Epithelial cells/progenitor cells in developing human lower respiratory tract: Characterization and transplantation to rat model of pulmonary injury.

Introduction: For cell-based therapies of lung injury, several cell sources have been extensively studied. However, the potential of human fetal respiratory cells has not been systematically explored for this purpose. Here, we hypothesize that these cells could be one of the top sources and hence, we extensively updated the definition of their phenotype. Methods: Human fetal lower respiratory tissues from pseudoglandular and canalicular stages and their isolated epithelial cells were evaluated by immunostaining, electron microscopy, flow cytometry, organoid assay, and gene expression studies. The regenerative potential of the isolated cells has been evaluated in a rat model of bleomycin-induced pulmonary injury by tracheal instillation on days 0 and 14 after injury and harvest of the lungs on day 28. Results: We determined the relative and temporal, and spatial pattern of expression of markers of basal (KRT5, KRT14, TRP63), non-basal (AQP3 and pro-SFTPC), and early progenitor (NKX2.1, SOX2, SOX9) cells. Also, we showed the potential of respiratory-derived cells to contribute to in vitro formation of alveolar and airway-like structures in organoids. Cell therapy decreased fibrosis formation in rat lungs and improved the alveolar structures. It also upregulated the expression of IL-10 (up to 17.22 folds) and surfactant protein C (up to 2.71 folds) and downregulated the expression of TGF-ß (up to 5.89 folds) and AQP5 (up to 3.28 folds). Conclusion: We provide substantial evidence that human fetal respiratory tract cells can improve the regenerative process after lung injury. Also, our extensive characterization provides an updated phenotypic profile of these cells.

Cross-linked acellular lung for application in tissue engineering: Effects on biocompatibility, mechanical properties and immunological responses.

The concept of providing tissue engineering scaffolds with natural physical properties and minimal immunogenicity has not been systematically approached for the lungs yet. Here, the rat acellular lung tissue (ALT) was cross-linked to provide either EDC/NHS cross-linked tissue (EDC/NHS-CLT) or tannic acid cross-linked tissue (TA-CLT). Young's modulus revealed that EDC/NHS-CLT had mechanical properties similar to the native lung and culture of lung mesenchymal cells showed a higher potential of cell proliferation on EDC/NHS-CLT versus TA-CLT and ALT. The in vitro immunogenicity tests showed a strong induction of T-cell proliferation by TA-CLT and an attenuated macrophage induction by TA-CLT. Processed rat lungs were implanted xenogenically into the mouse peritoneal cavity and the host-implant interactions showed that tannic acid is not released from TA-CLT in a physiologically effective dose. The profile of peritoneal fluid proinflammatory (TNFα, IL-1ß, IL-12p70 and IL-17) and anti-inflammatory (IL-10 and TGFß1) cytokines, and CD3+ T-lymphocytes and CD11b+ macrophages revealed that apart from induction of high levels of IL-17 during the first week and IL-10 during the second to third weeks after implantation by TA-CLT, other indicators of immune reactions to cross-linked tissues were not significantly different from ALT. Also, a high fibrotic reaction to TA-CLT was observed on the weeks 2-3, but alveolar structures were preserved in EDC/NHS-CLT. Our findings show that by controlled EDC/NHS cross-linking, an acellular lung scaffold could be provided with mechanical properties similar to native lung, which promotes mesenchymal lung cells proliferation and does not stimulate recipient's immune system more than a non-cross-linked tissue.

Long-term preservation effects on biological properties of acellular placental sponge patches.

Decellularization, preservation protocol and storage time influence the biomechanical and biological properties of allografts and xenografts. Here, we examined the consequences of storage time on the antibacterial, angiogenic and biocompatibility properties of the decellularized placental sponge (DPS) in vitro and in vivo. The DPS samples were preserved for one, three and six months at -20 °C. The decellularized scaffolds showed uniform morphology with interconnected pores compared with not decellularized sponges. Storage time did not interfere with collagen and vascular endothelial growth factor contents, and cytobiocompatibility for Hu02 fibroblast cells. Chorioallantoic membrane assay and subcutaneous implantation indicated a decreased new vessel formation and neovascularization in six months DPS sample compared with other experimental groups. The number of CD4+ and CD68+ cells infiltrated into the six months DPS on the implanted site showed a significant increase compared with one and three months sponges. The antibacterial activities and angiogenic properties of the DPS decreased over storage time. Three months preservation at -20 °C is suggested as the optimal storage period to retain its antibacterial activity and high stimulation of new vessel formation. This storage protocol could be considered for preservation of similar decellularized placenta-derived products with the aim of retaining their biological properties.

