded DNA (ssDNA)-amphiphiles were used as a delivery vehicle for doxorubicin (DOX) to target TNBC cells. Since DOX and other standard of care treatments such as radiation have been documented to induce senescence, the ability of the nanotubes to deliver the senolytic ABT-263 was also investigated. The ssDNA-amphiphiles were synthesized from a 10 nucleotide sequence attached to a dialkyl, (C₁₆)₂, tail via a C₁₂ alkyl spacer, and have been previously shown to self-assemble into hollow nanotubes and spherical micelles. Here, we demontrate that these ssDNA spherical micelles could transition into long nanotubes in the presence of excess tails. The nanotubes could then be shortened via probe sonication. The ssDNA nanotubes internalized into three different TNBC cell lines: Sum159, MDA-MB-231, and BT549, with minimal internalization in healthy Hs578Bst cells, suggesting an inherent targeting ability. Inhibition of different internalization mechanisms showed that the nanotubes internalized in the TNBC cells primarily through macropinocytosis and scavenger receptor-mediated endocytosis, both of which are upregulated pathways in TNBC. DOX was intercalated into the ssDNA nanotubes and delivered to TNBC cells. Compared to free DOX, DOX-intercalated nanotubes proved equally cytotoxic to TNBC cells. In order to demonstrate the potential for delivery of different therapeutics, ABT-263 was incorporated into the hydrophobic bilayer wall of the nanotubes and was delivered to a DOX-induced in vitro model of senescence. The ABT-263 encapsulating nanotubes demonstrated cytotoxicity to senescent TNBC cells as well as sensitization to further DOX treatment. Thus, our ssDNA nanotubes are a promising delivery vehicle that could be used for targeted delivery of therapeutics to TNBC cells.

Adenosine triphosphate (ATP) and other nucleotides can be irreversibly bound to the metal-organic framework (MOF) MIL-101(Cr). Analysis of X-ray diffraction data suggests that the location of the adsorbed ATP molecule is in proximity of the Cr3 clusters. Solid-state NMR and DFT calculations indicate that ATP is bound to MIL-101(Cr) through linkages of the terminal phosphate group with Cr(III) of the framework. In the presence of Cu(II) ions, the MOF-supported nucleotides can function as stable and reusable enantioselective heterogeneous catalysts for reactions like Diels-Alder and Michael addition. Compared to the corresponding homogeneous nucleotide-based artificial metalloenzymes (ArMs), the MOF-supported nucleotide-based ArMs exhibit significantly enhanced activity and selectivity in certain cases, demonstrating their potential as a new class of enantioselective heterogeneous catalysts.

Lipid nanoparticles (LNPs) are effective vehicles to deliver mRNA vaccines and therapeutics. It has been challenging to assess mRNA packaging characteristics in LNPs, including payload distribution and capacity, which are critical to understanding structure-property-function relationships for further carrier development. Here, we report a method based on the multi-laser cylindrical illumination confocal spectroscopy (CICS) technique to examine mRNA and lipid contents in LNP formulations at the single-nanoparticle level. By differentiating unencapsulated mRNAs, empty LNPs and mRNA-loaded LNPs via coincidence analysis of fluorescent tags on different LNP components, and quantitatively resolving single-mRNA fluorescence, we reveal that a commonly referenced benchmark formulation using DLin-MC3 as the ionizable lipid contains mostly 2 mRNAs per loaded LNP with a presence of 40%–80% empty LNPs depending on the assembly conditions. Systematic analysis of different formulations with control variables reveals a kinetically controlled assembly mechanism that governs the payload distribution and capacity in LNPs. These results form the foundation for a holistic understanding of the molecular assembly of mRNA LNPs.

In this work, we demonstrate the formation of supramolecular architectures from the assembly of single-tail single stranded DNA (ssDNA)-amphiphiles. Short ssDNA sequences of 10 nucleotides that were either unstructured or formed G-quadruplex secondary structures were conjugated to a single 4-(hexadecyloxy)benzamide tail, either directly or through a polycarbon (C-12) spacer. Conjugation of the ssDNA to the tail did not interfere with the G-quadruplex secondary structure of the ssDNA sequence. The ssDNA-amphiphiles self-assembled into ellipsoidal micelles, vesicles, nanotapes, and nanotubes. These nanotubes appeared to be formed by the rolling up of nanotapes. The increase of the hydrophobic block of the ssDNA-amphiphiles through the addition of a C-12 spacer led to an increase in wall thickness and nanotube diameter. The presence of pi-pi interactions, through the benzoic group, was verified via X-ray diffraction (XRD) and played a critical role in the formation of the different nanostructures. In contrast, ssDNA-amphiphiles with a single heptadecanoic acid tail self-assembled only into ellipsoidal micelles.

A DNA-based artificial metalloenzyme (ArM) consisting of a copper(II) complex of 4,4′-dimethyl-2,2′-bipyridine (dmbipy-Cu) bound to double-stranded DNA (dsDNA) as short as 8 base pairs with only 2 contiguous central pairs (G for guanine and C for cytosine) catalyzes the highly enantioselective Diels–Alder reaction, Michael addition, and Friedel–Crafts alkylation in water. Molecular simulations indicate that these minimal sequences provide a single site where dmbipy-Cu is groove-bound and able to function as an enantioselective catalyst. Enantioselective preference inverts when d-DNA is replaced with l-DNA. When the DNA is conjugated to a hydrophobic tail, the obtained ArMs exhibit enantioselective performance in a methanol–water mixture superior to that of non-amphiphilic dsDNA, and dsDNA-amphiphiles with more complex G•C-rich sequences.