Actual Molecular Weight Research Articles

Preparation of bacterial outer membrane vesicles with anti-GPC3 single-chain antibody and identification of their targeting effects on hepatocellular carcinoma

This study aims to prepare bacterial outer membrane vesicles (OMVs) with anti-glypican-3 (GPC3) single-chain antibody and analyze their targeting effects on Hep G2 hepatocellular carcinoma (HCC) cells and tissue. The recombinant plasmid pET28a-Hbp-hGC 33-scFv was constructed by ligating Hbp-hGC 33-scFv to pET28a. Western blotting was employed to determine the prokaryotic expression of the fusion protein Hbp-hGC 33-scFv, on the basis of which the optimal induction conditions were determined. Hbp-hGC 33-OMVs secreted from the recombinant expressing strains were collected by ultrafiltration concentration and then characterized. The localization of Hbp-hGC 33-scFv in bacteria and Hbp-hGC 33-OMVs was analyzed by immune electron microscopy. The binding of Hbp-hGC 33-scFv to Hep G2 cells was observed by immunofluorescence. The Hep G2 tumor-bearing mouse model was established, and the targeted retention of Hbp-hGC 33-OMVs in the tumor site of mice was observed by a fluorescence imaging system in vivo. The results showed that the actual molecular weight of the fusion protein was 175.3 kDa, and the optimal induction conditions were as follows: OD600=0.5, IPTG added at a final concentration of 0.5 mmol/L, and overnight induction at 16 ℃. The prepared Hbp-hGC 33-OMVs were irregular spherical structures with an average particle size of (112.3±4.6) nm, expressing OmpC, OmpA, and the fusion protein Hbp-hGC 33-scFv. The Hbp-hGC 33-OMVs prepared in this study demonstrated stronger ability of binding to Hep G2 cells than the wild-type OMVs (P=0.008). All the data indicated that Hbp-hGC 33-OMVs with anti-GPC3 single-chain antibody were successfully prepared and could be used for research on the targeted therapy of hepatocellular carcinoma.

Towards understanding the crystallization of photosystem II: influence of poly(ethylene glycol) of various molecular sizes on the micelle formation of alkyl maltosides.

The influence of poly(ethylene glycol) (PEG) polymers H-(O-CH2-CH2)p-OH with different average molecular sizes on the micelle formation of n-alkyl-β-D-maltoside detergents with the number of carbon atoms in the alkyl chain ranging from to is investigated with the aim to learn more about the detergent behavior under conditions suitable for the crystallization of the photosynthetic pigment-protein complex photosystem II. PEG is shown to increase the critical micelle concentration (CMC) of all three detergents in the crystallization buffer in a way that the free energy of micelle formation increases linearly with the concentration of oxyethylene units (O-CH2-CH2) irrespective of the actual molecular weight of the polymer. The CMC shift is modeled by assuming for simplicity that it is dominated by the interaction between PEG and detergent monomers and is interpreted in terms of an increase of the transfer free energy of a methylene group of the alkyl chain by 0.2kJmol-1 per 1mol L-1 increase of the concentration of oxyethylene units at 298K. Implications of this effect for the solubilization and crystallization of protein-detergent complexes as well as detergent extraction from crystals are discussed.

