Recent advances in lipid nanoparticles for delivery of nucleic acid,mRNA, and gene editing-based therapeutics

Lipid nanoparticles (LNPs) are becoming popular as a means of delivering therapeutics, including those based on nucleic acids and mRNA. The mRNA-based coronavirus disease 2019 vaccines are perfect examples to highlight the role played by drug delivery systems in advancing human health. The fundamentals of LNPs for the delivery of nucleic acid- and mRNA-based therapeutics, are well established. Thus, future research on LNPs will focus on addressing the following: expanding the scope of drug delivery to different constituents of the human body, expanding the number of diseases that can be targeted, and studying the change in the pharmacokinetics of LNPs under physiological and pathological conditions. This review article provides an overview of recent advances aimed at expanding the application of LNPs, focusing on the pharmacokinetics and advantages of LNPs. In addition, analytical techniques, library construction and screening, rational design, active targeting, and applicability to gene editing therapy have also been discussed.     

AuNP flares-capped mesoporous silica nanoplatform for MTH1 detection and inhibition

The human mutT homologue MTH1, a nucleotide pool sanitizing enzyme, represents a vulnerability factor and an attractive target for anticancer therapy. However, there is currently a lack of selective and effective platforms for the detection and inhibition of MTH1 in cells. Here, we demonstrate for the first time a gold nanoparticle (AuNP) flares-capped mesoporous silica nanoparticle (MSN) nanoplatform that is capable of detecting MTH1 mRNA and simultaneously suppressing MTH1 activity. The AuNP flares are made from AuNPs that are functionalized with a dense shell of MTH1 recognition sequences hybridized to short cyanine (Cy5)-labeled reporter sequences and employed to seal the pores of MSN to prevent the premature MTH1 inhibitors (S-crizotinib) release. Just like the pyrotechnic flares that produce brilliant light when activated, the resulting AuNP flares@MSN (S-crizotinib) undergo a significant burst of red fluorescence enhancement upon MTH1 mRNA binding. This hybridization event subsequently induces the opening of the pores and the release of S-crizotinib in an mRNA-dependent manner, leading to significant cytotoxicity in cancer cells and improved therapeutic response in mouse xenograft models. We anticipate that this nanoplatform may be an important step toward the development of MTH1-targeting theranostics and also be a useful tool for cancer phenotypic lethal anticancer therapy.     

Genotoxicity of synthetic amorphous silica nanoparticles in rats following short‐term exposure. Part 1: Oral route

Synthetic amorphous silica (SAS) in its nanosized form is now used in food applications although the potential risks for human health have not been evaluated. In this study, genotoxicity and oxidative DNA damage of two pyrogenic (NM‐202 and 203) and two precipitated (NM‐200 and ‐201) nanosized SAS were investigated in vivo in rats following oral exposure. Male Sprague Dawley rats were exposed to 5, 10, or 20 mg/kg b.w./day for three days by gavage. DNA strand breaks and oxidative DNA damage were investigated in seven tissues (blood, bone marrow from femur, liver, spleen, kidney, duodenum, and colon) with the alkaline and the (Fpg)‐modified comet assays, respectively. Concomitantly, chromosomal damage was investigated in bone marrow and in colon with the micronucleus assay. Additionally, malondialdehyde (MDA), a lipid peroxidation marker, was measured in plasma. When required, a histopathological examination was also conducted. The results showed neither obvious DNA strand breaks nor oxidative damage with the comet assay, irrespective of the dose and the organ investigated. Similarly, no increases in chromosome damage in bone marrow or lipid peroxidation in plasma were detected. However, although the response was not dose‐dependent, a weak increase in the percentage of micronucleated cells was observed in the colon of rats treated with the two pyrogenic SAS at the lowest dose (5 mg/kg b.w./day). Additional data are required to confirm this result, considering in particular, the role of agglomeration/aggregation of SAS NMs in their uptake by intestinal cells. Environ. Mol. Mutagen. 56:218–227, 2015. © 2014 Wiley Periodicals, Inc.     

Particulate endocytosis mediates biological responses of human mesenchymal stem cells to titanium wear debris.

