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![Self‐assembly process of LNPs within microfluidic systems. A) The self‐assembly process of nucleic acid–LNPs in a microfluidic mixer begins when lipid excipients (i.e., ionizable lipid, PEG lipid, helper lipid, and cholesterol) dissolved in ethanol are rapidly mixed with the nucleic acid payload (e.g., siRNA, mRNA, etc.) dissolved in an aqueous buffer. This initially occurs at a low buffer pH whereby the ionizable lipid will assume a protonated form and bind, via electrostatic interactions, to the negatively charged backbone of the nucleic acid. Concurrently, the increasingly polar environment drives the formation of vesicles and the encapsulation of the nucleic acid. As the mixing progresses, and with increasing buffer pH, the ionizable lipid becomes increasingly neutral, leading to the fusion of adjacent vesicles to form the interior core of the LNP. The extent of vesicle fusion is influenced by the amount of PEG lipid added due to its hydrophilicity as well as steric effects. Adapted with permission.[⁸¹] Copyright 2021, MDPI Publishing. B) Major microfluidic mixing architectures for LNP formulation (top to bottom): T‐junction mixer, staggered herringbone micromixer, bifurcating mixer, and baffle mixer. C) The effect of varying flow rate on the mixing profile of ethanol (red) and water (blue) within a baffle mixer, visualized through a computational fluid dynamics simulation. Adapted with permission.[⁸⁹] Copyright 2018, American Chemical Society. D) A diagrammatic representation of an example of microfluidics parallelization, showing (from top to bottom) the arrangement of mixing channels, each mixing unit, and each mixing cycle within a parallelized microfluidics device, which adopts the staggered herringbone architecture. Evaluation of the in vivo delivery performance of E) siRNA–LNPs and F) mRNA–LNPs obtained using the parallelized device compared to bulk mixing. The evaluation was performed by means of administering to mice. The mRNA encodes for luciferase and the in vivo delivery performance was evaluated by directly visualizing the luminescence from selected regions of the mice. The results clearly show that the in vivo delivery performance of nucleic acid–LNPs obtained via microfluidic mixing is superior to those obtained via bulk mixing. D–F) Adapted with permission.[⁸⁵] Copyright 2021, American Chemical Society.](profile/Hyeonjin-Park-2/publication/361356866/figure/fig4/AS:11431281173086444@1688754479823/Self-assembly-process-of-LNPs-within-microfluidic-systems-A-The-self-assembly-process.png)
Self‐assembly process of LNPs within microfluidic systems. A) The self‐assembly process of nucleic acid–LNPs in a microfluidic mixer begins when lipid excipients (i.e., ionizable lipid, PEG lipid, helper lipid, and cholesterol) dissolved in ethanol are rapidly mixed with the nucleic acid payload (e.g., siRNA, mRNA, etc.) dissolved in an aqueous buffer. This initially occurs at a low buffer pH whereby the ionizable lipid will assume a protonated form and bind, via electrostatic interactions, to the negatively charged backbone of the nucleic acid. Concurrently, the increasingly polar environment drives the formation of vesicles and the encapsulation of the nucleic acid. As the mixing progresses, and with increasing buffer pH, the ionizable lipid becomes increasingly neutral, leading to the fusion of adjacent vesicles to form the interior core of the LNP. The extent of vesicle fusion is influenced by the amount of PEG lipid added due to its hydrophilicity as well as steric effects. Adapted with permission.[⁸¹] Copyright 2021, MDPI Publishing. B) Major microfluidic mixing architectures for LNP formulation (top to bottom): T‐junction mixer, staggered herringbone micromixer, bifurcating mixer, and baffle mixer. C) The effect of varying flow rate on the mixing profile of ethanol (red) and water (blue) within a baffle mixer, visualized through a computational fluid dynamics simulation. Adapted with permission.[⁸⁹] Copyright 2018, American Chemical Society. D) A diagrammatic representation of an example of microfluidics parallelization, showing (from top to bottom) the arrangement of mixing channels, each mixing unit, and each mixing cycle within a parallelized microfluidics device, which adopts the staggered herringbone architecture. Evaluation of the in vivo delivery performance of E) siRNA–LNPs and F) mRNA–LNPs obtained using the parallelized device compared to bulk mixing. The evaluation was performed by means of administering to mice. The mRNA encodes for luciferase and the in vivo delivery performance was evaluated by directly visualizing the luminescence from selected regions of the mice. The results clearly show that the in vivo delivery performance of nucleic acid–LNPs obtained via microfluidic mixing is superior to those obtained via bulk mixing. D–F) Adapted with permission.[⁸⁵] Copyright 2021, American Chemical Society.
