Biodegradable nanogels prepared by atom transfer radical polymerization as potential drug delivery carriers: synthesis, biodegradation, in vitro release, and bioconjugation

Stable biodegradable nanogels cross-linked with disulfide linkages were prepared by inverse miniemulsion atom transfer radical polymerization (ATRP). These nanogels could be used for targeted drug delivery scaffolds for biomedical applications. The nanogels had a uniformly cross-linked network, which can improve control over the release of encapsulated agents, and the nanogels biodegraded into water-soluble polymers in the presence of a biocompatible glutathione tripeptide, which is commonly found in cells. The biodegradation of nanogels can trigger the release of encapsulated molecules including rhodamine 6G, a fluorescent dye, and Doxorubicin (Dox), an anticancer drug, as well as facilitate the removal of empty vehicles. Results obtained from optical fluorescence microscope images and live/dead cytotoxicity assays of HeLa cancer cells suggested that the released Dox molecules penetrated cell membranes and therefore could suppress the growth of cancer cells. Further, OH-functionalized nanogels were prepared to demonstrate facile applicability toward bioconjugation with biotin. The number of biotin molecules in each nanogel was determined to be 142,000, and the formation of bioconjugates of nanogels with avidin was confirmed using optical fluorescence microscopy.     

Tuning amphiphilicity/temperature‐induced self‐assembly and in‐situ disulfide crosslinking of reduction‐responsive block copolymers

New poly(ethylene oxide)‐based block copolymers (ssBCs) with a random copolymer block consisting of a reduction‐responsive disulfide‐labeled methacrylate (HMssEt) and a thermoresponsive di(ethylene glycol)‐containing methacrylate (MEO₂MA) units were synthesized. The ratio of HMssEt/MEO₂MA units in the random P(MEO₂MA‐co‐HMssEt) copolymer block enables the characteristics of well‐defined ssBCs to be amphiphilic or thermoresponsive and double hydrophilic. Their amphiphilicity or temperature‐induced self‐assembly results in nanoaggregates with hydrophobic cores having different densities of pendant disulfide linkages. The effect of disulfide crosslinking density on morphological variation of disulfide‐crosslinked nanogels is investigated. In response to reductive reactions, the partial cleavage of pendant disulfide linkages in the hydrophobic cores converts the physically associated aggregates to disulfide‐crosslinked nanogels. The occurrence of in‐situ disulfide crosslinks provides colloidal stability upon dilution. © 2014 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2014 , 52, 2057–2067     

A collagen(Col)/nano-hydroxyapatite (nHA) biological composite bone scaffold with double multi-level interface reinforcement

IntroductionBone scaffold is expected to possess excellent mechanical and biological properties similar to natural bone tissues. In this study, we aimed to prepare a biomineralized Col and hydroxyapatite composite scaffold consisting of biomimetic bone components and multi-level bionic bone structure to strengthen its mechanical properties.MethodsWe prepared a Col/nano-hydroxyapatite biological composite scaffold with multi-level structure (from nanofibers to micron bionic bone motif to bionc bone scaffold) of biomimetic bone tissue, and biomineralized the scaffold in simulated body fluid (SBF) preheated to 37 °C. X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy and Scanning electron microscope, were used to characterize the biomineralized products.ResultsMorphological study confirmed in situ deposition of nHA in the multi-scale hierarchical structure of the biomineralized scaffold. We explored the biomineralization nucleation mechanism of the scaffolds at the atomic level based on the first principles and the mechanisms for growth of mineralized nHA crystal array in its multi-scale structure, and how the double multiscales structure strengthened the mechanical properties of the material.ConclusionsThis synthetic bone scaffold, with bionic bone composition and double multi-level interface reinforcement, provides a new strategy for synthesizing bioactive bone scaffolds with enhanced biomechanical properties.     

Block copolymer micelles with enzyme molecules at the interfaces

This research provides an efficient method for the fabrication of hybrid micelles with enzyme molecules at the interfaces. Amphiphilic block copolymer is synthesized by reversible addition–fragmentation chain transfer (RAFT) polymerization, and thiol‐modified porcine pancreatic lipase (PPL‐SH) is obtained by treatment of native PPL with Traut's reagent. PPL‐SH is conjugated to the block copolymer chains by thiol‐disulfide exchange reaction. In phosphate buffered saline, the bioconjugate self‐assembles into micelles with enzyme molecules at the interfaces between hydrophobic cores and hydrophilic coronae. The bioactivity of the enzyme molecules on the micelles are compared with the native enzyme. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55, 2047–2052     