Self-assembled peptide nanoparticles for efficient delivery of methotrexate into cancer cells.

The low cellular uptake of Methotrexate (MTX), a commonly used anticancer drug, is a big challenge for efficient cancer therapy. Self-assembled peptide nanoparticles (SAPNs) are one of the major classes of peptide vectors that have gained much attention toward novel drug delivery systems. In the present study, different sequences of cell-penetrating peptides including R2W4R2 and W3R4W3 and their SAPNs (R2W4R2-E12 and W3R4W3-E12) were designed for efficient delivery of MTX into MCF7 breast cancer cells. Based on electron microscopy results, the obtained SAPNs were in nano scale with spherical shape. There was a positive relationship between the free energy of water to octanol transferring and cellular penetration of designed nanostructures. The R2W4R2 possessed proper free energy and ability to form a spherical structure and hydrophobic-hydrophobic interactions, therefore, exhibited more cellular penetration than W3R4W3. The cellular uptake of obtained nanoparticles was examined by flow cytometry and fluorescence microscopy, in which, R2W4R2 and R2W4R2-E12 showed more appropriate penetration into MCF7 cells than W3R4W3 and W3R4W3-E12. The cytotoxicity of MTX-loaded peptides and SAPNs was examined by MTT assay. As a result, at higher concentrations, the R2W4R2 and R2W4R2-E12 showed higher cytotoxic behavior than their counterparts. Despite their enhanced cellular internalization, the cytotoxic behavior of MTX-loaded SAPNs at lower concentrations was relatively less than free MTX, which could be ascribed to the gradual nature of drug detachment from these conjugates. Therefore, R2W4R2 could be considered as an efficient choice to enhance the therapeutic efficiency of MTX in cancer treatments.

Synthesis and cellular characterization of various nano-assemblies of cell penetrating peptide-epirubicin-polyglutamate conjugates for the enhancement of antitumor activity.

A new class of cell penetrating peptides (CPPs) named peptide amphiphile was designed to improve the intracellular uptake and the antitumor activity of epirubicin (EPR). Various amphiphilic CPPs were synthesized by solid phase peptide synthesis method and were chemically conjugated to EPR. Their corresponding nanoparticles (CPPs-E4 and CPPs-E8) were prepared via non-covalent binding of the peptides and polyanions. Cytotoxicity and anti-proliferative activity were evaluated by MTT assay. Cellular uptake was examined by flow cytometry and fluorescence microscopy. The CPPs exhibited slight cytotoxicity. Binding of polyglutamate to CPPs (CPPs-E4 and CPPs-E8 nanoparticles) decreased their cytotoxicity. CPPs-E8 nanoparticles showed lower cytotoxicity than CPPs-E4 nanoparticles. Cellular uptake of K3W4K3-E8, K2W4K2-E8 and W3K4W3-E8 reached 100% with no difference between each of the mentioned CPPs and its nanoparticles at 50 µM. The anti-proliferative activity of EPR was enhanced following conjugation to peptides and nanoparticles at 25 µM. CPPs-EPR-E4 and CPPs-E8-EPR nanoparticles displayed higher anti-proliferative activity than CPPs-EPR at 25 µM. CPPs-E8-EPR nanoparticles showed higher anti-proliferative activity than CPPs-E4-EPR. K3W4K3-E8-EPR nanoparticles exhibited the highest anti-proliferative activity at 25 µM. The synthesized peptide nanoparticles are proposed as suitable carriers for improving the intracellular delivery of EPR into tumor cells with low cytotoxicity and high antitumor activity.

Cellular uptake and anti-tumor activity of gemcitabine conjugated with new amphiphilic cell penetrating peptides.