Purification and Activity Evaluation of a Novel Thrombopoietin Mimetic Peptide

The emergence of thrombopoietin mimetic peptides presents a promising therapeutic strategy for addressing thrombocytopenia. This particular study aimed to establish a direct, expeditious, and efficient method for modifying and purifying a novel thrombopoietin mimetic peptide. Precursor proteins were subjected to modification utilizing three distinct fatty acids: C25H42O7N2, C39H66O15N4, and C41H70O15N4. Liquid chromatography analyses demonstrated that C41H70O15N4 yielded the most effective modification results. Mass spectrometry findings validated the correspondence between the theoretical and actual molecular weights of each sample. In vivo experiments conducted on normal mice showcased that the C41H70O15N4 modification group exhibited the highest platelet count, peaking at an impressive 5047 × 109/L. This count was approximately twice that of the peak platelet count observed in the dTMP group and four times higher than the control group. Pharmacokinetic investigations revealed that the C41H70O15N4 modification group displayed the lengthiest half-life among beagles, persisting for 128.5 h. This duration was approximately 28.5 times longer than that of the unmodified dTMP group. These findings underscore the effectiveness of the established C41H70O15N4 modification and purification method in preserving the biological activity of the thrombopoietin mimetic peptide. The novel thrombopoietin mimetic peptide showcased notable attributes of simplicity and cost-effectiveness, while also exhibiting a significant platelet-promoting effect and an extended half-life. Consequently, this novel peptide holds substantial significance for advancing the treatment of thrombocytopenia.

Synthesis and unique characteristics of biobased high Tg copolyesters with improved performance properties for flexible electronics and packaging applications

The synthesis and characterization of two series of biobased novel poly(1,4-cyclohexylenedimethylene terephthalate-co-1,4-cyclohexylene dimethylene 2,6-naphthalenedicarboxylate) copolyesters containing isosorbide (ISB) content (PCITN) ranging from 0 to 11 mol% relative to the total diol amount was carried out in this study. The Novel PCITN was containing 2,6-naphthalenedicarboxylic acid (NDA) content ranging from 0 to 100 mol% relative to the total diacid amount. The synthesized PCITN were thoroughly characterized for their actual chemical composition, molecular weight, thermal properties, thermal stability, dimensional stability, transmittance (%), and birefringence. All the copolyesters showed the characteristics of random copolyesters and their composition and thermal properties could be controlled by the molar feed ratio of diol and diacid moieties. Results indicate that ISB significantly improves the glass transition temperature (Tg) of resultant copolyesters. It was also found that synthesized copolyesters have a high Tg and wide processing window compared to conventional homopolyesters. Exceptional performance properties: such as high Tg, Tm, high thermal stability, dimensional stability, good moisture barrier, good transmittance, and low birefringence of fabricated PCI6TN70 film, make it suitable for versatile industrial applications especially for biomedical, packaging, and optical applications.

Genetic hybridization of highly active exogenous functional proteins into silk-based materials using “light-clothing” strategy

Genetic hybridization of highly active exogenous functional proteins into silk-based materials using “light-clothing” strategy

Structure–property relationship of poly(cyclohexane 1,4‐dimethylene terephthalate) modified with high trans‐1,4‐cyclohexanedimethanol and 2,6‐naphthalene dicarboxylicacid

ABSTRACTA series of novel poly(1,4‐cyclohexanedimthylene terephthalate‐co‐1,4‐cyclohxylenedimethylene 2,6‐naphthalenedicarboxylate) (PCTN) copolyesters were successfully melt polymerized using different content of trans‐ or cis‐isomers. Before evaluations, the performance properties, their actual chemical composition, chemical structure, and molecular weight were determined using proton nuclear magnetic resonance (1H‐NMR), Fourier transform infrared spectroscopy (FTIR), and intrinsic viscosity (IV) measurements. Thermal studies of obtained copolyesters were carried out using differential scanning calorimetry (DSC). Thermal degradation behaviors were analyzed by thermogravimetric analysis (TGA). Randomly oriented film specimens were developed using a hot‐press and their thermal, barrier, dimensional stability, and optical properties were analyzed and compared with conventional poly(ethylene terephthalate) (PET) and poly(ethylene naphthalate) (PEN). The results revealed that glass transition temperature (Tg), melting temperature (Tm), and crystallinity (Xc) of the synthesized copolyesters are increased in a linear trend by increasing the trans‐1,4‐cyclohexanedimethanol (trans‐CHDM) isomers. It was also found that synthesized films had better thermal, barrier, optical, and dimensional stability properties than conventional PET and PEN films. Results clearly indicated that high trans‐CHDM isomers significantly improve the performance properties of the fabricated films. © 2020 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2020, 137, 48950.