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Lipid‐based nanoparticles have emerged as a clinically viable platform technology to deliver nucleic acids for a wide range of healthcare applications. Within this scope, one of the most exciting areas of recent progress and future innovation potential lies in the material science of lipid‐based nanoparticles, both to refine existing nanoparticle s...
Citations
... Consequently, nanoarchitectonics boasts a plethora of applications, irrespective of the material or intended use. A cursory review of publications bearing the term "nanoarchitectonics" in their title reveals its extensive contributions to diverse fields, including the synthesis of functional materials [93][94][95][96][97], the formation of specific structures [98][99][100][101][102], the exploration of physical phenomena and principles [103][104][105][106][107], basic biochemistry [108][109][110][111][112], catalysts [113][114][115][116][117], photocatalysts [118][119][120][121][122], electrochemical catalysts [123][124][125][126][127], sensors [128][129][130][131][132], biosensors [133][134][135][136][137], devices [138][139][140][141][142], environmental purification [143][144][145][146][147], fuel cells [148][149][150][151][152], solar cells [153][154][155][156][157], various batteries [158][159][160][161][162], supercapacitors [163][164][165][166][167], other energy applications [168][169][170][171][172], drug delivery [173][174][175][176][177], cell engineering [178][179][180][181][182], and medical applications [183][184][185][186][187]. As previously stated, the applications of nanoarchitectonics are diverse, and correspondingly, the structures that can be assembled also have huge variety. In this review paper, the focus is on the construction of layered structures, which account for a significant proportion of functional materials. ...
The development of functional materials and the use of nanotechnology are ongoing projects. These fields are closely linked, but there is a need to combine them more actively. Nanoarchitectonics, a concept that comes after nanotechnology, is ready to do this. Among the related research efforts, research into creating functional materials through the formation of thin layers on surfaces, molecular membranes, and multilayer structures of these materials have a lot of implications. Layered structures are especially important as a key part of nanoarchitectonics. The diversity of the components and materials used in layer-by-layer (LbL) assemblies is a notable feature. Examples of LbL assemblies introduced in this review article include quantum dots, nanoparticles, nanocrystals, nanowires, nanotubes, g-C3N4, graphene oxide, MXene, nanosheets, zeolites, nanoporous materials, sol–gel materials, layered double hydroxides, metal–organic frameworks, covalent organic frameworks, conducting polymers, dyes, DNAs, polysaccharides, nanocelluloses, peptides, proteins, lipid bilayers, photosystems, viruses, living cells, and tissues. These examples of LbL assembly show how useful and versatile it is. Finally, this review will consider future challenges in layer-by-layer nanoarchitectonics.
... It is used in basic research areas such as material synthesis [56][57][58], structure control [59][60][61], fundamental physics research [62][63][64], and rather basic biochemistry research [65][66][67]. On the other hand, the concept is also used in applied fields such as catalysis [68][69][70], sensors [71][72][73], devices [74][75][76], energy production [77][78][79], energy storage [80][81][82], environmental handling [83][84][85], drug delivery [86][87][88], cell and tissue engineering [89][90][91], and biomedical applications [92][93][94]. ...
Nanotechnology has elucidated scientific phenomena of various materials at the nano-level. The next step in materials developments is to build up materials, especially condensed matter, based on such nanotechnology-based knowledge. Nanoarchitectonics can be regarded as a post-nanotechnology concept. In nanoarchitectonics, functional material systems are architected from nanounits. Here, this review would like to focus on layered structures in terms of structure formation. The unit structures of layered structures are mostly two-dimensional materials or thin-film materials. They are attractive materials that have attracted much attention in modern condensed matter science. By organizing them into layered structures, we can expect to develop functions based on communication between the layers. Building up layered functional structures by assembling nano-layers of units is a typical approach in nanoarchitectonics. The discussion will be divided into the following categories: hard matter, hybrid, soft matter, and living object. For each target, several recent research examples will be given to illustrate the discussion. This paper will extract what aspects are considered important in the creation of the layered structure of each component. Layering strategies need to be adapted to the characteristics of the components. The type of structural precision and functionality required is highly dependent on the flexibility and mobility of the component. Furthermore, what is needed to develop the nanoarchitectonics of layered structures is discussedas future perspectives.
