A molecular hybrid comprising AS1411 and PDGF-BB aptamer, cholesterol, and doxorubicin for inhibiting proliferation of SW480 cells.

Cancer treatment commonly relies on chemotherapy. This treatment faces many challenges, including treatment specificity and undesired side effects. To address these, a Dox-loaded Chol-aptamer molecular hybrid (Dox-CAH) was developed. This multivalent interaction system combines the key function of each integrated species: doxorubicin, cholesterol, and two aptamers binding to nucleolin and platelet-derived growth factor BB (PDGF-BB). The study has four stages: preparation of CAH via oligonucleotide hybridization, intercalation of doxorubicin into CAH, verification of CAH binding on SW480 by fluorescence microscopy and flow cytometry, and investigation of effect of Dox-CAH on SW480 proliferation. CAH was successfully prepared, as confirmed by electrophoresis. Flow cytometry and fluorescence microscopy demonstrated CAH binding to SW480, due to the presence of the AS1411 aptamer. This molecular hybrid exhibited specific binding because it did not bind to CCD 841 CoN. CAH binding to PDGF-BB compromises its function, as shown by enzyme-linked immunosorbent assay (ELISA) and cell assay. The DNA duplex in this molecular hybrid reduces the cytotoxicity of the Dox-CAH. Binding and the reduction of Dox-CAH toxicity may improve treatment specificity and minimize side effects. Dox-CAH is a model for more effective anticancer therapy, allowing incorporation of chemotherapeutic drugs and recognition elements.

3D-printed construct from hybrid suspension as spatially and temporally controlled protein delivery system.

Protein delivery systems have been extensively applied in controlled releasing of protein or polypeptides for therapeutic treatment or tissue regeneration. While 3 D printing technology shows great promise in novel dosage form with tailoring dose size and drug release profile, 3 D printable protein delivery system has to face many difficult challenges. In this study, we developed a hybrid suspension combining Eudragit polyacrylate colloid as matrix material and Pluronic polyether hydrogel as diffusion channel for protein release. This hybrid suspension can be 3 D-printed into construct with complex shape and inner structures thanks to its pseudoplastic and thixotropic rheological properties. The protein can be incorporated in hybrid suspension either in its original or nanoparticle capsulated form. The experiment shows that the protein release from construct is a function of drying time, molecular weight (MW) of chitosan, as well as their own structural/diffusional properties. Also, the theoretical derivation suggests polyacrylate matrix tortuosity, chitosan erosion rate as well as hydrogel diffusion coefficient all contributed to the extended duration of release profile. In addition, cytotoxicity test through cell culture confirmed that the construct fabricated from hybrid suspension exhibit relative good bio-compatibility. Finally, heterogeneous constructs with zoned design were fabricated as protein delivery system, which demonstrated the capability of hybrid suspension technique for spatial and temporal release of macromolecular drugs to realize pharmaceutical effectiveness or guild cell organization.

Polydopamine-assisted PDGF-BB immobilization on PLGA fibrous substrate enhances wound healing via regulating anti-inflammatory and cytokine secretion.

Platelet-derived growth factor-bb (PDGF-BB) is a potent chemokine and mitogen for fibroblasts, keratinocytes, and vascular endothelium in the injured area, believed to be effective in wound healing. However, the short half-life of PDGF-BB and its rapid release from the wound surface limited its efficacy in vivo and vitro. To evaluate the wound healing effects of dorsal skin in SD rats with polydopamine-assisted immobilized PDGF-BB on PLGA nanofibrous substrate. First, the effects of pDA-coating and PDGF-BB immobilization on the morphology, compositions, and hydrophilicity of substrates were evaluated in details. Second, the wound healing effect of pDA/PLGA/PDGF-BB substrate was assessed in the dorsal skin of SD rats. Last, the cytokine analysis by ELISA method was employed to evaluate the advantages of pDA/PLGA/PDGF-BB substrate on anti-inflammatory, angiogenesis, and cellular proliferation. This method significantly improved the immobilization amount and stability of PDGF-BB on the substrate (p<0.01), further improved the hydrophilicity of substrates (p<0.05). Furthermore, the wound closure process was much more accelerated in the pDA/PLGA/PDGF-BB group (p<0.05). H&E and CD31 staining informed that the wound treated by pDA/PLGA/PDGF-BB substrate showed a high degree of regeneration and angiogenesis. The cytokine analysis showed that pDA significantly reduced the high level of inflammatory cytokines such as TNF-α (p<0.05). And the immobilized PDGF-BB significantly elevated the level of TGF-ß and VEGF (p<0.05). The pDA/PLGA/PDGF-BB substrate showed great therapeutic effect on wound healing compared with other control groups via regulating anti-inflammatory, angiogenesis, and cellular proliferation. Absolutely, this report offered an available novel method for skin regeneration.