Colloidal toxic trace metals in urban riverine and estuarine waters of Yantai City, southern coast of North Yellow Sea

The environmental characteristics of colloidal toxic trace metals Cd, Cu and Pb in riverine and estuarine waters collected from two urban rivers of Yantai City in eastern China, the Guangdang and Xin'an Rivers, were investigated using a modified centrifugal ultrafiltration (CUF) method in conjunction with acid extraction and inductively coupled plasma mass spectrometry. The target metals in dissolved pool were divided into four CUF fractions, i.e. <1 kDa, 1–3 kDa, 3–10 kDa and 10 kDa–0.2 μm, and the results showed that colloidal Cd, Cu and Pb were dominated by 1–10 kDa (1–3 and 3–10 kDa), 1–3 kDa and 10 kDa–0.2 μm fractions, respectively. The coagulation/flocculation of low-molecular-weight (1–10 kDa) colloidal Cd and Cu in the estuaries was obvious and strong, while the enrichment of dissolved Pb in the 10 kDa–0.2 μm fraction may be mainly related to its biogeochemical interactions with Fe-oxides, which is easy to occur in macromolecular colloids. In addition, the actual molecular weight cutoffs (MWCOs) of the three used CUF units with nominal MWCOs of 1, 3 and 10 kDa were determined to be 4.9, 8.5 and 33.9 kDa, respectively, indicating that membrane calibration is essential for explaining the actual fraction of dissolved trace metals and verifying the integrity of ultrafiltration membrane. Overall, the results in this study provide a further understanding of the heterogeneity in biogeochemical features, migration and fate of toxic trace metals in aquatic ecosystems, especially that of the river-sea mixing zone.

What is the Actual Molecular Weight of Irisin Hormone According to Western Blot Analysis?

Irisin hormone, secreted mainly in skeletal, cardiac muscles, is proteolytically cleaved from the C-terminal moiety and secreted from the fibronectin domain-containing protein 5(FNDC5) receptor. This hormone carries carbohydrate moieties, which are glycosylated, and is a dimeric protein, and released as a hormone of 112 amino acids [1]. The dimerization of this hormone is not affected by glycosylation, although N-glycosylation is necessary for the stabilization of FNDC5 and secretion of irisin [2]. Quantitation of circulating human irisin by Tandem Mass Spectrometry was ∼ 3.6 ng/ml in sedentary individuals [3]. Irisin is secreted mainly in skeletal, cardiac muscles and adipose tissues.

Investigation of the effects of polycaprolactone molecular weight and graphene content on crystallinity, mechanical properties and shape memory behavior of polyurethane/graphene nanocomposites

In the following work, different shape memory polyurethanes (SMPUs) were synthesized using polycaprolactone (PCL) with various molecular weights, hexamethylene diisocyanate (HDI), and 1,4-butanediol (BDO). Afterward, polyurethane (PU)-based nanocomposites were prepared with different graphene nanosheets contents via solution casting method. Hydrogen nuclear magnetic resonance (1H-NMR) was used to confirm the chemical structure of PCLs and calculate their actual molecular weights. The chemical structure and hydrogen bonding content of PUs and their nanocomposites were investigated by Fourier-transform infrared spectroscopy (FTIR). According to the results, the hydrogen bonding contents of nanocomposites were reduced by graphene nanosheets inhibition from the formation of hydrogen bonds between polyurethane chains. Thermal properties and crystalline morphology of samples were studied using differential scanning calorimetry (DSC) and X-ray diffraction (XRD). The results indicated that the transition temperature and crystallinity of samples were changed by variation of the molecular weight of the PCL component and of the concentration of the graphene nanosheets. Graphene nanosheets dispersion in polyurethane matrix was investigated using the field emission scanning electron microscope (FE-SEM). The mechanical and shape memory properties of different PUs and their nanocomposites were determined at both 75 °C and room temperature. It can be deduced from the results that the modulus of the samples increased due to the rigidity of nanosheets. Furthermore, the restricted mobility of PCL chains, due to the presence of nanosheets, led to higher shape fixity ratio. Moreover, the nanosheets prevented the stress transfer on the hard segments which increased the shape recovery ratio.

