Targeting prostate cancer with docetaxel-loaded peptide 563-conjugated PEtOx-co-PEI30%-b-PCL polymeric micelle nanocarriers.

Prostate cancer is a global disease that negatively affects the quality of life. Although various strategies against prostate cancer have been developed, only a few achieved tumor-specific targeting. Therefore, a special emphasis has been placed on the treatment of cancer using nano-carrier-encapsulated chemotherapeutic agents conjugated with tumor-homing peptides. The targeting strategy coupling the drugs with nanotechnology helps to overcome the most common barriers, such as high toxicity and side effects. Prostate-specific membrane antigen has emerged as a promising target molecule for prostate cancer and shown to be targeted with high affinity by GRFLTGGTGRLLRIS peptide known as peptide 563 (P563). Here, we aimed to assess the in vitro and in vivo targeting efficiency, safety, and efficacy of P563-conjugated, docetaxel (DTX)-loaded polymeric micelle nanoparticles (P563-PEtOx-co-PEI30%-b-PCL-DTX) against prostate cancer. To this end, we analyzed the cytotoxic activity of P563-PEtOx-co-PEI30%-b-PCL and P563-PEtOx-co-PEI30%-b-PCL-DTX by a cell proliferation assay using PNT1A and 22Rv1 cells. We have also determined the targeting selectivity of P563-PEtOx-co-PEI30%-b-PCL-FITC by flow cytometry and assessed the induction of cell death by western blot and TUNEL assays for P563-PEtOx-co-PEI30%-b-PCL-DTX in 22Rv1 cells. To investigate the in vivo efficacy, we administered DTX in the free form or in polymeric micelle nanoparticles to athymic CD-1 nu/nu mice 22Rv1 xenograft models and performed histopathological analyses. Our study showed that targeting prostate cancer with P563-conjugated PEtOx-co-PEI30%-b-PCL polymeric micelles could exert a potent anti-cancer activity with low side effects.

In-Vitro Comparative Examination of the Effect of Stromal Vascular Fraction Isolated by Mechanical and Enzymatic Methods on Wound Healing.

BACKGROUND: Enzymatic digestion has been the gold standard for stromal vascular fraction (SVF) isolation but remains expensive and raises practical and legal concerns. Mechanical SVF isolation methods have been known to produce lower cell yields, but may potentially produce a more robust product by preserving the extracellular matrix niche. OBJECTIVES: The aim of this study was to compare mechanically dissociated SVF (M-SVF) and enzymatically digested SVF (E-SVF) in terms of wound-healing efficacy. METHODS: Lipoaspirate was partitioned into 2 equal groups and processed by either mechanical or enzymatic isolation methods. After SVF isolation, cell counts and viabilities were determined by flow cytometry and cell proliferation rates were measured by the WST-1 test. A wound-healing scratch assay test, which is commonly used to model in-vitro wound healing, was performed with both cell cocktails. Collagen type 1 (Col1A) gene expression level, which is known for its role in wound healing, was also measured for both groups. RESULTS: As predicted, E-SVF isolated more cells (mean [standard deviation], 1.74 [3.63] × 106/mL, n = 10, P = 0.015) than M-SVF (0.94 [1.69] × 106/mL, n = 10, P = 0.015), but no significant difference was observed in cell viability. However, M-SVF expressed over 2-fold higher levels of stem cell surface markers and a 10% higher proliferation rate compared with E-SVF. In addition, the migration rate and level of Col1A gene expression of M-SVF were found to be significantly higher than those of E-SVF. CONCLUSIONS: Although the cell yield of M-SVF was less than that of E-SVF, M-SVF appears to have superior wound-healing properties.

Comparison of Adipocyte Viability After Short-Term Cryopreservation of Adipose Aspirates Through 3 Different Techniques.

Background: Effective cryopreservation allows for the long-term storage of living cells or tissues with the possibility of later clinical applications. Unfortunately, no successful investigations on the long-term preservation of adipose aspirates for prospective autologous fat grafting have been conducted. Objectives: In this study, we aimed to compare 3 different freezing methods to preserve adipose aspirates obtained from conventional lipoplasty to determine the optimal cryopreservation technique. Methods: To determine the optimal cryopreservation technique, hematoxylin and eosin staining, MTS assay, and Annexin assay were performed on each of the 3 groups plus a fourth control group. Group 1 served as the control, and fat tissue was analyzed immediately after adipose harvesting with no cryopreservation. For experimental Group 2, 15 mL of adipose aspirates were directly frozen at -80°C for up to 2 weeks. For experimental Group 3, 15 mL of adipose aspirates were frozen inside the adi-frosty containing 100% isopropanol and stored at -80°C for up to 2 weeks. For experimental Group 4, 15 mL of adipose aspirates were frozen with freezing solution containing 90% fetal bovine serum (v/v) and 10% dimethyl sulfoxide (v/v). Results: The results demonstrated that the experimental Group 3 had significantly more live adipocytes and greater cellular function of adipose aspirates than the experimental Groups 2 and 4. Conclusion: Cryopreservation with adi-frosty containing 100% isopropanol appears to be the best means of cryopreservation of fat.

