Membrane-based inverse-transition purification facilitates a rapid isolation of various spider-silk elastin-like polypeptide fusion proteins from extracts of transgenic tobacco.

Plants can produce complex pharmaceutical and technical proteins. Spider silk proteins are one example of the latter and can be used, for example, as compounds for high-performance textiles or wound dressings. If genetically fused to elastin-like polypeptides (ELPs), the silk proteins can be reversibly precipitated from clarified plant extracts at moderate temperatures of ~ 30 °C together with salt concentrations > 1.5 M, which simplifies purification and thus reduces costs. However, the technologies developed around this mechanism rely on a repeated cycling between soluble and aggregated state to remove plant host cell impurities, which increase process time and buffer consumption. Additionally, ELPs are difficult to detect using conventional staining methods, which hinders the analysis of unit operation performance and process development. Here, we have first developed a surface plasmon resonance (SPR) spectroscopy-based assay to quantity ELP fusion proteins. Then we tested different filters to prepare clarified plant extract with > 50% recovery of spider silk ELP fusion proteins. Finally, we established a membrane-based purification method that does not require cycling between soluble and aggregated ELP state but operates similar to an ultrafiltration/diafiltration device. Using a data-driven design of experiments (DoE) approach to characterize the system of reversible ELP precipitation we found that membranes with pore sizes up to 1.2 µm and concentrations of 2-3 M sodium chloride facilitate step a recovery close to 100% and purities of > 90%. The system can thus be useful for the purification of ELP-tagged proteins produced in plants and other hosts.

Preparation of microfluidic-based pectin microparticles loaded carbon dots conjugated with BMP-2 embedded in gelatin-elastin-hyaluronic acid hydrogel scaffold for bone tissue engineering application.

The controlled delivery of the bone morphogenetic protein-2 (BMP-2) with tracking ability would overcome most of the side effects linked to the burst release and uncontrolled delivery of this growth factor for bone regeneration. Herein, BMP-2-conjugated carbon dots (CDs) was used as noninvasive detection platforms to deliver BMP-2 for therapeutic applications where osteogenesis and bioimaging are both required. With this in mind, the present work aimed to develop a controlled BMP-2-CDs release system using composite scaffolds containing BMP-2-CDs loaded pectin microparticles, which had been optimized for bone regeneration. By using microfluidic approach, we encapsulated BMP-2-CDs in pectin microparticles with narrow size distribution and then incorporated into composite scaffolds composed of gelatin, elastin, and hyaluronic acid. The BMP-2-CDs was released from the composite scaffolds in a sustained fashion for up to 21 days exhibited a high controlled delivery capacity. When tested in vitro with MG-63 cells, these extraction mediums showed the intercellular uptake of BMP-2-CDs and enhanced biological properties and pro-osteogenic effect. By utilizing the pectin microparticles carrying BMP-2-CDs as promising bioimaging agents for growth factor delivery and by tuning the composition of the scaffolds, this platform has immense potential in the field of bone tissue regeneration.

Improvement of the Bonding Properties of Mineral Trioxide Aggregate by Elastin-Like Polypeptide Supplementation.

INTRODUCTION: Elastin-like polypeptide (ELP) supplementation was previously reported to enhance the physical properties of mineral trioxide aggregate (MTA). The aim of this study was to investigate the effect of ELP supplementation on the bonding properties of MTA to dentin. METHODS: Two types of ELPs were synthesized and mixed with MTA in a 0.3 liquid/powder ratio. The push-out bond strength test and interfacial observation with scanning electron microscopy were performed for ELP-supplemented MTA. The porosity of MTA fillings in the cavity was observed with microcomputed tomography. The stickiness, flow rate, and contact angle were additionally measured for potential increased bonding properties. RESULTS: ELP supplementation improved the bond strength of MTA to dentin. MTA supplemented by a specific ELP exhibited a less porous structure, higher stickiness, and higher flow rate. ELPs also decreased the contact angle to dentin. CONCLUSIONS: This research data verifies that ELP improves the bonding properties of MTA to a tooth structure. The sticky and highly flowable characteristics of ELP-supplemented MTA may provide intimate contact with dentin and supply a less porous cement structure, which might improve the bonding properties of MTA.

