Optimization of Lipid Nanoparticles for saRNA Expression and Cellular Activation Using a Design-of-Experiment Approach.

Lipid nanoparticles (LNPs) are the leading technology for RNA delivery, given the success of the Pfizer/BioNTech and Moderna COVID-19 mRNA (mRNA) vaccines, and small interfering RNA (siRNA) therapies (patisiran). However, optimization of LNP process parameters and compositions for larger RNA payloads such as self-amplifying RNA (saRNA), which can have complex secondary structures, have not been carried out. Furthermore, the interactions between process parameters, critical quality attributes (CQAs), and function, such as protein expression and cellular activation, are not well understood. Here, we used two iterations of design of experiments (DoE) (definitive screening design and Box-Behnken design) to optimize saRNA formulations using the leading, FDA-approved ionizable lipids (MC3, ALC-0315, and SM-102). We observed that PEG is required to preserve the CQAs and that saRNA is more challenging to encapsulate and preserve than mRNA. We identified three formulations to minimize cellular activation, maximize cellular activation, or meet a CQA profile while maximizing protein expression. The significant parameters and design of the response surface modeling and multiple response optimization may be useful for designing formulations for a range of applications, such as vaccines or protein replacement therapies, for larger RNA cargoes.

Current Status and Future Perspectives on MRNA Drug Manufacturing.

The coronavirus disease of 2019 (COVID-19) pandemic launched an unprecedented global effort to rapidly develop vaccines to stem the spread of the novel severe acute respiratory syndrome coronavirus (SARS-CoV-2). Messenger ribonucleic acid (mRNA) vaccines were developed quickly by companies that were actively developing mRNA therapeutics and vaccines for other indications, leading to two mRNA vaccines being not only the first SARS-CoV-2 vaccines to be approved for emergency use but also the first mRNA drugs to gain emergency use authorization and to eventually gain full approval. This was possible partly because mRNA sequences can be altered to encode nearly any protein without significantly altering its chemical properties, allowing the drug substance to be a modular component of the drug product. Lipid nanoparticle (LNP) technology required to protect the ribonucleic acid (RNA) and mediate delivery into the cytoplasm of cells is likewise modular, as are technologies and infrastructure required to encapsulate the RNA into the LNP. This enabled the rapid adaptation of the technology to a new target. Upon the coattails of the clinical success of mRNA vaccines, this modularity will pave the way for future RNA medicines for cancer, gene therapy, and RNA engineered cell therapies. In this review, trends in the publication records and clinical trial registrations are tallied to show the sharp intensification in preclinical and clinical research for RNA medicines. Demand for the manufacturing of both the RNA drug substance (DS) and the LNP drug product (DP) has already been strained, causing shortages of the vaccine, and the rise in development and translation of other mRNA drugs in the coming years will exacerbate this strain. To estimate demand for DP manufacturing, the dosing requirements for the preclinical and clinical studies of the two approved mRNA vaccines were examined. To understand the current state of mRNA-LNP production, current methods and technologies are reviewed, as are current and announced global capacities for commercial manufacturing. Finally, a vision is rationalized for how emerging technologies such as self-amplifying mRNA, microfluidic production, and trends toward integrated and distributed manufacturing will shape the future of RNA manufacturing and unlock the potential for an RNA medicine revolution.

N/OFQ modulates orofacial pain induced by tooth movement through CGRP-dependent pathways.

BACKGROUND: Nociceptin/orphanin FQ (N/OFQ) has been revealed to play bidirectional roles in orofacial pain modulation. Calcitonin gene-related peptide (CGRP) is a well-known pro-nociceptive molecule that participates in the modulation of orofacial pain. We aimed to determine the effects of N/OFQ on the modulation of orofacial pain and on the release of CGRP. METHODS: Orofacial pain model was established by ligating springs between incisors and molars in rats for the simulation of tooth movement. The expression level of N/OFQ was determined and pain level was scored in response to orofacial pain. Both agonist and antagonist of N/OFQ receptor were administered to examine their effects on pain and the expression of CGRP in trigeminal ganglia (TG). Moreover, gene therapy based on the overexpression of N/OFQ was delivered to validate the modulatory role of N/OFQ on pain and CGRP expression. RESULTS: Tooth movement elicited orofacial pain and an elevation in N/OFQ expression. N/OFQ exacerbated orofacial pain and upregulated CGRP expression in TG, while UFP-101 alleviated pain and downregulated CGRP expression. N/OFQ-based gene therapy was successful in overexpressing N/OFQ in TG, which resulted in pain exacerbation and elevation of CGRP expression in TG. CONCLUSIONS: N/OFQ exacerbated orofacial pain possibly through upregulating CGRP.

