Microarray-Based CD38 Peptide Probe Screening for Multiple Myeloma Imaging.

Assessing CD38 expression in vivo has become a significant element in multiple myeloma (MM) therapy, as it can be used to detect lesions and forecast the effectiveness of treatment. Accurate diagnosis requires a multifunctional, high-throughput probe screening platform to develop molecular probes for tumor-targeted multimodal imaging and treatment. Here, we investigated a microarray chip-based strategy for high-throughput screening of peptide probes for CD38. We obtained two new target peptides, CA-1 and CA-2, from a 105 peptide library with a dissociation constant (KD) of 10-7 M. The specificity and affinity of the target peptides were confirmed at the molecular and cellular levels. Peptide probes were labeled with indocyanine green (ICG) dye and 68Ga-DOTA, which were injected into a CD38-positive Ramos tumor-bearing mouse via its tail vein, and small animal fluorescence and positron emission tomography (PET) imaging showed that the peptide probes could show specific enrichment in the tumor tissue. Our study shows that a microchip-based screening of peptide probes can be used as a promising imaging tool for MM diagnosis.

In vivo therapy of osteosarcoma using anion transporters-based supramolecular drugs.

BACKGROUND: Osteosarcoma represents a serious clinical challenge due to its widespread genomic alterations, tendency for drug resistance and distant metastasis. New treatment methods are urgently needed to address those treatment difficulties in osteosarcoma to improve patient prognoses. In recent years, small-molecule based anion transporter have emerged as innovative and promising therapeutic compound with various biomedical applications. However, due to a lack of efficient delivery methods, using ion transporters as therapeutic drugs in vivo remains a major challenge. RESULT: Herein, we developed self-assembled supramolecular drugs based on small-molecule anion transporters, which exhibited potent therapeutic effect towards osteosarcoma both in vitro and in vivo. The anion transporters can disrupt intracellular ion homeostasis, inhibit proliferation, migration, epithelial-mesenchymal transition process, and lead to osteosarcoma cell death. RNA sequencing, western blot and flow cytometry indicated reprogramming of HOS cells and induced cell death through multiple pathways. These pathways included activation of endoplasmic reticulum stress, autophagy, apoptosis and cell cycle arrest, which avoided the development of drug resistance in osteosarcoma cells. Functionalized with osteosarcoma targeting peptide, the assembled supramolecular drug showed excellent targeted anticancer therapy against subcutaneous xenograft tumor and lung metastasis models. Besides good tumor targeting capability and anti-drug resistance, the efficacy of the assembly was also attributed to its ability to regulate the tumor immune microenvironment in vivo. CONCLUSIONS: In summary, we have demonstrated for the first time that small-molecule anion transporters are capable of killing osteosarcoma cells through multiple pathways. The assemblies, OTP-BP-L, show excellent targeting and therapeutic effect towards osteosarcoma tumors. Furthermore, the supramolecular drug shows a strong ability to regulate the tumor immune microenvironment in vivo. This work not only demonstrated the biomedical value of small-molecule anion transporters in vivo, but also provided an innovative approach for the treatment of osteosarcoma.

Enhanced protection against hypoxia/reoxygenation-induced apoptosis in H9c2 cells by puerarin-loaded liposomes modified with matrix metalloproteinases-targeting peptide and triphenylphosphonium.