Temperature-responsive silk-elastinlike protein polymer enhancement of intravesical drug delivery of a therapeutic glycosaminoglycan for treatment of interstitial cystitis/painful bladder syndrome.

Interstitial cystitis (IC), also known as painful bladder syndrome, is a debilitating chronic condition with many patients failing to respond to current treatment options. Rapid clearance, mucosal coating, and tight epithelium create strong natural barriers that reduce the effectiveness of many pharmacological interventions in the bladder. Intravesical drug delivery (IDD) is the administration of therapeutic compounds or devices to the urinary bladder via a urethral catheter. Previous work in improving IDD for IC has focused on the sustained delivery of analgesics within the bladder and other small molecule drugs which do not address underlying inflammation and bladder damage. Therapeutic glycosaminoglycans (GAG) function by restoring the mucosal barrier within the bladder, promoting healing responses, and preventing irritating solutes from reaching the bladder wall. There is an unmet medical need for a therapy that provides both acute relief of symptoms while alleviating underlying physiological sources of inflammation and promoting healing within the urothelium. Semi-synthetic glycosaminoglycan ethers (SAGE) are an emerging class of therapeutic GAG with intrinsic anti-inflammatory and analgesic properties. To reduce SAGE clearance and enhance its accumulation in the bladder, we developed a silk-elastinlike protein polymer (SELP) based system to enhance SAGE IDD. We evaluated in vitro release kinetics, rheological properties, impact on bladder function, pain response, and bladder inflammation and compared their effectiveness to other temperature-responsive polymers including Poloxamer 407 and poly(lactic-co-glycolic acid)-poly(ethylene glycol). SAGE delivered via SELP-enhanced intravesical delivery substantially improved SAGE accumulation in the urothelium, provided a sustained analgesic effect 24 h after administration, and reduced inflammation.

Flow imaging microscopy as a novel tool for high-throughput evaluation of elastin-like polymer coacervates.

Biological and bioinspired polymer microparticles have broad biomedical and industrial applications, including drug delivery, tissue engineering, surface modification, environmental remediation, imaging, and sensing. Full realization of the potential of biopolymer microparticles will require methods for rigorous characterization of particle sizes, morphologies, and dynamics, so that researchers may correlate particle characteristics with synthesis methods and desired functions. Toward this end, we evaluated biopolymer microparticles using flow imaging microscopy. This technology is widely used in the biopharmaceutical industry but is not yet well-known among the materials community. Our polymer, a genetically engineered elastin-like polypeptide (ELP), self-assembles into micron-scale coacervates. We performed flow imaging of ELP coacervates using two different instruments, one with a lower size limit of approximately 2 microns, the other with a lower size limit of approximately 300 nanometers. We validated flow imaging results by comparison with dynamic light scattering and atomic force microscopy analyses. We explored the effects of various solvent conditions on ELP coacervate size, morphology, and behavior, such as the dispersion of single particles versus aggregates. We found that flow imaging is a superior tool for rapid and thorough particle analysis of ELP coacervates in solution. We anticipate that researchers studying many types of microscale protein or polymer assemblies will be interested in flow imaging as a tool for quantitative, solution-based characterization.

A Genetically Encoded Protein Polymer for Uranyl Binding and Extraction Based on the SpyTag-SpyCatcher Chemistry.

A defining goal of synthetic biology is to develop biomaterials with superior performance and versatility. Here we introduce a purely genetically encoded and self-assembling biopolymer based on the SpyTag-SpyCatcher chemistry. We show the application of this polymer for highly efficient uranyl binding and extraction from aqueous solutions, by embedding two functional modules-the superuranyl binding protein and the monomeric streptavidin-to the polymer via genetic fusion. We further provide a modeling strategy for predicting the polymer's physical properties, and experimentally demonstrate the autosecretion of component monomers from bacterial cells. The potential of multifunctionalization, in conjunction with the genetic design and production pipeline, underscores the advantage of the SpyTag-SpyCatcher biopolymers for applications beyond trace metal enrichment and environmental remediation.

Understanding Solar Skin Elastosis-Cause and Treatment.