Biomineralized Multifunctional Magnetite/Carbon Microspheres for Applications in Li‐Ion Batteries and Water Treatment

Advanced functional materials incorporating well‐defined multiscale architectures are a key focus for multiple nanotechnological applications. However, strategies for developing such materials, including nanostructuring, nano‐/microcombination, hybridization, and so on, are still being developed. Here, we report a facile, scalable biomineralization process in which Micrococcus lylae bacteria are used as soft templates to synthesize 3D hierarchically structured magnetite (Fe₃O₄) microspheres for use as Li‐ion battery anode materials and in water treatment applications. Self‐assembled Fe₃O₄ microspheres with flower‐like morphologies are systematically fabricated from biomineralized 2D FeO(OH) nanoflakes at room temperature and are subsequently subjected to post‐annealing at 400 °C. In particular, because of their mesoporous properties with a hollow interior and the improved electrical conductivity resulting from the carbonized bacterial templates, the Fe₃O₄ microspheres obtained by calcining the FeO(OH) in Ar exhibit enhanced cycle stability and rate capability as Li‐ion battery anodes, as well as superior adsorption of organic pollutants and toxic heavy metals.     

Highly Efficient Synthesis and Assay of Protein‐Imprinted Nanogels by Using Magnetic Templates

We report an approach integrating the synthesis of protein‐imprinted nanogels (“plastic antibodies”) with a highly sensitive assay employing templates attached to magnetic carriers. The enzymes trypsin and pepsin were immobilized on amino‐functionalized solgel‐coated magnetic nanoparticles (magNPs). Lightly crosslinked fluorescently doped polyacrylamide nanogels were subsequently produced by high‐dilution polymerization of monomers in the presence of the magNPs. The nanogels were characterised by a novel competitive fluorescence assay employing identical protein‐conjugated nanoparticles as ligands to reversibly immobilize the corresponding nanogels. Both nanogels exhibited K_d<10 pM for their respective target protein and low cross‐reactivity with five reference proteins. This agrees with affinities reported for solid‐phase‐synthesized nanogels prepared using low‐surface‐area glass‐bead supports. This approach simplifies the development and production of plastic antibodies and offers direct access to a practical bioassay.     

Efficient on-column conversion of IgG1 trisulfide linkages to native disulfides in tandem with Protein A affinity chromatography

Protein trisulfide linkages are generated by the post-translational insertion of a sulfur atom into a disulfide bond. Molecular heterogeneity was detected in a recombinant IgG₁ monoclonal antibody (mAb) and attributed to the presence of a protein trisulfide moiety. The predominant site of trisulfide modification was the bond between the heavy and light chains. The trisulfide was eliminated during purification of the IgG₁ mAb via a cysteine wash step incorporated into Protein A affinity column chromatography. Analysis of the cysteine-treated mAb by electrophoresis and peptide mapping indicated that the trisulfide linkages were efficiently converted to intact disulfide bonds (13% trisulfide decreased consistently to 1% or less) without disulfide scrambling or an increase in free sulfhydryls. The on-column trisulfide conversion caused no change in protein folding detectable by hydrogen/deuterium exchange or differential scanning calorimetry. Consistent with this, binding of the mAb to its antigen in vitro was insensitive to the presence of the trisulfide modification and to its removal by the on-column cysteine treatment. Similar, high efficiency trisulfide conversion was achieved for a second IgG₁ mAb using the column wash strategy (at least 7% trisulfide decreased to 1% or less). Therefore, trisulfide/disulfide heterogeneity can be eliminated from IgG₁ molecules via a convenient and inexpensive procedure compatible with routine Protein A affinity capture.     

Protein release from biodegradable dextran nanogels

The use of drugs with intracellular targets will strongly depend on the availability of delivery systems that are able to deliver them to specific intracellular sites at an optimal rate. Biodegradable dextran nanogels were prepared using liposomes as a nanoscaled reactor.1,2 These nanogels were obtained by UV polymerization of dextran hydroxyethylmethacrylate (dex-HEMA) containing 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) liposomes. We found the encapsulation efficiency of bovine serum albumin (BSA) and lysozyme in the dextran nanogels to be about 50%. Specifically, the release of BSA and lysozyme from the dextran nanogels was clearly governed by the cross-link density of the tiny gels. Depending on the size of the encapsulated protein, the cross-link density of the dextran network, and the presence or absence of a lipid coating, proteins were released from the nanogels over days to weeks. Interestingly, when sufficiently diluted, dextran nanogels did not aggregate in human serum, which is of major importance when one considers intravenous administration of such nanogels. Also, reconstitution of lyophilized dextran nanogels seemed perfectly possible, which is also an important finding since dextran nanogels will have to be stored in dry form. Because dextran nanogels can be taken up by cells, they are promising materials for controlled intracellular release of proteins.     