Based on the inhibition of mitochondrial permeability transition pore (mPTP) opening, puerarin (PUE) has a good potential to reduce myocardial ischemia/reperfusion injury (MI/RI). However, the lack of targeting of free PUE makes it difficult to reach the mitochondria. In this paper, we constructed matrix metalloproteinase-targeting peptide (MMP-TP) and triphenylphosphonium (TPP) cation co-modified liposomes loaded with PUE (PUE@T/M-L) for mitochondria-targeted drug delivery. PUE@T/M-L had a favorable particle size of 144.9 ± 0.8 nm, an encapsulation efficiency of 78.9 ± 0.6%, and a sustained-release behavior. The results of cytofluorimetric experiments showed that MMP-TP and TPP double-modified liposomes (T/M-L) enhanced intracellular uptake, escaped lysosomal capture, and promoted drug targeting into mitochondria. In addition, PUE@T/M-L enhanced the viability of hypoxia-reoxygenation (H/R) injured H9c2 cells by inhibiting mPTP opening and reactive oxygen species (ROS) production, reducing Bax expression and increasing Bcl-2 expression. It was inferred that PUE@T/M-L delivered PUE into the mitochondria of H/R injured H9c2 cells, resulting in a significant increase in cellular potency. Based on the ability of MMP-TP to bind the elevated expression of matrix metalloproteinases (MMPs), T/M-L had excellent tropism for Lipopolysaccharide (LPS) -stimulated macrophages and can significantly reduce TNF-α and ROS levels, thus allowing both drug accumulation in ischemic cardiomyocytes and reducing inflammatory stimulation during MI/RI. Fluorescence imaging results of the targeting effect using a DiR probe also indicated that DiR@T/M-L could accumulate and retain in the ischemic myocardium. Taken together, these results demonstrated the promising application of PUE@T/M-L for mitochondria-targeted drug delivery to achieve maximum therapeutic efficacy of PUE.

Cancer cells targeting with genetically engineered constructs based on a pH-dependent membrane insertion peptide and fluorescent protein.

The targeted delivery of nanodrugs to malignant neoplasm is one of the most pressing challenges in the development of modern medicine. It was reported earlier that a bacteriorhodopsin-derived pH low insertion peptide (pHLIP) targets acidic tumors and has the ability to translocate low molecular weight cargoes across the cancer cell membrane. Here, to better understand the potential of pHLIP-related technologies, we used genetically engineered fluorescent protein (EGFP) as a model protein cargo and examined targeting efficiencies of EGFP-pHLIP hybrid constructs in vitro with the HeLa cell line at different pHs. By two independent monitoring methods we observed an increased binding affinity of EGFP-pHLIP fusions to HeLa cells at pH below 6.8. Confocal images of EGFP-pHLIP-treated cells showed bright fluorescence associated with the cell membrane and fluorescent dots localized inside the cell, that became brighter with time. To elucidate the pHLIP-mediated EGFP cell entry mechanisms, we performed a series of experiments with specific inhibitors of endocytosis. Our results imply that EGFP-pHLIP internalization is realized by endocytosis of various types.

CSP I-plus modified rEndostatin inhibits hepatocellular carcinoma metastasis via down-regulation of VEGFA and integrinß1.

BACKGROUND: In our previous study, N end of the Circumsporozoite protein (CSP I-plus) modified recombinant human Endostatin (rEndostatin, endostar) (rES-CSP) was constructed, which had antiangiogenic capability and bound to hepatocellular carcinoma in vivo and in vitro. In this study, the inhibition of rES-CSP on hepatocellular carcinoma metastasis was verified in vivo and in vitro, and its possible mechanism was explored. METHODS: Firstly, the impact of rES-CSP on the migration, adhesion of hepatoma cell HCCLM3 was identified by wound healing, transwell, and on metastasis of orthotopic xenograft model was identified in nude mouse. Then the expression of metastasis-associated molecules (MMP2, E-cadherin, integrinß1) and angiogenesis-related factors (VEGFA) in vitro and in vivo were detected by real-time PCR, western blotting, immunohistochemistry. RESULTS: Finally, we found that rES-CSP could inhibit the migration and invasion of HCCLM3, and decrease tumor metastasis and growth in nude mouse orthotopic xenograft models. The tumor inhibiting rates of rES-CSP and Endostar were 42.46 ± 5.39% and 11.1 ± 1.88%. The lung metastasis rates of the control, Endostar and rES-CSP were 71, 50, and 42.8%, respectively. Compared with Endostar, rES-CSP significantly down-regulated the expression of VEGFA and integrinß1. Heparin, a competitive inhibitor of CSP I-plus, which can be bind to the highly-sulfated heparan sulfate proteoglycans (HSPGs) over-expressed in liver and hepatocellular carcinoma, alleviated the down-regulation of VEGFA and integrinß1. CONCLUSIONS: These indicate that rES-CSP may play a role in inhibiting tumor growth and metastasis by down-regulating the angiogenic factor VEGF and the metastasis-related molecules or by interfering with HSPGs-mediated tumor metastasis.