Photoageing, also called actinic ageing, is the main cause of prematurely aged skin. Our expertise in elastic fibers has led us to discover a process triggered in response to ultraviolet (UV) light and which upsets the balance of elastin fibers: there is too much elastin and insufficient lysyl oxidase (LOXL1) enzyme to form functional elastic fibers. This imbalance then leads to an accumulation of nonfunctional elastin, which forms aggregates. In addition to this imbalance, UV rays also induce elafin synthesis by fibroblasts. Known to be a marker of elastotic aggregates, elafin crystallizes the elastin fibers and stimulates the formation of aggregates that cannot be naturally eliminated by the skin. We developed a Hamamelis virginiana leaf extract that was able to restore both the balance between elastin and LOXL1 and to decrease the elafin synthesis to fight and correct the damage. This specific Hamamelis virginiana extract increased LOXL1 expression by twofold and decreased elafin synthesis. As a consequence, elastic fibers became functional and aggregates of unfunctional fibers decreased. The specific Hamamelis extract activity was confirmed in vivo with decreasing wrinkles and improving skin firmness.

Inhibition of Bromelain Activity During Enzymatic Debridement of Burn Wounds Pretreated With Frequently Used Products.

An enzyme mixture containing bromelain (NexoBrid®) was found to be suitable for enzymatic debridement of burn wounds, as determined by the criteria of patient comfort and pain, selectivity, and efficiency. Nevertheless, daily experience showed that pretreatment of burn wounds with several other clinical agents may inhibit debridement efficiency. Therefore, the current study was performed to identify those agents and evaluate their debridement inhibition capabilities. The impact of several common agents as well pH, on NexoBrid® debridement efficiency was evaluated in vitro. A collagen-based dermal substitute (MatriDerm®) was exposed to NexoBrid® in the presence of different agents of varying concentrations. Digestion was documented. The criteria used for judging digestion were independently classified by 3 investigators at least 3 times in succession. When a low concentration (1.0 mg/ml) of NexoBrid® was used, a ≥ 50% concentration of Prontosan® had an impact on enzymatic activity. Comparable results were obtained when even lower concentrations of Octenisept® (≥ 10%) were used. A 100-µmol/L concentration of copper inhibited the enzymatic activity of both a low (1.0 mg/ml) and high (10 mg/ml) concentration of NexoBrid®. Silver-sulfadiazine at concentrations of 10% and 90% inhibited the activity of 1 mg/ml NexoBrid®. No complete inhibition of NexoBrid® activity occurred at any concentration of iron. We recommend using polyhexanide-containing agents (Prontosan®) to rinse and presoak burn wounds. Pretreatment of burn wounds with agents containing silver and copper should be avoided. Experimentally, we found a partial inhibition of NexoBrid® activity at the distinct pH values of 3 and 11.

Novel hemostatic biomolecules based on elastin-like polypeptides and the self-assembling peptide RADA-16.

BACKGROUND: Safe and effective hemostatic materials are important for reducing mortality resulting from excessive hemorrhage. In this work, new biomaterials with hemostatic effects were created by fusing the gene coding for RADA-16, a self-assembling peptide with the sequence RADARADARADARADA, to the 3'-end of the open reading frame (ORF) encoding elastin-like polypeptides through gene recombination. RESULTS: The fusion proteins, termed 36R, 60R and 96R, were solubly over-expressed in Escherichia coli BL21 (DE3) based on genetic manipulation of the high-efficiency prokaryotic expression vector pET28a (+) and bacterial transformation. Western Blot analysis showed that the over-expressed proteins were the target fusion proteins. The target proteins 36R with 94.72% purity, 60R with 96.91% purity and 96R with 96.37% purity were prepared using an inverse phase transition cycle at 65 °C followed by His-tag affinity chromatography. The proliferation results of the mouse fibroblast cell line L929 and hippocampus neuron cell line HT22 indicated that the fusion proteins did not cause obvious cell toxicity. The lyophilized spongy film of the purified 36R, 60R and 96R could stop the hemorrhage of a 2 × 2 mm bleeding wound in the mouse liver after 27.21 ± 1.92 s, 18.65 ± 1.97 s and 15.85 ± 1.21 s, respectively. The hemostasis time was 21.23 ± 1.84 s for rat-tail collagen and 14.44 ± 1.33 s for RADA-16 lyophilized on gauze. The hemostatic time of three treated groups were all significantly superior to that of the negative control without any hemostasis treatment, which spontaneously stopped bleeding after 37.64 ± 1.34 s. Statistical analysis showed that the spongy film with purified 96R exhibited an exciting hemostatic effect that was superior to rat-tail collagen and close to that of RADA-16 lyophilized on gauze. CONCLUSIONS: These results revealed that the fusion proteins achieved by gene recombination technology could serve as a promising hemostatic material.