Preparation of hybrid triple‐stimuli responsive nanogels based on poly(L‐histidine)

A series of novel multi‐responsive disulfide cross‐linked polypeptide nanogels has been synthesized by a one‐step ring‐opening polymerization process. The pH‐responsive core of the prepared nanogels was based on poly(L‐histidine), the difunctional N‐carboxy anhydride of l ‐cystine (l ‐Cys‐NCA) was used as a reduction‐cleavable cross‐linking agent, while the outer hydrophilic corona was comprised of a poly(ethylene oxide) block. Extensive molecular characterization studies were conducted in order to confirm the formation of the desired polymeric nanostructures and also to prove their responsiveness to external stimuli within the physiological values of healthy and cancer tissues. Furthermore, the disruption of the disulfide‐bond linkages between the polymeric chains was achieved by the presence of the reductive tripeptide glutathione (GSH), leading to size variations that were monitored by dynamic light scattering (DLS) and size‐exclusion chromatography (SEC). “Stealth” properties of the formed nanostructures were examined by zeta potential measurements. The described nanogels are clearly promising candidates for drug delivery applications. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 1278–1288     

Reduction‐Triggered Self‐Cross‐Linked Hyperbranched Polyglycerol Nanogels for Intracellular Delivery of Drugs and Proteins

Owing to the unique advantages of combining the characteristics of hydrogels and nanoparticles, nanogels are actively investigated as a promising platform for advanced biomedical applications. In this work, a self‐cross‐linked hyperbranched polyglycerol nanogel is synthesized using the thiol–disulfide exchange reaction based on a novel disulfide‐containing polymer. A series of structural analyses confirm the tunable size and cross‐linking density depending on the type of polymer (homo‐ or copolymer) and the amount of reducing agent, dithiothreitol, used in the preparation of the nanogels. The nanogels retain not only small molecular therapeutics irrespective of hydrophilic and hydrophobic nature but also large enzymes such as β‐galactosidase by exploiting the self‐cross‐linking chemistry. Their superior biocompatibility together with the controllable release of active therapeutic agents suggests the applicability of nanogels in smart drug delivery systems.     

3-Diethylaminopropyl-bearing glycol chitosan as a protein drug carrier

Polysaccharidic nanogels were fabricated with bovine serum albumin (BSA) and a glycol chitosan (GCS) grafted with functional 3-diethylaminopropyl (DEAP) groups. These nanogels were investigated to evaluate their cellular uptake in HeLa cells and in vivo fate in nude mice tumor model. Unlike free BSA, GCS-g-DEAP/BSA nanogels improved cellular uptake of BSA. Furthermore, this system led to an enhanced blood circulation and a high accumulation of BSA in the tumor site. Our collective results strongly support that GCS-g-DEAP/BSA nanogel is a potential carrier system for high molecular weight proteins.     

Controlled Deformation of Soft Nanogel Particles Generates Artificial Biominerals with Ordered Internal Structure

Biominerals can exhibit exceptional mechanical properties owing to their hierarchically-ordered organic/inorganic nanocomposite structure. However, synthetic routes to oriented artificial biominerals of comparable complexity remain a formidable technical challenge. Herein we design a series of soft, deformable nanogels that are employed as particulate additives to prepare nanogel@calcite nanocomposite crystals. Remarkably, such nanogels undergo a significant morphological change—from spherical to pseudo-hemispherical—depending on their degree of cross-linking. This deformation occurs normal to the growth direction of the (104) face of the calcite and the underlying occlusion mechanism is revealed by in situ atomic force microscopy studies. This model system provides new mechanistic insights regarding the formation of oriented structures during biomineralization and offers new avenues for the design of synthetic nanocomposites comprising aligned anisotropic nanoparticles.     

