Bioactive Macromolecules Research Articles

Sponge Phases and Nanoparticle Dispersions in Aqueous Mixtures of Mono- and Diglycerides.

The lipid liquid crystalline sponge phase (L3) has the advantages that it is a nanoscopically bicontinuous bilayer network able to accommodate large amounts of water and it is easy to manipulate due to its fluidity. This paper reports on the detailed characterization of L3 phases with water channels large enough to encapsulate bioactive macromolecules such as proteins. The aqueous phase behavior of a novel lipid mixture system, consisting of diglycerol monooleate (DGMO), and a mixture of mono-, di- and triglycerides (Capmul GMO-50) was studied. In addition, sponge-like nanoparticles (NPs) stabilized by Polysorbate 80 (P80) were prepared based on the DGMO/GMO-50 system, and their structure was correlated with the phase behavior of the corresponding bulk system. These NPs were characterized by dynamic light scattering (DLS), cryo-transmission electron microscopy (Cryo-TEM) and small angle X-ray scattering (SAXS) to determine their size, shape, and inner structure as a function of the DGMO/GMO-50 ratio. In addition, the effect of P80 as stabilizer was investigated. We found that the NPs have aqueous pores with diameters up to 13 nm, similar to the ones in the bulk phase.

Engineering Intracellular Delivery Nanocarriers and Nanoreactors from Oxidation-Responsive Polymersomes via Synchronized Bilayer Cross-Linking and Permeabilizing Inside Live Cells.

Reactive oxygen species (ROS) and oxidative stress are implicated in various physiological and pathological processes, and this feature provides a vital biochemical basis for designing novel therapeutic and diagnostic nanomedicines. Among them, oxidation-responsive micelles and vesicles (polymersomes) of amphiphilic block copolymers have been extensively explored; however, in previous works, oxidation by ROS including H2O2 exclusively leads to microstructural destruction of polymeric assemblies. For oxidation-responsive polymersomes, fast release of encapsulated hydrophilic drugs and bioactive macromolecules will occur upon microstructural disintegration. Under certain application circumstances, this does not meet design requirements for sustained-release drug nanocarriers and long-acting in vivo nanoreactors. Also note that conventional polymersomes possess thick hydrophobic bilayers and compromised membrane permeability, rendering them as ineffective nanocarriers and nanoreactors. We herein report the fabrication of oxidation-responsive multifunctional polymersomes exhibiting intracellular milieu-triggered vesicle bilayer cross-linking, permeability switching, and enhanced imaging/drug release features. Mitochondria-targeted H2O2 reactive polymersomes were obtained through the self-assembly of amphiphilic block copolymers containing arylboronate ester-capped self-immolative side linkages in the hydrophobic block, followed by surface functionalization with targeting peptides. Upon cellular uptake, intracellular H2O2 triggers cascade decaging reactions and generates primary amine moieties; prominent amidation reaction then occurs within hydrophobic bilayer membranes, resulting in concurrent cross-linking and hydrophobic-to-hydrophilic transition of polymersome bilayers inside live cells. This process was further utilized to achieve integrated functions such as sustained drug release, (combination) chemotherapy monitored by fluorescence and magnetic resonance (MR) imaging turn-on, and to construct intracellular fluorogenic nanoreactors for cytosolic thiol-containing bioactive molecules.