Huc-MSC-derived exosomes modified with the targeting peptide of aHSCs for liver fibrosis therapy.

BACKGROUND: Effective therapeutics to stop or reverse liver fibrosis have not emerged, because these potential agents cannot specifically target activated hepatic stellate cells (aHSCs) or are frequently toxic to parenchymal cells. Human umbilical cord mesenchymal stem cell (Huc-MSC)-derived exosomes show promise in nanomedicine for the treatment of liver fibrosis. However, systemic injection showed that unmodified exosomes were mainly taken up by the mononuclear phagocyte system. The discovery of ligands that selectively bind to a specific target plays a crucial role in clinically relevant diagnostics and therapeutics. Herein, we aimed to identify the targeting peptide of aHSCs by screening a phage-displayed peptide library, and modify Huc-MSC-derived exosomes with the targeting peptide. RESULTS: In this study, we screened a phage-displayed peptide library by biopanning for peptides preferentially bound to HSC-T6 cells. The identified peptide, HSTP1, also exhibited better targeting ability to aHSCs in pathological sections of fibrotic liver tissues. Then, HSTP1 was fused with exosomal enriched membrane protein (Lamp2b) and was displayed on the surface of exosomes through genetic engineering technology. The engineered exosomes (HSTP1-Exos) could be more efficiently internalized by HSC-T6 cells and outperformed both unmodified exosomes (Blank-Exos) and Lamp2b protein overexpressed exosomes (Lamp2b + Exos) in enhancing the ability of exosomes to promote HSC-T6 reversion to a quiescent phenotype. In vivo results showed HSTP1-Exos could specifically target to the aHSC region after intravenous administration, as demonstrated by coimmunofluorescence with the typical aHSCs marker α-SMA, and enhance the therapeutic effect on liver fibrosis. CONCLUSION: These results suggest that HSTP1 is a reliable targeting peptide that can specifically bind to aHSCs and that HSTP1-modified exosomes realize the precise treatment for aHSCs in complex liver tissue. We provide a novel strategy for clinical liver fibrosis therapy.

Selection and identification of a specific peptide binding to ovarian cancer cells from a phage-displayed peptide library.

OBJECTIVES: Ovarian cancer is one of the most fatal gynecological malignancies. It is emergently needed to select a novel molecular fragment as a targeting element for the future development of molecular imaging diagnosis and targeting chemotherapy to ovarian cancer. RESULTS: After five rounds of biopanning, a total of 44 positive phage clones were selected from final phage displayed peptide library. Nine consensus sequences were found based on the assay of sequencing results, then one clone of each consensus group was characterized and identified further by immunofluorescence assay. The result showed the phage clone R20 presents best targeting capacity. Then we synthesized peptide (OSP2) clone R20 displayed, it was characterized with high specificity and sensitivity binding to human ovarian cancer by a tissue chip assay. The target of OSP2 was predicted and docked as human carbonic anhydrase XII (CA12), an important protein usually deregulated in cancer. CONCLUSIONS: Taken together, OSP2 and its target indicate a novel investigation way in future to develop novel agent or drug delivery formulation for molecular imaging diagnosis and targeting chemotherapy of ovarian cancer.

Identification and Validation of a New Peptide Targeting Pancreatic Beta Cells.