Recovery, overloading, and protein interactions in asymmetrical flow field-flow fractionation

In asymmetrical flow field-flow fractionation (AF4), similar to other separation techniques, mass recovery and overloading require special attention in order to obtain quantitative results. We conducted a systematic study with five globular proteins of different molecular weight (36.7–669 kDa) and isoelectric point (4.0–6.5), and ultrafiltration membranes that are commonly used in aqueous AF4, regenerated cellulose (RC) and polyethersulfone (PES). Phosphate-buffered saline (PBS) with ionic strength 0.15 M and pH 7.2 was used as the carrier liquid in this study. The actual molecular weight cutoff (MWCO) was found to be higher than the nominal value and varied between membranes of different chemistry but the same nominal MWCO. Adsorption on the membrane was found to be dependent on the membrane chemistry (RC had lower adsorption compared to PES), and independent of the protein standard for the examined proteins. On the other hand, the mass overloading effects (i.e., higher retention times, peak broadening, and fronting peaks) were significantly more pronounced for γ-globulin than for the other proteins. The overloading effects could be rationalized with the increase of the local viscosity close to the membrane, depending on the properties of the proteins, and we derived theoretical equations that related the dependency of the migration velocity on the protein concentration through this non-ideal viscosity effect.

Identification of six grouper species under the genus Epinephelus (Bloch, 1793) from Indian waters using PCR-RFLP of cytochrome c oxidase I (COI) gene fragment

Groupers are biologically diverse, commercially important and highly regarded of all fish species associated with reef fisheries globally. Given the huge demand and high value in live fish trade, restaurants and supermarkets around the world, the accurate identification of the closely related grouper species is crucial to prevent any adulteration. Partial nucleotide sequence data of the mitochondrial cytochrome c oxidase I (COI) gene, which is globally accepted as fish DNA barcoding gene, was generated for six commercially important grouper species belonging to the genus, Epinephelus viz. E. fuscoguttatus, E. coioides, E. logispinis, E. malabaricus, E. bleekeri and E. tauvina collected from Indian waters. Based on the analysis of 704 bp COI gene sequences, a PCR-RFLP method was developed to identify all the six species accurately. The PCR products of COI gene fragment was digested with selected restriction enzymes viz. HinfI, AseI, HindIII and BsaJI. Restriction endonuclease HinfI was enabled to produce species-specific fragments for E. coioides, E. malabaricus, E. logispinis and E. bleekeri. The result shows that AseI restriction enzyme could be used to differentiate E. fuscoguttatus from other five species. The expected and actual molecular weight of the digested products was similar indicating the authenticity of the methods followed. This is the first report on differentiating closely related grouper species using above mentioned restriction endonucleases and COI gene fragment. PCR-RFLP profiles generated could be useful for detecting seafood fraud by identifying the grouper species under the genus Epinephelus using samples from fresh, frozen, cooked and even processed products.

Effects of selected processing treatments on antigenicity of banana prawn (Fenneropenaeus merguiensis) tropomyosin

SummaryDifferent processing techniques were assessed to attenuate the antigenicity of banana prawn tropomyosin (Fenneropenaeus merguiensis). Frying and boiling at different temperatures, acid treatment using different acids and storage at −20 °C up to 3 months were investigated. Untreated prawn sample was used as a control. Frying significantly increased antigenicity (6–8 times) in temperature‐dependent manner, whereas the trend was fundamentally reversed with boiling. Boiling at 121 °C resulted in the lowest antigenicity (12.99 mg mL−1) among all heat‐treated samples, yet higher than the control (5.06 mg mL−1). Freezing had initially very minor impact, although prolonged storage at −20 °C increased antigenicity slightly (2.29 mg mL−1) compared with control. Antigenicity was impacted the most by a reduction in pH independent on the type of acid as both acetic and HCl acids significantly reduced antigenicity of tropomyosin by ~90% compared with control. This could be considered as a new approach to processing that may potentially reduce tropomyosin‐derived antigenicity in prawns and prawn products. This study also confirmed the actual molecular weight of banana prawn tropomyosin was 37 kDa implementing a new methodology using unstained gel.

