Nonviral delivery of self-amplifying RNA vaccines.

Despite more than two decades of research and development on nucleic acid vaccines, there is still no commercial product for human use. Taking advantage of the recent innovations in systemic delivery of short interfering RNA (siRNA) using lipid nanoparticles (LNPs), we developed a self-amplifying RNA vaccine. Here we show that nonviral delivery of a 9-kb self-amplifying RNA encapsulated within an LNP substantially increased immunogenicity compared with delivery of unformulated RNA. This unique vaccine technology was found to elicit broad, potent, and protective immune responses, that were comparable to a viral delivery technology, but without the inherent limitations of viral vectors. Given the many positive attributes of nucleic acid vaccines, our results suggest that a comprehensive evaluation of nonviral technologies to deliver self-amplifying RNA vaccines is warranted.

Assessment of occlusion changes during laboratory phase of relining: An in vitro study.

AIM: Assessment of occlusion changes during laboratory phase of relining is essential to evaluate the occlusal discrepancies that could get incorporated in the denture with the use of different relining materials. Since the long term stability and functional success of the denture is heavily influenced by occlusion, an In-vitro study to assess these changes after relining is warranted. The aim of the study is to evaluate the changes in occlusion during laboratory phase of relining procedure. SETTINGS AND DESIGN: This is an in vitro study with a total of 30 specimen. MATERIALS AND METHODOLOGY: A total of 30 maxillary standardized dentures were fabricated after mounting on a semi adjustable articulator. These samples will be divided into three groups based on the relining material used (Autopolymerizing resin, Heat-cure resin, Tissue conditioner). The vertical dimension, Centric contact points and eccentric contact points were measured before and after relining. STATISTICAL ANALYSIS USED: The variables were tested to see if they had a normal distribution using the Shapiro-Wilk test. Parametric distribution was seen for ECP leading to further comparison using one way analysis of variance (ANOVA). Non-parametric distribution was found while testing the VD, CCP leading to adoption of Kruskal-wallis test for comparison of groups. Dunn Bonferroni test was done for VD since results were significant. RESULTS: The results of this in-vitro study showed statistically significant difference with respect to change in vertical dimension in all groups pre and post relining (P = 0.005). The centric contact points showed lesser variation in position when comparing the pre to the post relining phase with the use of autopolymerising resins, whereas heat cure resins and tissue conditioners showed statistically significant difference in the centric point contacts post relining. No statistically significant changes were seen in eccentric occlusion post relining in all groups. Tissue conditioners showed minimum mean changes in eccentric contacts. CONCLUSION: Within the limitations of this study, the use of autopolymerising resins depicted the most stable results with respect to occlusion, for relining of dentures.

A Review on Multiplicity in Multi-Material Additive Manufacturing: Process, Capability, Scale, and Structure.

Additive manufacturing (AM) has experienced exponential growth over the past two decades and now stands on the cusp of a transformative paradigm shift into the realm of multi-functional component manufacturing, known as multi-material AM (MMAM). While progress in MMAM has been more gradual compared to single-material AM, significant strides have been made in exploring the scientific and technological possibilities of this emerging field. Researchers have conducted feasibility studies and investigated various processes for multi-material deposition, encompassing polymeric, metallic, and bio-materials. To facilitate further advancements, this review paper addresses the pressing need for a consolidated document on MMAM that can serve as a comprehensive guide to the state of the art. Previous reviews have tended to focus on specific processes or materials, overlooking the overall picture of MMAM. Thus, this pioneering review endeavors to synthesize the collective knowledge and provide a holistic understanding of the multiplicity of materials and multiscale processes employed in MMAM. The review commences with an analysis of the implications of multiplicity, delving into its advantages, applications, challenges, and issues. Subsequently, it offers a detailed examination of MMAM with respect to processes, materials, capabilities, scales, and structural aspects. Seven standard AM processes and hybrid AM processes are thoroughly scrutinized in the context of their adaptation for MMAM, accompanied by specific examples, merits, and demerits. The scope of the review encompasses material combinations in polymers, composites, metals-ceramics, metal alloys, and biomaterials. Furthermore, it explores MMAM's capabilities in fabricating bi-metallic structures and functionally/compositionally graded materials, providing insights into various scale and structural aspects. The review culminates by outlining future research directions in MMAM and offering an overall outlook on the vast potential of multiplicity in this field. By presenting a comprehensive and integrated perspective, this paper aims to catalyze further breakthroughs in MMAM, thus propelling the next generation of multi-functional component manufacturing to new heights by capitalizing on the unprecedented possibilities of MMAM.