Noninvasive targeted visualization of pancreatic beta cells or islets is becoming the focus of molecular imaging application in diabetes and islet transplantation studies. In this study, we aimed to produce the beta-cell-targeted peptide for molecular imaging of islet. We used phage display libraries to screen a beta-cell-targeted peptide, LNTPLKS, which was tagged with fluorescein isothiocyanate (FITC). This peptide was validated for targeting beta-cell with in vitro and in vivo studies. Immunocytochemistry (ICC) and fluorescence-activated cell sorting (FACS) analysis were used to validate the target specificity of the peptide. FITC-LNTPLKS displayed much higher fluorescence in beta cells vs. control cells in ICC. This discrimination was consistently observed using primary rodent islet. FACS analysis showed right shift of peak point in beta cells compared to control cells. The specific bind to in situ islet was verified by in vitro experiments using rodent and human pancreatic slices. The peptide also showed high affinity of islet grafts under the renal capsule. In the insulinoma animal model, we could find FITC-LNTPLKS accumulated specifically to the tumor, thus indicating a potential clinical application of molecular imaging of insulinoma. In conclusion, LNTPLKS showed a specific probe for beta-cells, which might be further utilized in targeted imaging/monitoring beta cells and theragnosis for beta-cells-related disease (diabetes, insulinoma, etc.).

Identification of a peptide binding to cancer antigen Kita-kyushu lung cancer antigen 1 from a phage-display library.

Kita-kyushu lung cancer antigen 1 (KK-LC-1) is a kind of cancer-testis antigen with anti-tumor potential for clinical application. As a class of small-molecule antigen conjugate, tumor-targeting peptides have broad application prospects in gastric cancer diagnosis, imaging, and biological treatment. Here, we screened specific cyclic nonapeptides from a phage-display library. The targeting peptide with the best affinity was selected and further verified in ex vivo tissue sections. Finally, enrichment of targeting peptides in tumor tissues was observed in vivo, and the dynamic biodistribution process was also observed with micro-positron emission tomography (micro-PET)/computed tomography (CT) imaging. Studies showed that the specific cyclic nonapeptide had a high binding capacity for KK-LC-1 protein. It has a strong affinity and specificity for KK-LC-1-expressing positive tumor cells. Targeting peptides were significantly enriched at tumor sites in vivo, with very low normal tissue background. These findings demonstrated that the KK-LC-1 targeting peptide has high clinical potential.

SPECT Imaging of Hepatocellular Carcinoma Detection by the GPC3 Receptor.

The glypican-3 (GPC3) receptor is a membrane protein that is highly expressed in tumor tissues but rarely expressed in the normal liver and can be used as a target for early diagnosis of hepatocellular carcinoma (HCC). Herein, we developed a GPC3-targeted 99mTc-labeled probe for SPECT imaging in HCC. 99mTc-HPG was rapidly radiosynthesized within 20 min with an excellent radiochemical purity (>98%), possessing good stability. Results from in vitro cell binding assays indicated that the binding specificity of 99mTc-HPG to GPC3-positive HepG2 cells was acceptable. For SPECT/CT imaging, the HepG2 tumors were clearly visualized with the highest tumor/muscle ratio (11.55 ± 0.54) at 1 h post-injection, and the tumor uptake of 99mTc-HPG reduced from 2.99 ± 0.15 to 1.17 ± 0.09% ID/g in the blocking study. Convenient preparation, excellent GPC3 specificity in HCC, rapid clearance from normal organs, and good biosafety profiles of 99mTc-HPG warrant further investigations for clinical translation.

Interference-free SERS nanoprobes for labeling and imaging of MT1-MMP in breast cancer cells.

The expression of membrane type-1 matrix metalloproteinase (MT1-MMP) in cancer cells is critical for understanding the development, invasion and metastasis of cancers. In this study, we devised an interference-free surface-enhanced Raman scattering (SERS) nanoprobe with high selectivity and specificity for MT1-MMP. The nanoprobe was comprised of silver core-silica shell nanoparticle with a Raman reporter tag (4-mercaptobenzonitrile) embedded in the interface. Moreover, the nitrile group in 4-mercaptobenzonitrile shows a unique characteristic peak in the Raman-silent region (1800-2800 cm-1), which eliminates spectral overlapping or background interference in the Raman fingerprint region (500-1800 cm-1). After surface modification with a targeting peptide, the nanoprobe allowed visualization and evaluation of MT1-MMP in breast cancer cells via SERS spectrometry. This interference-free, peptide-functionalized SERS nanoprobe is supposed to be conducive to early diagnosis and invasive assessment of cancer in clinical settings.