Exogenous Signaling Molecules Released from Aptamer-Functionalized Hydrogels Promote the Survival of Mesenchymal Stem Cell Spheroids.

Mesenchymal stem cells (MSCs) have a very low survival rate after in vivo delivery, which limits their great promise for treating human diseases. Various strategies have been studied to overcome this challenge. However, an overlooked but important potential is to apply exogenous signaling molecules as biochemical cues to promote MSC survival, presumably because it is well-known that MSCs themselves can release a variety of potent signaling molecules. Thus, the purpose of this work was to examine and understand whether the release of exogenous signaling molecules from hydrogels can promote the survival of MSC spheroids. Our data show that more vascular endothelial growth factor (VEGF) but not platelet-derived growth factor BB (PDGF-BB) were released from MSC spheroids in comparison with 2D cultured MSCs. Aptamer-functionalized fibrin hydrogel (aFn) could release exogenous VEGF and PDGF-BB in a sustained manner. PDGF-BB-loaded aFn promoted MSC survival by ∼70% more than VEGF-loaded aFn under the hypoxic condition in vitro. Importantly, PDGF-BB-loaded aFn could double the survival rate of MSC spheroids in comparison with VEGF-loaded aFn during the one-week test in vivo. Therefore, this work demonstrated that defined exogenous signaling molecules (e.g., PDGF-BB) can function as biochemical cues for promoting the survival of MSC spheroids in vivo.

Bioactive proteins delivery through core-shell nanofibers for meniscal tissue regeneration.

Mimicking the ultrastructural morphology of the meniscus with nanofiber scaffolds, coupled with controlled growth-factor delivery to the appropriate cells, can help engineer tissue with the potential to grow, mature, and regenerate after in vivo implantation. We electrospun nanofibers encapsulating platelet-derived growth factor (PDGF-BB), which is a potent mitogen and chemoattractant in a core of serum albumin contained within a shell of polylactic acid. We controlled the local PDGF-BB release by adding water-soluble polyethylene glycol to the polylactic acid shell to serve as a porogen. The novel core-shell nanofibers generated 3D scaffolds with an interconnected macroporous structure, with appropriate mechanical properties and with high cell compatibility. Incorporating PDGF-BB increased cell viability, proliferation, and infiltration, and upregulated key genes involved in meniscal extracellular matrix synthesis in human meniscal and synovial cells. Our results support proof of concept that these core-shell nanofibers can create a cell-favorable nanoenvironment and can serve as a system for sustained release of bioactive factors.

Transgenic PDGF-BB/sericin hydrogel supports for cell proliferation and osteogenic differentiation.

Sericin has been exploited as a biomaterial due to its biocompatibility, biodegradability, and low-immunogenicity as an isolated polymer and support for cell adhesion. In the present study, human platelet-derived growth factor (PDGF-BB)-functionalized sericin hydrogels were generated using transgenic silkworms, where the as-spun silk incorporated engineered PDGF-BB (termed PDGFM) in the sericin layers of the cocoons. Sericin and PDGFM were simultaneously extracted from the silk fibroin cocoon fibers, and the soluble extract was then formed into a hydrogel via thermal exposure. The PDGFM sericin hydrogels exhibited increased ß-sheet content and a compressive modulus of 74.91 ± 2.9 kPa comparable to chemically crosslinked sericin hydrogels (1.68-55.53 kPa) and a porous microstructure, which contributed to cell adhesion and growth. A 13.1% of total extracted PDGFM from the initial silk fibers was incorporated and immobilized in the sericin hydrogels during material processing, and 1.33% of PDGFM was released over 30 days from the hydrogels in vitro. The remaining PDGFM achieved long-term storage/stability in the sericin hydrogels for more than 42 days at 37 °C. In addition, the PDGFM sericin hydrogels were not immunogenic, were biocompatible and bioactive in promoting the support of cell proliferation. When combined with BMP-9, the PDGFM sericin hydrogels provided synergy to support the osteoblastic differentiation of mesenchymal stem cells (hMSCs) in vitro and in vivo. This study demonstrates that genetically functionalized PDGFM sericin hydrogels can provide useful biomaterials to support cell and tissue outcomes, here with a focus on osteogenesis.