Continual loading and articulation cycles undergone by metallic (e.g., titanium) alloy arthroplasty prostheses lead to liberation of a large number of metallic debris particulates, which have long been implicated as a primary cause of periprosthetic osteolysis and postarthroplasty aseptic implant loosening. Long-term stability of total joint replacement prostheses relies on proper integration between implant biomaterial and osseous tissue, and factors that interfere with this integration are likely to cause osteolysis. Because multipotent mesenchymal stem cells (MSCs) located adjacent to the implant have an osteoprogenitor function and are critical contributors to osseous tissue integrity, when their functions or activities are compromised, osteolysis will most likely occur. To date, it is not certain or sufficiently confirmed whether MSCs endocytose titanium particles, and if so, whether particulate endocytosis has any effect on cellular responses to wear debris. This study seeks to clarify the phenomenon of titanium endocytosis by human MSCs (hMSCs), and investigates the influence of endocytosis on their activities. hMSCs incubated with commercially pure titanium particles exhibited internalized particles, as observed by scanning electron microscopy and confocal laser scanning microscopy, with time-dependent reduction in the number of extracellular particles. Particulate endocytosis was associated with reduced rates of cellular proliferation and cell-substrate adhesion, suppressed osteogenic differentiation, and increased rate of apoptosis. These cellular effects of exposure to titanium particles were reduced when endocytosis was inhibited by treatment with cytochalasin D, and no significant effect was seen when hMSCs were treated only with conditioned medium obtained from particulate-treated cells. These findings strongly suggest that the biological responses of hMSCs to wear debris are triggered primarily by the direct endocytosis of titanium particulates, and not mediated by secreted soluble factors. In this manner, therapeutical approaches that suppress particle endocytosis could reduce the bioreactivity of hMSCs to particulates, and enhance long-term orthopedic implant prognosis by minimizing wear-debris periprosthethic osteolysis.     

Hepatic Disposition Characteristics of Electrically Charged Macromolecules in Rat in Vivo and in the Perfused Liver

The effect of electric charge on the hepatic disposition of macromolecules was studied in the rat. Charged derivatives of dextran (T-70) and bovine serum albumin (BSA), mitomycin C–dextran conjugates (MMC-D), and lactosaminated BSA (Lac-BSA) were employed as model macromolecules. After intravenous injection, cationic macromolecules were rapidly eliminated from plasma because of their extensive hepatic uptake, while anionic and neutral macromolecules were slowly eliminated. Cationic macromolecules were recovered from parenchymal and nonparenchymal hepatic cells at a cellular uptake (per unit cell number) ratio of 1.4–3.2, while that of Lac-BSA was 14. During liver perfusion using a single-pass constant infusion mode, cationic macromolecules were continuously extracted by the liver, with extraction ratios at steady-state (E _ss) ranging between 0.03 and 0.54, whereas anionic and neutral macromolecules were almost completely recovered in the outflow at steady state. The E _ss for cationized BSA (Cat-BSA) and cationic MMC-Dcat were concentration dependent and decreased at low temperatures and in the presence of colchicine and cytochalasin B. The possible participation of the internalization process in the uptake of cationic macromolecules by hepatocytes was suggested.     

Fibronectin inhibits endocytosis of calcium oxalate crystals by renal tubular cells

BACKGROUND: Fibronectin (FN; 230 kDa) is a multifunctional alpha2-glycoprotein distributed throughout the extracellular matrix and body fluids. We recently reported that FN has a protective effect against injury of renal tubular cells by exposure to oxalate and calcium oxalate (CaOX) crystals and inhibits the adhesion of CaOX crystals to renal tubular cells. In the study presented here, we investigated whether FN has inhibitory effect on crystal endocytosis by renal tubular cells. METHODS: The inhibitory effect of FN on endocytosis of CaOX crystals by MDCK cells was examined by using a radioactivity uptake assay. Also, crystal endocytosis by cells was morphologically assessed by means of transmission electron microscopy (TEM). RESULTS: FN had inhibitory effects on CaOX crystal endocytosis by MDCK cells. The morphological TEM study showed that few crystals were taken into cells when FN was added compared to the number of crystals when FN was not added. CONCLUSION: We found that FN had the inhibitory effects on the interaction between crystals and renal tubular cells, including the adhesion or endocytosis of crystals by cells.     

Short-term enzyme replacement in the murine model of Sanfilippo syndrome type B

The Sanfilippo syndrome type B (MPS III B) is an autosomal recessive disease caused by deficiency of alpha-N-acetylglucosaminidase (EC 3. 2.1.50), one of the lysosomal enzymes required for the degradation of heparan sulfate. The disease is characterized by profound neurodegeneration but relatively mild somatic manifestations, and is usually fatal in the second decade. A mouse model had been generated by disruption of the Naglu gene in order to facilitate the study of pathogenesis and the development of therapy for this currently untreatable disease. Recombinant human alpha-N-acetylglucosaminidase (rhNAGLU) was prepared from secretions of Lec1 mutant Chinese hamster ovary cells. The enzyme, which has only unphosphorylated high-mannose carbohydrate chains, was endocytosed by mouse peritoneal macrophages via mannose receptors, with half-maximal uptake at ca. 10(-7) M. When administered intravenously to 3 month-old mice, rhNAGLU was taken up avidly by liver and spleen but marginally if at all by thymus, lung, kidney, heart, and brain (in order of diminishing uptake). The half-life of the enzyme was 2.5 days in liver and spleen. Immunohistochemistry and electron microscopy showed that only macrophages were involved in enzyme uptake and correction in these two organs, yet the storage of glycosaminoglycan was reduced to almost normal levels. The results show that the macrophage-targeted rhNAGLU can substantially reduce the body burden of glycosaminoglycan storage in the mouse model of Sanfilippo syndrome III B.     