Improvement of physical properties of calcium phosphate cement by elastin-like polypeptide supplementation.

Calcium phosphate cements (CPCs) are synthetic bioactive cements widely used as hard tissue substitutes. Critical limitations of use include their poor mechanical properties and poor anti-washout behaviour. To address those limitations, we combined CPC with genetically engineered elastin-like polypeptides (ELPs). We investigated the effect of the ELPs on the physical properties and biocompatibility of CPC by testing ELP/CPC composites with various liquid/powder ratios. Our results show that the addition of ELPs improved the mechanical properties of the CPC, including the microhardness, compressive strength, and washout resistance. The biocompatibility of ELP/CPC composites was also comparable to that of the CPC alone. However, supplementing CPC with ELPs functionalized with octaglutamate as a hydroxyapatite binding peptide increased the setting time of the cement. With further design and modification of our biomolecules and composites, our research will lead to products with diverse applications in biology and medicine.

Magnetic Separation of Elastin-like Polypeptide Receptors for Enrichment of Cellular and Molecular Targets.

Protein-conjugated magnetic nanoparticles (mNPs) are promising tools for a variety of biomedical applications, from immunoassays and biosensors to theranostics and drug-delivery. In such applications, conjugation of affinity proteins (e.g., antibodies) to the nanoparticle surface many times compromises biological activity and specificity, leading to increased reagent consumption and decreased assay performance. To address this problem, we engineered a biomolecular magnetic separation system that eliminates the need to chemically modify nanoparticles with the capture biomolecules or synthetic polymers of any kind. The system consists of (i) thermoresponsive magnetic iron oxide nanoparticles displaying poly(N-isopropylacrylamide) (pNIPAm), and (ii) an elastin-like polypeptide (ELP) fused with the affinity protein Cohesin (Coh). Proper design of pNIPAm-mNPs and ELP-Coh allowed for efficient cross-aggregation of the two distinct nanoparticle types under collapsing stimuli, which enabled magnetic separation of ELP-Coh aggregates bound to target Dockerin (Doc) molecules. Selective resolubilization of the ELP-Coh/Doc complexes was achieved under intermediate conditions under which only the pNIPAm-mNPs remained aggregated. We show that ELP-Coh is capable of magnetically separating and purifying nanomolar quantities of Doc as well as eukaryotic whole cells displaying the complementary Doc domain from diluted human plasma. This modular system provides magnetic enrichment and purification of captured molecular targets and eliminates the requirement of biofunctionalization of magnetic nanoparticles to achieve bioseparations. Our streamlined and simplified approach is amenable for point-of-use applications and brings the advantages of ELP-fusion proteins to the realm of magnetic particle separation systems.

Förster Resonance Energy Transfer-Paired Hydrogel Forming Silk-Elastin-Like Recombinamers by Recombinant Conjugation of Fluorescent Proteins.

In the last decades, recombinant structural proteins have become very promising in addressing different issues such as the lack of traceability of biomedical devices or the design of more sensitive biosensors. Among them, we find elastin-like recombinamers (ELRs), which can be designed to self-assemble into diverse structures, such as hydrogels. Furthermore, they might be combined with other protein polymers, such as silk, to give silk-elastin-like recombinamers (SELRs), holding the properties of both proteins. In this work, due to their recombinant nature, we have fused two different fluorescent proteins (FPs), i.e., the green Aequorea coerulescens enhanced green fluorescent protein and the near-infrared eqFP650, to a SELR able to form irreversible hydrogels through physical cross-linking. These recombinamers showed an emission of fluorescence similar to the single FPs, and they were capable of forming hydrogels with different stiffness (G' = 60-4000 Pa) by varying the concentration of the SELR-FPs. Moreover, the absorption spectrum of SELR-eqFP650 showed a peak greatly overlapping the emission spectrum of the SELR-Aequorea coerulescens enhanced green fluorescent protein. Hence, this enables Förster resonance energy transfer (FRET) upon the interaction between two SELR molecules, each one containing a different FP, due to the stacking of silk domains at any temperature and to the aggregation of elastin-like blocks above the transition temperature. This effect was studied by different methods, and a FRET efficiency of 0.06-0.2 was observed, depending on the technique used for its calculation. Therefore, innovative biological applications arise from the combination of SELRs with FPs, such as enhancing the traceability of hydrogels based on SELRs intended for tissue engineering, the development of biosensors, and the prediction of FRET efficiencies of novel FRET pairs.

