Anticancer and Chemosensitizing Effects of Menadione-Containing Peptide-Targeted Solid Lipid Nanoparticles.

Vitamin K derivatives such as menadione (MD) have been recognized as promising redox-modulating and chemosensitizing agents for anticancer therapy, however, their cellular activities in peptide-targeted nanocarriers have not been elucidated to date. This study provides the guidelines for developing MD-loaded solid lipid nanoparticles (SLN) modified with extracellular matrix (ECM)-derived peptides. Relationships between RGD peptide concentration and changes in DLS characteristics as well as accumulation of SLN in cancer cells were revealed to adjust the peptide-lipid ratio. SLN system maintained adequate nanoparticle concentration and low dispersity after introduction of MD and MD/RGD, whereas formulated MD was protected from immediate conjugation with reduced glutathione (GSH). RGD-modified MD-containing SLN showed enhanced prooxidant, GSH-depleting and cytotoxic activities toward PC-3 prostate cancer cells attributed to improved cellular pharmacokinetics of the targeted formulation. Furthermore, this formulation effectively sensitized PC-3 cells and OVCAR-4 ovarian cancer cells to free doxorubicin and cisplatin so that cell growth was inhibited by MD-drug composition at nontoxic concentrations of the ingredients. These results provide an important background for further improving chemotherapeutic methods based on combination of conventional cytostatics with peptide-targeted SLN formulations of MD.

Evaluation of GHK peptide-heparin interactions in multifunctional liposomal covering.

Small biospecific peptides with defined chemical structure and cellular responses are promising alternatives to full-length therapeutic proteins. Identification of these peptides solely or in combination with other bioactive factors and determination of their targets are of substantial interest in current drug delivery research. This study is aimed at the development of new liposomal formulations of ECM-derived GHK peptide known for its multiple regeneration-related activities but poorly recognized cellular targets. In situ association of membranotropic GHK derivative with unilamellar liposomes was performed to prepare GHK-modified liposomes with defined properties. According to DLS, the GHK component on the liposomal surface interacted with heparin in a specific manner compared to other polysaccharides and RGD counterpart, whereas ITC analysis of such interactions was complicated. The results provide a useful tool for screening of bio-interactions of synthetic peptide-presenting liposomes by the DLS technique. They were also employed to produce a multi-functional nanosized GHK-heparin covering for liposomes. The resulting composite liposomes possessed low size dispersity, increased anionic charge, and mechanical rigidity. The heparin component significantly promoted the accumulation of GHK-modified liposomes in 3T3 fibroblasts so that the composite liposomes exhibited the highest cell-penetrating activity. Furthermore, the latter formulation stimulated cell proliferation and strongly inhibited ROS production and GSH depletion under oxidative stress conditions. Together, the results support that cell-surface glycosaminoglycans can be involved in GHK-mediated liposomal delivery, which can be further greatly enhanced by association with heparin. The composite liposomes with GHK-heparin covering can be considered as an advanced GHK-based formulation for therapeutic and cosmeceutical applications.

Metal-Chelating Self-Assembling Peptide Nanofiber Scaffolds for Modulation of Neuronal Cell Behavior.

Synthetic peptides are promising structural and functional components of bioactive and tissue-engineering scaffolds. Here, we demonstrate the design of self-assembling nanofiber scaffolds based on peptide amphiphile (PA) molecules containing multi-functional histidine residues with trace metal (TM) coordination ability. The self-assembly of PAs and characteristics of PA nanofiber scaffolds along with their interaction with Zn, Cu, and Mn essential microelements were studied. The effects of TM-activated PA scaffolds on mammalian cell behavior, reactive oxygen species (ROS), and glutathione levels were shown. The study reveals the ability of these scaffolds to modulate adhesion, proliferation, and morphological differentiation of neuronal PC-12 cells, suggesting a particular role of Mn(II) in cell-matrix interaction and neuritogenesis. The results provide a proof-of-concept for the development of histidine-functionalized peptide nanofiber scaffolds activated with ROS- and cell-modulating TMs to induce regenerative responses.

Oligo (Poly (Ethylene Glycol) Fumarate)-Based Multicomponent Cryogels for Neural Tissue Replacement.

