Configuration of two cysteine residues in a ring within a stapled Bim peptide affects the secondary structure and apoptotic activity.

Many reports have shown that stabilization of secondary structure by stapling functional peptides enhances the intracellular bioactivity. However, no report has discussed the correlation between stabilization and biological activity based on the configuration of amino acid residues used as anchors for stapling. To clarify this, we investigated the helix content and apoptotic efficiency of an apoptosis-inducing peptide, Bim, and four stapled Bim peptides containing stapling-related Cys residues introduced with different configurations within the sequence. The results demonstrated that the configuration of Cys residues in stapled Bim peptides affected the secondary structure and intracellular activity of the peptides, and furthermore, there was a correlation between these latter two variables.

In Vitro Study of Tumor-Homing Peptide-Modified Magnetic Nanoparticles for Magnetic Hyperthermia.

Cancer cells have higher heat sensitivity compared to normal cells; therefore, hyperthermia is a promising approach for cancer therapy because of its ability to selectively kill cancer cells by heating them. However, the specific and rapid heating of tumor tissues remains challenging. This study investigated the potential of magnetic nanoparticles (MNPs) modified with tumor-homing peptides (THPs), specifically PL1 and PL3, for tumor-specific magnetic hyperthermia therapy. The synthesis of THP-modified MNPs involved the attachment of PL1 and PL3 peptides to the surface of the MNPs, which facilitated enhanced tumor cell binding and internalization. Cell specificity studies revealed an increased uptake of PL1- and PL3-MNPs by tumor cells compared to unmodified MNPs, indicating their potential for targeted delivery. In vitro hyperthermia experiments demonstrated the efficacy of PL3-MNPs in inducing tumor cell death when exposed to an alternating magnetic field (AMF). Even without exposure to an AMF, an additional ferroptotic pathway was suggested to be mediated by the nanoparticles. Thus, this study suggests that THP-modified MNPs, particularly PL3-MNPs, hold promise as a targeted approach for tumor-specific magnetic hyperthermia therapy.

FRET probe for detecting two mutations in one EGFR mRNA.

Technologies for visualizing and tracking RNA are essential in molecular biology, including in disease-related fields. In this study, we propose a novel probe set (DAt-probe and T-probe) that simultaneously detects two mutations in the same RNA using fluorescence resonance energy transfer (FRET). The DAt-probe carrying the fluorophore Atto488 and the quencher Dabcyl were used to detect a cancer mutation (exon19del), and the T-probe carrying the fluorophore Tamra was used to detect drug resistance mutations (T790M) in epidermal growth factor receptor (EGFR) mRNA. These probes were designed to induce FRET when both mutations were present in the mRNA. Gel electrophoresis confirmed that the two probes could efficiently bind to the mutant mRNA. We measured the FRET ratios using wild-type and double-mutant RNAs and found a significant difference between them. Even in living cells, the FRET probe could visualize mutant RNA. As a result, we conclude that this probe set provides a method for detecting two mutations in the single EGFR mRNA via FRET.

Novel Self-Forming Nanosized DDS Particles for BNCT: Utilizing A Hydrophobic Boron Cluster and Its Molecular Glue Effect.

BNCT is a non-invasive cancer therapy that allows for cancer cell death without harming adjacent cells. However, the application is limited, owing to the challenges of working with clinically approved boron (B) compounds and drug delivery systems (DDS). To address the issues, we developed self-forming nanoparticles consisting of a biodegradable polymer, namely, "AB-type Lactosome (AB-Lac)" loaded with B compounds. Three carborane isomers (o-, m-, and p-carborane) and three related alkylated derivatives, i.e., 1,2-dimethy-o-carborane (diC1-Carb), 1,2-dihexyl-o-carborane (diC6-Carb), and 1,2-didodecyl-o-carborane (diC12-Carb), were separately loaded. diC6-Carb was highly loaded with AB-Lac particles, and their stability indicated the "molecular glue" effect. The efficiency of in vitro B uptake of diC6-Carb for BNCT was confirmed at non-cytotoxic concentration in several cancer cell lines. In vivo/ex vivo biodistribution studies indicated that the AB-Lac particles were remarkably accumulated within 72 h post-injection in the tumor lesions of mice bearing syngeneic breast cancer (4T1) cells, but the maximum accumulation was reached at 12 h. In ex vivo B biodistribution, the ratios of tumor/normal tissue (T/N) and tumor/blood (T/Bl) of the diC6-Carb-loaded particles remained stably high up to 72 h. Therefore, we propose the diC6-Carb-loaded AB-Lac particles as a promising candidate medicine for BNCT.