Binding of nucleosomes to a cell surface receptor: Redistribution and endocytosis in the presence of lupus antibodies

In the present study, we sought evidence for a surface nucleosome receptor in the fibroblastic cell line CV-1, and questioned whether anti-double-stranded (ds)DNA and/or anti-histone autoantibodies could recognized and influence the fate of cell surface-bound nucleosomes. ¹²⁵I-labeled mononucleosomes were shown to bind to the cell layer in a specific, concentration-dependent and a saturable manner. Scatchard analysis revealed the presence of two binding sites: a high-affinity site with a Kd of ～ 7nM and a low-affinity site (Kd ～ 400 nM) with a high capacity of 9 × 10⁷ sites. Visualization of bound mononucleosomes by fluorescence revealed staining on both the cell surface and the extracellular matrix (ECM). Purified mononucleosome-derived dsDNA (180–200 bp) was found to compete for binding of ¹²⁵I-mononucleosomes on the low-affinity site, to stain exclusively the ECM in immunofluorescence, and to precipitate three specific proteins of 43, 180 and 240 kDa from ¹²⁵-I-labeled cell lysates. Nucleosomes were found to precipitate not only the 180-kDa dsDNA-reactive component, but also a unique protein of 50 kDa, suggesting that this protein is a cell surface receptor for nucleosomes on these fibroblasts. Once bound on the cell surface, mononucleosomes were recognized and secondarily complexed by lupus anti-dsDNA or anti-histone antibodies (i.e. anti-nucleosome antibodies), thus forming immune complexes in situ. The presence of these complexing auto-antibodies was found dramatically to enhance the kinetics of mononucleosome internalization. Following the internalization of the nucleosome-anti-nucleosome complexes by immunofluorescence, we observed the formation of vesicles at the edge of the cells by 5–10 min which moved toward the perinuclear region by 20–30 min. By means of double-fluorescence labeling and proteolytic treatment, these fluorescent vesicles were shown to be in the cytoplasm, suggesting true endocytosis of nucleosome-anti-nucleosome immune complexes. As shown by confocal microscopy, at no stage of this endocytic process was there any indication that coated pits or coated vesicles participated. Co-distribution of the endocytic vesicles with regions rich in actin filaments and inhibition of endocytosis of nucleosome-anti-nucleosome complexes by disruption of the micro-filament network with cytochalasin D suggest a mechanism mediated by the cytoskeleton. Taken together, our data provide evidence for the presence of a surface nucleosome receptor. We also show that anti-dsDNA and anti-histone antibodies can form nucleosome-anti-nucleosome immune complexes in situ at the cell surface, and thus dramatically enhance the kinetics of nucleosome endocytosis.     

利用纳米颗粒的光核反应实现原位活化治疗的可行性研究

目的:利用蒙特卡罗程序Geant4模拟13.5 MeV和6 MeV X射线照射细胞内的纳米颗粒，分析其光核反应的剂量贡献份额。方法:以纳米金颗粒（GNP）为例，分别模拟6 MeV和13.5 MeV照射细胞内的GNP，给出各自条件下由GNP造成的剂量贡献。创建水模体（0.426 mm×0.426 mm×0.426 mm），包含1 103个细胞，作为GNP的载体。在6 MeV和13.5 MeV下分别模拟细胞中包含和不包含GNP所造成的剂量沉积。结果:13.5 MeV X射线照射，其由GNP造成的剂量贡献为5.12 cGy，细胞总能量沉积为25.37 cGy，由GNP引起的剂量贡献占20.19%；6 MeV X射线照射，其由GNP造成的剂量贡献为2.87 cGy，细胞总能量沉积为23.05 cGy，由GNP造成剂量贡献约为12.46%。与6 MeV相比，13.5 MeV下由GNP光核反应造成的剂量贡献占7.7%。结论:对于细胞模型内纳米金的研究表明，GNP确实能引起额外的剂量贡献。由于GNP光核反应引起的剂量贡献很低，难以作为能够被原位激活的放射源使用。     