Engineering a microcarrier based on a polysaccharide-growth factor complex for enhancing the proliferation of mesenchymal stem cells.

Mesenchymal stem cell (MSC) delivery has been broadly investigated as a cell-based therapy strategy towards various diseases and tissue injury. In these applications, the cell-delivery vehicle plays a crucial role in determining the therapeutic performance of MSCs and their fate post-implantation. We report here the development of a microcarrier system combining platelet-derived growth factor-BB (PDGF-BB) and a PDGF-BB-binding polysaccharide - Eucommia ulmoides (EUP3) - for MSC cultivation. First, we investigated the optimal conditions to prepare the EUP3-PDGF-BB complex, by comparing its i) diameter, ii) morphology, and iii) bioactivity to promote MSC proliferation and fibroblast migration in vitro, under different PDGF-BB/EUP3 ratios. Then, we fabricated microspheres using gelatin and EUP3 as the matrix while stabilizing PDGF-BB at the optimal ratio for MSC adhesion and growth. Live staining and SEM observation indicated that the prepared microspheric carrier supported MSC growth and maintained cell stemness. We suggest that the EUP3/PDGF-gelatin microcarriers can potentially serve as a cell-delivery vehicle for tissue engineering.

Sustained delivery of growth factors with high loading efficiency in a layer by layer assembly.

Layer by layer (LBL) assembly has garnered considerable interest due to its ability to generate multifunctional films with high tunability and versatility in terms of substrates and polyelectrolytes, allowing the option to use complex devices and drugs. Polyelectrolytes, such as growth factors (GFs), are essential, but costly, delicate, biological molecules that have been used in various tissue regeneration applications. For this reason, the controlled drug delivery of efficiently loaded GFs via LBL assembly (GF-LBL) can contribute to the establishment of cost-effective biologically triggered biomedical applications. We have developed an LBL method to load GFs (specifically, transforming growth factor beta 1, platelet-derived growth factor ßß, and insulin growth factor 1), with up to 90% efficiency approximately, by gas plasma surface activation and tuning the pH to increase the ionic strength of polyelectrolytes. Poly(styrenesulfonate) (PSS) and poly(ethyleneimine) (PEI) have been used to provide the initial necessary charge for multilayer build-up. Heparin and dextran sulphate have been investigated as counter polyelectrolytes to enhance the activity of GFs by protecting their ligands, where heparin resulted in the highest achievable loading efficiency for all GFs. Oxygen gas plasma and acidic pH levels also resulted in a significant increase in GF loading efficiency. The three GFs were released by diffusion and erosion in a controlled manner over lengthy time scales and the bioactivity was maintained for up to 14 days. When tested as implants in vitro, GF-LBL constructs increased fibroblast proliferation, influenced cell morphology and migration, and enhanced myofibroblast differentiation, indicating that the biological functionalities of the GFs were preserved. In conclusion, this developed LBL assembly method can provide a simple drug delivery system, which may yield more effective applications for tissue regeneration as well as biomedical sciences at large.

The Phenylethanol Glycoside Liposome Inhibits PDGF-Induced HSC Activation via Regulation of the FAK/PI3K/Akt Signaling Pathway.