Fluorescence ratiometric DNA detection by peptide nucleic acid-pyrene binary probes.

We developed a method for detecting DNA by excimer fluorescence from two peptide nucleic acids (PNAs) modified with a pyrene (Pyr). The two PNA-Pyr probes were prepared by solid-phase peptide synthesis, and we assessed fluorescence from the mixture of probes with DNA. From the results, excimer fluorescence derived from the two PNA-Pyr probes forming hybrids with the complementary DNA was observed, and the two probes showed the maximum excimer/monomer ratio when the probes and DNA were hybridized at a 1:1:1 ratio, indicating that the PNA-Pyr probes can detect target DNA. Furthermore, we adjusted the spatial arrangement between the two PNA-Pyr hybrids formed on the DNA to promote optimal excimer formation. As a result, optimal excimer formation was achieved by spacing the two nucleobases between the formed two hybrids and further inserting a hexamethylene linker (C6) between the PNA and Pyr of the PNA-Pyr probe on one side.

Potential of a Novel Chemical Compound Targeting Matrix Metalloprotease-13 for Early Osteoarthritis: An In Vitro Study.

Osteoarthritis is a progressive disease characterized by cartilage destruction in the joints. Matrix metalloproteinases (MMPs) and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTSs) play key roles in osteoarthritis progression. In this study, we screened a chemical compound library to identify new drug candidates that target MMP and ADAMTS using a cytokine-stimulated OUMS-27 chondrosarcoma cells. By screening PCR-based mRNA expression, we selected 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide as a potential candidate. We found that 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide attenuated IL-1ß-induced MMP13 mRNA expression in a dose-dependent manner, without causing serious cytotoxicity. Signaling pathway analysis revealed that 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide attenuated ERK- and p-38-phosphorylation as well as JNK phosphorylation. We then examined the additive effect of 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide in combination with low-dose betamethasone on IL-1ß-stimulated cells. Combined treatment with 2-(8-methoxy-2-methyl-4-oxoquinolin-1(4H)-yl)-N-(3-methoxyphenyl) acetamide and betamethasone significantly attenuated MMP13 and ADAMTS9 mRNA expression. In conclusion, we identified a potential compound of interest that may help attenuate matrix-degrading enzymes in the early osteoarthritis-affected joints.

Cannabidiol inhibits SARS-CoV-2 replication through induction of the host ER stress and innate immune responses.

The spread of SARS-CoV-2 and ongoing COVID-19 pandemic underscores the need for new treatments. Here we report that cannabidiol (CBD) inhibits infection of SARS-CoV-2 in cells and mice. CBD and its metabolite 7-OH-CBD, but not THC or other congeneric cannabinoids tested, potently block SARS-CoV-2 replication in lung epithelial cells. CBD acts after viral entry, inhibiting viral gene expression and reversing many effects of SARS-CoV-2 on host gene transcription. CBD inhibits SARS-CoV-2 replication in part by up-regulating the host IRE1α RNase endoplasmic reticulum (ER) stress response and interferon signaling pathways. In matched groups of human patients from the National COVID Cohort Collaborative, CBD (100 mg/ml oral solution per medical records) had a significant negative association with positive SARS-CoV-2 tests. This study highlights CBD as a potential preventative agent for early-stage SARS-CoV-2 infection and merits future clinical trials. We caution against use of non-medical formulations including edibles, inhalants or topicals as a preventative or treatment therapy at the present time.

Selective Preparation and High Dynamic-Range Analysis of Cannabinoids in "CBD Oil" and Other Cannabis sativa Preparations.