含银介孔二氧化硅-壳聚糖复合材料的制备与性能研究

目的探讨含银介孔二氧化硅-壳聚糖复合材料(Ag/MSN-Chi)的制备方法及其微观表征、细胞毒性、吸水性能、抗菌性能及止血性能。 方法以正硅酸乙酯为前驱体，十六烷基三甲基溴化铵为致孔剂，采用离子交换法在介孔二氧化硅纳米粒子(MSN)中引入银离子，制备出具有抗菌作用的新型有序的含银介孔二氧化硅纳米粒子(Ag/MSN)材料。再利用烷基化壳聚糖负载Ag/MSN，制备出Ag/MSN-Chi。根据所用材料不同将实验分为实验组和空白对照组，实验组又分为3个亚组：MSN组、Ag/MSN组、Ag/MSN-Chi组，空白组为不加任何材料的阳性对照。计算MSN和Ag/MSN的比表面积、孔容、孔径和Ag/MSN与Ag/MSN-Chi的电荷。并通过吸水实验、体外凝血实验、抗菌实验对MSN、Ag/MSN和Ag/MSN-Chi的细胞毒性、吸水性能、止血性能及抗菌性能进行评价，计算细胞相对存活率、吸水率、凝血酶原时间(PT)、凝血活酶时间(APTT)及抑菌率。取健康成年新西兰大白兔18只，随机分成3组：对照组(采用医用纱布处理)、Ag/MSN组(采用Ag/MSN处理)、Ag/MSN-Chi组(采用Ag/MSN-Chi处理)，每组6只，建立肝创伤出血模型，计算止血时间。数据比较采用方差分析和t检验。 结果MSN的比表面积为(523.8±12.4) m²/g、孔容为(1.2±0.4) m³/g、孔径为(3.5±0.9) nm；Ag/MSN的比表面积为(521.6±11.7) m²/g、孔容为(1.15±0.5) m³/g、孔径为(3.6±0.7) nm，2种材料的比表面积、孔容、孔径比较差异均无统计学意义(t＝0.224、0.135、0.015，P值均大于0.05)。经测量，Ag/MSN的Zeta电位为－19.7 mV，Ag/MSN-Chi的Zeta电位为10.27 mV，表明Ag/MSN表面电荷从负值变为正值。Ag/MSN-Chi组、Ag/MSN组和MSN组与小鼠成肌细胞共培养1、4、7 d的细胞相对存活率比较，差异均无统计学意义(F＝2.61、4.72、3.52, P值均大于0.05)。Ag/MSN组吸水率分别与MSN组和Ag/MSN-Chi组比较，差异均无统计学意义(t＝0.482、1.159，P值均大于0.05)。经检测，Ag/MSN-Chi组、Ag/MSN组、MSN组和空白对照组的PT比较，差异无统计学意义(F＝10.28，P>0.05)；Ag/MSN-Chi组、Ag/MSN组、MSN组和空白对照组APTT分别为(20.9±2.1)、(28.5±3.4)、(31.4±2.6)、(38.7±2.5) s，4组比较差异有统计学意义(F＝8.70，P<0.05)；Ag/MSN-Chi组、Ag/MSN组、MSN组APTT分别与空白对照组比较，差异均有统计学意义(t＝9.443、4.186、3.506，P值均小于0.05)；Ag/MSN-Chi组APTT与Ag/MSN组比较，差异有统计学意义(t＝3.294，P<0.05)。MSN组在培养0.5、2、4、6、24 h 5个时间点抑菌率比较差异无统计学意义(F＝5.437，P>0.05)；培养0.5 h，Ag/MSN组和Ag/MSN-Chi组抑菌率分别为(99.7±5.2)%、(97.1±5.4)%，与培养0.5 h MSN组抑菌率(11.2±5.8)%比较，差异均有统计学意义(t＝19.678、18.775, P值均小于0.05)；培养24 h，Ag/MSN组和Ag/MSN-Chi组抑菌率分别为(73.2±5.1)%和(72.9±6.9)%，与MSN组(11.8±5.7)%比较，差异均有统计学意义(t＝13.904、11.825, P值均小于0.05)。Ag/MSN-Chi组、Ag/MSN组和对照组止血时间分别为(12.3±1.5)、(17.2±3.4)、(28.1±3.8) s，3组比较差异有统计学意义(F＝5.892，P<0.05)；Ag/MSN-Chi组和Ag/MSN组止血时间分别与对照组比较，差异均有统计学意义(t＝9.473、5.236, P值均小于0.05)；且Ag/MSN-Chi组与Ag/MSN组止血时间比较，差异有统计学意义(t＝3.230，P<0.05)。 结论Ag/MSN-Chi在不增加细胞毒性的基础上具有有较好的吸水性能、止血性能及抗菌性能。