Gemcitabine (Gem) is used as a single agent or in combination with other anticancer agents to treat many types of solid tumors. However, it has many limitations such as a short plasma half-life, dose-limiting toxicities and drug resistance. Cell-penetrating peptides (CPPs) are short peptides which may deliver a large variety of cargo molecules into the cancerous cells. The current study was designed to evaluate the antiproliferative activity of gemcitabine chemically conjugated to CPPs. The peptides were synthesized using solid phase synthesis procedure. The uptake efficiency of CPPs into cells was examined by flow cytometry and fluorescent microscopy. The synthesized peptides were chemically conjugated to Gem and the in vitro cytotoxicity of conjugates was tested by MTT assay on A594 cell line. According to the obtained results, cellular uptake was increased with increasing the concentration of CPPs. On the other hand the coupling of Gem with peptides containing block sequence of arginine (R5W3R4) and some alternating sequences (i.e. [RW]6 and [RW]3) exhibited improved antitumor activity of the drug. The findings in this study support the advantages of using cell-penetrating peptides for improving intracellular delivery of Gem into tumor as well as its activity.

Effect of poly-glutamate on uptake efficiency and cytotoxicity of cell penetrating peptides.

Cell penetrating peptides (CPPs) were developed as vehicles for efficient delivery of various molecules. An ideal CPP-peptide should not display any toxicity against cancer cells as well as healthy cells and efficiently enter into the cell. Because of the cationic nature and the intrinsic vector capabilities, these peptides can cause cytotoxicity. One of the possible reasons for toxicity of CPPs is direct translocation and consequently, pore formation on the plasma membrane. In this study it was demonstrated that interaction of poly-glutamate with CPP considerably reduced their cytotoxicity in A549 cell. This strategy could be useful for efficient drug delivery mediated by CPP.

The Relation Between Thermodynamic and Structural Properties and Cellular Uptake of Peptides Containing Tryptophan and Arginine.

PURPOSE: Cell-penetrating peptides (CPPs) are used for delivering drugs and other macromolecular cargo into living cells. In this paper, we investigated the relationship between the structural/physicochemical properties of four new synthetic peptides containing arginine-tryptophan in terms of their cell membrane penetration efficiency. METHODS: The peptides were prepared using solid phase synthesis procedure using FMOC protected amino acids. Fluorescence-activated cell sorting and fluorescence imaging were used to evaluate uptake efficiency. Prediction of the peptide secondary structure and estimation of physicochemical properties was performed using the GOR V method and MPEx 3.2 software (Wimley-White scale, helical wheel projection and total hydrophobic moment). RESULTS: Our data showed that the uptake efficiency of peptides with two tryptophans at the C- and N-terminus were significantly higher (about 4-fold) than that of peptides containing three tryptophans at both ends. The distribution of arginine at both ends also increased the uptake efficiency 2.52- and 7.18-fold, compared with arginine distribution at the middle of peptides. CONCLUSION: According to the obtained results the value of transfer free energies of peptides from the aqueous phase to membrane bilayer could be a good predictor for the cellular uptake efficiency of CPPs.

Synthesis and in vitro evaluation of amphiphilic peptides and their nanostructured conjugates.

PURPOSE: Breast cancer is the second leading cancer type among people of advanced countries. Various methods have been used for cancer treatment such as chemotherapy and radiotherapy. In the present study we have designed and synthesized a new group of drug delivery systems (DDS) containing a new class of Cell Penetrating Peptides (CPPs) named Peptide Amphiphiles (PAs). METHODS: Two PAs and anionic peptides were synthesized using solid phase peptide synthesis (SPPS), namely [KW]4, [KW]5, E4 and E8. Then nano-peptides were synthesized by non-covalent binding between PAs and poly anions as [KW]4-E4, [KW]4-E8, [KW]5-E4 and [KW]5-E8. RESULTS: Flow cytometry studies showed that increased chain length of PAs with a higher ratio between hydrophobicity and net charge results in increased intracellular uptake by MCF7 cells after 2h incubation. Moreover, nano-peptides showed greater intracellular uptake compared to PAs. Anti-proliferative assay revealed that by increasing chain length of PAs, the toxicity effect on MCF7 cells is reduced, however nano-peptides did not show significant toxicity on MCF7 cells even at high concentration levels. CONCLUSION: These data suggest that due to the lack of toxicity effect at high concentration levels and also high cellular uptake, nano-peptides are more suitable carrier compared to PAs for drug delivery.