Cistanche tubulosa is a traditional Chinese herbal medicine that is widely used to regulate immunity, and phenylethanol glycosides (CPhGs) are among the primary components responsible for this activity. However, the application of CPhGs is negatively affected by their poor absorption and low oral utilization. Targeted drug delivery is an important development direction for pharmaceutics. Previous studies have indicated that CPhGs could block the conduction of the signaling pathways in TGF-ß1/smad and inhibit the activation of hepatic stellate cells (HSCs). The aim of this study was to evaluate the anti-hepatic fibrosis effect of CPhG liposomes by inhibiting HSC activation, promoting apoptosis, blocking the cell cycle, suppressing the conduction of signaling pathways in focal adhesion kinase(FAK)/phosphatidylinositol-3-kinase(PI3K)/protein kinase B(Akt), and determining their in vitro hepatoprotective activity. In vitro release studies demonstrated that CPhG liposomes have a sustained release effect compared to drug CPhGs. HSC proliferation was inhibited after treatment with the CPhG liposomes (29.45, 14.72, 7.36 µg/mL), with IC50 values of 42.54 µg/mL in the MTT assay. Different concentrations of the CPhG liposomes could inhibit HSC proliferation, promote apoptosis, and block the cell cycle. The MTT method showed an obvious inhibition of HSC proliferation after CPhG liposome and Recombinant Rat Platelet-derived growth factor-BB(rrPDGF-BB) treatment. The levels of collagen-1, metallopeptidase inhibitor 1 (TIMP-1), α smooth muscle actin (α-SMA), and phosphorylated PI3K/Akt were downregulated, and matrix metalloproteinase-1 (MMP-1) was upregulated, by pretreatment with different concentrations of CPhG liposomes. Moreover, 29.45 µg/mL of CPhG liposomes could decrease the expression of the FAK protein and the phosphorylated PI3K and Akt protein downstream of FAK by overexpression of the FAK gene. This experiment suggests that CPhG liposomes may inhibit the activation of HSCs by inhibiting FAK and then reducing the expression of phosphorylated Akt/PI3K, thereby providing new insights into the application of CPhGs for liver fibrosis.

Inhibition of polycomb repressor complex 2 ameliorates neointimal hyperplasia by suppressing trimethylation of H3K27 in vascular smooth muscle cells.

BACKGROUND AND PURPOSE: The increased proliferation and migration of vascular smooth muscle cells (VSMCs) after arterial injury contributes greatly to the pathogenesis of neointimal hyperplasia. As a major component of epigenetics, histone methylation plays an important role in several cardiovascular diseases. However, its role in restenosis is still unclear. EXPERIMENTAL APPROACH: Human aortic VSMCs were challenged with PDGF-BB, and total histones were extracted and analysed by HPLC/MS. For the in vivo study, rats were subjected to wire-guided common carotid injury. KEY RESULTS: PDGF-BB markedly increased the H3K27me3 level, as demonstrated by use of HPLC/MS and confirmed by western blot analysis. Enhancer of zeste homologue 2 (EZH2), the histone H3K27 methyltransferase component of polycomb repressive complex 2, was also up-regulated by PDGF-BB in VSMCs, and in the neointimal hyperplasia induced by wire injury of the rat carotid artery. Furthermore, inhibiting H3K27me3 by treatment with 3-µM UNC1999, an EZH2/1 inhibitor, significantly suppressed PDGF-BB-induced VSMC proliferation compared with the PDGF-BB-treated group. Consistently, neointimal formation was significantly attenuated by oral or perivascular administration of UNC1999 compared with the sham group. Mechanistically, the increase in H3K27me3 inhibited the transcription of the cyclin-dependent kinase inhibitor p16INK4A and thus promoted VSMC proliferation. CONCLUSIONS AND IMPLICATIONS: Vascular injury elevated the expression of EZH2 and the downstream target H3K27me3, which suppressed p16INK4A expression in VSMCs and promoted VSMC proliferation and neointimal hyperplasia. EZH2 inhibition might be a potential therapeutic target for restenosis.

Ranking migration cue contributions to guiding individual fibroblasts faced with a directional decision in simple microfluidic bifurcations.