Screening and identification of a specific peptide binding to breast cancer cells from a phage-displayed peptide library.

OBJECTIVES: Breast cancer is a popular fatal malignant tumor for women with high of rates incidence and mortality. Development of the new approaches for breast cancer targeted diagnosis and chemotherapy is emergently needed by the current clinical practice, the important first step is finding a breast cancer specifically binding molecule or fragment as early clinical indicators. RESULTS: By a phage-displayed peptide library, a 12-mer peptide, CSB1 was screened out using MCF-7 cells as the target. The consequently results under immunofluorescence and laser scanning confocal microscope (LSCM) indicated that CSB1 bound MCF-7 cells and breast cancer tissues specifically and sensitively with high affinity. Bioinformatics analysis suggested that the peptide CSB1 targets the 5-Lipoxygenase-Activating Protein (FLAP), which has been implicated in breast cancer progression and prognosis. CONCLUSIONS: The peptide, CSB1 is of the potential as a candidate to be used for developing the new approaches of molecular imaging detection and targeting chemotherapy of breast cancer in the future.

PPR20 Is Required for the cis-Splicing of Mitochondrial nad2 Intron 3 and Seed Development in Maize.

Pentatricopeptide repeat (PPR) proteins are helical repeat RNA-binding proteins that function in RNA processing by conferring sequence-specific RNA-binding activity. Owing to the lethality of PPR mutants, functions of many PPR proteins remain obscure. In this study, we report the function of PPR20 in intron splicing in mitochondria and its role in maize seed development. PPR20 is a P-type PPR protein targeted to mitochondria. The ppr20 mutants display slow embryo and endosperm development. Null mutation of PPR20 severely reduces the cis-splicing of mitochondrial nad2 intron 3, resulting in reduction in the assembly and activity of mitochondrial complex I. The ppr20-35 allele with a Mu insertion in the N-terminal region shows a much weaker phenotype. Molecular analyses revealed that the mutant produces a truncated transcript, coding for PPR20ΔN120 lacking the N-terminal 120 amino acids. Subcellular localization revealed that PPR20ΔN120:GFP is able to target to mitochondria as well, suggesting the sequence diversity of the mitochondrial targeting peptides. Another mutant zm_mterf15 was also found to be impaired in the splicing of mitochondrial nad2 intron 3. Further analyses are required to identify the exact function of PPR20 and Zm_mTERF15 in the splicing of nad2 intron 3.

Endocytosis and Trafficking of Heparan Sulfate Proteoglycans in Triple-Negative Breast Cancer Cells Unraveled with a Polycationic Peptide.

The process of heparan sulfate proteoglycan (HSPG) internalization has been described as following different pathways. The tumor-specific branched NT4 peptide has been demonstrated to bind HSPGs on the plasma membrane and to be internalized in tumor cell lines. The polycationic peptide has been also shown to impair migration of different cancer cell lines in 2D and 3D models. Our hypothesis was that HSPG endocytosis could affect two important phenomena of cancer development: cell migration and nourishment. Using NT4 as an experimental tool mimicking heparin-binding ligands, we studied endocytosis and trafficking of HSPGs in a triple-negative human breast cancer cell line, MDA-MB-231. The peptide entered cells employing caveolin- or clathrin-dependent endocytosis and macropinocytosis, in line with what is already known about HSPGs. NT4 then localized in early and late endosomes in a time-dependent manner. The peptide had a negative effect on CDC42-activation triggered by EGF. The effect can be explained if we consider NT4 a competitive inhibitor of EGF on HS that impairs the co-receptor activity of the proteoglycan, reducing EGFR activation. Reduction of the invasive migratory phenotype of MDA-MB-231 induced by NT4 can be ascribed to this effect. RhoA activation was damped by EGF in MDA-MB-231. Indeed, EGF reduced RhoA-GTP and NT4 did not interfere with this receptor-mediated signaling. On the other hand, the peptide alone determined a small but solid reduction in active RhoA in breast cancer cells. This result supports the observation of few other studies, showing direct activation of the GTPase through HSPG, not mediated by EGF/EGFR.

