Optimization of polyethylene glycol shielding and mannose density on the lipid nanoparticles for efficient delivery to macrophages and spleens.

This study compared the effects of polyethylene glycol (PEG) shielding and mannose-conjugated ligands density on lipid nanoparticles (LNPs) for intracellular uptake to macrophages in vitro and accumulation in spleens in vivo. Fabricated phosphatidyl serine-incorporated LNPs (sLNPs) was physically decorated with mannose-conjugated DSPE-PEG (DPM) at different DPM/LNP molar ratios achieving the DPM density from 0 to 0.6 PEGs/nm2. We demonstrated that low PEG shielding sLNPs with mannose ligands (sLNP-DPMs) displayed superior uptake to macrophages (RAW 264.7 cells) compared with high PEG shielding sLNP-DPMs in vitro. However, high PEG shielding sLNP-DPMs showed significant spleen accumulation compared with low PEG shielding sLNP-DPMs in vivo after intravenous injection. In particular, high PEG shielding sLNPs coated with DSPE-methoxyPEG (DP) and DPM mixture at DP/DPM molar ratios of 5/5 exhibited greater accumulation in red pulp of spleens than naked sLNPs by 2.7-folds in vivo. These results suggested that the optimal PEG shielding and mannose densities per a particle might be different between in vitro cellular uptake to macrophages and in vivo spleen accumulation after systemic administration. Taken together, precision-tailored LNP-surface modifications achieved through optimization of PEG shielding and mannose density can greatly enhance accumulation of LNPs in red pulp of spleens, which could be applied for the delivery of nucleic acid-based drugs and vaccines to spleens in vivo.

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.

Role of ginseng in the neurovascular unit of neuroinflammatory diseases focused on the blood-brain barrier.

Ginseng has long been considered as an herbal medicine. Recent data suggest that ginseng has anti-inflammatory properties and can improve learning- and memory-related function in the central nervous system (CNS) following the development of CNS neuroinflammatory diseases such as Alzheimer's disease, cerebral ischemia, and other neurological disorders. In this review, we discuss the role of ginseng in the neurovascular unit, which is composed of endothelial cells surrounded by astrocytes, pericytes, microglia, neural stem cells, oligodendrocytes, and neurons, especially their blood-brain barrier maintenance, anti-inflammatory effects and regenerative functions. In addition, cell-cell communication enhanced by ginseng may be attributed to regeneration via induction of neurogenesis and angiogenesis in CNS diseases. Thus, ginseng may have therapeutic potential to exert cognitive improvement in neuroinflammatory diseases such as stroke, traumatic brain injury, multiple sclerosis, Parkinson's disease, and Alzheimer's disease.

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.

Mannose-Modified Serum Exosomes for the Elevated Uptake to Murine Dendritic Cells and Lymphatic Accumulation.

The surface of bovine serum-derived exosomes (EXOs) are modified with α-d-mannose for facile interaction with mannose receptors on dendritic cells (DCs) and for efficient delivery of immune stimulators to the DCs. The surface of the EXOs is modified with polyethylene glycol (PEG) without particle aggregation (≈50 nm) via the incorporation of 1,2-distearoyl-sn-glycero-3-phosphoethanolamine (DSPE) into the lipid layer of the EXO, compared to chemical conjugation by N-hydroxysuccinimide activated PEG (NHS-PEG). PEG modification onto the exosomal surface significantly decreases the non-specific cellular uptake of the EXOs into the DCs. However, the EXOs with mannose-conjugated PEG-DSPE (EXO-PEG-man) exhibit excellent intracellular uptake into the DCs and boost the immune response by the incorporation of adjuvant, monophosphoryl lipid A (MPLA) within the EXO. After an intradermal injection, a higher retention of EXO-PEG-man is observed in the lymph nodes, which could be used for the efficient delivery of immune stimulators and antigens to the lymph nodes in vivo.

Byakangelicin as a modulator for improved distribution and bioactivity of natural compounds and synthetic drugs in the brain.

BACKGROUND: The elucidation of the biological roles of individual active compounds in terms of their in vivo bio-distribution and bioactivity could provide crucial information to understand how natural compounds work together as treatments for diseases. PURPOSE: We examined the functional roles of Byakangelicin (Byn) to improve the brain accumulation of active compounds, e.g., umbelliferone (Umb), curcumin (Cur), and doxorubicin (Dox), and consequently to enhance their biological activities. METHODS: Active compounds were administered intravenously to mice, with or without Byn, after which organs were isolated and visualized for their ex vivo fluorescence imaging to determine the bio-distribution of each active compound in vivo. For the in vivo bioactivity, Cur, either with or without Byn, was administered to a lipopolysaccharide (LPS)-induced neuro-inflammation model for 5 days, and its anti-inflammatory effects were examined by ELISA using a brain homogenate and serum. RESULTS: We successfully demonstrated that the levels of active compounds (Umb, Cur, and Dox) in the brain, lung, and pancreas were greatly elevated by the addition of Byn via direct ex vivo fluorescence monitoring. In addition, sufficient accumulation of the active compound, Cur, greatly reduced LPS-induced neuro-inflammation in vivo. CONCLUSION: Byn could serve as a modulator to allow improved brain accumulation of diverse active compounds (Umb, Cur, and Dox) and enhanced therapeutic effects.

Enhanced intracellular uptake and stability of umbelliferone in compound mixtures from Angelica gigas in vitro.