Directed cell migration in complex micro-environments, such as in vivo pores, is important for predicting locations of artificial tissue growth and optimizing scaffold architectures. Yet, the directional decisions of cells facing multiple physiochemical cues have not been characterized. Hence, we aim to provide a ranking of the relative importance of the following cues to the decision-making of individual fibroblast cells: chemoattractant concentration gradient, channel width, mitosis, and contact-guidance. In this study, bifurcated micro-channels with branches of different widths were created. Fibroblasts were then allowed to travel across these geometries by following a gradient of platelet-derived growth factor-BB (PDGF-BB) established inside the channels. Subsequently, a combination of statistical analysis and image-based diffusion modeling was used to report how the presence of multiple complex migration cues, including cell-cell influences, affect the fibroblast decision-making. It was found that the cells prefer wider channels over a higher chemoattractant gradient when choosing between asymmetric bifurcated branches. Only when the branches were symmetric in width did the gradient become predominant in directing which path the cell will take. Furthermore, when both the gradient and the channels were symmetric, contact guidance became important for guiding the cells in making directional choices. Based on these results we were able to rank these directional cues from most influential to the least as follows: mitosis > channel width asymmetry > chemoattractant gradient difference > and contact-guidance. It is expected that these results will benefit the fields of regenerative medicine, wound healing and developmental biology.

Dual Aptamer-Functionalized in Situ Injectable Fibrin Hydrogel for Promotion of Angiogenesis via Codelivery of Vascular Endothelial Growth Factor and Platelet-Derived Growth Factor-BB.

In situ injectable hydrogels hold great potential for in vivo applications such as drug delivery and regenerative medicine. However, it is challenging to ensure stable sequestration and sustained release of loaded biomolecules in these hydrogels. As aptamers have high binding affinities and specificities against target biomolecules, we studied the capability of aptamers in functionalizing in situ injectable fibrin (Fn) hydrogels for in vivo delivery of two growth factors including vascular endothelial growth factor (VEGF) and platelet-derived growth factor-BB (PDGF-BB). The results show that aptamer-functionalized fibrinogen (Fg) could form in situ injectable Fn hydrogels with porous structures. The aptamer-functionalized Fn hydrogels could sequester more VEGF and PDGF-BB than the native Fn and release these growth factors in a sustained manner with high bioactivity. After the aptamer-functionalized Fn hydrogels were subcutaneously injected into mice, the codelivery of VEGF and PDGF-BB could promote the growth of mature blood vessels. Therefore, this study has successfully demonstrated that aptamer-functionalized in situ injectable hydrogels hold great potential for in vivo codelivery of multiple growth factors and promotion of angiogenesis .

Platelet-derived growth factor stabilises vascularisation in collagen-glycosaminoglycan scaffolds in vitro.

Collagen-glycosaminoglycan (CG) scaffolds have been widely developed for a range of regenerative medicine applications. To enhance their efficacy, CG scaffolds have previously been prevascularised in vitro using human umbilical vein endothelial cells and human mesenchymal stromal cells (hMSCs); however, at later timepoints, a regression of vascularisation is observed. This is undesirable for longer preculture periods (e.g., for partial/full organ regeneration) and for in vitro vascularised tissue model systems (e.g., for drug testing/modelling). We hypothesised that delayed platelet-derived growth factor (PDGF)-BB addition could stabilise vessels, preventing their regression. In 2D, we identified 25 ng/ml as a suitable dose that enhanced hMSC metabolic activity and proliferation, without affecting endothelial cells, or migration in either cell type. In our 3D model of CG scaffold vascularisation, early addition of PDGF (Day 3) behaved similarly to no PDGF controls. However, PDGF addition at later timepoints (i.e., Days 4 and 5), with a second addition on Day 10, prevented vascular regression. In quantifying our observations, we identified a need for a tool to measure in vitro vascularisation in porous scaffolds. This was a second key objective of this work. A novel ImageJ macro was developed, which allowed us to analyse vessel-like structures, evaluating their number and morphology, and confirmed our qualitative observations. Finally, upregulation of angiogenic genes (ANG1, KDR, and TEK2) involved in vessel maturation illustrated how PDGF addition contributed to vascular stability. Taken together, the results suggest that addition of PDGF at specific timepoints can be used to stabilise vasculature in CG scaffolds.

Aptamer Conformation Switching-Induced Two-Stage Amplification for Fluorescent Detection of Proteins.