Diet supplementation with rice bran enzymatic extract restores endothelial impairment and wall remodelling of ApoE(-/-) mice microvessels.

BACKGROUND AND AIMS: Small mesenteric artery resistance and functionality are key factors for the maintenance of blood homeostasis. We attained to evaluate the effects of a rice bran enzymatic extract (RBEE) on structural, mechanic and myogenic alterations and endothelial dysfunction secondary to atherosclerosis disease. METHODS: Seven week-old ApoE(-/-) mice were fed on standard (ST) or high fat (HF) diets supplemented or not with 1 or 5% RBEE (w/w) for 23 weeks. Wild-type C57BL/6J mice fed on ST diet served as controls. Small mesenteric arteries were mounted in a pressure myograph in order to evaluate structural, mechanical and myogenic properties. Vascular reactivity was assessed in the presence of different combinations of inhibitors: l-NAME, indometacin, apamin and charybdotoxin. RESULTS: ApoE(-/-) mice fed on ST and HF diets showed different structural and mechanical alterations, alleviated by RBEE supplementation of ST and HF diets. C57BL/6J was characterized by increased expression of IKCa (199.3%, p = 0.023) and SKCa (133.2%, p = 0.026), resulting in higher EDHF participation (p = 0.0001). However, NO release was more relevant to ApoE(-/-) mice vasodilatation. HF diet reduced the amount of NO released due to 2-fold increase of eNOS phosphorylation in the inhibitory residue Thr495 (p = 0.034), which was fully counteracted by RBEE supplementation (p = 0.028), restoring ACh-induced vasodilatation (p = 0.00006). Dihydroethidium fluorescence of superoxide and picrosirius red staining of collagen were reduced by RBEE supplementation of HF diet by 76.91% (p = 0.022) and 65.87% (p = 0.030), respectively. CONCLUSION: RBEE supplemented diet reduced vessel remodeling and oxidative stress. Moreover, RBEE supplemented diet increased NO release by downregulating p-eNOS(Thr495), thus, protecting the endothelial function.

Structural, mechanical and myogenic properties of small mesenteric arteries from ApoE KO mice: characterization and effects of virgin olive oil diets.

OBJECTIVE: We analyzed the structural, mechanical, myogenic and functional properties of resistance arteries of ApoE KO compared to wild type (WT) mice. We also determined the influence of saturated fat in comparison to virgin olive oil-enriched diets in vascular wall abnormalities. METHODS: Male ApoE KO (ApoE) and WT mice (8-weeks-old) were assigned to the groups: standard chow diet (SD), high fat diet (HFD), virgin olive oil (VOO) and high polyphenol-VOO-enriched diet (Oleaster(®)) (OT) (15% w/w). After 20 weeks, structural, mechanical and myogenic properties of isolated small mesenteric arteries (SMA) were analyzed by pressure myography. For functional studies, vasodilatation to acetylcholine was assessed. Arterial superoxide anion production was measured by ethidium fluorescence. RESULTS: Hypertrophic remodeling and distensibility in ApoE KO SMA was lower compared to WT mice, suggesting an alteration in the autoregulation mechanisms aimed to compensate disease progression. However, ApoE deficiency resulted in a lower impairment in myogenic tone in response to intraluminal pressure, in addition to an improved endothelium-dependent hyperpolarizing vasodilatation. Also, we evidenced the beneficial effects of VOO in contrast to a saturated fat-enriched diet on SMA wall disorders. Only the endothelial function improvement induced by olive oil was dependent on polyphenols content. CONCLUSION: Resistance arteries structure, mechanic, myogenic and functional responses from ApoE KO mice significantly differ from WT mice, evidencing the influence of the type of diet on these disorders. These results are particularly useful to determine the contribution of resistance arteries during the atherosclerotic process and to provide novel insights into the Mediterranean dietary pattern to reduce the burden of atherosclerotic disease.