The Effect of Hybrosome (Umbilical Cord Blood Exosome-Liposome Hybrid Vesicles) on Human Dermal Cells In Vitro.

Background: Wound healing is a process that involves multiple physiological steps, and despite the availability of various wound treatment methods, their effectiveness is still limited due to several factors, including cost, efficiency, patient-specific requirements, and side effects. In recent years, nanovesicles called exosomes have gained increasing attention as a potential wound care solution due to their unique cargo components which enable cell-to-cell communication and regulate various biological processes. Umbilical cord blood plasma (UCBP) exosomes have shown promise in triggering beneficial signaling pathways that aid in cell proliferation and wound healing. However, there is still very limited information about the wound-healing effect of UCBP exosomes in the literature. Objectives: The primary objective of this study was to investigate the "hybrosome" technology generated with calf UCBP-derived exosome-liposome combination. Methods: The authors developed hybrosome technology by fusing cord blood exosome membranes with liposomes. Nanovesicle characterization, cell proliferation assay, wound-healing scratch assay, immunohistochemistry analysis, anti-inflammation assay, real-time polymerase chain reaction (RT-PCR), enzyme-linked immunosorbent assay, and cellular uptake studies were performed using the novel hybrid exosomes. Results: Experimental results showed that hybrosome increases cell proliferation and migration by 40% to 50%, depending on the dose, and induces an anti-inflammatory effect on different cell lines as well as increased wound healing-related gene expression levels in dermal cells in vitro. All in all, this research widens the scope of wound-healing therapeutics to the novel hybrosome technology. Conclusions: UCBP-based applications have the potential for wound treatments and are promising in the development of novel therapies. This study shows that hybrosomes have outstanding abilities in wound healing using in vitro approaches.

The apoptotic effect of ozone therapy on mitochondrial activity of highly metastatic breast cancer cell line MDA-MB-231 using in vitro approaches.

Ozone (O3) gas is the triatomic state of oxygen and it is used as a disinfection agent due to its strong oxidizing effect, since its discovery in the mid-nineteenth century. Ozone therapy is also an alternative therapeutic approach for some diseases like circulatory disorders, AIDS, asthma, cardiovascular diseases, and certain types of cancer by increasing the oxygen levels in the blood by external addition of ozone to the body. In this study, the therapeutic potential of ozone therapy was examined by inhibiting the growth of breast cancer cells in a dose-dependent procedure. Ozone concentrations varying from 5 to 20 â€‹µg/ml were applied to the MDA-MB-231, human breast adenocarcinoma and HUVEC, human umbilical vein endothelium, cell lines, and MDA cells demonstrated an increased rate of death while its migration potential decreases. RT-PCR analysis showed mRNA expression levels of pro-apoptotic genes demonstrated higher folds in MDA cells after 10 â€‹µg/ml treatment. In the same context, Annexin V/PI and cell cycle analysis also concluded that ozone therapy causes apoptotic cell death on breast tumor cells. The use of ozone therapy for cancer treatment requires further and extensive research. However, this research has shown that ozone therapy is a promising source for cancer treatment in a way by inhibiting the proliferation of breast tumor cells.

Selection of natural biomaterials for micro-tissue and organ-on-chip models.

The desired organ in micro-tissue models of organ-on-a-chip (OoC) devices dictates the optimum biomaterials, divided into natural and synthetic biomaterials. They can resemble biological tissues' biological functions and architectures by constructing bioactivity of macromolecules, cells, nanoparticles, and other biological agents. The inclusion of such components in OoCs allows them having biological processes, such as basic biorecognition, enzymatic cleavage, and regulated drug release. In this report, we review natural-based biomaterials that are used in OoCs and their main characteristics. We address the preparation, modification, and characterization methods of natural-based biomaterials and summarize recent reports on their applications in the design and fabrication of micro-tissue models. This article will help bioengineers select the proper biomaterials based on developing new technologies to meet clinical expectations and improve patient outcomes fusing disease modeling.

Hybrid Stromal Vascular Fraction (Hybrid-SVF): A New Paradigm in Mechanical Regenerative Cell Processing.

