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Recent technical advances related to the CRISPR/Cas9-based genome editing system have enabled sophisticated genome editing in microalgae. Although the demand for research on genome editing in microalgae has increased over time, methodological research has not been established to date for the delivery of a ribonucleoprotein (Cas9/sgRNA complex) using a cell penetrating peptide into microalgal cell lines. Here, we present a ribonucleoprotein delivery system for Chlamydomonas reinhardtii mediated by the cell penetrating peptide pVEC (LLIILRRRIRKQAHAHSK) which is in a non-covalent form. Using this technically simple method, the ribonucleoprotein was successfully delivered into C. reinhardtii . Gene Maa7 and FKB12 were disrupted, and their distinguishing patterns of Indel mutations were analyzed with the observation of several insertions of sequences not originating from the genome DNA, such as chloroplast DNA, into the expected loci. In addition, the cytotoxicity of Cas9 and the ribonucleoprotein was investigated according to the concentration and time in the algal cells. It was observed that Cas9 alone without the sgRNA induces a more severe cytotoxicity compared to the ribonucleoprotein. Our study will not only contribute to algal cell biology and its genetic engineering for further applications involving various organisms but will also provide a deeper understating of the basic science of the CRISPR/Cas9 system.

Immunogenic cell death (ICD) is distinguished by the release of tumor‐associated antigens (TAAs) and danger‐associated molecular patterns (DAMPs). This cell death has been studied in the field of cancer immunotherapy due to the ability of ICD to induce antitumor immunity. Herein, endoplasmic reticulum (ER) stress‐mediated ICD inducing fluorinated mitochondria‐disrupting helical polypeptides (MDHPs) are reported. The fluorination of the polypeptide provides a high helical structure and potent anticancer ability. This helical polypeptide destabilizes the mitochondrial outer membrane, leading to the overproduction of intracellular reactive oxygen species (ROS) and apoptosis. In addition, this oxidative stress triggers ER stress‐mediated ICD. The in vivo results show that cotreatment of fluorinated MDHP and antiprogrammed death‐ligand 1 antibodies (αPD‐L1) significantly regresses tumor growth and prevents metastasis to the lungs by activating the cytotoxic T cell response and alleviating the immunosuppressive tumor microenvironment. These results indicate that fluorinated MDHP synergizes with the immune checkpoint blockade therapy to eliminate established tumors and to elicit antitumor immune responses. Endoplasmic reticulum (ER) stress‐mediated immunogenic cell death (ICD) inducing fluorinated mitochondria‐disrupting helical polypeptides (MDHPs) are developed. Intracellular oxidative stress by mitochondrial outer membrane destabilization triggers ER stress‐mediated ICD. In addition, combination regimen of fluorinated MDHP and immune checkpoint inhibitor significantly regresses tumor growth and prevents metastasis to the lungs by activating the cytotoxic T cell response.

This study showed that drug-coated PLLA (Poly (L-lactide)) microneedle arrays can induce rapid and painless local anesthesia. Microneedle arrays were fabricated using a micro-molding technique, and the needle tips were coated with 290.6 ± 45.9 μg of lidocaine, the most widely used local anesthetic worldwide. A dip-coating device was newly designed for the coating step using an optimized coating formulation. Lidocaine coated on the arrays was released rapidly into PBS within 2 min, and its stability in storage lasted 3 weeks at 4, 25, and 37°C. Furthermore, the microneedle arrays showed consistent in vitro skin penetration and delivered 200.8 ± 43.9, 224.2 ± 39.3, and 244.1 ± 19.6 μg of lidocaine into the skin 1, 2, and 5 min after application with a high delivery efficiency of 69, 77, and 84%. Compared to a commercially available topical anesthetic EMLA® cream, a 22.0, 13.6, and 14.0-fold higher amount of lidocaine was delivered into the skin. Note, in vitro skin permeation of Lidocaine was also notably enhanced by a 2-min-application of the lidocaine-coated microneedle arrays. Altogether, these results suggest that the biocompatible lidocaine-coated PLLA microneedle arrays could provide significantly rapid local anesthesia in a painless manner without any of the issues from topical applications or hypodermic injections of local anesthetics.