An efficient MRI agent targeting extracellular markers in prostate adenocarcinoma.

PURPOSE: Prostate cancer (PCa) is the most widespread tumor affecting males in Western countries. We propose a novel MRI molecular tetrameric probe based on the heptadentate gadolinium (Gd)-AAZTA (6-amino-6-methylperhydro-1,4-diazepinetetraacetic acid) that is able to in vivo detect PCa through the recognition of the fibrin-fibronectin (FB-FN) complex. METHODS: The peptide CREKA (Cys-Arg-Glu-Lys-Ala), targeting the FB-FN complex in the reactive stroma of the tumor, was synthesized by solid phase peptide synthesis (SPPS) and conjugated to the tetramer dL-(Gd-AAZTA)4 . The resulting probe was characterized by 1 H relaxometry, tested in vitro on FB clots and in vivo on an orthotopic mouse model of PCa. RESULTS: CREKA-dL-(Gd-AAZTA)4 showed a remarkable relaxivity of 18.2 m MGd-1s-1 (0.47 T, 25°C) because of the presence of 2 water molecules (q = 2) in the inner coordination sphere of each Gd3+ ion, whose rotational motion (τR ) is lengthened as the result of the relatively high molecular weight. The probe displayed a detectable affinity for plasma-derived FB clots. On intravenous injection of the probe in an orthotopic mouse model of PCa, a significant increase in the prostate T1 contrast (~40%) was observed. The MRI signal appears statistically higher either with respect to the one observed for the control probes and to the one detected when CREKA-dL-(Gd-AAZTA)4 was administered to healthy animals. CONCLUSIONS: This study demonstrated the ability of the CREKA-dL-(Gd-AAZTA)4 probe to specifically localize in prostate tumor after injection. The high relaxivity of the probe allows the reduction of the injected dose to 20 µmolGd /kg, yielding a good in vivo contrast enhancement in the region of prostate tumor.

The peptidases involved in plant mitochondrial protein import.

The endosymbiotic origin of the mitochondrion and the subsequent transfer of its genome to the host nucleus has resulted in intricate mechanisms of regulating mitochondrial biogenesis and protein content. The majority of mitochondrial proteins are nuclear encoded and synthesized in the cytosol, thus requiring specialized and dedicated machinery for the correct targeting import and sorting of its proteome. Most proteins targeted to the mitochondria utilize N-terminal targeting signals called presequences that are cleaved upon import. This cleavage is carried out by a variety of peptidases, generating free peptides that can be detrimental to organellar and cellular activity. Research over the last few decades has elucidated a range of mitochondrial peptidases that are involved in the initial removal of the targeting signal and its sequential degradation, allowing for the recovery of single amino acids. The significance of these processing pathways goes beyond presequence degradation after protein import, whereby the deletion of processing peptidases induces plant stress responses, compromises mitochondrial respiratory capability, and alters overall plant growth and development. Here, we review the multitude of plant mitochondrial peptidases that are known to be involved in protein import and processing of targeting signals to detail how their activities can affect organellar protein homeostasis and overall plant growth.

Development of Targeted siRNA Nanocomplexes to Prevent Fibrosis in Experimental Glaucoma Filtration Surgery.

RNAi induced by double-stranded small interfering RNA (siRNA) molecules has attracted great attention as a naturally occurring approach to silence gene expression with high specificity. The myocardin-related transcription factor/serum response factor (MRTF/SRF) pathway is a master regulator of cytoskeletal gene expression and, thus, represents a promising target to prevent fibrosis. A major hurdle to implementing siRNA therapies is the method of delivery, and we have, thus, optimized lipid-peptide-siRNA (LPR) nanoparticles containing MRTF-B siRNAs as a targeted approach to prevent conjunctival fibrosis. We tested 15 LPR nanoparticle formulations with different lipid compositions, surface charges, and targeting or non-targeting peptides in human conjunctival fibroblasts. In vitro, the LPR formulation of the DOTMA/DOPE lipid with the targeting peptide Y (LYR) was the most efficient in MRTF-B gene silencing and non-cytotoxic compared to the non-targeting formulation. In vivo, subconjunctival administration of LYR nanoparticles containing MRTF-B siRNAs doubled bleb survival in a pre-clinical rabbit model of glaucoma filtration surgery. Furthermore, MRTF-B LYR nanoparticles reduced the MRTF-B mRNA by 29.6% in rabbit conjunctival tissues, which led to significantly decreased conjunctival scarring with no adverse side effects. LYR-mediated delivery of siRNA shows promising results to increase bleb survival and to prevent conjunctival fibrosis after glaucoma filtration surgery.