Construction and Characterization of Cell-Penetrating Peptide-Fused Fibroblast Growth Factor and Vascular Endothelial Growth Factor for an Enhanced Percutaneous Delivery System.

The exogenous administration of growth factors has been examined for treating wounds and such factors serve as cosmetic agents for skin regeneration. However, the topical application of growth factors is hampered by their limited percutaneous absorption. In this study, we genetically modified basic fibroblast growth factor (bFGF) and vascular endothelial growth factor-A (VEGF-A) using a cell-penetrating peptide to facilitate their permeation into skin and to avoid multiple steps in the chemical or physical modification of biomaterials. Low-molecular-weight protamine (LMWP)-fused bFGF and VEGF-A (LMWP-bFGF and LMWP-VEGF-A) were designed by serial polymerase chain reaction (PCR)-mediated addition of the LMWP codons onto the bFGF and VEGF-A genes and then produced in Escherichia coli. The purified LMWP-bFGF and LMWP-VEGF-A represented >90% of the total proteins, as determined by SDS-PAGE and densitometric quantification, and their actual molecular weights, determined by mass spectroscopy, were 19,026 and 15,715 Da, respectively, corresponding to the theoretical values. N-terminal amino acid sequencing analyses confirmed the N-terminal conjugation of LMWP to bFGF and VEGF-A. Immunoblotting analyses using antibodies against bFGF and VEGF-A, together with the proliferative effects of LMWP-bFGF and LMWP- VEGF-A on human keratinocytes and fibroblasts, demonstrated that the original biological activity of each growth factor was not altered by LMWP conjugation. In addition, the LMWP conjugation did not induce further cytotoxic effects on the skin cells, while the cell membrane-penetrating activities of LMWP-bFGF and LMWP-VEGF-A were significantly enhanced compared with the respective unconjugated growth factors. These results suggest that LMWP-bFGF and LMWP-VEGF-A can be used as effective topical therapeutic or cosmetic agents for skin regeneration and anti-aging treatments.

The role of macro-aggregation in regulating enzymatic depolymerization of soil organic nitrogen

Extracellular enzymatic depolymerization of polymeric organic nitrogen (PON) is a rate-limiting step in N mineralization. However, enzymatic accessibility to PON might be regulated by physical occlusion of the PON resulting from the architectural packing of soil minerals during aggregate formation. To examine the extent to which enzymatic accessibility to PON is regulated by soil aggregation, we put forward a new approach involving the comparison of relationships between potential N depolymerase activity (protease and β-glucosaminidase; as an estimate of the potential to produce depolymerized products) and net N mineralization (as a bioassay for actual low molecular weight dissolved ON production) in aggregated and corresponding disaggregated soil. Soils were sampled from grassland (GL) and arable land (AL), separated by dry sieving into fractions (4.75–2, 2–0.25 and 0.25–0.063 mm) and fractions mixed (4:4:1 by mass, respectively) to obtain constructed aggregated soils. Corresponding disaggregated soils were prepared using a mortar and pestle. This procedure mainly disrupted the 4.75–2 mm (large macro-aggregate) fraction. Disaggregation significantly promoted (p < 0.05) net N mineralization rates by 1.3 times and 1.5 times in GL and AL soil, respectively. When net N mineralization - potential N depolymerase relationships for GL were examined, a greater slope parameter for disaggregated compared to aggregated soil (p = 0.001; ANCOVA) quantified the extent to which this promoted N mineralization could be attributed to disruption of macroaggregate-increased enzymatic accessibility to PON. For AL, which had low protease and β-glucosaminidase activity, promoted N mineralization rate could not be attributed to increased protease + β-glucosaminidase accessibility to PON reflecting a possible role for other N depolymerases and/or osmolyte/lysate effects. By proposing how differences between mineralization-depolymerase relationships for soils differing in aggregation status might, with assumptions, be interpreted to identify the role of physical occlusion in protection of PON, we give new insight on the regulation of enzymatic depolymerization by physical protection through macro-aggregation for soils from contrasting land use.

