Light emitting diode (LED) irradiation of liposomes enhances drug encapsulation and delivery for improved cancer eradication.

Liposomes are widely used as drug delivery nanoplatforms because of their versatility and biocompatibility; however, their ability to load certain drugs may be suboptimal. In this study, we generated liposomes using a combination of DSPE and DSPE-PEG-2 k lipids and loaded them with doxorubicin (DOX) and paclitaxel (PTX), to investigate the effects of light emitting diode (LED) irradiation on liposome structure and drug loading efficiency. Scanning and transmission electron microscopy revealed that the surface of liposomes irradiated with blue or near-infrared LEDs (LsLipo) was rougher and more irregular than that of non-LED-irradiated liposomes (NsLipo). Nuclear magnetic resonance analysis showed that the hydrogen peak originating from the lipid head groups was lower in LsLipo than in NsLipo preparations, indicating that LED irradiation changed the chemical and physical properties of the liposome. Structural changes, such as reduced rigidity, induced by LED irradiation, increased the loading efficiency of DOX and PTX. In vitro and in vivo experiments showed that LsLipo were more effective at inhibiting the growth of cancer cells than NsLipo. Our findings suggest that LED irradiation enhances the drug delivery efficacy of liposomes and offer new possibilities for improving drug delivery systems.

Nano-chemical priming strategy to enhance TGF-ß resistance and anti-tumor activity of natural killer cells.

Immunotherapy based on adoptive transfer of natural killer (NK) cells is a promising strategy for circumventing the limitations of cancer treatments. However, components of the immunosuppressive tumor microenvironment (TME), such as transforming growth factor-beta (TGF-ß), compromise the therapeutic efficacy of NK cells significantly. To address these limitations, we developed a novel method of engineering NK cells for adaptive transfer. The method is based on nanogels that serve two functions: (1) they overcome the TGF-ß-mediated stress environment of the TME, and (2) they enhance the direct anti-tumor activity of NK cells. Previously, we demonstrated that cationic compounds such as 25 K branched polyethylenimine (25 K bPEI) prime NK cells, putting them in a 'ready-to-fight' state. Based on these findings, we designed nanogels that have two primary characteristics: (1) they encapsulate galunisertib (Gal), which is used clinically to inhibit TGF-ß receptor activity, thereby blocking TGF-ß signaling; and (2) they provide cells with a surface coating of 25 K bPEI. When grown in culture medium containing TGF-ß, nanogel-treated NK cells demonstrated greater migration ability, degranulation activity, and cytotoxicity towards cancer cells than untreated NK cells. Additionally, the in vivo efficacy of nanogel-treated NK cells against PC-3 xenografts was significantly greater than that of Chem_NK cells primed by 25 K bPEI alone. These findings suggest that Gal-loaded 25 K bPEI-coated nanogels exert anti-tumor effects via chemical priming, as well suppressing the effects of TGF-ß on NK cells. We also expect 25 K bPEI-based nanogels to have great potential to overcome the suppressive effects of the TME through their NK cell-priming activity and delivery of the desired chemicals.

Upconverting-photon quenching-mediated perforation influx as an intracellular delivery method using posAuNP@UCNPs nanocomposites for osteoarthritis treatment.

Photoporation techniques based on plasmonic nanoparticles such as gold nanoparticles have been extensively studied for the intracellular delivery of substances via cell membrane disruption. However, the clinical application of AuNP is challenging due to its absorption in the 500 nm region of the light spectrum. To overcome this challenge, upconversion nanoparticles were employed to stimulate AuNP at NIR wavelengths. posAuNP@UCNPs nanocomposites were produced by coating 30 nm UCNPs on 80 nm AuNPs using DOPA-PEI, which were then irradiated with 980 nm NIR light to facilitate their intracellular delivery. TEM and DLS confirmed that posAuNP and UCNP combine to form nanocomposites. Additionally, multiphysics simulation was used to analyze the distribution of the posAuNP electric field based on morphological differences that change as the UCNP ratio increases. Next, effective LED irradiation conditions were established by applying upconverting-photon quenching-mediated perforation influx to C28/I2 cells as suspensions or spheroids. posAuNP@UCNP nanocomposites were confirmed to be effective for the delivery of baricitinib as a treatment for osteoarthritis in a three-dimensional osteoarthritis model. Finally, chondrocyte differentiation was induced through intracellular delivery of baricitinib using posAuNP@UCNPs. The findings suggest that posAuNP@UCNPs have great potential as a tool for non-invasive drug delivery via UCPPin.