Solid nano-in-nanoparticles for potential delivery of siRNA

siRNA-based therapeutics possess great potential to treat a wide variety of genetic disorders. However, they suffer from low cellular uptake and short half-lives in blood circulation; issues that remain to be addressed. This work is, to the best of our knowledge, the first to report the production of solid nano-in-nanoparticles, termed double nano carriers (DNCs) by means of the innovative technology of nano spray drying. DNCs (with a median size of 580–770nm) were produced by spraying at low temperatures (50°C) to prevent damage to heat-sensitive biomacromolecules like siRNA. DNCs consisting of Poly (d,l-lactide-co-glycolide) used as a wall material, encapsulating 20% human serum albumin primary nanoparticles (PNPs) loaded with siRNA, were obtained as a dry nanoparticulate powder with smooth spherical surfaces and a unique inner morphology. Incubation of pegylated or non-pegylated DNCs under sink conditions at 37°C, elicited a controlled release profile of the siRNA for up to 12 or 24h, respectively, with a minimal burst effect. Prolonged incubation of pegylated DNCs loaded with active siRNA (anti EGFR) in an A549 epithelial cell culture monolayer did not induce any apparent cytotoxicity. A slow degradation of the internalized DNCs by the cells was also observed resulting in the progressive release of the siRNA for up to 6days, as corroborated by laser confocal microscopy. The structural integrity and silencing activity of the double encapsulated siRNA were fully preserved, as demonstrated by HPLC, gel electrophoresis, and potent RNAi activity of siRNA extracted from DNCs. These results demonstrate the potential use of DNCs as a nano drug delivery system for systemic administration and controlled release of siRNA and potentially other sensitive bioactive macromolecules.

Ultrasonic extraction, antioxidant and anticancer activities of novel polysaccharides from Chuanxiong rhizome

Ultrasonic-assisted extraction technology was employed to prepare Ligusticum chuanxiong Hort polysaccharide. Single factor test and orthogonal experimental design were used to optimize the extraction conditions. The results showed that the optimal extraction conditions consisted of ultrasonic temperature of 80°C, ultrasonic time of 40min and water to raw material ratio of 30mL/g. Three novel polysaccharides fractions, LCX0, LCX1 and LCX2, were isolated and purified from the crude polysaccharides using DEAE-52 cellulose and Sephadex G-100 column chromatography. The molecular weight and monosaccharide composition of three LCX polysaccharides fractions were analyzed with gel permeation chromatography (GPC) and HPLC analysis, respectively. Furthermore, the antioxidant and in vitro anticancer activities of the polysaccharides were investigated. Compared with LCX0, LCX2 and LCX1 showed relative higher antioxidant activity and inhibitory activity to the growth of HepG2, SMMC7721, A549 and HCT-116 cells. It is suggested that the novel polysaccharides from rhizome of L. chuanxiong could be promising bioactive macromolecules for biomedical use.

Interaction between gut immunity and polysaccharides

ABSTRACTThe human gut is colonized with a vast and diverse microbial ecosystem, and these bacteria play fundamental roles in the well being of our bodies. Gut-associated lymphoid tissues, the largest mucosal immune system, should never be overlooked for their profound effect in maintaining the host immunity. Therefore, we discussed the relationship between gut immunity and host health, primarily from two aspects: the homeostasis of gut microbiota, and the function of gut-associated lymphoid tissues. Polysaccharides, widely concerned as bioactive macromolecules in recent centuries, have been proved to benefit the intestinal health. Dietary polysaccharides can improve the ratio of probiotics, regulate the intestinal microenvironment like decreasing the gut pH, and stimulate the macrophages or lymphocytes in gut tissues to fight against diseases like cancer. Based on various experimental and clinical evidence, the impacts of dietary polysaccharides on intestinal health are summarized, in order to reveal the possible immunomodulatory mechanisms of polysaccharides.

Antioxidant capacity and cytotoxicity of sulfated polysaccharide TLH-3 from Tricholoma lobayense

Polysaccharide TLH-3 from the fruit body of Tricholoma lobayense Heim has shown outstanding antioxidant activity. In order to further explore it, TLH-3 was successfully modified to obtain a sulfated derivative (STLH-3). The chemical characteristics of STLH-3 and TLH-3 were determined by high performance liquid chromatography and infrared spectroscopy. Antioxidant activity and cytotoxicity of the samples were investigated in vitro. The antioxidant activities of STLH-3 were significantly improved. In the four indicators of antioxidant activity, the 1, 1-diphenyl-2-picrylhydrazyl (DPPH) and superoxide anion scavenging activities of STLH-3 have exceeded that of Vitamin C (Vc). The cytotoxicity against tumor cells including human cervical carcinoma cell line (HeLa) and human breast cancer cell line (MCF-7) of STLH-3 was obviously stronger than that of TLH-3. STLH-3 had no toxic on human embryonic lung fibroblasts (HELF) cells. These results suggested that STLH-3 would be a promising bioactive macromolecule for potential applications in the field of health care and pharmaceutical.