Size does matter – Determination of the critical molecular size for the uptake of chemicals across the chorion of zebrafish (Danio rerio) embryos

In order to identify the upper limits of the molecular size of chemicals to cross the chorion of zebrafish, Danio rerio, differently sized, non-toxic and chemically inert polyethylene glycols (PEGs; 2000–12,000Da) were applied at concentrations (9.76mM) high enough to provoke osmotic pressure. Whereas small PEGs were expected to be able to cross the chorion, restricted uptake of large PEGs was hypothesized to result in shrinkage of the chorion. Due to a slow, but gradual uptake of PEGs over time, molecular size-dependent equilibration in conjunction with a regain of the spherical chorion shape was observed. Thus, the size of molecules able to cross the chorion could be narrowed down precisely to ≤4000Da, and the time-dependency of the movement across the chorion could be described. To account for associated alterations in embryonic development, fish embryo toxicity tests (FETs) according to OECD test guideline 236 (OECD, 2013) were performed with special emphasis to changes in chorion shape.FETs revealed clear-cut size-effects: the higher the actual molecular weight (=size) of the PEG, the more effects (both acutely toxic and sublethal) were found. No effects were seen with PEGs of 2000 and 3000Da. In contrast, PEG 8000 and PEG 12,000 were found to be most toxic with LC50 values of 16.05 and 16.40g/L, respectively. Likewise, the extent of chorion shrinkage due to increased osmotic pressure strictly depended on PEG molecular weight and duration of exposure. A reflux of water and PEG molecules into the chorion and a resulting re-shaping of the chorion could only be observed for eggs exposed to PEGs ≤4000Da. Results clearly indicate a barrier function of the zebrafish chorion for molecules larger than 3000 to 4,000Da.

Influence of hydrogen bonding on the melt rheology of polypropylene

The rheological behavior of hydroxyl-functionalized polypropylene (PPOH) copolymers modified with up to 3.9 mol% hydroxyl groups was compared with unmodified polypropylene (PP) in the melt state. The hydroxyl groups interacted via hydrogen (H-) bonding, the strength of which increased with increasing hydroxyl concentration. The hydrogen bonds persisted at temperatures as high as 250 °C, even though the H-bonding strength decreased with increasing temperature. A direct consequence of H-bonding was an increase in the elasticity, viscosity and relaxation time of the PPOH copolymers compared to PP, and also an increase in these properties with increasing hydroxyl concentration. The zero-shear viscosity exhibited a power-law relationship with the hydroxyl concentration, η0 ∝ [Mv]3.6 [OH]2, which was found to be consistent with the predictions from Leibler-Rubinstein-Colby (LRC) theory for associating polymers. The PPOH copolymers exhibited apparent molecular weights 2–8 times higher than the actual molecular weights. The PPOH copolymer with 3.9 mol% hydroxyl groups displayed a gel-like behavior that suggested the formation of an elastic network in the melt presumably due to the phase separation of H-bonded OH groups into nanoclusters. The network, however, was weak since non-Newtonian viscous flow occurred under non-linear deformations.