Basing on the conformation change of aptamer caused by proteins, a simple and sensitive protein fluorescent assay strategy is proposed, which is assisted by the isothermal amplification reaction of polymerase and nicking endonuclease. In the presence of platelet-derived growth factor (PDGF-BB), the natural conformation of a DNA aptamer would change into a Y-shaped complex, which could hybridize with a molecular beacon (MB) and form a DNA duplex, leading to the open state of the MB and generating a fluorescence signal. Subsequently, with further assistance of isothermal recycling amplification strategies, the designed aptamer sensing platform showed an increment of fluorescence. As a benefit of this amplified strategy, the limit of detection (LOD) was lowered to 0.74 ng/mL, which is much lower than previous reports. This strategy not only offers a new simple, specific, and efficient platform to quantify the target protein in low concentrations, but also shows a powerful approach without multiple washing steps, as well as a precious implementation that has the potential to be integrated into portable, low-cost, and simplified devices for diagnostic applications.

Electrospun Patch Functionalized with Nanoparticles Allows for Spatiotemporal Release of VEGF and PDGF-BB Promoting In Vivo Neovascularization.

The use of nanomaterials as carriers for the delivery of growth factors has been applied to a multitude of applications in tissue engineering. However, issues of toxicity, stability, and systemic effects of these platforms have yet to be fully understood, especially for cardiovascular applications. Here, we proposed a delivery system composed of poly(dl-lactide- co-glycolide) acid (PLGA) and porous silica nanoparticles (pSi) to deliver vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF). The tight spatiotemporal release of these two proteins has been proven to promote neovascularization. In order to minimize tissue toxicity, localize the release, and maintain a stable platform, we conjugated two formulations of PLGA-pSi to electrospun (ES) gelatin to create a combined ES patch releasing both PDGF and VEGF. When compared to freely dispersed particles, the ES patch cultured in vitro with neonatal cardiac cells had significantly less particle internalization (2.0 ± 1.3%) compared to free PLGA-pSi (21.5 ± 6.1) or pSi (28.7 ± 2.5) groups. Internalization was positively correlated to late-stage apoptosis with PLGA-pSi and pSi groups having increased apoptosis compared to the untreated group. When implanted subcutaneously, the ES patch was shown to have greater neovascularization than controls evidenced by increased expression of α-SMA and CD31 after 21 days. Quantitative reverse transcription-polymerase chain reaction results support increased angiogenesis by the upregulation of VEGFA, VEGFR2, vWF, and COL3A1, exhibiting a synergistic effect with the release of VEGF-A164 and PDGF-BB after 21 days in vivo. The results of this study proved that the ES patch reduced cellular toxicity and may be tailored to have a dual release of growth factors promoting localized neovascularization.

In vitro fibroblast migration by sustained release of PDGF-BB loaded in chitosan nanoparticles incorporated in electrospun nanofibers for wound dressing applications.

Migration of fibroblasts into wound area is a critical phenomenon in wound healing process. We used an appropriate system to fabricate an electrospun bioactive scaffold with controlled release of PDGF-BB in order to induce migration of primary skin fibroblast cells. First of all, protein-loaded chitosan nanoparticles based on ionic gelation interaction between chitosan and sodium tripolyphosphate were prepared. Then polycaprolactone electrospun fibers containing chitosan nanoparticles or PDGF-BB-loaded chitosan nanoparticles were prepared. Cellular attachment and morphology of cells seeded on scaffolds with or without PDGF-BB were evaluated by using a fluorescence microscope and scanning electron microscopy. Cells were well-oriented 72 h after seeding on the scaffolds containing PDGF-BB. The mean aspect ratio of populations on scaffold containing PDGF-BB-loaded chitosan nanoparticles was significantly greater than those on the scaffold containing chitosan nanoparticles but no PDGF-BB. Furthermore, the Arp2 gene, which is involved in cell protrusion formation, showed about three times more expression at mRNA level, in cells seeding on PDGF-BB-containing scaffold compared to cells seeding on scaffold containing only chitosan nanoparticles, using Real Time PCR test. Finally, under agarose migration assay results demonstrated that cells' chemotaxic behavior was more toward scaffold containing PDGF-BB compared to the PDGF-BB alone or FBS group. In addition, in terms of distance, the cell mass could grow faster, in response to scaffold containing PDGF-BB compared to FBS or PDGF-BB alone; however, the number of migrating cells might be the same or significantly higher in the latter groups.