The Effects of Static Magnetic Field on Orthodontic Tooth Movement in Mice.

The application of static magnetic field (SMF) has been considered an effective and noninvasive method to accelerate orthodontic tooth movement. The objective of this study was to explore the effects of SMF on orthodontic tooth movement in mice. A total of 105 Balb/c mice (body mass: 25-30 g) were divided into experimental group (SMF + force, 48), control group (force only, 48), and blank group (neither SMF nor force, 9). After the placement of orthodontic appliances, the experimental group was exposed to the SMF environment generated by Neodymium-iron-boron (NdFeB) magnets with an intensity of 20-204 mT. At 1, 3, 7, 14, 21, and 28 days after appliance insertion, eight animals in both experimental and control groups were sacrificed and the left maxillae were dissected to measure the distance of tooth movement, respectively. Meanwhile, the width of periodontal ligament (PDL), length of hyalinized zone, and the number of osteoclasts were evaluated by hematoxylin-eosin and tartrate-resistant acid phosphatase staining. We finally found that the experimental group demonstrated an enhanced rate and greater cumulative amount of tooth movement than the control group (0.2887 ± 0.0041 mm vs. 0.2114 ± 0.0089 mm, P < 0.05). On Days 7, 14, and 28, the experimental group also displayed a significantly greater width of PDL. Earlier formation and removal of the hyalinized zone, and significantly more osteoclasts were observed in the experimental group as well. The results suggested that SMF may be a promising nonsurgical intervention to accelerate orthodontic tooth movement. © 2021 Bioelectromagnetics Society.

A Soluplus/Poloxamer 407-based self-nanoemulsifying drug delivery system for the weakly basic drug carvedilol to improve its bioavailability.

Carvedilol (CAR), a ß-adrenoceptor and α1-receptor blocker, has pH-dependent solubility, which greatly limits its oral bioavailability. In this work, a precipitation inhibitor-based self-nanoemulsifying drug delivery system (PI-SNEDDS) was developed by employing Soluplus and Poloxamer 407 to improve drug dissolution and to inhibit drug precipitation in the gastrointestinal tract. In vitro phase distribution and in vivo dissolution studies indicated that PI-SNEDDS significantly increased drug content in the oil phase of the nanoemulsions in the stomach and greatly inhibited the subsequent precipitation of CAR in the intestine compared with the carvedilol self-nanoemulsifying drug delivery system (CAR SNEDDS) and the carvedilol tablets. Moreover, a 1.56-fold increase in the relative bioavailability of CAR was observed for the CAR PI-SNEDDS (397.41%) compared to a CAR SNEDDS (254.09%) with commercial capsules as a reference. Therefore, our developed PI-SNEDDS is a promising vehicle for improving the dissolution and bioavailability of poorly soluble drugs with pH-dependent solubility.

[Correlation of dry granulation process parameters and granule quality based on multiple regression analysis].

In this paper, microcrystalline cellulose WJ101 was used as a model material to investigate the effect of various process parameters on granule yield and friability after dry granulation with a single factor and the effect of comprehensive inspection process parameters on the effect of granule yield and friability, then the correlation between process parameters and granule quality was established. The regress equation was established between process parameters and granule yield and friability by multiple regression analysis, the affecting the order of the size of the order of the process parameters on granule yield and friability was: rollers speed > rollers pressure > speed of horizontal feed. Granule yield was positively correlated with pressure and speed of horizontal feed and negatively correlated rollers speed, while friability was on the contrary. By comparison, fitted value and real value, fitted and real value are basically the same of no significant differences (P > 0.05) and with high precision and reliability.