... For the drug-loaded lipid vesicles (Fig.3 46,47 . LNPs are typically inverted micelles whose inner cores are occupied by cationic or ionizable lipids 64 . ...
... LNPs are typically inverted micelles whose inner cores are occupied by cationic or ionizable lipids 64 . LNPs are often formed by the direct coassembly of lipids and nucleic acids, a format of passive loading 46 . Conversely, lipoplexes are vesiclelike complexes formed by attaching nucleic acids to the surface of preformed liposomes 46 . ...
... LNPs are often formed by the direct coassembly of lipids and nucleic acids, a format of passive loading 46 . Conversely, lipoplexes are vesiclelike complexes formed by attaching nucleic acids to the surface of preformed liposomes 46 . Owing to the active loading without destroying the preformed liposomes, lipoplexes retain the continuous bilayer structure of their precursor liposomes 64 . ...
Encapsulating both biological and non-biological materials in lipid vesicles presents significant potential in both industrial and academic settings. When smaller than 100 nm, lipid vesicles and lipid nanoparticles are ideal...
... 4) Some nanocarriers can be engineered to combine functionalities like delivering therapeutic molecules and imaging capabilities, offering a more comprehensive approach to treatment and monitoring (257). 5) Nanocarriers can be designed to release their cargo in response to specific stimuli like pH changes or light exposure (237). This allows for more controlled and localized release of nucleic acids within the body. ...
Nucleic acid (NA) based therapeutics have witnessed tremendous progress and breakthroughs in treating pathological conditions, including viral infections, neurological disorders, genetic diseases, and metabolic disorders. NAs such as plasmid DNA (pDNA), short interfering RNA (siRNA), microRNA (miRNA), and antisense oligonucleotides (ASOs) can be modified to revolutionize personalized medicine. Despite the great potential of NA-based therapeutics, their clinical transformation is significantly hampered by instability, degradation, and inefficient delivery to the targeted site in the in vivo system. Lipid-based delivery systems hold great potential to overcome these shortcomings to enhance the delivery and bioavailability, improve stability, and increase the therapeutic effect of the NAs by delivering them to the active site. This review emphasized various nucleic acid-based therapeutics and their enhanced and improved delivery using different nanocarriers. Ultimately, the importance of lipid-based nanocarriers for delivering NAs is discussed and provides perspective in this field.
... For example, recent papers advocating nanoarchitectonics can be found in a wide range of fields. In addition to application-oriented areas such as catalysis [131][132][133][134][135], sensors [136][137][138][139][140], devices [141][142][143][144][145], energy production [146][147][148][149][150], energy storage [151][152][153][154][155], environmental response [156][157][158][159][160], drug delivery [161][162][163][164][165], and biomedical applications [166][167][168][169][170], there are also fundamental areas such as material synthesis [171][172][173][174][175][176], structural control [177][178][179][180][181], the exploration of physical phenomena [182][183][184][185][186], relatively basic biochemical studies [187][188][189][190][191], and research on cellular interactions [192][193][194][195][196]. Since all matter is principally composed of atoms and molecules, the methodology of building matter from atoms and molecules is applicable to all material synthesis. It could be likened to the ultimate theory of everything in physics [197], and nanoarchitectonics could be called a method for everything in materials science [198,199]. ...
The characteristic feature of a biofunctional system is that components with various functions work together. These multi-components are not simply mixed together, but are rationally arranged. The fundamental technologies to do this in an artificial system include the synthetic chemistry of the substances that make the component unit, the science and techniques for assembling them, and the technology for analyzing their nanoostructures. A new concept, nanoarchitectonics, can play this role. Nanoarchitectonics is a post-nanotechnology concept that involves building functional materials that reflect the nanostructures. In particular, the approach of combining and building multiple types of components to create composite materials is an area where nanoarchitectonics can be a powerful tool. This review summarizes such examples and related composite studies. In particular, examples are presented in the areas of catalyst & photocatalyst, energy, sensing & environment, bio & medical, and various other functions and applications to illustrate the potential for a wide range of applications. In order to show the various stages of development, the examples are not only state-of-the-art, but also include those that are successful developments of existing research. Finally, a summary of the examples and a brief discussion of future challenges in nanoarchitectonics will be given. Nanoarchitectonics is applicable to all materials and aims to establish the ultimate methodology of materials science.