An injectable thermosensitive polymeric hydrogel for sustained release of Avastin® to treat posterior segment disease

Delivery of drugs, especially bioactive macromolecules such as proteins and nucleic acids, to the posterior segment is still a significant challenge for pharmaceutical scientists. In the present study, we developed an injectable thermosensitive polymeric hydrogel for sustained release of Avastin® to treat posterior segment disorders. The payload of Avastin® to poly(lactic acid-co-glycolic acid)-poly(ethylene glycol)-poly(lactic acid-co-glycolic acid) (PLGA-PEG-PLGA) hydrogel did not influence its inherent sol–gel transition behavior, but shifted the sol–gel transition to a lower temperature. The resulting Avastin®/PLGA-PEG-PLGA hydrogels had a porous structure (pore size, 100∼150μm) as determined by scanning electron microcopy (SEM), facilitating sustained Avastin® release over a period of up to 14 days in vitro. The PLGA-PEG-PLGA hydrogel was immediately formed in the vitreous humor after intravitreal injection, followed by slow clearance over an 8 week study period. The PLGA-PEG-PLGA hydrogel exhibited no apparent toxicity against retinal tissue, as indicated by the absence of inflammation, retinal necrosis, and stress responses, using optical coherence tomography (OCT) and histological/immunochemical analyses. Electrophysiology (ERG) examination also showed that the PLGA-PEG-PLGA hydrogel did not affect retinal function. In vivo pharmacokinetic studies indicated that the use of the PLGA-PEG-PLGA hydrogel greatly extended the release of Avastin® over time in the vitreous humor and retina after intravitreal injection. Together, these results demonstrated that the PLGA-PEG-PLGA hydrogel was a promising candidate for ocular drug delivery of Avastin®via intravitreal injection.

A Huaier polysaccharide restrains hepatocellular carcinoma growth and metastasis by suppression angiogenesis

Hepatocellular carcinoma (HCC) is a highly metastatic cancer. Huaier polysaccharide (TP-1) is a naturally occurring bioactive macromolecule, found in Huaier fungus and has been shown to exert in vitro antitumor and antimetastasis for HCC, but no study has addressed in vivo efficacy and mechanisms of action. Presently, we found that TP-1 at doses of 0.5, 1 and 2mg/kg significantly inhibited tumor growth and metastasis to the lung in mice bearing HCC SMMC-7721 tumors without toxicity. The analysis of tumors by immunohistochemistry demonstrated that TP-1 inhibited PCNA expression, increased the number of TUNEL-positive cells and reduced microvessel density (MVD) to achieve this effect. Furthermore, TP-1 administration reduced the protein expression of hypoxia-inducible factor (HIF)-1alpha, vascular endothelial growth factor (VEGF), AUF-1 and AEG-1, in tumor tissues. Taken together, our data suggested that the antitumor and anti-metastatic activities of TP-1 might be at least partially through down-regulation of HIF-1alpha/VEGF and AUF-1/AEG-1 signaling pathways.

Intracellular Delivery of Histidine and Arginine Rich Cell Penetrating Peptides into HeLa cell Line

Most bioactive macromolecules, such as DNA and RNA, cannot permeate into cells freely from outside the plasma membrane. Cell penetrating peptides are a group of short peptide sequences that are able to transverse the cell membrane for mediating gene into living cells. In this study we demonstrate two cell penetrating peptides 5H-R9-C and 7H-R9–7H-C. These peptides are synthesized by solid phase methodology and labelled these peptides by FITC and purified them by RP-HPLC. FITC labelled peptides are efficiently internalize into HeLa cells confirmed by fluorescence microscopy and flow cytometry. Further studies are being carried out to deliver cargo molecule (siRNA, oligonucleotide and pDS RED) by these peptides. These results suggest that these peptides appear to be a promising tool for drug delivery.