A noninvasive and selective therapy, photodynamic therapy (PDT) is widely researched in clinical fields; however, the lower efficiency of PDT can induce unexpected side effects. Mitochondria are extensively researched as target sites to maximize PDT effects because they play crucial roles in metabolism and can be used as cancer markers due to their high transmembrane potential. Here, a mitochondria targeting photodynamic therapeutic agent (MitDt) is developed. This photosensitizer is synthesized from heptamethine cyanine dyes, which are conjugated or modified as follows. The heptamethine meso‐position is conjugated with a triphenylphosphonium derivative for mitochondrial targeting, the N‐alkyl side chain is modified for regulation of charge balance and solubility, and the indolenine groups are brominated to enhance reactive oxygen species generation (ROS) after laser irradiation. The synthesized MitDt increases the cancer uptake efficiency due to the lipo‐cationic properties of the triphenylphosphonium, and the PDT effects of MitDt are amplified after laser irradiation because mitochondria are susceptible to ROS, the response to which triggers an apoptotic anticancer effect. Consequently, these hypotheses are demonstrated by in vitro and in vivo studies, and the results indicate strong potential for use of MitDts as efficient single‐molecule‐based PDT agents for cancer treatment. Mitochondria targeting photodynamic therapeutic agents (MitDt) including triphenylphosphonium, which can be guided to mitochondrial membranes, are devised. Among them, the brominated indolenine structure can boost reactive oxygen species production after near‐infrared (NIR) laser irradiation, which elicits outstanding cellular apoptotic death in vitro and in vivo. Therefore, MitDt suggests a promising therapeutic probe for cancer therapy.

Background Growth factors (GFs) are signaling proteins that affect cellular processes such as growth, proliferation, and differentiation. GFs are used as cosmeceuticals, exerting anti-wrinkle, anti-aging, and whitening effects, and also as pharmaceuticals to treat wounds, growth failure, and oral mucositis. However, in mammalian and bacterial cells, low productivity and expression in inclusion bodies, respectively, of GFs does not satisfy the consumer demand. Here, we aimed to develop a bacterial expression system that produces high yields of soluble GFs that can be purified in their native forms. Results We present Fh8, an 8-kDa peptide from Fasciola hepatica with an N-terminal hexa-histidine (6HFh8), as a fusion partner for enhanced human GF production in recombinant Escherichia coli . The fusion partner harboring a tobacco etch virus (TEV) protease cleavage site was fused to the N-terminus of 10 human GFs: acidic and basic fibroblast growth factors (aFGF and bFGF, respectively), epidermal growth factor (EGF), human growth hormone (hGH), insulin-like growth factor 1 (IGF-1), vascular endothelial growth factor 165 (VEGF165), keratinocyte growth factor 1 (KGF-1), placental growth factor (PGF), stem cell factor (SCF), and tissue inhibitor of metalloproteinase 1 (TIMP-1). The fusion proteins were expressed in E. coli under the control of T7 promoter at three temperatures (25 °C, 30 °C, and 37 °C). All individual fusion proteins, except for SCF and TIMP-1, were successfully overexpressed in cytoplasmic soluble form at more than one temperature. Further, the original aFGF, IGF-1, EGF, and VEGF165 proteins were cleaved from the fusion partner by TEV protease. Five-liter fed-batch fermentation approaches for the 6HFh8-aFGF (lacking disulfide bonds) and 6HFh8-VEGF165 (a cysteine-rich protein) were devised to obtain the target protein at concentrations of 9.7 g/l and 3.4 g/l, respectively. The two GFs were successfully highly purified (> 99% purity). Furthermore, they exerted similar cell proliferative effects as those of their commercial equivalents. Conclusions We demonstrated that 6HFh8-GF fusion proteins could be overexpressed on a g/l scale in the cytoplasm of E. coli , with the GFs subsequently highly purified and maintaining their biological activity. Hence, the small protein 6HFh8 can be used for efficient mass-production of various GFs.

This work aimed at formulating paclitaxel (PTX) loaded cell penetrating peptide (CPP) coated Mn doped ZnS nanoparticles (Mn:ZnS NPs) for improved anti-cancer efficacy in vitro and in vivo . The developed PTX loaded Mn:ZnS NPs with different CPPs (PEN, pVEC and R9) showed enhanced anti-cancer effect compared to bare PTX, which has been validated by MTT assay followed by apoptosis assay and DNA fragmentation analysis. The in vivo bio-distribution and anti-cancer efficacy was studied on breast cancer xenograft model showing maximum tumor localization and enhanced therapeutic efficacy with R9 coated Mn:ZnS NPs (R9:Mn:ZnS NPs) and was confirmed by H/E staining. Thus, R9:Mn:ZnS NPs could be an ideal theranostic nano-carrier for PTX with enhanced  the rapeutic efficacy toward cancer cells, where penetration and sustainability of therapeutics are essential.