Effect of surface properties on nanoparticle-cell interactions.

The interaction of nanomaterials with cells and lipid bilayers is critical in many applications such as phototherapy, imaging, and drug/gene delivery. These applications require a firm control over nanoparticle-cell interactions, which are mainly dictated by surface properties of nanoparticles. This critical Review presents an understanding of how synthetic and natural chemical moieties on the nanoparticle surface (in addition to nanoparticle shape and size) impact their interaction with lipid bilayers and cells. Challenges for undertaking a systematic study to elucidate nanoparticle-cell interactions are also discussed.

Surface-structure-regulated cell-membrane penetration by monolayer-protected nanoparticles.

Nanoscale objects are typically internalized by cells into membrane-bounded endosomes and fail to access the cytosolic cell machinery. Whereas some biomacromolecules may penetrate or fuse with cell membranes without overt membrane disruption, no synthetic material of comparable size has shown this property yet. Cationic nano-objects pass through cell membranes by generating transient holes, a process associated with cytotoxicity. Studies aimed at generating cell-penetrating nanomaterials have focused on the effect of size, shape and composition. Here, we compare membrane penetration by two nanoparticle 'isomers' with similar composition (same hydrophobic content), one coated with subnanometre striations of alternating anionic and hydrophobic groups, and the other coated with the same moieties but in a random distribution. We show that the former particles penetrate the plasma membrane without bilayer disruption, whereas the latter are mostly trapped in endosomes. Our results offer a paradigm for analysing the fundamental problem of cell-membrane-penetrating bio- and macro-molecules.

Water-soluble amphiphilic gold nanoparticles with structured ligand shells.

Highly water-soluble mixed monolayer protected "rippled" gold nanoparticles were synthesized through a one step reaction with sodium 11-mercaptoundecanesulfonate and octanethiol ligands at various ratios.

Binding and templation of nanoparticle receptors to peptide alpha-helices through surface recognition.

Nanoparticles featuring highly flexible sidechains template to peptides, demonstrating substantial pre-organization of the particle monolayer.

"Cleaning" of nanoparticle inhibitors via proteolysis of adsorbed proteins.

Cytochrome c adsorbed to anionic nanoparticles is selectively proteolyzed by trypsin, providing a mechanism for the catalytic degradation of proteins.

Model systems for flavoenzyme activity: recognition and redox modulation of flavin mononucleotide in water using nanoparticles.

We have used mixed monolayer protected gold clusters (MMPCs) to provide flavoenzyme model systems with a high affinity and ability to modulate cofactor reduction potential.

Engineering the nanoparticle-biomacromolecule interface.

Monolayer-protected nanoparticles feature tunable size, surface functionality and core material, providing scaffolds for targeting biomacromolecules. This review highlights recent advances in nanoparticle-biomacromolecule interactions, focusing on two key areas: (1) The modulation of structure and function of biomacromolecules through engineered interactions with nanoparticle surfaces; (2) The use of biomacromolecules as building blocks for nanostructured materials.

Surface recognition of biomacromolecules using nanoparticle receptors.

Nanoparticles present a versatile scaffold to target biomacromolecule surfaces via complementary interactions. This review highlights some unique features of nanoparticles that make them particularly attractive resources for biomacromolecular recognition, and displays their use in modulation of structure and function of biomacromolecules.

Controlled recovery of the transcription of nanoparticle-bound DNA by intracellular concentrations of glutathione.