... Over the past decades, liposomes or lipid nanoparticles (LNPs) have become the most effective drug delivery systems available [1][2][3][4][5][6][7]. They have been used to protect nucleic acid drugs, such as small interfering RNAs or microRNAs, from endonuclease degradation, achieve specific organ targeting, and reduce biotoxicity in vivo [8][9][10][11]. ...
Lipid nanoparticles (LNPs) have emerged as highly effective delivery systems for nucleic acid-based therapeutics. However, the broad clinical translation of LNP-based drugs is hampered by the lack of robust and scalable synthesis techniques that can consistently produce formulations from early development to clinical application. In this work, we proposed a method to achieve scalable synthesis of LNPs by scaling inertial microfluidic mixers isometrically in three dimensions. Moreover, a theoretical predictive method, which controls the mixing time to be equal across different chips, is developed to ensure consistent particle size and size distribution of the synthesized LNPs. LNPs loaded with small interfering RNA (siRNA) were synthesized at different flow rates, exhibiting consistent physical properties, including particle size, size distribution and encapsulation efficiency. This work provides a practical approach for scalable synthesis of LNPs consistently, offering the potential to accelerate the transition of nucleic acid drug development into clinical application.
... This partly agrees with published literature which argues that LNP size is primarily influenced by concentration, flow rate (for particles synthesized using a microfluidic system), and PEG-lipid concentration. However, there has been speculation that differences in cargo size and morphology should affect LNP size 8 . This is because compartmental organization of lipids and cargo within an LNP differs based on cargo size 39,40 . ...
With the recent success of lipid nanoparticle (LNP) based SARS-CoV-2 mRNA vaccines, the potential for RNA therapeutics has gained widespread attention. LNPs are promising non-viral delivery vectors to protect and deliver delicate RNA therapeutics, which are ineffective and susceptible to degradation alone. While food and drug administration (FDA) approved formulations have shown significant promise, benchmark lipid formulations still require optimization and improvement. In addition, the translatability of these formulations for several different RNA cargo sizes has not been compared under the same conditions. Herein we analyze “gold standard” lipid formulations for encapsulation efficiency of various non-specific RNA cargo lengths representing antisense oligonucleotides (ASO), small interfering RNA (siRNA), RNA aptamers, and messenger RNA (mRNA), with lengths of 10 bases, 21 base pairs, 96 bases, 996 bases, and 1929 bases, respectively. We evaluate encapsulation efficiency as the percentage of input RNA encapsulated in the final LNP product (EEinput%), which shows discrepancy with the traditional calculation of encapsulation efficiency (EE%). EEinput% is shown to be < 50% for all formulations tested, when EE% is consistently > 85%. We also compared formulations for LNP size (Z-average) and polydispersity index (PDI). LNP size does not appear to be strongly influenced by cargo size, which is a counterintuitive finding. Thoughtful characterization of LNPs, in parallel with consideration of in vitro or in vivo behavior, will guide design and optimization for better understanding and improvement of future RNA therapeutics.
... 31,32 Inspired by nature's use of the lyotropic mesophases during fat digestion, 33,34 researchers have developed lipid nanoparticles containing responsive mesophases for drug delivery purposes and a few are in clinical trials. 5,35 Inverse self-assembled structures of RNA-lipid complexes are widely acknowledged to play a vital role during endosomal escape by destabilizing the membranes of endosomes/lysosomes and thus facilitating the successful release and transfection of RNA therapeutics. 5,17,22,36 In the past few years, many endogenous and synthetic lipids, including the clinically used ionizable lipids, possessing a conelike molecular shape have been shown to self-assemble into inverse mesophases. ...
... However, the bioavailability of RNA-based therapeutics is limited because of their poor permeability across cell membranes and susceptibility to degradation by RNAses in the plasma. To overcome these limitations, the RNA therapeutics are commonly encapsulated in a carrier, among which lipid nanodiscs (LNDs), and have been gaining recognition as superior to conventional lipid nanoparticles (LNPs) for vascular delivery [7]. Discoidal particles exhibit greater cellular uptake than spherical particles for two reasons. ...