A Straightforward Estimation of Activation and Deactivation Parameters for ATRP Systems from Actual Polymerization Rate and Molecular Weight Distribution Data

The accuracy of a model prediction relies heavily on the quality of the parameters used. Some of the available methods to estimate the activation and deactivation rate constants in atom transfer radical polymerization (ATRP) are done in the absence of monomer, hence they may not be representative of the polymerization conditions. Others offer great accuracy but requires data that are not commonly measured in experiments. In this study, a simple method is proposed to estimate activation and deactivation rate constants through correlating the recently reported theoretical equations with experimental data of monomer conversion and polymer molecular weight dispersity. The kinetics and dispersity equations used are straightforward and explicit, thus can be easily calculated. Only two parameters (activation and deactivation rate constant) are involved in the correlation of two data sets without any other adjustable parameters. In addition, the experimental data required are some of the most often measured quantities in polymerization, thus no additional experiments or measurements are required. Since the conversion and dispersity are obtained from actual polymerization, the reaction rate constants estimated are representative of the real reaction conditions. The applicability of this method is demonstrated by estimating parameters using reported experimental data of ATRP of methyl methacrylate conducted at various conditions. image

Choosing the dialyzer type in order to perform optimum hemodialysis in dogs

In order to identify the upper limits of the molecular size of chemicals to cross the chorion of zebrafish, Danio rerio, differently sized, non-toxic and chemically inert polyethylene glycols (PEGs; 2000–12,000 Da) were applied at concentrations (9.76 mM) high enough to provoke osmotic pressure. Whereas small PEGs were expected to be able to cross the chorion, restricted uptake of large PEGs was hypothesized to result in shrinkage of the chorion. Due to a slow, but gradual uptake of PEGs over time, molecular size-dependent equilibration in conjunction with a regain of the spherical chorion shape was observed. Thus, the size of molecules able to cross the chorion could be narrowed down precisely to ≤4000 Da, and the time-dependency of the movement across the chorion could be described. To account for associated alterations in embryonic development, fish embryo toxicity tests (FETs) according to OECD test guideline 236 (OECD, 2013) were performed with special emphasis to changes in chorion shape.FETs revealed clear-cut size-effects: the higher the actual molecular weight (= size) of the PEG, the more effects (both acutely toxic and sublethal) were found. No effects were seen with PEGs of 2000 and 3000 Da. In contrast, PEG 8000 and PEG 12,000 were found to be most toxic with LC50 values of 16.05 and 16.40 g/L, respectively. Likewise, the extent of chorion shrinkage due to increased osmotic pressure strictly depended on PEG molecular weight and duration of exposure. A reflux of water and PEG molecules into the chorion and a resulting re-shaping of the chorion could only be observed for eggs exposed to PEGs ≤4000 Da. Results clearly indicate a barrier function of the zebrafish chorion for molecules larger than 3000 to 4,000 Da.

Leaveraging heat shock protein analogues to induce the expression of Interleukin 10. Smart design of emerging anti-inflammatory biomaterials