Enzymatic digestion of extracellular matrix (ECM) from lipoaspirate is the conventional form of harvesting stromal vascular fraction (SVF) called enzymatically digested SVF (E-SVF). Mechanical SVF (M-SVF) isolation has emerged as an alternative method, but it has also some limitations in terms of lower cell viability and diminished cell counts. To enhance the SVF qualitatively and quantitatively, we propose a novel concept called "hybrid-SVF," in which we combine M-SVF with the concentrated parts of adipose tissue after centrifugation, which is called stromal vascular matrix (SVM). Methods: Hybrid-SVF injection was applied as an adjunctive therapy to fat grafting in 88 patients and 11 samples were evaluated in the laboratory for cell count, viability and cell activity. Results: Experimental results determined that SVM part showed higher cellular activity. SVM and M-SVF showed higher cellular potency than E-SVF. Clinically, none of the patients required an additional session for fat grafting since there was no significant graft resorption. However, seven patients asked for further volume augmentation due to their individual preferences. No major complication was encountered. Conclusions: The usage of hybrid-SVF has a very high regenerative potential due to the ECM support and exceptionally high cell yield in addition to preserved cell potency. Although there are ongoing studies focusing on optimizing cell counts and further clinical applications, we believe that our preliminary results might create a paradigm shift in the area of regenerative fat grafting.

Hybrid extracellular vesicles-liposome incorporated advanced bioink to deliver microRNA.

In additive manufacturing, bioink formulations govern strategies to engineer 3D living tissues that mimic the complex architectures and functions of native tissues for successful tissue regeneration. Conventional 3D-printed tissues are limited in their ability to alter the fate of laden cells. Specifically, the efficient delivery of gene expression regulators (i.e. microRNAs (miRNAs)) to cells in bioprinted tissues has remained largely elusive. In this study, we explored the inclusion of extracellular vesicles (EVs), naturally occurring nanovesicles (NVs), into bioinks to resolve this challenge. EVs show excellent biocompatibility, rapid endocytosis, and low immunogenicity, which lead to the efficient delivery of miRNAs without measurable cytotoxicity. EVs were fused with liposomes to prolong and control their release by altering their physical interaction with the bioink. Hybrid EVs-liposome (hEL) NVs were embedded in gelatin-based hydrogels to create bioinks that could efficiently encapsulate and deliver miRNAs at the target site in a controlled and sustained manner. The regulation of cells' gene expression in a 3D bioprinted matrix was achieved using the hELs-laden bioink as a precursor for excellent shape fidelity and high cell viability constructs. Novel regulatory factors-loaded bioinks will expedite the translation of new bioprinting applications in the tissue engineering field.

Garlic (Allium sativum)-derived SEVs inhibit cancer cell proliferation and induce caspase mediated apoptosis.

As a key component of the cell-to-cell communication, small extracellular vesicles (SEVs) released from various sources are known to be affecting the physiological conditions of the target cells. Although it has been suggested that edible plant-derived nanoparticles contributes to the cross kingdom communication with the mammalian cells, the effect of these particles on cancer cell progression still needs a further exploration. Here, we isolated and then characterized garlic derived SEVs by nanoparticle tracking analysis, electron microscopy and SEV surface antibodies. In order to investigate anti-cancer property of garlic SEVs A498 human kidney carcinoma, A549 human lung carcinoma were used as cell models along with the normal human dermal fibroblast cell lines. Annexin V/pI staining and analysis of apoptotic mRNA and protein expression levels suggested that garlic SEVs induced apoptosis through activation of intrinsic pathway. Furthermore, angiogenic VEGF protein expression levels significantly decreased in response to SEVs treatment in cancer cells. Our results support that garlic derived SEVs could cause apoptotic cell death among cancer cells while normal cells remain unaffected with the treatment. This study revealed for the first time that plant SEVs possess anti-cancer affects by inducing caspase mediated apoptosis and provided a new alternative for cancer treatment.

The Utilization of Poly(2-ethyl-2-oxazoline)-b-Poly(ε-caprolactone) Ellipsoidal Particles for Intracellular BIKDDA Delivery to Prostate Cancer.

Prostate cancer is the most common cancer, which is about 15-20% among male cancers worldwide. As most common strategies such as radiotherapy, chemotherapy, or surgery alone can be unsuccessful in the treatment of prostate cancer, this study aims to develop a new approach to deliver newly generated proapoptotic gene, BIKDDA, to androgen independent prostate cancer cells, 22RV1, using new generation nanocarriers called ellipsoids. As far as it is known, this is the first study that assesses the ability of proapoptotic gene BIKDDA to induce apoptosis in prostate cancer cell. BIKDDA encapsulating PEtOx-b-PCL-based ellipsoids are fabricated by solvent-switch method, and their morphology, size, and BIKDDA content are characterized. Gene delivery efficiency of BIKDDA loaded PEtOx-b-PCL ellipsoids is demonstrated by analysis of BIK mRNA expression with real-time PCR. The apoptotic effect of PEtOx-b-PCL ellipsoids loaded with BIKDDA (EPs-BIKDDA) on 22RV1 is shown by Annexin V staining. The obtained results demonstrate that the treatment of 22RV1 cells with EPs-BIKDDA can significantly increase BIK mRNA levels by 4.5-fold leading to cell death. This study not only represents BIKDDA as a potential therapeutic strategy in prostate cancer but also the capacity of ellipsoids as promising in vivo gene delivery vehicles.