Development of lignin hydrogel reinforced polypyrrole rich electrode material for supercapacitor and sensing applications.

The preparation of natural polymer-based highly conductive hydrogels with reliable durability for applications in supercapacitors (SCs) is still challenging. Herein, a facile method to prepare alkaline lignin (AL)-based polypyrrole (PPy)-rich, high-conductive PPy@AL/PEGDGE gel was reported, where AL was used as a dopant, polyethylene glycol diglycidyl ether (PEGDGE) as a cross-linking agent, and PPy as a conducting polymer. The PPy@AL/PEGDGE gel electrode materials with hollow structures were prepared by electrochemical deposition and chemical etching method and then assembled into sandwich-shaped SCs. Cyclic voltammetry (CV), galvanotactic charge discharge (GCD), electrochemical impedance spectroscopy (EIS) and cycling stability tests of the PPy@AL/PEGDGE SCs were performed. The results demonstrated that the SCs can achieve a conductivity of 25.9 S·m-1 and a specific capacitance of 175 F·g-1, which was 127.4 % higher compared to pure PPy (77 F·g-1) electrode. The highest energy density and power density for the SCs were obtained at 23.06 Wh·kg-1 and 5376 W·kg-1, respectively. In addition, the cycling performance was also higher than that of pure PPy assembled SCs (50 %), and the capacitance retention rate can reach 72.3 % after 1000 cycles. The electrode materials are expected to be used as sensor and SCs devices.

Denture use and risk for cardiometabolic disease: observational and Mendelian randomization analyses.

AIMS: Denture use may potentially increase the risk of cardiometabolic diseases (CMDs), but the casual relevance and strength of the associations are currently unknown. METHODS AND RESULTS: A total of 495 938 participants from the UK Biobank were included in the observational analyses. Linkage disequilibrium score (LDSC) regression and Mendelian randomization analyses were employed to estimate genetic correlation and the associations between the genetic liability for denture use with coronary artery disease, myocardial infarction, heart failure (HF), any stroke (AS), ischaemic stroke, haemorrhagic stroke, type 2 diabetes (T2D), and related clinical risk factors. In observational analysis, denture use was associated with 14-25% higher risks of various CMDs. The LDSC analysis found that denture use showed a positive genetic correlation with CMDs (rg 0.21-0.38). Genetic liability for denture use was associated with an elevated risk of HF [odds ratio: 1.49 (1.20-1.83)] and T2D [1.11 (1.01-1.24)]. By integrating genetic summary data of denture use with the sum of decayed, missing, and filled tooth surfaces (DMFS), a clinical measure of dental caries obtained from an independent source, genetically determined denture use/DMFS was also associated with an elevated risk of AS [1.21 (1.04-1.40)]. Furthermore, genetically predicted denture use/DMFS was significantly associated with established cardiometabolic risk factors, including HDL cholesterol, triglycerides, waist circumference, waist-to-hip ratio, and height. CONCLUSION: Our study supported potential causal associations between the genetic liability for denture use and risks for HF, AS, T2D, and related clinical risk factors. These findings may inform prevention and intervention strategies targeting dental diseases and CMDs.

This study examined the association of denture use with cardiometabolic diseases (CMDs) and related clinical risk factors through Mendelian randomization analyses using data from UK Biobank and published consortia. Genetic liability for denture use was associated with an 11­49% higher risk of heart failure, stroke, and type 2 diabetes.The potential causal relationship between denture use and CMDs was further strengthened by the associations of denture use with HDL cholesterol, triglycerides, waist circumference, waist-to-hip ratio, and height, which are among the major risk factors of CMDs.

Targeted nanoparticles assembled via complexation of boronic-acid-containing targeting moieties to diol-containing polymers.