Single-domain magnetic nanoparticles in an alternating magnetic field as mediators of local deformation of the surrounding macromolecules

The forces, deformations, and stresses generated in macromolecules attached to single-domain magnetic nanoparticles under the influence of a low-frequency (nonheating) magnetic field have been analyzed analytically and numerically. It has been shown that, in bioactive macromolecules, an alternating magnetic field with an induction of 0.1–1.0 T and a circular frequency of ≲104 s−1 can induce forces up to several hundred piconewtons, absolute deformations up to a few tens of nanometers, as well as compressive and shear stresses exceeding 107 Pa. These mechanical stimuli are sufficient for a significant change of interatomic distances in active centers, conformation of macromolecules, and even a breaking of some bonds, which makes it possible to develop a new technological platform for targeted delivery of drugs, remote control of their activity, and cancer-cell destruction.

The electrically conductive scaffold as the skeleton of stem cell niche in regenerative medicine

Stem cells with multipotent and self-renewal abilities play a vital role in regenerative medicine and tissue engineering. They can assist tissue reconstruction through specific differentiation and secretion of various bioactive macromolecules. More and more studies confirm that the cell-fate commitment can be manipulated via constructing a specific stem cell niche. The construction of specific niches with conductive materials (conducting polymers, carbon nanotubes and graphene) can promote stem cell differentiation towards electro-active lineages and emphasize the promising role of stem cells in electro-active tissue regeneration (e.g., nerve and heart). In this review, we summarize the commonly applied conductive materials for scaffold construction and evaluate their contributions in the regeneration of electro-active tissues.

Bone defect repair in mice by mesenchymal stem cells.

Adult bone marrow niche contains rare primitive but highly functional multipotent progenitors (e.g., mesenchymal stem cells; MSCs) capable of differentiating into specific mesenchymal tissues like bone, cartilage, muscle, fat tissues, ligament, dermis, bone marrow stroma, tendon, and other connective tissues. Upon in vivo transplantation, MSCs also secrete a wide range of growth factors, immunomodulatory cytokines, and important bioactive macromolecules including cell-derived exosomes to structure regenerative microenvironments. This protocol describes a mouse model to study bone formation/regeneration from adult mesenchymal stem cells.

Recent Trends in Bioorthogonal Click-Radiolabeling Reactions Using Fluorine-18

The increasing application of positron emission tomography (PET) in nuclear medicine has stimulated the extensive development of a multitude of novel and versatile bioorthogonal conjugation techniques especially for the radiolabeling of biologically active high molecular weight compounds like peptides, proteins or antibodies. Taking into consideration that the introduction of fluorine-18 (t1/2 = 109.8 min) proceeds under harsh conditions, radiolabeling of these biologically active molecules represents an outstanding challenge and is of enormous interest. Special attention has to be paid to the method of 18F-introduction. It should proceed in a regioselective manner under mild physiological conditions, in an acceptable time span, with high yields and high specific activities. For these reasons and due to the high number of functional groups found in these compounds, a specific labeling procedure has to be developed for every bioactive macromolecule. Bioorthogonal strategies including the Cu-assisted Huisgen cycloaddition and its copper-free click variant, both Staudinger Ligations or the tetrazine-click reaction have been successfully applied and represent valuable alternatives for the selective introduction of fluorine-18 to overcome the afore mentioned obstacles. This comprehensive review deals with the progress and illustrates the latest developments in the field of bioorthogonal labeling with the focus on the preparation of radiofluorinated building blocks and tracers for molecular imaging.