Event Abstract Back to Event Leaveraging heat shock protein analogues to induce the expression of Interleukin 10. Smart design of emerging anti-inflammatory biomaterials Mariagemiliana Dessi1, Louise Topping1 and Matteo Santin1 1 University of Brighton, School of Pharmacy and Biomolecular Sciences, United Kingdom INTRODUCTION In tissue engineering and regenerative medicine applications, there is a compelling need of novel therapeutics for the treatment of chronic inflammation. Nowadays, the therapeutic approaches mainly help relieve the symptoms of the inflammation. The high rates of non-responders and the evidence of systemic immunosuppression have evolved in the development of selective anti-inflammatory materials. Among the family of heat shock proteins (HSPs), the heat shock protein 70 (HSP70) is the most evolutionary conserved and is widely recognized for its great ability to treat and prevent inflammatory conditions [1]. In the current scenario, the exploitation of the immunoregulatory nature of peptides derived from heat shock proteins offers a potential alternative for the treatment of inflammatory conditions. The peptide MtHSP70 derived from Mycobacterium tuberculosis HSP70 is of clinical relevance due to its immunoregulatory features. In this study, for the first time, the MtHSP70 peptide has been conjugated to a poly-L-lysine (PLL) used as coating biopolymer in tissue engineering applications. We demonstrate that the presence of MtHSP70 is able to encouraging lymphocyte to release anti-inflammatory cytokines (i.e.IL-10) causing the repolarization of macrophages, from a pro-inflammatory (M1) to an anti-inflammatory skew (M2). Aim of the study is to create an advanced and clinically-reflective coating biomaterial with anti-inflammatory cues to locally treat or prevent chronic inflammations. EXPERIMENTAL METHODS Structurally well-defined MtHSP70 mimicking peptide was synthetized through a bottom up strategy based on a multistep sequence of coupling and deprotection reactions on a solid support. The complete molecule was characterized by mass spectrometry (MS) and Fourier Transformed Infrared spectroscopy (FTIR). The peptide was conjugated by carbodiimide chemistry to poly-L-lysine at different concentrations and the functionalized materials has been analysed by FTIR to evaluate the efficacy of peptide conjugation. The cytotoxicity of the biomaterial was assessed by MTS assay and the viability of cells was evaluated by HPI test. ELISA sandwich test was performed to test the ability of lymphocyte to produce the IL-10 cytokine. Co-colture study was performed to simulate the body conditions and immunocytochemistry assessed the expression of CD206 (M2 specific surface receptor). RESULTS AND DISCUSSION Physico-chemical analysis confirmed the successful synthesis of peptide. The accurate mass measurements of MS (Fig.1) confirmed that the actual molecular weight matches the theoretical value (1611 m/z). The absorption bands of FTIR spectra revealed the characteristic elements present on the molecule backbone. As confirmed by FTIR the chemical grafting of the peptide to PLL was successful. Cell viability analysis reveals that after 24hrs in contact with functionalized substrates, both adherent and non-adherent cells are viable. ELISA test showed that lymphocytes incubated upon the functionalized material release significantly high level of IL-10. Type 1 macrophages co-cultured on PLL showed a low amount of CD206 marker and exhibited a rounded outspread cytoplasm (Fig.2). The expression of CD206 is higher on the macrophages co-coltured on the MtHSP70 functionalized coating and their morphology is evolving towards an elongated cytoskeleton (Fig.3) Figure 2. Expression of CD206 and morphology of macrophages co-coltured on PLL. Figure 3. Expression of CD206 and morphology of macrophages co-coltured on functionalized PLL. CONCLUSION In the current study, a new successful route to obtain anti-inflammatory biomaterials is demonstrated. For the first time, the heat shock mimicking peptide MtHSP70 was covalently attached to a biopolymer. The presence of peptide was able to elicit an anti-inflammatory response by stimulating the IL-10 production at levels that could induce a skewing in macrophages phenotype from M1 (inflammatory state) to M2 (anti-inflammatory state). These results are paving the way for the potential use of MtHSP70 analogues as promising molecules to functionalize biomaterials to locally treat chronic inflammatory conditions.

Chitosan amphiphiles provide new drug delivery opportunities

Chitosan, an acid soluble and renewable biopolymer, was first studied as a drug delivery agent in 1990. This review focuses on the relatively newer self‐assembling chitosan amphiphiles and their use as drug delivery agents. Chitosan amphiphiles, first introduced in the late 1990s, are prepared by conjugating hydrophobic and sometimes additional hydrophilic units to chitosan. These amphiphiles self‐assemble in aqueous media at neutral pH to form micelles, with critical micellar concentrations (CMCs) ranging from 0.09 to 700 µg mL−1. The CMCs depend on the actual molecular architecture, molecular weight and hydrophobic character, but are typically lower than the values reported for block copolymer amphiphiles. As well as linear amphiphiles in which chitosan is derivatised with hydrophobic pendant groups, new claw amphiphiles have been prepared in which chitosan amphiphiles radiate geometrically from a dendrimer core. These chitosan amphiphiles (linear and claw) have been exploited as drug delivery agents and they increase the oral bioavailability of hydrophobic drugs up to sixfold, deliver hydrophobic drugs to tumours, genes to the liver via the intravenous route, genes to the muscle via the intramuscular routes and small interfering RNAs to tumours via the intratumoural route. Chitosan amphiphile nanoparticles also deliver peptides to the brain via the intravenous and oral routes. © 2014 Society of Chemical Industry