Much confusion exists about the chemical composition of widely sold Cannabis sativa products that utilize the cannabidiol (CBD) acronym and related terms such as "CBD oil", "CBD plus hemp oil", "full spectrum CBD", "broad spectrum CBD", and "cannabinoids". Their rational chemical and subsequent biological assessment requires both knowledge of the chemical complexity and the characterization of significant individual constituents. Applicable to hemp preparations in general, this study demonstrates how the combination of liquid-liquid-based separation techniques, NMR analysis, and quantum mechanical-based NMR interpretation facilitates the process of natural product composition analysis by allowing specific structural characterization and absolute quantitation of cannabinoids present in such products with a large dynamic range. Countercurrent separation of a commercial "CBD oil" yielded high-purity CBD plus a more polar cannabinoid fraction containing cannabigerol and cannabidivarin, as well as a less polar cannabinoid fraction containing cannabichromene, trans-Δ9-tetrahydrocannabinol, cis-Δ9-tetrahydrocannabinol, and cannabinol. Representatives of six cannabinoid classes were identified within a narrow range of polarity, which underscores the relevance of residual complexity in biomedical research on cannabinoids. Characterization of the individual components and their quantitation in mixed fractions were undertaken by TLC, HPLC, 1H (q)NMR spectroscopy, 1H iterative full spin analysis (HiFSA), 13C NMR, and 2D NMR. The developed workflow and resulting analytical data enhance the reproducible evaluation of "CBD et al." products, which inevitably represent complex mixtures of varying molecular populations, structures, abundances, and polarity features.

Photocontrolled apoptosis induction using precursor miR-664a and an RNA carrier-conjugated with photosensitizer.

Methods to spatially induce apoptosis are useful for cancer therapy. To control the induction of apoptosis, methods using light, such as photochemical internalization (PCI), have been developed. We hypothesized that photoinduced delivery of microRNAs (miRNAs) that regulate apoptosis could spatially induce apoptosis. In this study, we identified pre-miR-664a as a novel apoptosis-inducing miRNA via mitochondrial apoptotic pathway. Further, we demonstrated the utility of photoinduced cytosolic dispersion of RNA (PCDR), which is an intracellular RNA delivery method based on PCI. Indeed, apoptosis is spatially regulated by pre-miR-664a and PCDR. In addition, we found that apoptosis induced by pre-miR-664a delivered by PCDR was more rapid than that by lipofection. These results suggest that pre-miR-664a is a nucleic acid drug candidate for cancer therapy and PCDR and pre-miR-664a-based strategies have potential therapeutic uses for diseases affecting various cell types.

Osteopontin silencing attenuates bleomycin-induced murine pulmonary fibrosis by regulating epithelial-mesenchymal transition.

Idiopathic pulmonary fibrosis (IPF) is the most common and most deadly form of interstitial lung disease. Osteopontin (OPN), a matricellular protein with proinflammatory and profibrotic properties, plays a major role in several fibrotic diseases, including IPF; OPN is highly upregulated in patients' lung samples. In this study, we knocked down OPN in a bleomycin (BLM)-induced pulmonary fibrosis (PF) mouse model using small interfering RNA (siRNA) to determine whether the use of OPN siRNA is an effective therapeutic strategy for IPF. We found that fibrosing areas were significantly smaller in specimens from OPN siRNA-treated mice. The number of alveolar macrophages, neutrophils, and lymphocytes in bronchoalveolar lavage fluid was also reduced in OPN siRNA-treated mice. Regarding the expression of epithelial-mesenchymal transition (EMT)-related proteins, the administration of OPN-siRNA to BLM-treated mice upregulated E-cadherin expression and downregulated vimentin expression. Moreover, in vitro, we incubated the human alveolar adenocarcinoma cell line A549 with transforming growth factor (TGF)-ß1 and subsequently transfected the cells with OPN siRNA. We found a significant upregulation of Col1A1, fibronectin, and vimentin after TGF-ß1 stimulation in A549 cells. In contrast, a downregulation of Col1A1, fibronectin, and vimentin mRNA levels was observed in TGF-ß1-stimulated OPN knockdown A549 cells. Therefore, the downregulation of OPN effectively reduced pulmonary fibrotic and EMT changes both in vitro and in vivo. Altogether, our results indicate that OPN siRNA exerts a protective effect on BLM-induced PF in mice. Our results provide a basis for the development of novel targeted therapeutic strategies for IPF.

Inhibition of HSF1 and SAFB Granule Formation Enhances Apoptosis Induced by Heat Stress.