A 3-step Mechanical Digestion Method to Harvest Adipose-derived Stromal Vascular Fraction.

BACKGROUND: Adipose stromal vascular fraction (SVF) isolation with enzymatic digestion is the gold standard, but is expensive, having practical and legal concerns. The alternative mechanical SVF isolation methods provide lower cell yields as they employ either centrifugation, emulsification, or digestion steps alone. We combined mechanical processing with buffer incubation and centrifugation steps into an isolation method called "mechanical digestion" and compared the cell yields with that of enzymatic digestion. METHODS: A total of 40-mL lipoaspirate was harvested from 35 women undergoing liposuction and was submitted to conventional enzymatic digestion for SVF isolation or mechanical digestion using a closed unit harnessing 3 ports with blades, followed by buffer incubation and centrifugation. Culture of the SVFs and flow cytometry were performed. RESULTS: The SVF cell yield obtained by enzymatic digestion was significantly higher 3.38 × 106/mL (±3.63; n = 35) than that obtained by mechanical digestion 1.34 × 106/mL (±1.69; n = 35), P = 0.015. The average cell viability was 82.86% ± 10.68 after enzymatic digestion versus 85.86% ± 5.74 after mechanical digestion, which was not significant. Mechanical digested SVF expressed 2-fold higher stem cell surface markers compared with enzymatically digested SVF. Mechanical digestion was less time consuming, cost effective, and did not require a specific laboratory environment. CONCLUSIONS: Mechanically digested SVF was comparable to enzymatically digested SVF in terms of stromal cell composition and viability. With mechanical digestion, we can isolate 30%-50% SVF cells of that isolated with enzymatic digestion. Further studies are warranted to determine the clinical outcomes.

Engineering Smart Targeting Nanovesicles and Their Combination with Hydrogels for Controlled Drug Delivery.

Smart engineered and naturally derived nanovesicles, capable of targeting specific tissues and cells and delivering bioactive molecules and drugs into them, are becoming important drug delivery systems. Liposomes stand out among different types of self-assembled nanovesicles, because of their amphiphilicity and non-toxic nature. By modifying their surfaces, liposomes can become stimulus-responsive, releasing their cargo on demand. Recently, the recognized role of exosomes in cell-cell communication and their ability to diffuse through tissues to find target cells have led to an increase in their usage as smart delivery systems. Moreover, engineering "smarter" delivery systems can be done by creating hybrid exosome-liposome nanocarriers via membrane fusion. These systems can be loaded in naturally derived hydrogels to achieve sustained and controlled drug delivery. Here, the focus is on evaluating the smart behavior of liposomes and exosomes, the fabrication of hybrid exosome-liposome nanovesicles, and the controlled delivery and routes of administration of a hydrogel matrix for drug delivery systems.

In Vitro Wound Healing Activity of Wheat-Derived Nanovesicles.

Triticum aestivum plant extracts are often used as a natural healer in traditional medicine but which particles mainly have role in these processes are not scientifically proven. In other words, no attempts have been made to investigate the effects of wheat exosomes in regenerative medicine applications or drug development up to now. The current study was first time performed to demonstrate the activity of wheat exosomes in wound healing process using in vitro approaches. Although its fundamental wound healing process remains a mystery, in the current study, the efficiency of wheat grass juice-derived exosomes on cell viability and migration was examined. Increasing concentrations up to 200 µg/mL of the wheat exosome have yielded astonishing proliferative and migratory effects on endothelial, epithelial, and dermal fibroblast cells. RT-PCR analysis also showed collagen type I; mRNA levels were approximately twofold higher in expression after treating with 200 µg/mL wheat exosome. Additionally, Annexin V staining of apoptotic cells accompanied with the cell cycle analysis resulted with the reduction of the apoptotic cell number with no dispersion to the cell cycle analysis while plant exosomes have also increased tube-like structure formation of the endothelial cells. All in all, this research suggests a brand-new opening for skin wound healing therapy strategy by using wheat-derived exosomes due to its proliferative and migratory characteristics. Plant exosomes require a further research both clinically and in in vivo for wound healing drug development. Moreover, plant exosome therapy strategies would be safer and economical alternative for clinical wound healing.