Effects of Histidine Oligomers in Lipid Nanoparticles on siRNA Delivery.

In this study, histidine oligomer (oHis; 10mer)-incorporating LNPs (H10LNPs) are developed as a novel carrier for efficient siRNA delivery. Notably, the unmodified oHis (10mer) is greatly incorporated within LNPs through ionic interaction with siRNAs, which serves as an endosome escape enhancer. H10LNPs with a size of ≈65 nm demonstrate a significantly enhanced extent of endosomal escape, as evidenced by calcein assay and confocal microscopy images of intracellular fluorescence, surpassing conventional LNPs. Furthermore, the half inhibitory concentration (IC50) of the human endogenous globotriaosylceramide synthase (Gb3 synthase) gene in H10LNPs-treated cells exhibits a significant threefold decrease, compared to that in LNP-treated cells. Notably, H10LNPs maintain comparable biocompatibility and biodistribution both in vitro and in vivo. Considering that the fabricated siRNA H10LNPs exhibit excellent biocompatibility and superior gene silencing activity over conventional LNPs, these particles can be harnessed for the safe delivery of therapeutic siRNAs. Additionally, this study introduces promising, feasible, simple, and alternative formulation processes for integrating unmodified functional cationic peptides into LNPs to enhance the delivery efficiency of a wide range of nucleic acid-based drugs.

Efficient Delivery of Globotriaosylceramide Synthase siRNA using Polyhistidine-Incorporated Lipid Nanoparticles.

In this study, a novel polyhistidine-incorporated lipid nanoparticle (pHis/LNP) is developed for the delivery of therapeutic globotriaosylceramide (Gb3) synthase siRNAs using a microfluidic device with pHis as a biocompatible method of endosome escape. To inhibit the expression of Gb3 synthase, six siRNAs against Gb3 synthase are designed and an optimal siRNA sequence is selected. Selected Gb3 synthase siRNA is incorporated into pHis/LNP to prepare a spherical siRNA pHis/LNP with a size of 62.5 ± 1.9 nm and surface charge of -13.3 ± 4.2 mV. The pHis/LNP successfully protects siRNAs from degradation in 50% serum condition for 72 h. Prepared pHis/LNP exhibits superior stability for 20 days and excellent biocompatibility for A549 cells. After treatment with fluorescence-labeled LNPs, dotted fluorescent signals are co-localized with Lysotracker in cells with LNPs, whereas strong and diffused fluorescence intensity is observed in cells with pHis/LNPs probably due to successful endosomal escape. The extent of Gb3 synthase gene silencing by siRNA pHis/LNP is greatly improved (6.0-fold) compared to that by siRNA/LNP. Taken together, considering that the fabricated siRNA pHis/LNP exhibits excellent biocompatibility and superior gene silencing activity over conventional LNP, these particles can be utilized for the delivery of a wide range of therapeutic siRNAs.

Protection of Skin Fibroblasts from Infrared-A-Induced Photo-Damage Using Ginsenoside Rg3(S)-Incorporated Soybean Lecithin Liposomes.

Protection of skin cells from chronic infrared-A (IRA) irradiation is crucial for anti-photoaging of the skin. In this study, we investigated the protective activity of Rg3(S) and Rg3(S)-incorporated anionic soybean lecithin liposomes (Rg3/Lipo) with a size of approximately 150 nm against IRA-induced photodamage in human fibroblasts. The formulated Rg3/Lipo showed increased solubility in aqueous solution up to a concentration of 200 µg/ml, compared to free Rg3(S). In addition, Rg3/Lipo exhibited superior colloidal stability in aqueous solutions and biocompatibility for normal human dermal fibroblasts (NHDFs). After repeated IRA irradiation on NHDFs, elevated levels of cellular and mitochondrial reactive oxygen species (ROS) were greatly reduced by Rg3(S) and Rg3/Lipo. In addition, cells treated with Rg3/Lipo exhibited noticeably reduced apoptotic signals following IRA irradiation compared to untreated cells. Thus, considering aqueous solubility and cellular responses, Rg3/Lipo could serve as a promising infrared protector for healthy aging of skin cells.

Different Influences of Biotinylation and PEGylation on Cationic and Anionic Proteins for Spheroid Penetration and Intracellular Uptake to Cancer Cells.