Stress resistance mechanisms include upregulation of heat shock proteins (HSPs) and formation of granules. Stress-induced granules are classified into stress granules and nuclear stress bodies (nSBs). The present study examined the involvement of nSB formation in thermal resistance. We used chemical compounds that inhibit heat shock transcription factor 1 (HSF1) and scaffold attachment factor B (SAFB) granule formation and determined their effect on granule formation and HSP expression in HeLa cells. We found that formation of HSF1 and SAFB granules was inhibited by 2,5-hexanediol. We also found that suppression of HSF1 and SAFB granule formation enhanced heat stress-induced apoptosis. In addition, the upregulation of HSP27 and HSP70 during heat stress recovery was suppressed by 2,5-hexanediol. Our results suggested that the formation of HSF1 and SAFB granules was likely to be involved in the upregulation of HSP27 and HSP70 during heat stress recovery. Thus, the formation of HSF1 and SAFB granules was involved in thermal resistance.

A Novel 89Zr-labeled DDS Device Utilizing Human IgG Variant (scFv): "Lactosome" Nanoparticle-Based Theranostics for PET Imaging and Targeted Therapy.

"Theranostics," a new concept of medical advances featuring a fusion of therapeutic and diagnostic systems, provides promising prospects in personalized medicine, especially cancer. The theranostics system comprises a novel 89Zr-labeled drug delivery system (DDS), derived from the novel biodegradable polymeric micelle, "Lactosome" nanoparticles conjugated with specific shortened IgG variant, and aims to successfully deliver therapeutically effective molecules, such as the apoptosis-inducing small interfering RNA (siRNA) intracellularly while offering simultaneous tumor visualization via PET imaging. A 27 kDa-human single chain variable fragment (scFv) of IgG to establish clinically applicable PET imaging and theranostics in cancer medicine was fabricated to target mesothelin (MSLN), a 40 kDa-differentiation-related cell surface glycoprotein antigen, which is frequently and highly expressed by malignant tumors. This system coupled with the cell penetrating peptide (CPP)-modified and photosensitizer (e.g., 5, 10, 15, 20-tetrakis (4-aminophenyl) porphyrin (TPP))-loaded Lactosome particles for photochemical internalized (PCI) driven intracellular siRNA delivery and the combination of 5-aminolevulinic acid (ALA) photodynamic therapy (PDT) offers a promising nano-theranostic-based cancer therapy via its targeted apoptosis-inducing feature. This review focuses on the combined advances in nanotechnology and material sciences utilizing the "89Zr-labeled CPP and TPP-loaded Lactosome particles" and future directions based on important milestones and recent developments in this platform.

Lactosome-Conjugated siRNA Nanoparticles for Photo-Enhanced Gene Silencing in Cancer Cells.

The A3B-type Lactosome comprised of poly(sarcosine)3-block-poly(l-lactic acid), a biocompatible and biodegradable polymeric nanomicelle, was reported to accumulate in tumors in vivo via the enhanced permeability and retention (EPR) effect. Recently, the cellular uptake of Lactosome particles was enhanced through the incorporation of a cell-penetrating peptide (CPP), L7EB1. However, the ability of Lactosome as a drug delivery carrier has not been established. Herein, we have developed a method to conjugate the A3B-type Lactosome with ATP-binding cassette transporter G2 (ABCG2) siRNA for inducing in vitro apoptosis in the cancer cell lines PANC-1 and NCI-H226. The L7EB1 peptide facilitates the cellular uptake efficiency of Lactosome but does not deliver siRNA into cytosol. To establish the photoinduced cytosolic dispersion of siRNA, a photosensitizer loaded L7EB1-Lactosome was prepared, and the photosensitizer 5,10,15,20-tetra-kis(pentafluorophenyl)porphyrin (TPFPP) showed superiority in photoinduced cytosolic dispersion. We exploited the combined effects of enhanced cellular uptake by L7EB1 and photoinduced endosomal escape by TPFPP to efficiently deliver ABCG2 siRNA into the cytosol for gene silencing. Moreover, the silencing of ABCG2, a protoporphyrin IX (PpIX) transporter, also mediated photoinduced cell death via 5-aminolevulinic acid (ALA)-mediated PpIX accumulated photodynamic therapy (PDT). The synergistic capability of the L7EB1/TPFPP/siRNA-Lactosome complex enabled both gene silencing and PDT.

Complementary leucine zippering system for effective intracellular delivery of proteins by cell-penetrating peptides.