A simple methodology for the synthesis of heterotelechelic protein–polymer–biomolecule conjugates

A synthetic protocol for the preparation of hetero‐biofunctional protein–polymer conjugates is described. A chain transfer agent, S,S‐bis (α,α′‐dimethyl‐α″‐acetic acid) trithiocarbonate was functionalized with α,ω‐pyridyl disulfide (PDS) groups, Subsequently, one of the PDS groups was covalently attached to bovine serum albumin (BSA) at the specific free thiol group on the cysteine residue through a disulfide linkage. The second PDS group remained intact, as it was found to be inaccessible to further BSA functionalization. The BSA‐macro‐reversible addition‐fragmentation chain transfer (RAFT) agent was then used to prepare BSA‐polymer conjugates via in situ polymerization of oligo (ethyleneglycol) acrylate and N‐(2‐hydroxypropyl) methacrylamide using an ambient temperature initiator, 4,4′‐azobis [2,9‐imidazolin‐2‐ethyl)propane] dihydrochloride in an aqueous medium. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS‐PAGE) confirmed that the in situ polymerization occurred at the protein surface where the RAFT agent was attached and the molecular weights of the BSA–polymer conjugates were found to increase concomitantly with monomer conversion and polymerization time. After polymerization the remaining terminal PDS groups were then utilized to attach thiocholesterol and a flurophore, rhodamine B to the protein–polymer conjugates via disulfide coupling. UV–Vis and fluorescence analyses revealed that ～80% of the protein conjugates were found to retain integral PDS end groups for further attachment to free thiol‐tethered precursors. © 2010 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 48: 1399–1405, 2010     

Competitive Process of Binding Between the Anionic Surfactants Sodium Dodecyl Sulfate and Sodium Cholate in Bovine Serum Albumin

Summary: In this study sodium cholate (NaC) was used as a representative bile salt for the competitive binding between NaC and sodium dodecyl sulfate (SDS) in bovine serum albumin (BSA), in 0.02 M tris-HCl buffer solution at pH 7.50 and 25 °C. The NaC and SDS associations with BSA were monitored at low surfactant concentrations where only this specific binding process can develop. The applied method to monitor the binding was based on the analysis of the effect of SDS and NaC concentrations and their mixtures upon the fluorescence intensity of the BSA tryptophan residues. This consists of the measurement of the surfactant monomer partitioning between the dispersion medium and the microaggregates on the protein molecule where the binding is indicated by the quenching of the fluorescence chromophores. Experimentally, varying the protein concentration, the surfactant concentration needed to reach a given I_o/I ratio (I_o and I are the intensities with and without protein, respectively) was measured. The analyses, based on the average number of surfactant molecules bound on the protein, indicated that the SDS is a more efficient quencher than the bile salt. The need for 4–6 NaC bound molecules to give the same protein quenching efficiency by a single molecule of SDS was estimated. We concluded that the differences in the competitive binding on the protein are exclusively related to the quenching efficiency in the formation of the nonfluorescent fluorophore-quencher complex via a physical contact and static quenching process.     

Poly(PEGMA) magnetic nanogels: preparation via photochemical method,characterization and application as drug carrier

One-pot synthesis of magnetic nanogels with excellent biocompatibility via the photochemical method is reported in this paper. Poly(PEGMA) modified superparamagnetic nanogels (poly(PEGMA) magnetic nanogels) were synthesized by in-situ polymerization using poly(ethylene glycol) methacrylate (PEGMA) as the monomer and N, N′-methylene-bis-(acrylamide) (MBA) as the cross-linking agent in magnetite aqueous suspension under UV irradiation. The surface functional groups and components of magnetic nanogels were analyzed by Fourier transform infrared spectroscopy (FTIR) and a thermogravimetric analyzer (TGA). The results indicated that the poly(PEGMA) magnetic nanogels were synthesized successfully by coating poly(PEGMA) on the Fe₃O₄ nanoparticles under UV irradiation, and the Fe₃O₄ nanoparticles content in this nanogels was above 50 wt%. The morphology, size, zeta-potential and magnetic property were also characterized. The magnetic nanogels had a nearly spherical shape and core-shell structure, the average size in aqueous system measured by photon correlation spectroscopy (PCS) was 68.4 nm, which was much bigger than that in the dry state, the nanogels behaved superparamagnetically with saturated magnetization of 58.6 emu/g, and the zeta-potential was −16.3–−17.3 mV at physiological pH (6.8–7.4) which could help to maintain stability in blood. The preliminary application as drug carrier was made and the doxorubicin-loaded magnetic nanogels had an excellent property in slow-release. The experiment indicated that the magnetic nanogel was an ideal candidate carrier in target drug delivery systems and other biomedical application. Supported by the Natural Science Foundation of Shandong Province (Grant No. Q2006F01), Scientific and Technological Project of Shandong Province (Grant No. 2007GG3WZ02066) and Scientific and Technological Project of Department of Education, Shandong (Grant No. J07WC01)     

仿生矿化的机理和应用研究进展

碳酸钙、磷酸钙为代表的生物矿物广泛分布于自然界中,经过不同的矿化过程,在生物体内呈现出多样的结构、形貌和功能,构成生物体多种组织和器官.在人工材料合成领域,仿生矿化通过调控碳酸钙、磷酸钙等矿物的成核与生长,获得具有复杂高级结构和特殊生物功能的无机或无机/有机复合材料.本文重点介绍仿生矿化机理和应用的最近研究进展,包括仿...     