To better understand the effects of PEGylation and biotinylation on the delivery efficiency of proteins, the cationic protein lysozyme (LZ) and anionic protein bovine serum albumin (BSA) were chemically conjugated with poly(ethylene glycol) (PEG) and biotin-PEG to primary amine groups of proteins using N-hydroxysuccinimide reactions. Four types of protein conjugates were successfully prepared: PEGylated LZ (PEG-LZ), PEGylated BSA (PEG-BSA), biotin-PEG-conjugated LZ (Bio-PEG-LZ), and biotin-PEG-conjugated BSA (Bio-PEG-BSA). PEG-LZ and Bio-PEG-LZ exhibited a lower intracellular uptake than that of LZ in A549 human lung cancer cells (in a two-dimensional culture). However, Bio-PEG-BSA showed significantly improved intracellular delivery as compared to that of PEG-BSA and BSA, probably because of favorable interactions with cells via biotin receptors. For A549/fibroblast coculture spheroids, PEG-LZ and PEG-BSA exhibited significantly decreased tissue penetration as compared with that of unmodified proteins. However, Bio-PEG-BSA showed tissue penetration comparable to that of unmodified BSA. In addition, citraconlyated LZ (Cit-LZ) showed reduced spheroid penetration as compared to that of LZ, probably owing to a decrease in protein charge. Taken together, chemical conjugation of targeting ligands-PEG to anionic proteins could be a promising strategy to improve intracellular delivery and in vivo activity, whereas modifications of cationic proteins should be more delicately designed.

Formulation of Glycyrrhizic Acid-based Nanocomplexes for Enhanced Anti-cancer and Anti-inflammatory Effects of Curcumin.

In this study, nanocomplexes composed of glycyrrhizic acid (GA) derived from the root of the licorice plant (Glycyrrhiza glabra) were formulated for the delivery of curcumin (CUR). Sonication of amphiphilic GA solution with hydrophobic CUR resulted in the production of nanosized complexes with a size of 164.8 ± 51.7 nm, which greatly enhanced the solubility of CUR in aqueous solution. A majority of the CURs were released from these GA/ CUR nanocomplexes within 12 h. GA/CUR nanocomplexes exhibited excellent intracellular uptake in human breast cancer cells (Michigan cancer foundation-7/multi-drug resistant cells), indicating enhanced anti-cancer effects compared to that of free CUR. In addition, GA/CUR nanocomplexes demonstrated high intracellular uptake into macrophages (RAW264.7 cells), consequently reducing the release of the pro-inflammatory cytokine tumor necrosis factor-α. Furthermore, GA/CUR nanocomplexes successfully reduced the levels of serum pro-inflammatory cytokines and splenomegaly in a rheumatoid arthritis model.

Chronic infrared-A irradiation-induced photoaging of human dermal fibroblasts from different donors at physiological temperature.

BACKGROUND: In this study, we examined cellular responses to acute and chronic IRA irradiation at mild and natural levels of exposure in two types of human fibroblasts, each isolated from a different donor, at physiological temperature (34°C). METHOD: Two types of human dermal fibroblasts (derived from a 20- and 50-year-old women, respectively) were exposed to different repeat numbers of IRA exposure (3, 6, 10, and 14 times; 42 mW/cm2 ) at a frequency of 3-4 times per week (4 h per irradiation). Cellular responses to acute and chronic IRA irradiation were examined by reactive oxygen species (ROS) level, apoptotic signals, cellular morphology, and collagen level. RESULTS: We demonstrated that chronic IRA irradiation-induced severe cellular damage, including prolonged cell proliferation, increased intracellular ROS levels, activated cellular apoptosis, and elongated cell morphology, whereas acute IRA irradiation had negligible effects at 34°C. In addition, it was evident that the degree of cellular damage due to IRA irradiation differed according to the type of fibroblasts. CONCLUSIONS: Considering the severe cellular damage induced by chronic IRA irradiation without heat, continuous exposure of skin to IRA irradiation during daily life may be harmful enough to induce photoaging.

Exosome-mediated delivery of transforming growth factor-ß receptor 1 kinase inhibitors and toll-like receptor 7/8 agonists for combination therapy of tumors.