A heterodimeric leucine zipper composed of a pair of leucine zipper peptides containing acidic or basic amino acid residues at appropriate positions in each peptide was used as a molecular glue to connect protein cargos to a cell-penetrating peptide (CPP) carrier. To investigate the hybridization properties by fluorescence experiments, we prepared an enhanced green fluorescent protein (EGFP) fused with an acidic leucine zipper (LzK), EGFP-LzK, and a basic leucine zipper (LzE) modified with a CPP, LzE-CPP. The LzK and LzE formed a 1:1 hybrid when EGFP-LzK and LzE-CPP were mixed in phosphate buffer saline, thereby conjugating the EGFP with the CPP. The formation of the 1:1 hybrid was confirmed by fluorescence spectra and fluorescence titration curves. Results from fluorescence microscopy experiments showed that EGFP was successfully delivered into cells by conjugating with the CPP via formation of the LzK/LzE hybrid. We also fused the apoptotic protein p53 with LzK (p53-LzK) and investigated the inhibition of cell proliferation of various cell lines by incubation with the p53-LzK/LzE-CPP hybrid. This hybrid was found to localize in nuclei and successfully inhibited cell-specific proliferation. The LzE/LzK zipper system inhibited cell proliferation more efficiently than the directly fused conjugate, p53-CPP. Our method will be a useful drug delivery system for delivering bioactive proteins to treat various diseases.

Minimization of apoptosis-inducing CPP-Bim peptide.

Pro-apoptotic peptides may be promising agents for cancer therapy owing to their ability to induce apoptosis in cancer cells. TatBim, a fusion peptide of Tat cell-penetrating peptide (CPP) and the BH3 domain derived from Bim apoptosis-inducing protein, is a pro-apoptotic peptide. In this study, based on the TatBim sequence, we attempted to minimize the CPP-Bim peptide while retaining apoptosis-inducing activity. The CPP and Bim parts were systematically shortened, and the pro-apoptotic activities of the shortened peptides were examined. We obtained TatBim-N1C2 and R8Bim-N1C2 as minimized peptides with efficient apoptotic activity. These peptides may have potential applications in future biomedical studies, such as cancer therapeutics.

Cell cycle dependence of apoptosis photo-triggered using peptide-photosensitizer conjugate.

Investigation of the relevance between cell cycle status and the bioactivity of exogenously delivered biomacromolecules is hindered by their time-consuming cell internalization and the cytotoxicity of transfection methods. In this study, we addressed these problems by utilizing the photochemical internalization (PCI) method using a peptide/protein-photosensitizer conjugate, which enables immediate cytoplasmic internalization of the bioactive peptides/proteins in a light-dependent manner with low cytotoxicity. To identify the cell-cycle dependent apoptosis, a TatBim peptide-photosensitizer conjugate (TatBim-PS) with apoptotic activity was photo-dependently internalized into HeLa cells expressing a fluorescent ubiquitination-based cell cycle indicator (Fucci2). Upon irradiation, cytoplasmic TatBim-PS internalization exceeded 95% for all cells classified in the G1, S, and G2/M cell cycle phases with no significant differences between groups. TatBim-PS-mediated apoptosis was more efficiently triggered by photoirradiation in the G1/S transition than in the G1 and S/G2/M phases, suggesting high sensitivity of the former phase to Bim-induced apoptosis. Thus, the cell cycle dependence of Bim peptide-induced apoptosis was successfully investigated using Fucci2 indicator and the PCI method. Since PCI-mediated cytoplasmic internalization of peptides is rapid and does not span multiple cell cycle phases, the Fucci-PCI method constitutes a promising tool for analyzing the cell cycle dependence of peptides/protein functions.

Analysis of Single Nucleotide-Mutated Single-Cancer Cells Using the Combined Technologies of Single-Cell Microarray Chips and Peptide Nucleic Acid-DNA Probes.

Research into cancer cells that harbor gene mutations relating to anticancer drug-resistance at the single-cell level has focused on the diagnosis of, or treatment for, cancer. Several methods have been reported for detecting gene-mutated cells within a large number of non-mutated cells; however, target single nucleotide-mutated cells within a large number of cell samples, such as cancer tissue, are still difficult to analyze. In this study, a new system is developed to detect and isolate single-cancer cells expressing the T790M-mutated epidermal growth factor receptor (EGFR) mRNA from multiple non-mutated cancer cells by combining single-cell microarray chips and peptide nucleic acid (PNA)-DNA probes. The single-cell microarray chip is made of polystyrene with 62,410 microchambers (31-40 µm diameter). The T790M-mutated lung cancer cell line, NCI-H1975, and non-mutated lung cancer cell line, A549, were successfully separated into single cells in each microchambers on the chip. Only NCI-H1975 cell was stained on the chip with a fluorescein isothiocyanate (FITC)-conjugated PNA probe for specifically detecting T790M mutation. Of the NCI-H1975 cells that spiked into A549 cells, 0-20% were quantitatively analyzed within 1 h, depending on the spike concentration. Therefore, our system could be useful in analyzing cancer tissue that contains a few anticancer drug-resistant cells.