Understanding how natural compounds work together for disease treatments can improve their clinical efficacy and therapeutic effects. To elucidate the mechanisms of synergistic biological effects in natural compound mixtures, umbelliferone (UMB, 7-hydroxycoumarin), derived from Angelica (A.) gigas, was selected as active compound with fluorescent characteristic to examine bioactivities in vitro in the presence of other compounds from Angelica gigas. Antioxidant effects of UMB in biochemical assays and cellular reactive oxygen species (ROS) levels in RAW264.7 cells were not significantly improved by addition of other compounds. However, intracellular uptake, inhibition of the efflux pump P-glycoprotein (P-gp), and physiological stability of UMB were greatly enhanced by the addition of other compounds, specifically Angelicin (ANG) and Byakangelicin (BYN). Taken together, enhanced intracellular localization and enzymatic stability in compound mixtures might lead to superior synergistic bioactivity of UMBs in compound mixtures.

PLGA Microspheres Coated with Cancer Cell-Derived Vesicles for Improved Internalization into Antigen-Presenting Cells and Immune Stimulation.

Microspheres (MS; 1-3 µm) with different degrees of surface roughness were prepared to assess the effects of surface topology on internalization into antigen-presenting cells (APCs; macrophages and dendritic cells). In this study, we demonstrated that the intracellular uptake of MS is readily enhanced by surface modification with nanoparticles or cancer cell-derived vesicles (VE) to modulate their surface topology. MS coated with nanovesicles (MS-VE) with high surface roughness was more successfully and efficiently engulfed by APCs, compared with bare MS and those with low surface roughness. Incorporated MPLA within MS-VEs (M/MS-VE) triggered greatly elevated release of immune stimulating cytokines, interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α), from macrophages and dendritic cells, compared to free MPLA. Taken together, this MS-VE could serve as a platform system for the delivery of immune stimulators and antigens to APCs with negligible toxicity.

Platelet-derived nanovesicles for hemostasis without release of pro-inflammatory cytokines.

Natural platelet-derived nanovesicles with a vacant core were prepared by hypotonic sonication. The nanovesicles efficiently formed platelet-like aggregates using membrane protein in the presence of thrombin and calcium chloride without a notable release of pro-inflammatory cytokines. These natural and biocompatible platelet-derived nanovesicles have great potential as biomaterials for inflammation-free injectable hemostasis.

Correction: CpG incorporated DNA microparticles for elevated immune stimulation for antigen presenting cells.

[This corrects the article DOI: 10.1039/C7RA13293J.].

Efficient Delivery of Tyrosinase Related Protein-2 (TRP2) Peptides to Lymph Nodes using Serum-Derived Exosomes.

Exosomes (EXO) are considered to be versatile carriers for biomolecules; however, the delivery of therapeutic peptides using EXOs poses several challenges. In this study, the efficiency of serum-derived EXOs in delivering tyrosinase-related protein-2 (TRP2) peptides to lymph nodes is determined. TRP2 peptides are successfully incorporated into EXOs, which show a uniform and narrow size distribution of around 45 nm. The TRP2-incorporated exosomes (EXO-TRP2) are efficiently internalized into macrophages and dendritic cells, and are seen to display a punctate distribution. EXOs loaded with TRP2 together with MPLA, (EXO-MPLA-TRP2) result in a strong release of proinflammatory cytokines (TNF-α and IL-6) from both RAW264.7 and DC2.4 cells. Finally, subcutaneous injection of fluorescently labeled EXO-TRP2 followed by ex vivo imaging using in vivo imaging system (IVIS) show a strong fluorescent signal in the lymph nodes after only 1 h, which is maintained until at least 4 h after injection. Taken together, the findings suggest that serum-derived EXOs can serve as promising carriers to deliver therapeutic peptides to lymph nodes for immunotherapy.

Efficient Enrichment and Analysis of Vicinal-Diol-Containing Flavonoid Molecules Using Boronic-Acid-Functionalized Particles and Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry.

Detection and quantitation of flavonoids are relatively difficult compared to those of other small-molecule analytes because flavonoids undergo rapid metabolic processes, resulting in their elimination from the body. Here, we report an efficient enrichment method for facilitating the analysis of vicinal-diol-containing flavonoid molecules using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. In our strategy, boronic-acid-functionalized polyacrylamide particles were used, where boronic acids bound to vicinal diols to form boronate monoesters at basic pH. This complex remained intact during the enrichment processes, and the vicinal-diol-containing flavonoids were easily separated by centrifugation and subsequent acidic treatments. The selectivity and limit of detection of our strategy were confirmed by mass spectrometry analysis, and the validity was assessed by performing the detection and quantitation of quercetin in mouse organs.

Comparative evaluation of cell- and serum-derived exosomes to deliver immune stimulators to lymph nodes.

To determine whether exosomes are efficient carriers for immune stimulating molecules into lymph nodes, comparative studies of exosomes (EXOs) derived from different origins (cells and serums) in terms of physicochemical properties and delivery efficiency were performed. Serum-derived EXOs were of a preferable size and generated higher yields than RAW264.7 cell-derived exosomes (RAW-EXO). In particular, fetal bovine serum-derived exosomes (bo-EXO), with a size below 50 nm, were delivered not only to surface zones (subcapsular sinus (SCS) macrophage zone) but also to inner paracortex zones (T cell zone) of lymph nodes, which allowed an efficient delivery of immune stimulating molecules to antigen presenting cells and T cells. The encapsulation of immune stimulating biomolecules (monophosphoryl lipid A (MPLA) and CpG oligodeoxynucleotides (CpG ODN)) within EXOs greatly increased intracellular delivery to macrophages via phagocytic pathways, which induced higher TNF-α and IL-6 secretion than free MPLA and free CpG ODN. MPLA-incorporated exosomes activated and differentiated T cells after subcutaneous injection, which elevated cytokine IFN-γ and TNF-α induction for CD3+ T cells. Taken together, bo-EXOs might serve as efficient carrier systems of immune stimulators to lymph nodes for desired immune responses.