A microneedle vaccine printer for thermostable COVID-19 mRNA vaccines.

Decentralized manufacture of thermostable mRNA vaccines in a microneedle patch (MNP) format could enhance vaccine access in low-resource communities by eliminating the need for a cold chain and trained healthcare personnel. Here we describe an automated process for printing MNP Coronavirus Disease 2019 (COVID-19) mRNA vaccines in a standalone device. The vaccine ink is composed of lipid nanoparticles loaded with mRNA and a dissolvable polymer blend that was optimized for high bioactivity by screening formulations in vitro. We demonstrate that the resulting MNPs are shelf stable for at least 6 months at room temperature when assessed using a model mRNA construct. Vaccine loading efficiency and microneedle dissolution suggest that efficacious, microgram-scale doses of mRNA encapsulated in lipid nanoparticles could be delivered with a single patch. Immunizations in mice using manually produced MNPs with mRNA encoding severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein receptor-binding domain stimulate long-term immune responses similar to those of intramuscular administration.

Virus-Mimicking Polymer Nanoparticles Targeting CD169+ Macrophages as Long-Acting Nanocarriers for Combination Antiretrovirals.

Monosialodihexosylganglioside (GM3)-presenting lipid-coated polymer nanoparticles (NPs) that recapitulate the sequestration of human immunodeficiency virus-1 (HIV-1) particles in CD169+ virus-containing compartments (VCCs) of macrophages were developed as carriers for delivery and sustained release of a combination of two antiretrovirals (ARVs), rilpivirine (RPV) and cabotegravir (CAB). RPV and CAB were co-loaded into GM3-presenting lipid-coated polylactic acid (PLA) and poly(lactic-co-glycolic acid) (PLGA) NPs without loss in potency of the drugs. GM3-presenting PLA NPs demonstrated the most favorable release properties and achieved inhibition of HIV-1 infection of primary human macrophages for up to 35 days. Intracellular localization of GM3-presenting PLA NPs in VCCs correlated with retention of intracellular ARV concentrations and sustained inhibition of HIV-1 infection. This work elucidates the design criteria of lipid-coated polymer NPs to utilize CD169+ macrophages as cellular drug depots for eradicating the viral reservoir sites or to achieve long-acting prophylaxis against HIV-1 infection.

Stiffness of HIV-1 Mimicking Polymer Nanoparticles Modulates Ganglioside-Mediated Cellular Uptake and Trafficking.

The monosialodihexosylganglioside, GM3, and its binding to CD169 (Siglec-1) have been indicated as key factors in the glycoprotein-independent sequestration of the human immunodeficiency virus-1 (HIV-1) in virus-containing compartments (VCCs) in myeloid cells. Here, lipid-wrapped polymer nanoparticles (NPs) are applied as a virus-mimicking model to characterize the effect of core stiffness on NP uptake and intracellular fate triggered by GM3-CD169 binding in macrophages. GM3-functionalized lipid-wrapped NPs are assembled with poly(lactic-co-glycolic) acid (PLGA) as well as with low and high molecular weight polylactic acid (PLAlMW and PLAhMW) cores. The NPs have an average diameter of 146 ± 17 nm and comparable surface properties defined by the self-assembled lipid layer. Due to differences in the glass transition temperature, the Young's modulus (E) differs substantially under physiological conditions between PLGA (E PLGA = 60 ± 32 MPa), PLAlMW (E PLA lMW = 86 ± 25 MPa), and PLAhMW (E PLA hMW = 1.41 ± 0.67 GPa) NPs. Only the stiff GM3-presenting PLAhMW NPs but not the softer PLGA or PLAlMW NPs avoid a lysosomal pathway and localize in tetraspanin (CD9)-positive compartments that resemble VCCs. These observations suggest that GM3-CD169-induced sequestration of NPs in nonlysosomal compartments is not entirely determined by ligand-receptor interactions but also depends on core stiffness.

Physiologically Relevant Mechanics of Biodegradable Polyester Nanoparticles.

Despite the extensive use of biodegradable polyester nanoparticles for drug delivery, and reports of the strong influence of nanoparticle mechanics on nano-bio interactions, there is a lack of systematic studies on the mechanics of these nanoparticles under physiologically relevant conditions. Here, we report indentation experiments on poly(lactic acid) and poly(lactide-co-glycolide) nanoparticles using atomic force microscopy. While dried nanoparticles were found to be rigid at room temperature, their elastic modulus was found to decrease by as much as 30 fold under simulated physiological conditions (i.e., in water at 37 °C). Differential scanning calorimetry confirms that this softening can be attributed to the glass transition of the nanoparticles. Using a combination of mechanical and thermoanalytical characterization, the plasticizing effects of miniaturization, molecular weight, and immersion in water were investigated. Collectively, these experiments provide insight for experimentalists exploring the relationship between polymer nanoparticle mechanics and in vivo behavior.

Membrane-wrapped nanoparticles probe divergent roles of GM3 and phosphatidylserine in lipid-mediated viral entry pathways.

Gold nanoparticles (NPs) wrapped in a membrane can be utilized as artificial virus nanoparticles (AVNs) that combine the large nonblinking or bleaching optical cross-section of the NP core with the biological surface properties and functionalities provided by a self-assembled lipid membrane. We used these hybrid nanomaterials to test the roles of monosialodihexosylganglioside (GM3) and phosphatidylserine (PS) for a lipid-mediated targeting of virus-containing compartments (VCCs) in macrophages. GM3-presenting AVNs bind to CD169 (Siglec-1)-expressing macrophages, but inclusion of PS in the GM3-containing AVN membrane decreases binding. Molecular dynamics simulations of the AVN membrane and experimental binding studies of CD169 to GM3-presenting AVNs reveal Na+-mediated interactions between GM3 and PS as a potential cause of the antagonistic action on binding by the two negatively charged lipids. GM3-functionalized AVNs with no or low PS content localize to tetherin+, CD9+ VCC in a membrane composition-depending fashion, but increasing amounts of PS in the AVN membrane redirect the NP to lysosomal compartments. Interestingly, this compartmentalization is highly GM3 specific. Even AVNs presenting the related monosialotetrahexosylganglioside (GM1) fail to achieve an accumulation in VCC. AVN localization to VCC was observed for AVN with gold NP core but not for liposomes, suggesting that NP sequestration into VCC has additional requirements beyond ligand (GM3)-receptor (CD169) recognition that are related to the physical properties of the NP core. Our results confirm AVN as a scalable platform for elucidating the mechanisms of lipid-mediated viral entry pathways and for selective intracellular targeting.