Penetrating the cell membrane, thermal targeting and novel anticancer drugs: the development of thermally targeted, elastin-like polypeptide cancer therapeutics.

Therapeutic peptides offer important cancer treatment approaches. Designed to inhibit oncogenes and other oncoproteins, early therapeutic peptides applications were hampered by pharmacokinetic properties now addressed through tumor targeting strategies. Active targeting with environmentally responsive biopolymers or macromolecules enhances therapeutics accumulation at tumor sites; passive targeting with macromolecules, or liposomes, exploits angiogenesis and poor lymphatic drainage to preferentially accumulate therapeutics within tumors. Genetically engineered, thermally-responsive, elastin-like polypeptides use both strategies and cell-penetrating peptides to further intratumoral cell uptake. This review describes the development and application of cell-penetrating peptide-elastin-like polypeptide therapeutics for the thermally targeted delivery of therapeutic peptides.

Elastin-like polypeptides: the influence of its molecular weight on local hyperthermia-induced tumor accumulation.

Elastin-like polypeptides (ELP) are thermally responsive polypeptides that are soluble in solutions at 37°C, but which aggregate above 42°C. ELP can be used as effective carrier systems of anticancer molecules, because they can be targeted to tumor sites through the application of local hyperthermia. Since molecular size largely influences how successfully therapeutic agents can cross the vasculatures of tumors, it was crucial to determine an optimal molecular size. In this study, we designed and evaluated three ELP macromolecules with varying molecular weights (43, 63, and 122 kDa), with the goal of determining which would optimize the ELP drug delivery system. The N-terminus of the ELP macromolecule was modified with the cell penetrating peptide Bac to enhance intratumoral and intracellular uptake, and it was also confirmed that each polypeptide had the target transition temperature of 37-42°C and the results of the studies, using tumor-bearing mice, showed that the tumor accumulations increased in the case of all three peptides when local hyperthermia was applied, but that the elimination patterns from these tumors varied according to peptide size. Local hyperthermia was found to produce prolonged retention of all ELP conjugates in tumors except Bac-ELP43. In addition, the pharmacokinetic analysis showed that two larger polypeptides with 63 and 122 kDa have increased AUC in comparison with the 43 kDa polypeptide. These results suggest that, when combined with local hyperthermia, the larger ELP conjugates (63 and 122 kDa) have advantages over the smaller Bac-ELP43 polypeptide in terms of enhanced permeability and higher retention effects.

Rational design of "heat seeking" drug loaded polypeptide nanoparticles that thermally target solid tumors.

This paper demonstrates the first example of targeting a solid tumor that is externally heated to 42 °C by "heat seeking" drug-loaded polypeptide nanoparticles. These nanoparticles consist of a thermally responsive elastin-like polypeptide (ELP) conjugated to multiple copies of a hydrophobic cancer drug. To rationally design drug-loaded nanoparticles that exhibit thermal responsiveness in the narrow temperature range between 37 and 42 °C, an analytical model was developed that relates ELP composition and chain length to the nanoparticle phase transition temperature. Suitable candidates were designed based on the predictions of the model and tested in vivo by intravital confocal fluorescence microscopy of solid tumors, which revealed that the nanoparticles aggregate in the vasculature of tumors heated to 42 °C and that the aggregation is reversible as the temperature reverts to 37 °C. Biodistribution studies showed that the most effective strategy to target the nanoparticles to tumors is to thermally cycle the tumors between 37 and 42 °C. These nanoparticles set the stage for the targeted delivery of a range of cancer chemotherapeutics by externally applied mild hyperthermia of solid tumors.

How to help the skin cope with glycoxidation.