Heart failure (HF) is a coronary disease that affects people worldwide and has a high mortality rate. N-terminal pro-brain natriuretic peptide (NT-proBNP) has been proven to be a useful and accurate biomarker for diagnosing systolic HF. Here, we report a strategy for the high-level production of recombinant (r)NT-proBNP in Escherichia coli . An Fh8 tag with six histidines was fused to the N terminus of NT-proBNP along with the recognition site of tobacco etch virus (TEV) protease; the 6HFh8-NT-proBNP fusion peptide was expressed in flask cultures of E. coli in almost completely soluble form. The peptide was purified by HisTrap affinity chromatography, and the N-terminal tag was cleaved by TEV protease. After a second round of HisTrap affinity chromatography to remove the TEV protease and N-terminal tag, rNT-proBNP was isolated with high purity (≥ 98%) by carboxymethyl cation exchange chromatography. The final yield of purified rNT-proBNP (97.5 mg/l of bacterial culture; 3.25 mg/g of wet cell) was 55-fold higher than that reported in previous studies (0.5–1.75 mg/l of bacterial culture). Furthermore, the high cell density E. coli fed-batch culture enabled high-level production of rNT-proBNP in the order of grams per liter. The purified rNT-proBNP was detected by enzyme-linked immunosorbent assay and chemiluminescence enzyme immunoassay using commercial monoclonal antibodies recognizing different epitopes, showing a linear dose-response relationship in the range of tested concentrations (slope = 3.58 and r 2  = 0.995). These results demonstrate the efficiency of our process for mass producing (gram-to-liter level) rNT-proBNP with acceptable analytical performance.

Perturbation of potassium homeostasis can affect various cell functions and lead to the onset of programmed cell death. Although ionophores have been intensively used as an ion homeostasis disturber, the mechanisms of cell death are unclear and the bioapplicability is limited. In this study, helical polypeptide‐based potassium ionophores are developed to induce endoplasmic reticulum (ER) stress‐mediated apoptosis. The polypeptide‐based potassium ionophores disturb ion homeostasis and then induce prolonged ER stress in the cells. The ER stress results in oxidative environments that accelerate the activation of mitochondria‐dependent apoptosis. Moreover, ER stress‐mediated apoptosis is triggered in a tumor‐bearing mouse model that suppresses tumor proliferation. This study provides the first evidence showing that helical polypeptide‐based potassium ionophores trigger ER stress‐mediated apoptosis by perturbation of potassium homeostasis. The perturbation of potassium homeostasis is a promising strategy for the induction of apoptosis. Novel potassium ionophores disturbing ion homeostasis, thereby inducing endoplasmic reticulum stress‐mediated apoptosis in vitro and in vivo, are developed. This work reports the apoptotic mechanism by perturbing ion homeostasis, and also shows the bioapplicability as a potential anticancer agent.

Antimicrobial peptides are potential therapeutics as anti-bacteria, anti-viruses, anti-fungi, or anticancers. However, they suffer from a short half-life and drug resistance which limit their long-term clinical usage. Herein, we captured the encapsulation of antimicrobial peptide HA-FD-13 into boron nitride nanotube (BNNT) (20,20) and its release due to subsequent insertion of BNNT (14,14) with molecular dynamics simulation. The peptide-BNNT (20,20) van der Waals (vdW) interaction energy decreased to -270 kcal·mol at the end of the simulation (15 ns). However, during the period of 0.2-1.8 ns, when half of the peptide was inside the nanotube, the encapsulation was paused due to an energy barrier in the vicinity of BNNT and subsequently the external intervention, such that the self-adjustment of the peptide allowed full insertion. The free energy of the encapsulation process was -200.12 kcal·mol , suggesting that the insertion procedure occurred spontaneously. Once the BNNT (14,14) entered into the BNNT (20,20), the peptide was completely released after 83.8 ps. This revealed that the vdW interaction between the BNNT (14,14) and BNNT (20,20) was stronger than between BNNT (20,20) and the peptide; therefore, the BNNT (14,14) could act as a piston pushing the peptide outside the BNNT (20,20). Moreover, the sudden drop in the vdW energy between nanotubes to the value of the -1300 Kcal·mol confirmed the self-insertion of the BNNT (14,14) into the BNNT (20,20) and correspondingly the release of the peptide.

The stratum corneum is the outermost skin layer that obstructs the delivery of active ingredients found in cosmeceutical products. Chemical peels and microbeads have been used to overcome this layer, but these methods can cause side effects and are not environmentally friendly. While microneedles do not share the dangers mentioned above, they are currently only available as patches, which makes them unsuitable to be used with products that are usually applied onto a large area of the skin surface. Therefore, the aim of this study was to develop microneedle-like particles (MLP) whose needles would disrupt the skin during the rubbing process. A modified approach taken from conventional micromolding techniques was used to make the MLPs. The experimental results show that the fabricated structures had the required mechanical strength. Furthermore, after the application of the MLPs, the permeability of two fluorescent dyes, fluorescein sodium salt and sulforhodamine B increased to 217.6% ± 25.6% and 251.7% ± 12.8% respectively. Additionally, the permeability of a model drug, niacinamide, was shown to have increased to 193.8% ± 29.9%. Cryosectioned porcine slices also confirmed the ability of MLPs to enhance skin permeability by revealing a deeper penetration of the applied fluorescent dye. Altogether, the results demonstrate the potential of MLPs to be used as safe skin permeability enhancers that can be applied all over the skin.