Blood-brain barrier (BBB) dysfunction is prevalent in Alzheimer’s disease and other neurological disorders. Restoring normal BBB function through RNA therapy is a potential avenue for addressing cerebrovascular changes in these disorders that may lead to cognitive decline. Although lipid nanoparticles have been traditionally used as drug carriers for RNA, bicelles have been emerging as a better alternative because of their higher cellular uptake and superior transfection capabilities. Cationic bicelles composed of DPPC/DC7PC/DOTAP at molar ratios of 63.8/25.0/11.2 were evaluated for the delivery of RNA in polarized hCMEC/D3 monolayers, a widely used BBB cell culture model. RNA-bicelle complexes were formed at five N/P ratios (1:1 to 5:1) by a thin-film hydration method. The RNA-bicelle complexes at N/P ratios of 3:1 and 4:1 exhibited optimal particle characteristics for cellular delivery. The cellular uptake of cationic bicelles laced with 1 mol% DiI-C18 was confirmed by flow cytometry and confocal microscopy. The ability of cationic bicelles (N/P ratio 4:1) to transfect polarized hCMEC/D3 with FITC-labeled control siRNA was tested vis-a-vis commercially available Lipofectamine RNAiMAX. These studies demonstrated the higher transfection efficiency and greater potential of cationic bicelles for RNA delivery to the BBB endothelium.
... LNP manufacturing is typically performed in a series of discrete unit operations, including the following: dissolving lipids in an organic solvent (e.g., ethanol), dissolving nucleic acid(s) in an acidic aqueous solution, mixing of the organic and aqueous solutions to form the LNP, solution exchange to remove ethanol and replace it with a storage solution near physiological pH, concentration, and sterile filtration. 1,14,18 If mixing is slow or incomplete during the LNP formation step, then this may result in a larger nanoparticle size, broader particle size distribution, and decreased nucleic acid encapsulation. 18,19 Consequently, the rate and manner of mixing are critical to the formation of uniform LNPs with controlled physicochemical properties and structure. ...
... 1,14,18 If mixing is slow or incomplete during the LNP formation step, then this may result in a larger nanoparticle size, broader particle size distribution, and decreased nucleic acid encapsulation. 18,19 Consequently, the rate and manner of mixing are critical to the formation of uniform LNPs with controlled physicochemical properties and structure. The presence of residual ethanol and changes in the pH, ionic content, and osmolality of the surrounding environment can further modulate LNP physicochemical properties and structure. ...
... Process scaleup can be achieved through parallelization of the microfluidic chambers, longer flow duration, or careful redesign of the mixer geometry to enable comparable mixing at higher flow rates. 18,21,26,27 Potential limitations associated with microfluidic mixing approaches include environmental waste and cost associated with single-use disposable cartridges, cartridgeto-cartridge variability, and incompatibility of the cartridge materials with certain solvents. However, it is worth noting that commercially available cartridges are compatible with ethanol, the most used organic solvent, and that variation between cartridges typically results in minimal differences in LNP quality attributes. ...
The recent clinical and commercial success of lipid nanoparticles (LNPs) for nucleic acid delivery has incentivized the development of new technologies to manufacture LNPs. As new technologies emerge, researchers must determine which technologies to assess and how to perform comparative evaluations. In this article, we use a quality-by-design approach to systematically investigate how the mixer technology used to form LNPs influences LNPstructure. Specifically, a coaxial turbulent jet mixer and a staggered herringbone microfluidic mixer were systematically compared via matched formulation and process conditions. A full-factorial design-of-experiments study with three factors and three levels was executed for each mixer to compare process robustness in the production of antisense oligonucleotide (ASO) LNPs. ASO-LNPs generated with the coaxial turbulent jet mixer were consistently smaller, had a narrower particle size distribution, and had a higher ASO encapsulation as compared to the microfluidic mixer, but had a greater variation in internal structure with less ordered cores. A subset of the study was replicated for mRNA-LNPs with comparable trends in particle size and encapsulation, but more frequent bleb features for LNPs produced by the coaxial turbulent jet mixer. The study design used here provides a road map for how researchers may compare different mixer technologies (or process changes more broadly) and how such studies can inform process robustness and manufacturing control strategies.