The delivery of therapeutics via nanoscaled vehicles for solid cancer treatment can be enhanced by the incorporation of a targeting capability. Here, we describe a new method for assembling a targeted nanoparticle that utilizes the reversible covalent complexation between boronic acids and diols to achieve a targeted nanoparticle for the delivery of the anticancer drug camptothecin (CPT). CPT is conjugated to a biocompatible, hydrophilic copolymer of mucic acid and PEG (MAP). When this polymer-drug conjugate is placed in water, it self-assembles into MAP-CPT nanoparticles of ca. 30 nm (diameter) and slightly negative zeta potential. The antibody Herceptin is attached to a boronic acid via a polyethylene glycol (PEG) spacer, and this boronic acid-containing targeting moiety is complexed with the diol-containing MAP to form a targeted MAP-CPT nanoparticle. The addition of Herceptin targeting agent to the MAP-CPT nanoparticles yields targeted MAP-CPT nanoparticles with increased nanoparticle size to ca. 40 nm (diameter). The main mechanisms of CPT release from MAP-CPT nanoparticles are found by in vitro analysis to be hydrolysis and nanoparticle disruption by fat. Cellular uptake of nanoparticles is enhanced by 70% compared to nontargeted version by the incorporation of a single Herceptin antibody targeting agent per nanoparticle. This single Herceptin antibody targeted MAP-CPT nanoparticle system carries ca. 60 CPT molecules per nanoparticle and shows prolonged plasma circulation with an elimination half-life of 21.2 h and AUC value of 2766 µg.h/mL at a 10 mg CPT/kg tail vein injection in mice.

Single-antibody, targeted nanoparticle delivery of camptothecin.

We have developed a new method for assembling targeted nanoparticles that utilizes the complexation between targeting agents that contain boronic acids and polymer-drug conjugates that possess diols. Here, we report the first in vivo, antitumor results of a nanoparticle formed via this new assembly methodology. A nanoparticle consisting of a mucic acid polymer conjugate of camptothecin (CPT), MAP-CPT, and containing on average one Herceptin antibody is investigated in nude mice bearing HER2 overexpressing BT-474 human breast cancer tumors. Nontargeted MAP-CPT and antibody-containing MAP-CPT nanoparticles of ca. 30-40 nm diameter and slightly negative zeta potential show prolonged in vivo circulation and similar biodistributions after intravenous tail vein injections in mice. The maximum tolerated dose (MTD) of the nontargeted and Herceptin-containing MAP-CPT nanoparticles is found to be 10 and 8 mg of CPT/kg, respectively, in mice. Mice bearing BT-474 human breast tumors treated with nontargeted MAP-CPT nanoparticles at 8 mg of CPT/kg show significant tumor growth inhibition (mean tumor volume of 63 mm(3)) when compared to irinotecan at 80 mg/kg (mean tumor volume of 575 mm(3)) and CPT at 8 mg/kg (mean tumor volume of 808 mm(3)) at the end of the study. Herceptin antibody treatment at 5.9 mg/kg results in complete tumor regressions in 5 out of 8 mice, with a mean tumor volume of 60 mm(3) at the end of the study. Mice treated with MAP-CPT nanoparticles at 1 mg of CPT/kg do not show tumor inhibition. However, all mice receiving administrations of MAP-CPT nanoparticles (1 mg of CPT/kg) that contain on average a single Herceptin molecule per nanoparticle (5.9 mg of Herceptin equivalent/kg) show complete tumor regression by the end of the study. These results demonstrate that the antitumor efficacy of nanoparticles carrying anticancer drugs can be enhanced by incorporating on average a single antibody.

Diet Evolution and Habitat Contraction of Giant Pandas via Stable Isotope Analysis.