Mechanical strain attenuates cytokine-induced ADAMTS9 expression via transient receptor potential vanilloid type 1.

The synovial fluids of patients with osteoarthritis (OA) contain elevated levels of inflammatory cytokines, which induce the expression of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) and of the matrix metalloproteinase (MMP) in chondrocytes. Mechanical strain has varying effects on organisms depending on the strength, cycle, and duration of the stressor; however, it is unclear under inflammatory stimulation how mechanical strain act on. Here, we show that mechanical strain attenuates inflammatory cytokine-induced expression of matrix-degrading enzymes. Cyclic tensile strain (CTS), as a mechanical stressor, attenuated interleukin (IL)-1ß and tumor necrosis factor (TNF)-α-induced mRNA expression of ADAMTS4, ADAMTS9, and MMP-13 in normal chondrocytes (NHAC-kn) and in a chondrocytic cell line (OUMS-27). This effect was abolished by treating cells with mechano-gated channel inhibitors, such as gadolinium, transient receptor potential (TRP) family inhibitor, ruthenium red, and with pharmacological and small interfering RNA-mediated TRPV1 inhibition. Furthermore, nuclear factor κB (NF-κB) translocation from the cytoplasm to the nucleus resulting from cytokine stimulation was also abolished by CTS. These findings suggest that mechanosensors such as the TRPV protein are potential therapeutic targets in treating OA.

Imaging analysis of EGFR mutated cancer cells using peptide nucleic acid (PNA)-DNA probes.

Lung cancer cells harbor various gene mutations in the mRNA sequence of the Epidermal Growth Factor Receptor (EGFR), especially the mutations of exon19del E746-A750, T790M, and L858R. This results in cancer progression and resistance to anticancer drugs (tyrosine kinase inhibitor; TKI). Therefore, the imaging analysis of EGFR mutations is required for the treatment planning for non-small cell lung cancers. This study focused on the imaging analysis of a single nucleotide substitute in EGFR mutated cancer cells. We developed three novel peptide nucleic acid (PNA)-DNA probes for recognizing and detecting the following three gene mutations in EGFR gene mutations. The PNA-DNA probes consist of fluorescein isothiocyanate (FITC) conjugated PNA as a detection probe and Dabcyl conjugated DNA as a quencher probe. The PNA-DNA probes were used to validate the feasibility for detecting three EGFR mutated sequences: exon19del E746-A750, T790M, and L858R. The three probes emitted fluorescent dose-dependent signals against three target DNA and RNA. Using the three PNA-DNA probes, we succeeded in distinguishing three kinds of lung-cancer cell lines (H1975, PC-9, and A549) which have different EGFR mutations by the fluorescence in situ hybridization (FISH) method.

Combined apoptotic effects of peptide and miRNA in a peptide/miRNA nanocomplex.

The present study investigated combined biological effects of peptide and miRNA in a peptide/miRNA nanocomplex. We utilized TatBim peptide as a cell-penetrating peptide-based RNA carrier with apoptotic activity. miRNA with apoptotic activity (miR-34a) was used for complex formation to investigate the additional effects of the combination with TatBim peptide. TatBim peptide and the miRNA formed nanocomplexes (approximately 250 nm in diameter), and these complexes were efficiently internalized by cells. Despite its efficient cell internalization, apoptotic activity of the nanocomplex decreased with increasing RNA content. However, photosensitizer-attachment to TatBim and photoirradiation significantly improved the apoptotic activity of the nanocomplex by facilitating dispersion of the peptide and RNA in the cytoplasm. Combined apoptotic activity of both TatBim peptide and miR-34a in the nanocomplex was demonstrated by substituting TatBim with Lipofectamine and by substituting miR-34a with scrambled siRNA.