BACKGROUND: Protein glycation refers to the spontaneous reaction of reducing sugars with proteins and the subsequent formation of stable advanced glycation end products (AGEs). Glycation is linked with oxidative stress, and this association is called "glycoxidation". Glycoxidation alters the protein structure and function and causes tissue aging, as seen in human skin. Therefore, research on substances inhibiting glycoxidation appears to be crucial in the prevention of skin aging. With this aim, several plant extracts have been screened for antiglycation activity, and the results of the best candidates are presented in this article. METHODS: Glycation was studied on human skin proteins (collagen, elastin, and albumin) and on a model of reconstructed skin. Oxidative stress has been addressed by testing the copper-induced low-density lipoprotein oxidation, ultraviolet irradiation of glycated dermis, and carbonyl activation of human dermal fibroblasts. A clinical test evaluated the extent of oxidative stress induced by ultraviolet A irradiation. RESULTS: Among the tested products, several plant extracts have decreased the glycation effects on skin proteins collagen, elastin, and albumin. In addition, a plant extract has significantly inhibited the different forms of oxidative stress associated with protein glycation. CONCLUSIONS: We have demonstrated that plant extracts can relieve the deleterious effects of glycation on human skin. Moreover, a plant extract rich in antioxidant molecules has also significantly preserved the human skin from glycoxidation attacks.

Thermal targeting of an acid-sensitive doxorubicin conjugate of elastin-like polypeptide enhances the therapeutic efficacy compared with the parent compound in vivo.

Elastin-like polypeptides (ELP) aggregate in response to mild hyperthermia, but remain soluble under normal physiologic conditions. ELP macromolecules can accumulate in solid tumors because of the enhanced permeability and retention effect. Tumor retention of ELPs can be further enhanced through hyperthermia-induced aggregation of ELPs by local heating of the tumor. We evaluated the therapeutic potential of ELPs in delivering doxorubicin in the E0771 syngeneic mouse breast cancer model. The ELP-Dox conjugate consisted of a cell-penetrating peptide at the N-terminus and the 6-maleimidocaproyl hydrazone derivative of doxorubicin at the C-terminus of ELP. The acid-sensitive hydrazone linker ensured release of doxorubicin in the lysosomes/endosomes after cellular uptake of the drug conjugate. ELP-Dox dosed at 5 mg doxorubicin equivalent/kg, extended the plasma half-life of doxorubicin to 5.5 hours. In addition, tumor uptake of ELP-Dox increased 2-fold when hyperthermia was applied, and was also enhanced compared to free doxorubicin. Although high levels of doxorubicin were found in the heart of animals treated with free doxorubicin, no detectable levels of doxorubicin were found in ELP-Dox-treated animals, indicating a correlation between tumor targeting and reduction of potential cardiac toxicity by ELP-Dox. At an optimal dose of 12 mg doxorubicin equivalent/kg, ELP-Dox in combination with hyperthermia induced a complete tumor growth inhibition, which was distinctly superior to free drug that only moderately inhibited tumor growth. In summary, our findings show that thermal targeting of ELP increases the potency of doxorubicin underlying the potential of exploiting ELPs to enhance the therapeutic efficacy of conventional anticancer drugs.

Effects of selenium on the vessel walls and anti-elastin antibodies in spontaneously hypertensive rats.

Selenium (Se) is an exogenous antioxidant that performs its role via expression of selenoproteins. Pathological changes of the structure of the vessel wall, elastin turnover and collagen production may lead to increased stiffness of the vessels with decreased blood flow to the peripheries. The level of anti-elastin antibodies (AEABs) may give information for elastin metabolism. The aim of the study is to investigate the influence of Se intake on the vessel wall changes and production of AEABs in spontaneously hypertensive rats (SHR). Twenty-four male, 32-week-old SHR were used, divided into three groups, G1, G2 and G3. Before blood and morphological testing, G1 received a low-Se diet for eight weeks, G2 received a diet with adequate Se content and G3 received a diet with Se supplementation. The Se nutritional status was assessed by determination of glutathione peroxidase-1 (GPx-1) activity in whole blood, using the 'Ransel' kit. The rats from group G3 showed higher GPx-1 activity and lower level of AEABs than the other groups (P = 0.021), and the aortic wall histology showed slight degenerative changes compared with other rats. A low-Se diet caused severe changes to the aortic wall's ultrastructure, whereas Se supplementation slowed the changes down. The morphometry revealed a thicker abdominal aortic wall in rats of G1 compared with the other groups, and reduced thickness of the wall of the left coronary artery in G3 compared with the other groups (P < 0.05). Our results have shown that low Se intake leads to severe changes in the vessel walls in SHR, whereas selenium supplementation slows down the elastin degradation and degenerative changes of the vessel walls.