Synthetic hydrogels provide a promising platform to produce neural tissue analogs with improved control over structural, physical, and chemical properties. In this study, oligo (poly (ethylene glycol) fumarate) (OPF)-based macroporous cryogels were developed as a potential next-generation alternative to a non-porous OPF hydrogel previously proposed as an advanced biodegradable scaffold for spinal cord repair. A series of OPF cryogel conduits in combination with PEG diacrylate and 2-(methacryloyloxy) ethyl-trimethylammonium chloride (MAETAC) cationic monomers were synthesized and characterized. The contribution of each component to viscoelastic and hydration behaviors and porous structure was identified, and concentration relationships for these properties were revealed. The rheological properties of the materials corresponded to those of neural tissues and scaffolds, according to the reviewed data. A comparative assessment of adhesion, migration, and proliferation of neuronal cells in multicomponent cryogels was carried out to optimize cell-supporting characteristics. The results show that OPF-based cryogels can be used as a tunable synthetic scaffold for neural tissue repair with advantages over their hydrogel counterparts.

Probing Cell Redox State and Glutathione-Modulating Factors Using a Monochlorobimane-Based Microplate Assay.

Thiol compounds including predominantly glutathione (GSH) are key components of redox homeostasis, which are involved in the protection and regulation of mammalian cells. The assessment of cell redox status by means of in situ analysis of GSH in living cells is often preferable over established assays in cell lysates due to fluctuations of the GSH pool. For this purpose, we propose a microplate assay with monochlorobimane (MCB) as an available fluorescent probe for GSH, although poorly detected in the microplate format. In addition to the new procedure for improved MCB-assisted GSH detection in plate-grown cells and its verification with GSH modulators, this study provides a useful methodology for the evaluation of cell redox status probed through relative GSH content and responsiveness to both supplemented thiols and variation in oxygen pressure. The roles of extracellular interactions of thiols and natural variability of cellular glutathione on the assay performance were emphasized and discussed. The results are of broad interest in cell biology research and should be particularly useful for the characterization of pathological cells with decreased GSH status and increased oxidative status as well as redox-modulating factors.

Regenerative Activities of ROS-Modulating Trace Metals in Subcutaneously Implanted Biodegradable Cryogel.

Divalent trace metals (TM), especially copper (Cu), cobalt (Co) and zinc (Zn), are recognized as essential microelements for tissue homeostasis and regeneration. To achieve a balance between therapeutic activity and safety of administered TMs, effective gel formulations of TMs with elucidated regenerative mechanisms are required. We studied in vitro and in vivo effects of biodegradable macroporous cryogels doped with Cu, Co or Zn in a controllable manner. The extracellular ROS generation by metal dopants was assessed and compared with the intracellular effect of soluble TMs. The stimulating ability of TMs in the cryogels for cell proliferation, differentiation and cytokine/growth factor biosynthesis was characterized using HSF and HUVEC primary human cells. Multiple responses of host tissues to the TM-doped cryogels upon subcutaneous implantation were characterized taking into account the rate of biodegradation, production of HIF-1α/matrix metalloproteinases and the appearance of immune cells. Cu and Zn dopants did not disturb the intact skin organization while inducing specific stimulating effects on different skin structures, including vasculature, whereas Co dopant caused a significant reorganization of skin layers, the appearance of multinucleated giant cells, along with intense angiogenesis in the dermis. The results specify and compare the prooxidant and regenerative potential of Cu, Co and Zn-doped biodegradable cryogels and are of particular interest for the development of advanced bioinductive hydrogel materials for controlling angiogenesis and soft tissue growth.

Design and Assessment of Biodegradable Macroporous Cryogels as Advanced Tissue Engineering and Drug Carrying Materials.

Cryogels obtained by the cryotropic gelation process are macroporous hydrogels with a well-developed system of interconnected pores and shape memory. There have been significant recent advancements in our understanding of the cryotropic gelation process, and in the relationship between components, their structure and the application of the cryogels obtained. As cryogels are one of the most promising hydrogel-based biomaterials, and this field has been advancing rapidly, this review focuses on the design of biodegradable cryogels as advanced biomaterials for drug delivery and tissue engineering. The selection of a biodegradable polymer is key to the development of modern biomaterials that mimic the biological environment and the properties of artificial tissue, and are at the same time capable of being safely degraded/metabolized without any side effects. The range of biodegradable polymers utilized for cryogel formation is overviewed, including biopolymers, synthetic polymers, polymer blends, and composites. The paper discusses a cryotropic gelation method as a tool for synthesis of hydrogel materials with large, interconnected pores and mechanical, physical, chemical and biological properties, adapted for targeted biomedical applications. The effect of the composition, cross-linker, freezing conditions, and the nature of the polymer on the morphology, mechanical properties and biodegradation of cryogels is discussed. The biodegradation of cryogels and its dependence on their production and composition is overviewed. Selected representative biomedical applications demonstrate how cryogel-based materials have been used in drug delivery, tissue engineering, regenerative medicine, cancer research, and sensing.