In this study, combination therapy with the transforming growth factor-ß receptor I (TGFßRI) kinase inhibitor SD-208 and a toll-like receptor (TLR)-7/8 agonist resiquimod (R848) was examined along with serum-derived exosomes (EXOs) as versatile carriers. SD-208-encapsulated EXOs (SD-208/EXOs) and R848-encapsulated EXOs (R848/EXOs) were successfully prepared with a size of 87 ± 8 nm and 51 ± 4 nm, respectively, which were stable in aqueous solution at pH 7.4. SD-208/EXOs and R848/EXOs reduced the migration of cancer cells (B16F10 and PC-3) and triggered the release of proinflammatory cytokines from stimulated macrophages and dendritic cells, respectively. The fluorescent dye-labeled EXOs showed significantly improved penetration through the PC-3/fibroblast co-culture spheroids and enhanced accumulation in the B16F10 mouse tumor model compared with the free fluorescent dye. In addition, the combination therapy of R848/EXOs (R848 dose of 0.36 mg/kg) and SD-208/EXOs (SD-208 dose of 0.75 mg/kg) reduced tumor growth and improved survival rate at low doses in the B16F10 tumor xenograft model. Taken together, the combination therapy using the TGFßRI kinase inhibitor and TLR 7/8 agonist with EXOs may serve as a promising strategy to treat melanoma and prostate cancer. STATEMENT OF SIGNIFICANCE: Owing to the prevalence of several non-responding cancers that resist treatment, it is necessary to identify a novel combined treatment strategy with biomaterials to maximize therapeutic efficacy and minimize the undesirable side effects. In this study, we aimed to examine the use of the TGFßRI kinase inhibitor SD-208 and the TLR7/8 agonist resiquimod (R848) encapsulated within serum-derived EXOs for their synergistic antitumor effects. We first demonstrated that combined treatment with SD-208 and R848 can be a convincing strategy to circumvent tumor growth in vivo using serum-derived exosomes as promising carriers. Therefore, we believe this manuscript would be of great interest to the biomaterial communities especially who are studying immunotherapy.

Cancer-Cell-Derived Hybrid Vesicles from MCF-7 and HeLa Cells for Dual-Homotypic Targeting of Anticancer Drugs.

Here, as a proof of concept, hybrid vesicles (VEs) are developed from two types of cancer cells, MCF-7 and HeLa, for the dual targeting of the anticancer drug doxorubicin (Dox) to cancer cells via homotypic interactions. Hybrid VEs with a size of 181.8 ± 28.2 nm and surface charge of -27.8 ± 1.9 mV are successfully prepared by the fusion of MCF-7 and HeLa VEs, as demonstrated by the fluorescence resonance energy transfer assay. The hybrid VEs exhibit enhanced intracellular uptake both in MCF-7 and HeLa cells. Dox-encapsulated hybrid VEs (Dox-hybrid VEs) also exhibit promising anticancer and antiproliferative activities against MCF-7/multidrug-resistant cells and HeLa cells. In addition, compared to free Dox, the Dox-hybrid VEs exhibit low intracellular uptake and reduced cytotoxicity for RAW264.7 cells. Thus, hybrid VEs with dual-targeting activity toward two types of cancer cells may be useful for the specific targeting of anticancer drugs for improved anticancer effects with reduced nonspecific toxicity.

Activated Platelet-Derived Vesicles for Efficient Hemostatic Activity.

In this study, activated platelet-derived vesicles (Act-VEs) are developed as a novel hemostatic biomaterial. Spherical Act-VEs (114.40 ± 11.69 nm in size) with surface charges of -24.73 ± 1.32 mV are successfully prepared from thrombin-activated murine platelets with high surface expression of active glycoprotein IIb/IIIa (GP IIb/IIIa, also known as αIIbß3) and P-selectin. Although nanosized vesicles from resting platelets (VEs) and Act-VEs showed similar sizes and surface charges, Act-VEs formed much larger aggregates in the presence of thrombin and CaCl2 , compared to VEs. After incubation with fibrinogen, Act-VEs formed much denser fibrin networks compared to platelets or VEs, probably due to active αIIbß3 on the surfaces of the Act-VEs. After intravenous injection of the Act-VEs, tail bleeding time and the blood loss are greatly reduced by Act-VEs in vivo. In addition, Act-VEs showed approximately sevenfold lower release of pro-inflammatory interleukin-1ß (IL-1ß) during incubation for 4 days, compared to platelets. Taken together, the formulated Act-VEs can serve as a promising hemostatic biomaterial for the efficient formation of fibrin clots without releasing pro-inflammatory cytokine.