Microstructure characterization through mechanical, electrokinetic and spectroscopic methods of polyampholyte gelatin hydrogels crosslinked with poly(vinyl alcohol)

The microstructure of polyampholyte gelatin hydrogels crosslinked covalently with fully hydrolyzed poly(vinyl alcohol) is characterized. The experimental methodology involves simple mechanical extension, spectroscopic and thermal methods (FTIR, XRD, DSC, TGA) and equilibrium swelling test to quantify relevant microstructural parameters and electrokinetic properties of this polyampholyte-polypeptide hydrogel, for a well defined range of crosslinker to gelatin mass ratios. These polyampholyte crosslinked matrices having different positive effective electrical charge are studied within an experimental and theoretical framework of their elastic and swelling properties. Estimations of average mesh size and toughness of the swollen hydrogels are provided. The feasibility of polyion complexation between positive crosslinked gelatin chains and negative bioactive macromolecules to be delivered through hydrogel biodegradation is analyzed. Several additional hydrogel properties are estimated like shear modulus, average molecular mass of network strands, swelling ratio, Flory–Huggins interaction parameter, hydrogel electrostatic zeta-potential and average effective charge number per chain before and after the crosslinking process to quantify gelatin chain cationization.

Oligoarginine-modified biodegradable nanoparticles improve the intestinal absorption of insulin

The strategy of oral administration of bioactive macromolecules using cell-penetrating peptides (CPPs) is restricted to covalent linkage or electrostatic interaction between the cargo and CPPs. In the present study, we devised an approach utilizing CPP-functionalized poly(lactic-co-glycolic acid) (PLGA) nanoparticles as a carrier for oral delivery of insulin. Pegylated PLGA nanoparticles were modified with poly(arginine)8 enantiomers (l-R8 and d-R8) via a maleimide-mediated covalent conjugating procedure. The physical and chemical features of the nanoparticles were characterized, which confirmed the successful immobilization of R8 to the nanoparticles. Using a Caco-2 cell monolayer model, R8-modified nanoparticles were found to exhibit significantly increased cellular uptake and transportation. Pharmacokinetics and pharmacodynamics of the insulin-loaded nanoparticles were evaluated with rats by intestinal administration. Compared to the unmodified nanoparticles, l-R8 and d-R8 modified-nanoparticles increased the relative bioavailabilities of insulin by 3.2- and 4.4-times, meanwhile, improved the hypoglycemic effects by 2.5- and 3.7-times, respectively. Neither of the R8-modified nanoparticles caused perceptible histological toxicities. The results implied that surface modification of biodegradable nanoparticles with poly(arginine)8, especially with the d-form enantiomer, showed remarkable advancement in promoting the intestinal absorption of insulin. This delivery system is also promising for the delivery of a wide variety of bioactive macromolecules by oral administration.

Pathophysiological relevance of aldehydic protein modifications

There is growing body of evidence that oxidative stress, i.e. excess in production of reactive oxygen species, can lead to covalent modification of proteins with bioactive aldehydes that are mostly produced under lipid peroxidation of polyunsaturated fatty acids. Thus generated reactive aldehydes are considered as second messengers of free radicals because they react with major bioactive macromolecules, in particular with various humoral and cellular proteins changing their structure and functions. Therefore, the aldehydic-protein adducts, in particular those involving 4-hydroxy-2-nonenal, malondialdehyde and acrolein can be valuable biomarkers of numerous pathophysiological processes. The development of immunochemical methods is increasing the possibilities to study such non-enzymatic protein modifications, on the one hand, while on the other hand the increase of knowledge on bioactivities of the aldehydes and their protein adducts might lead to better prevention, diagnosis and treatments of pathophysiological processes associated with lipid peroxidation and oxidative stress in general. This article is part of a Special Issue entitled: Posttranslational Protein modifications in biology and Medicine.