Enhanced angiogenic effects of RGD, GHK peptides and copper (II) compositions in synthetic cryogel ECM model.

Synthetic oligopeptides are a promising alternative to natural full-length growth factors and extracellular matrix (ECM) proteins in tissue regeneration and therapeutic angiogenesis applications. In this work, angiogenic properties of dual and triple compositions containing RGD, GHK peptides and copper (II) ions (Cu2+) were for the first time studied. To reveal specific in vitro effects of these compositions in three-dimensional scaffold, adamantyl group bearing peptides, namely Ada-Ahx-GGRGD (1) and Ada-Ahx-GGGHK (2), were effectively immobilized in bioinert pHEMA macroporous cryogel via host-guest ß-cyclodextrin-adamantane interaction. The cryogels were additionally functionalized with Cu2+ via the formation of GHK-Cu complex. Angiogenic responses of HUVECs grown within the cryogel ECM model were analyzed. The results demonstrate that the combination of RGD with GHK and further with Cu2+ dramatically increases cell proliferation, differentiation, and production of a series of angiogenesis related cytokines and growth factors. Furthermore, the level of glutathione, a key cellular antioxidant and redox regulator, was altered in relation to the angiogenic effects. These results are of particular interest for establishing the role of multiple peptide signals on regeneration related processes and for developing improved tissue engineering materials.

In Situ functionalization of Poly(hydroxyethyl methacrylate) Cryogels with Oligopeptides via ß-Cyclodextrin-Adamantane Complexation for Studying Cell-Instructive Peptide Environment.

Oligopeptides are versatile cell modulators resembling pleiotropic activities of ECM proteins and growth factors. Studying the role of cell-instructive peptide signals within 3D scaffolds, yet poorly known, requires effective approaches to introducing bioactive sequences into appropriate materials. We synthesized RGD and GHK motif based peptides 1 and 2 linked to the terminal adamantyl group (Ad) and their fluorescent derivatives 3 and 4. Poly(hydroxyethyl methacrylate) (pHEMA) cryogels with additional PEG/ß-cyclodextrin (CD) units were prepared as an inert macroporous scaffold capable to bind the adamantylated peptides via affinity CD-Ad complexation. According to toluidine blue staining, the CD moieties were effectively and stably incorporated in the pHEMA cryogels at nanomolar amounts per milligram of material. The CD component gradually increased the thickness and swelling ability of the polymer walls of cryogels, resulting in a noticeable decrease in macropore size and modulation of viscoelastic properties. The labeled peptides exhibited fast kinetics of specific binding to the CD-modified cryogels and were simultaneously immobilized by coincubation. The peptide loading approached ca. 0.31 mg per cm2 of cryogel sheet. A well-defined mitogenic effect of the immobilized peptides (2 < 1≪ 1 + 2) was revealed toward 3T3 and PC-12 cells. The synergistic action of RGD and GHK peptides induced a profound change in cell behavior/morphology attributed to a growth-factor-like activity of the composition. Altogether, our results provide an effective procedure for the preparation of CD-modified pHEMA cryogels and their uniform in situ functionalization with bioactive peptide(s) of interest and an informative study of cellular responses in the functionalized scaffolds.

Transition metal-doped cryogels as bioactive materials for wound healing applications.

Transition metals (TM) are essential microelements with various biological functions demanded in tissue regeneration applications. Little is known about therapeutic potential of TM within soft hydrogel biomaterials. The soluble divalent TM, such as Zn, Cu, Mn and Co, were stably incorporated into gelatin network during cryogelation. TM content in the resultant cryogels varied from 0.1 × 103 to 11.8 × 103 ppm, depending on the TM type and concentration in the reaction solution. Zn component was uniformly complexed with the gelatin scaffold according to elemental imaging, increasing the swelling of polymer walls and the G'/G″ values and also decreasing the size of cryogel macro-pores. Zn-doped cryogels supported migration of human skin fibroblasts (HSF); only upper Zn content of 11.8 × 103 ppm in the scaffold caused c.a. 50% inhibition of cell growth. Zn ions solubilized in culture medium were more active towards HSF (IC50 ≈ 0.3 mM). Treatment of splinted full-skin excisional wounds in rats with the Zn-doped and non-doped cryogels showed that Zn considerably promoted passing inflammatory/proliferation phases of healing process, inducing more intense dermis formation and structuration. The results show the feasibility of development of cryogel based formulations with different TM and support high phase-specific ability of the Zn-gelatin cryogels to repair acute wounds.