Lipid-peptide bioconjugation through pyridyl disulfide reaction chemistry and its application in cell targeting and drug delivery.

BACKGROUND: The design of efficient drug delivery vectors requires versatile formulations able to simultaneously direct a multitude of molecular targets and to bypass the endosomal recycling pathway of cells. Liposomal-based vectors need the decoration of the lipid surface with specific peptides to fulfill the functional requirements. The unspecific binding of peptides to the lipid surface is often accompanied with uncontrolled formulations and thus preventing the molecular mechanisms of a successful therapy. RESULTS: We present a simple synthesis pathway to anchor cysteine-terminal peptides to thiol-reactive lipids for adequate and quantitative liposomal formulations. As a proof of concept, we have synthesized two different lipopeptides based on (a) the truncated Fibroblast Growth Factor (tbFGF) for cell targeting and (b) the pH sensitive and fusogenic GALA peptide for endosomal scape. CONCLUSIONS: The incorporation of these two lipopeptides in the liposomal formulation improves the fibroblast cell targeting and promotes the direct delivery of cargo molecules to the cytoplasm of the cell.

An antimicrobial origin of transit peptides accounts for early endosymbiotic events.

Primary endosymbiosis, which gave rise to mitochondria or chloroplasts, required successful targeting of a number of proteins from the host cytosol to the endosymbiotic organelles. A survey of studies published in separate fields of biological research over the past 40 years argues for an antimicrobial origin of targeting peptides. It is proposed that mitochondria and chloroplast derive from microbes that developed a resistance strategy to antimicrobial peptides that consisted in their rapid internalization and proteolytic disposal by microbial peptidases.

Cardiac-specific delivery by cardiac tissue-targeting peptide-expressing exosomes.

Naturally occurring RNA carriers such as exosomes might be an untapped source of effective delivery vehicles. However, if exosomes are to be exploited for therapeutic applications, they must target specific tissues or cell types to avoid off-target effects. This study evaluated whether genetic modification of exosomes could enhance exosome delivery to heart cells and heart tissue without toxicity. Exosomes expressing cardiac-targeting peptide (CTP)-Lamp2b on the exosomal membrane (CTP-Exo) were generated by introducing vectors encoding CTP-Lamp2b into HEK 293 cells. The expression of CTP-Lamp2b peptide on exosomes was stabilized by attaching glycosylation sequences. Exosomes expressing only Lamp2b on exosomal membranes (CTL-Exo) were generated as a control. The in vitro and in vivo uptake of CTL-Exo and CTP-Exo was evaluated in cell lines and mice. Both exosomes were delivered to HEK 293 and H9C2 cells. The delivery of the exosome was not different between CTP-Exo and CTL-Exo in HEK 293 cells, whereas the delivery of CTP-Exo was 16% greater than that of CTL-Exo in H9C2 cells (P = 0.047). Cell viability was maintained at almost 100% with different dosages of both CTL-Exo and CTP-Exo. Moreover, compared with CTL-Exo, the in vivo delivery of exosomes to the hearts of mice was increased by 15% with CTP-Exo (P = 0.035). The delivery to livers and spleens was not different between the two exosomes. Genetic modification of exosomes by expressing CTP-Lamp2b on the exosomal membrane enhanced exosome delivery to heart cells and the heart tissue. These results suggested that CTP-Exo might be used as a therapeutic tool for heart disease.