Microparticle-Mediated Transfer of the Viral Receptors CAR and CD46, and the CFTR Channel in a CHO Cell Model Confers New Functions to Target Cells

Cell microparticles (MPs) released in the extracellular milieu can embark plasma membrane and intracellular components which are specific of their cellular origin, and transfer them to target cells. The MP-mediated, cell-to-cell transfer of three human membrane glycoproteins of different degrees of complexity was investigated in the present study, using a CHO cell model system. We first tested the delivery of CAR and CD46, two monospanins which act as adenovirus receptors, to target CHO cells. CHO cells lack CAR and CD46, high affinity receptors for human adenovirus serotype 5 (HAdV5), and serotype 35 (HAdV35), respectively. We found that MPs derived from CHO cells (MP-donor cells) constitutively expressing CAR (MP-CAR) or CD46 (MP-CD46) were able to transfer CAR and CD46 to target CHO cells, and conferred selective permissiveness to HAdV5 and HAdV35. In addition, target CHO cells incubated with MP-CD46 acquired the CD46-associated function in complement regulation. We also explored the MP-mediated delivery of a dodecaspanin membrane glycoprotein, the CFTR to target CHO cells. CFTR functions as a chloride channel in human cells and is implicated in the genetic disease cystic fibrosis. Target CHO cells incubated with MPs produced by CHO cells constitutively expressing GFP-tagged CFTR (MP-GFP-CFTR) were found to gain a new cellular function, the chloride channel activity associated to CFTR. Time-course analysis of the appearance of GFP-CFTR in target cells suggested that MPs could achieve the delivery of CFTR to target cells via two mechanisms: the transfer of mature, membrane-inserted CFTR glycoprotein, and the transfer of CFTR-encoding mRNA. These results confirmed that cell-derived MPs represent a new class of promising therapeutic vehicles for the delivery of bioactive macromolecules, proteins or mRNAs, the latter exerting the desired therapeutic effect in target cells via de novo synthesis of their encoded proteins.

A Highly Tunable Biocompatible and Multifunctional Biodegradable Elastomer

Biodegradable elastomers have emerged as promising materials for their potential to mimic the viscoelastic properties of several tissues and exhibit compliance with dynamic environments without damaging the surrounding tissue.[1, 2] Several elastomers have been recently proposed;[3–8] however, the development of highly tunable biodegradable elastomers that can effectively and controllably present biological and physical signals and withstand repeated cycles of physiologic loads, has remained elusive. Such materials should be useful for a broad range of clinically-relevant applications, such as cardiac therapy. For example, following myocardial infarction, the local controlled delivery of bioactive cues[9] or the physical support of the left ventricle wall[10] have been shown to improve cardiac function. The synergistic therapeutic effect of biochemical and biophysical cues has not yet been explored using degradable materials given the absence of materials that can simultaneously deliver bioactive cues and maintain mechanical integrity in a dynamic environment such as the beating heart. Here, we describe a novel biocompatible and mechanically tunable elastomer, poly(glycerol sebacate urethane) (PGSU), suitable for efficient encapsulation and controlled delivery of bioactive macromolecules and with the potential to be applied to cardiac drug delivery.

Phosphatidylethanolamine is externalized at the surface of microparticles

Microparticles (MPs) are membrane-bound vesicles shed normally or as a result of various (pathological) stimuli. MPs contain a wealth of bio-active macromolecules. Aminophospholipid phosphatidylserine (PS) is present on the surface of many MPs. As PS and phosphatidylethanolamine (PE) are related, yet distinct aminophospholipids, the purpose of this study was to systematically and directly assess PE exposure on MPs. We examined MPs from various human cellular sources (human breast cancer, endothelial, red and white blood cells) by flow cytometry using a PE-specific probe, duramycin, and two PS-specific probes, annexin V and lactadherin. PS and PE exposure percentage was comparable on vascular and blood cell-derived MPs (80–90% of MP-gated events). However, the percentage of malignant breast cancer MPs exposing PE (~90%) was significantly higher than PS (~50%). Thus, while PS and PE exposure can result from a general loss of membrane asymmetry, there may also be distinct mechanisms of PE and PS exposure on MPs that vary by cellular source.

ChemInform Abstract: Reactions in Dynamic Self‐Assemblies

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