Fusogenic liposomes encapsulating mitochondria as a promising delivery system for osteoarthritis therapy.

Many attempts have been made to use mitochondria (MT) to treat human diseases; however, MT are large, making them difficult to deliver effectively. Therefore, a transfer strategy based on membrane fusion was established. Fusogenic mitochondrial capsules (FMCs) comprising a neutral lipid (PE), a cationic lipid (DOTAP), an aromatic lipid (Liss Rhod PE), and three types of liposome (FMC0, FMC1, and FMC2), were designed and synthesized. The amount of DOTAP, which affects membrane fusion efficiency, differed between FMC preparations. The characteristics of these FMCs were analyzed by DLS, TEM, and AFM, and the encapsulation and fusion efficiency between FMC-MT and FMC-chondrocytes were confirmed by FRET, mtDNA copy number, and CLSM, respectively. Compared with naked MT, delivery of FMCs to chondrocytes was faster and more efficient. Moreover, fusion was a more stable delivery method than endocytosis, as evidenced by reduced induction of mitophagy. In vitro and in vivo experiments revealed that FMCs reduced expression of inflammatory cytokines and MMP13, increased expression of extracellular matrix components, and promoted cartilage regeneration. These findings suggest that FMCs are a highly effective and promising strategy for delivery of MT to promote cartilage regeneration, and highlight their potential as a novel platform for MT transfer therapy.

Melatonin loaded PLGA nanoparticles effectively ameliorate the in vitro maturation of deteriorated oocytes and the cryoprotective abilities during vitrification process.

Almost all cells can be exposed to stress, but oocytes, which are female germ cells, are particularly vulnerable to damage. In this study, melatonin, a well-known antioxidant, was loaded into biodegradable poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) and delivered to damaged oocytes in order to improve their quality and restoration. Etoposide (ETP)-induced deteriorated oocytes show poor maturity, mitochondrial aggregation, and DNA damage. Treatment of NPs not only reduced DNA damage but also improved mitochondrial stability, as evidenced by increased ATP levels and mitochondrial homogeneity. When melatonin was added to the culture medium at the same concentration as that present in NPs, DNA and mitochondrial repair was insignificant due to the half-life of melatonin, whereas DNA repair in damaged oocytes upon multiple treatments with melatonin was similar to that observed with melatonin-loaded NPs. Next, we evaluated whether the oocytes treated with NPs could have cryoprotective abilities during vitrification/thawing. Vitrified-oocytes were stored at -196 °C for 0.25 h (T1) or 0.5 h (T2). After thawing, live oocytes were subjected to in vitro maturation. The NP-treated group showed maturity similar to the control group (77.8% in T1, 72.7% in T2) and the degree of DNA damage was reduced compared to the ETP-induced group (p < 0.05).

Strategies for accelerating osteogenesis through nanoparticle-based DNA/mitochondrial damage repair.

The efficiency of gene therapy is often dictated by the gene delivery system. Cationic polymers are essential elements of gene delivery systems. The relatively cheap cationic polymer, polyethyleneimine, has high gene delivery efficiency and is often used for gene delivery. However, the efficiency of gene therapy with polyethyleneimine-pDNA polyplex (PEI) is low. Human mesenchymal stem cells transfected with polyethyleneimine and a plasmid carrying the important osteogenic differentiation gene runt-related transcription factor 2 (RUNX2) accumulated DNA double-strand breaks and mitochondrial damage proportional to the amount of polyethyleneimine, reducing viability. Genomic/cellular stabilizer mediating RUNX2 delivery (GuaRD), a new reagent incorporating RS-1 NPs developed in this study, promoted DNA repair and prevented the accumulation of cell damage, allowing the delivery of pRUNX2 into hMSCs. while maintaining genome and mitochondrial stability. DNA damage was significantly lower and the expression of DNA repair-related genes significantly higher with GuaRD than with PEI. In addition, GuaRD improved mitochondrial stability, decreased the level of reactive oxygen species, and increased mitochondrial membrane potential. Osteogenic extracellular matrix (ECM) expression and calcification were higher with GuaRD than with PEI, suggesting improved osteogenic differentiation. These results indicate that lowering the cytotoxicity of PEI and improving cell stability are key to overcoming the limitations of conventional gene therapy, and that GuaRD can help resolve these limitations.