The ancestral panda Ailurarctos lufengensis, excavated from the late Miocene, is thought to be carnivorous or omnivorous [1]. Today, giant pandas exclusively consume bamboo and have distinctive tooth, skull, and muscle characteristics adapted to a tough and fibrous bamboo diet during their long evolution [1, 2]. A special feature, the pseudo-thumb, has evolved to permit the precise and efficient grasping of bamboo [3, 4]. Unlike those of extant pandas, little is known about the diet and habitat preferences of extinct pandas. Prevailing studies suggest that the panda shifted to specialized bamboo feeding in the Pleistocene [5, 6]; however, this remains questionable. Pandas now survive in a fraction of their historical habitat [7], but no specific information has been reported. Stable isotope analyses can be used to understand diet- and habitat-related changes in animals [8]. Isotopic signals in bone collagen reflect dietary compositions of ancient human diets [9, 10] and dietary changes between historical and modern animal populations [11, 12]. Here, we conduct stable isotope analyses of bone and tooth samples from ancient and modern pandas and from sympatric fauna. We show that pandas have had a diet dominated by C3 resources over time and space and that trophic niches of ancient and modern pandas are distinctly different. The isotopic trophic and ecological niche widths of ancient pandas are approximately three times larger than those of modern pandas, suggesting that ancient pandas possibly had more complex diets and habitats than do their modern counterparts. Our findings provide insight into the dietary evolution and habitat contraction of pandas.

Statistical optimization for the production of recombinant cold-adapted superoxide dismutase in E. coli using response surface methodology.

Cold-adapted superoxide dismutase (SOD) with higher catalytic activity at lower temperature has great amount of applications in many aspects as an industrial enzyme. The application of recombinant enzyme in gene engineering and microbial fermentation technology is an effective way to obtain high-yield product. In this study, to obtain the recombinant SOD in E. coli (rPsSOD) with the highest activity, the Box-Behnken design was first applied to optimize the important parameters (lactose, tryptone and Tween-80) affecting the activity of rPsSOD. The results showed that the optimal fermentation conditions were Tween-80 (0.047%), tryptone (6.16 g/L), lactose (11.38 g/L). The activity of rPsSOD was 71.86 U/mg (1.54 times) as compared with non-optimized conditions. Such an improved production will facilitate the application of the cold-adapted rPsSOD.

[Biomechanical effect of 2 anchorages on maxillary protraction with cleft lip and palate: a finite element analysis].

PURPOSE: To establish cranio-maxillary complex finite element models (FEMs) in cleft lip and palate (CLP) patients with miniplate and tooth-borne anchorage, and to investigate the biomechanical effects on maxillary protraction. METHODS: The protracting process was simulated on FEMs established from a male patient's cone beam CT (CBCT) data to analyze the displacement and stress from different appliances. RESULTS: Four FEMs of unilateral cleft lip and palate (UCLP) or bilateral cleft lip and palate (BCLP) patients' cranio-maxillary complex with MA and TA were established. The maxillary arch on both sides of the cleft moved towards the cleft side during protraction, which was more obvious in the miniplate anchorage three-dimensional finite element models(FEM-MA) than the tooth-borne anchorage three-dimensional finite element models (FEM-TA).The amount of forward, downward displacement of the inferior orbital rim, paranasal area and middle maxilla point in the FEM-MA was larger than that in the FEM-TA, while the amount of forward displacement of premaxilla and upper canine in the FEM-TA was larger than that in the FEM-MA. The FEM-MA exhibited an orthopedic effect with more favorable stress distribution on the middle maxilla point while the FEM-TA showed a dentoalveolar effect with prominent stress distribution on the upper canine point. In addition, the FEM-MA showed a larger stress distribution area and sutural stress value than did the FEM-TA. The UCLP models showed an asymmetric pattern in stress distribution and displacement, which was larger on the cleft side than that on the non-cleft side. The amount of displacement of premaxilla in the BCLP models was smaller than that in the UCLP models. CONCLUSIONS: The established models have high geomagic and biomechanical similarities. It would be more advantageous to obtain more aesthetic outcomes and better stability using the miniplate anchorage.

The Cardiovascular and Neurotoxic Effects of the Venoms of Six Bony and Cartilaginous Fish Species.