Positively charged trimethylammonium-functionalized mixed monolayer protected clusters (MMPCs) bind DNA through complementary electrostatic interactions, resulting in complete inhibition of DNA transcription of T7 RNA polymerase. DNA was released from the nanoparticle by intracellular concentrations of glutathione, resulting in efficient transcription. The restoration of RNA production was dose-dependent in terms of GSH, with considerable control of the release process possible through variation in monolayer structure. This work presents a new approach to controlled release of DNA, with potential applications in the creation of transfection vectors and gene regulation systems.

Effect of ionic strength on the binding of alpha-chymotrypsin to nanoparticle receptors.

Negatively charged carboxylate-functionalized mixed monolayer protected clusters (MMPCs) effectively bind and inhibit alpha-chymotrypsin based on complementary electrostatic surface recognition. We demonstrate that this binding can be disrupted by varying the ionic strength of the medium. Enzyme activity in the presence of MMPCs increases from 5% to 97% of native activity as salt concentration is increased from 0 to 1.5 M. Variation of ionic strengths after complete binding over 13 h results only in a modest restoration of enzymatic activity (< 35%). The conformation of chymotrypsin was characterized using circular dichroism and fluorescence spectroscopy, correlating structure with enzymatic activity. This work provides a useful insight of the electrostatic influence on protein--MMPC interactions and can be applied toward MMPC-based controlled release of proteins in vivo.

Tunable reactivation of nanoparticle-inhibited beta-galactosidase by glutathione at intracellular concentrations.

Positively charged trimethylammonium-functionalized mixed monolayer protected clusters (MMPCs) of different chain lengths (C(8) and C(11)) have been used to bind beta-galactosidase through complementary electrostatic interactions, resulting in complete enzyme inhibition. This inhibition can be reversed in vitro by intracellular concentrations of glutathione (GSH), the main thiol component of the cell. The restoration of activity depends on the chain length of the monolayer. The activity of enzyme bound to particles with C(8) monolayer was completely restored by intracellular concentrations (1-10 mM) of GSH; however, little or no release was observed at extracellular GSH concentrations. In contrast, no restoration was observed for enzyme bound to the C(11) particles at any of the concentrations studied. Taken together, these studies demonstrate that the GSH-mediated release of enzymes bound to MMPCs can be tuned through the structure of the monolayer, a significant tool for protein and drug delivery applications.

Reversible "irreversible" inhibition of chymotrypsin using nanoparticle receptors.

Anionically functionalized amphiphilic nanoparticles efficiently inhibit chymotrypsin through electrostatic binding followed by protein denaturation. We demonstrate the ability to disrupt this "irreversible" inhibition of chymotrypsin through modification of the nanoparticle surface using cationic surfactants. Up to 50% of original chymotrypsin activity is rescued upon long-chain surfactant addition. Dynamic light-scattering studies demonstrate that chymotrypsin is released from the nanoparticle surface. The conformation of the rescued chymotrypsin was characterized by fluorescence and fluorescence anisotropy, indicating that chymotrypsin regains a high degree of native structure upon surfactant addition.

Recognition and stabilization of peptide alpha-helices using templatable nanoparticle receptors.

alpha-Helices are important structural elements in proteins. To provide a scaffold for the facial recognition of peptides, we have explored the interaction of cationic mixed monolayer protected clusters (MMPCs) with a tetra-aspartate peptide in water. In these studies, substantial enhancement of peptide helicity was observed upon addition of the MMPC. Significantly, this stabilization increased with time, demonstrating templation of the monolayer to the peptide helix.

Control of protein structure and function through surface recognition by tailored nanoparticle scaffolds.

Thioalkyl and thioalkylated oligo(ethylene glycol) (OEG) ligands with chain-end functionality were used to fabricate water-soluble CdSe nanoparticle scaffolds. Surface recognition of chymotrypsin (ChT) was achieved using these functionalized nanoparticle scaffolds, with three levels of interaction demonstrated: no interaction (OEG terminated with hydroxyl group), inhibition with denaturation (carboxylate-terminated thioalkyl ligands), and inhibition with retention of structure (carboxylate-terminated OEG). The latter process was reversible upon an increase in ionic strength, with essentially complete restoration of enzymatic activity.