Efficient CRISPR-Cas9-based knockdown of RUNX2 to induce chondrogenic differentiation of stem cells.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system recognizes and deletes specific nucleotide sequences in cells for gene editing. This study aimed to edit and knockdown the RUNX2 gene, a key transcription factor that is directly involved in all stages of stem cell differentiation into osteoblasts. The RUNX2 gene was depleted using the CRISPR-Cas9 system to inhibit osteoblast differentiation of stem cells. shRNA vectors targeting RUNX2 were used as a control. The surface of nanoparticles (NPs) was coated with the cationic polymer linear polyethyleneimine. Thereafter, negatively charged CRISPR-Cas9 and shRNA vectors were complexed with positively charged NPs via ionic interactions. Several analytical methods were used to determine the size, surface charge, and morphology of NPs and to characterize the complexed genes. NPs complexed with CRISPR-Cas9 and shRNA vectors were delivered into human mesenchymal stem cells (hMSCs) via endocytosis. The mRNA and protein expression patterns of various genes in hMSCs were measured over time following internalization of NPs complexed with CRISPR-Cas9 and shRNA vectors in two- and three-dimensional culture systems. Knockdown of the RUNX2 gene decreased osteogenic differentiation and increased chondrogenic differentiation of hMSCs. As a result of investigating the efficiency of NPs complexed with CRISPR-Cas9 (CASP-NPs), Runx2 effectively knocked down in mesenchymal stem cells to enhance differentiation into chondrocytes, therefore CASP-NPs proved to be an effective gene carrier in hMSCs.

Organelle targeting using a fluorescent probe that selectively penetrates the zona pellucida.

The characteristics of oocytes, which are female germ cells, have not been studied using optical materials. The structural layers (zona pellucida, ZP) around oocytes make it difficult to deliver drugs aimed at treating infertility. Here, we investigated whether the fluorescent probes sulforhodamine, fluorescein 5(6)-isothiocyanate, tetramethylrhodamine isothiocyanate, cyanine 3 carboxylic acid, and cyanine 5 carboxylic acid penetrate oocytes. By targeting the ZP layer of the oocyte, the characteristics of the model drug, a fluorescent probe, were analyzed, and the position of the probe in the oocyte was confirmed for differences in the characteristics. Penetration of the ZP and delivery into the cytoplasm differed between the fluorescent probes. This was due to their different physiochemical properties, including hydrophobicity (contact angle and surface tension), surfactant activity, and electrical charge. Among the fluorescent probes delivered to cytoplasm, unlike TRITC, Cy3 and Cy5 perturbed oocyte development. These results suggest that in oocytes with high physical barriers (cell membrane, zona pellucida), the delivery efficiency can be estimated by considering the properties (molecular weight and structure, solubility and functional structure, etc.) of the drug. In addition, it suggests that an encapsulated or bound carrier of a drug with properties similar to that of a fluorescent probe can be efficiently delivered into oocytes.

Development of a three-layer consecutive gene delivery system for enhanced bone regeneration.

This study developed a three-layer consecutive gene delivery system (T-CGDS) for timely gene delivery into human mesenchymal stem cells (hMSCs). The timing of transcription factor expression is important to effectively induce bone differentiation. Therefore, a three-layered nanocomposite was fabricated using differently sized gold nanoparticles to promote bone regeneration and osteogenic differentiation. The core layer comprised 80 nm gold nanoparticles coupled with ATF4 pDNA. Following coating with heparin-conjugated Pluronic F-127 (HP-F127), 50 nm gold nanoparticles coupled with SP7 pDNA were added to fabricate a bi-layer system. After further coating with HP-F127, 20 nm gold nanoparticles combined with RUNX2 pDNA were added. Consequently, a T-CGDS measuring 350-450 nm was fabricated. Genes were released for more than 8 days, while the size of the T-CGDS decreased over time. When the T-CGDS was applied to hMSCs, the gene in the outer layer (RUNX2) was expressed first, followed by those in the middle (SP7) and core (ATF4) layers. The T-CGDS effectively induced bone differentiation and regeneration in vitro and in vivo. Timely delivery of the ATF4 gene to stem cells via the T-CGDS can greatly assist osteogenic differentiation involved in bone regeneration.