Fish venoms are often poorly studied, in part due to the difficulty in obtaining, extracting, and storing them. In this study, we characterize the cardiovascular and neurotoxic effects of the venoms from the following six species of fish: the cartilaginous stingrays Neotrygon kuhlii and Himantura toshi, and the bony fish Platycephalus fucus, Girella tricuspidata, Mugil cephalus, and Dentex tumifrons. All venoms (10-100 µg/kg, i.v.), except G. tricuspidata and P. fuscus, induced a biphasic response on mean arterial pressure (MAP) in the anesthetised rat. P. fucus venom exhibited a hypotensive response, while venom from G. tricuspidata displayed a single depressor response. All venoms induced cardiovascular collapse at 200 µg/kg, i.v. The in vitro neurotoxic effects of venom were examined using the chick biventer cervicis nerve-muscle (CBCNM) preparation. N. kuhlii, H. toshi, and P. fucus venoms caused concentration-dependent inhibition of indirect twitches in the CBCNM preparation. These three venoms also inhibited responses to exogenous acetylcholine (ACh) and carbachol (CCh), but not potassium chloride (KCl), indicating a post-synaptic mode of action. Venom from G. tricuspidata, M. cephalus, and D. tumifrons had no significant effect on indirect twitches or agonist responses in the CBCNM. Our results demonstrate that envenoming by these species of fish may result in moderate cardiovascular and/or neurotoxic effects. Future studies aimed at identifying the molecules responsible for these effects could uncover potentially novel lead compounds for future pharmaceuticals, in addition to generating new knowledge about the evolutionary relationships between venomous animals.

The Evolution of Fangs, Venom, and Mimicry Systems in Blenny Fishes.

Venom systems have evolved on multiple occasions across the animal kingdom, and they can act as key adaptations to protect animals from predators [1]. Consequently, venomous animals serve as models for a rich source of mimicry types, as non-venomous species benefit from reductions in predation risk by mimicking the coloration, body shape, and/or movement of toxic counterparts [2-5]. The frequent evolution of such deceitful imitations provides notable examples of phenotypic convergence and are often invoked as classic exemplars of evolution by natural selection. Here, we investigate the evolution of fangs, venom, and mimetic relationships in reef fishes from the tribe Nemophini (fangblennies). Comparative morphological analyses reveal that enlarged canine teeth (fangs) originated at the base of the Nemophini radiation and have enabled a micropredatory feeding strategy in non-venomous Plagiotremus spp. Subsequently, the evolution of deep anterior grooves and their coupling to venom secretory tissue provide Meiacanthus spp. with toxic venom that they effectively employ for defense. We find that fangblenny venom contains a number of toxic components that have been independently recruited into other animal venoms, some of which cause toxicity via interactions with opioid receptors, and result in a multifunctional biochemical phenotype that exerts potent hypotensive effects. The evolution of fangblenny venom has seemingly led to phenotypic convergence via the formation of a diverse array of mimetic relationships that provide protective (Batesian mimicry) and predatory (aggressive mimicry) benefits to other fishes [2, 6]. Our results further our understanding of how novel morphological and biochemical adaptations stimulate ecological interactions in the natural world.

Distinctive diet-tissue isotopic discrimination factors derived from the exclusive bamboo-eating giant panda.

Stable isotope analysis is very useful in animal ecology, especially in diet reconstruction and trophic studies. Differences in isotope ratios between consumers and their diet, termed discrimination factors, are essential for studies of stable isotope ecology and are species-specific and tissue-specific. Given the specialized bamboo diet and clear foraging behavior, here, we calculated discrimination factors for carbon and nitrogen isotopes from diet to tissues (tooth enamel, hair keratin and bone collagen) for the giant panda (Ailuropoda melanoleuca), a species derived from meat-eating ancestors. Our results showed that carbon discrimination factor obtained from giant panda tooth enamel (ε 13 Cdiet-enamel = 10.0‰) and nitrogen discrimination factors from hair keratin (Δ15 Ndiet-hair = 2.2‰) and bone collagen (Δ15 Ndiet-collagen = 2.3‰) were lower, and carbon discrimination factors from hair keratin (Δ13 Cdiet-hair = 5.0‰) and bone collagen (Δ13 Cdiet-collagen = 6.1‰) were higher than those of other mammalian carnivores, omnivores and herbivores. Such distinctive values are likely the result of a low-nutrient and specialized bamboo diet, carnivore-like digestive system and exceptionally low metabolism in giant pandas.