单分散磁性纳米粒子固定化猪胰脂肪酶的研究

借助溶热法制备了一种亲水及生物相容良好的Fe3O4磁性纳米粒子,用γ-氨丙基三乙氧基硅烷直接对所得磁性粒子表面改性,然后用戊二醛偶联法制得了固定化猪胰脂肪酶.表征研究显示,所得磁性粒子粒径约200 nm,具有良好的单分散性和磁响应性.考察了戊二醛浓度、给酶量和反应时间对脂肪酶固定化过程的影响,并通过游离酶与固定化酶的比...     

Impact of imidazolium-based ionic liquid surfactant additions on dilational rheology properties of different protein adsorption layer

The interfacial behavior of β-casein and BSA solutions have been investigated in the presence of imidazolium-based ionic liquid surfactant ([C₁₄mim]Br) at the decane/water interface with the oscillating the drop and interfacial tension relaxation measurements. Both the electrostatic and the hydrophobic interaction between protein and [C₁₄mim]Br played crucial roles as [C₁₄mim]Br concentration increases. Furthermore, it was found that the dilational rheology parameters provided information of the adsorbed layers structure, and the dynamics properties of the adsorbed layers depend on the bulk [C₁₄mim]Br concentration. Moreover, with the concentration of [C₁₄mim]Br increasing, β-casein in the interfacial layer was subject to conformational changes where it gave space to [C₁₄mim]Br molecules in the form of co-adsorb; for BSA/[C₁₄mim]Br solutions, the globule protein BSA deformed and then co-adsorb with [C₁₄mim]Br molecules at the decane/water interface. These results will contribute to elucidation of the influence of the surfactant on the different structure proteins and the wide applications of protein/surfactant systems in practice.     

Study of interaction of Potassium Dodecatangestato Cobaltate(III) with Bovine Serum Albumin using Fluorescence Spectroscopy

The binding of potassium dodecatangestato cobaltate(III) (PDC) as a water-soluble polyoxometal with bovine serum albumin (BSA) as a major transporting protein of plasma, has been investigated at pH 7.2, 5?mM phosphate buffer, 27°C and various ionic strength by fluorescence spectroscopy.

The results show that the binding of PDC to BSA quenches BSA emission and the Stern–Volmer linear relationship can be applied for the quenching process.

The values of Stern–Volmer constant, K _sv, which can be considered as a binding constant for formation of 1:1 complex at 0.01, 0.1 and 0.2?M NaCl are 8.56 × 10⁵, 5.72 × l0⁵ and 9.60 × 10⁵, respectively. The interpretation of the results represents that binding affinity depends on both electrostatic forces and conformational stability of BSA. A step-by-step aggregation model, which has been developed by Borissevich et al., was used to determine the average aggregation number of BSA, ?J?, from the fluorescence quenching. The results show that the binding of PDC to BSA does not induce any considerable aggregation in BSA molecules. Therefore, it can be concluded that there are no considerable conformational changes in BSA molecules during its interaction with PDC.     

Synthesis and characterization of PDEAEMA‐based magneto‐nanogels: Preliminary results on the biocompatibility with cells of human peripheral blood

Nanogels based on biocompatible, dual pH‐ and temperature‐sensitive poly(2‐(diethylamino)ethyl) methacrylate (PDEAEMA) have been successfully used as nanocontainers for the encapsulation of magnetite, Fe₃O₄ magnetic nanoparticles (MNPs). For this purpose, citric acid‐coated MNPs were encapsulated into previously synthesized PDEAEMA‐based nanogels using a poly(ethyleneglycol)‐based stabilizer. After the encapsulation of the magnetite MNPs, the so‐called magneto‐nanogels (MNGs) were proved to be multiresponsive on temperature, pH, and magnetic field and colloidally stable. Moreover, preliminary studies on the biocompatibility of these MNGs with cells of human peripheral blood were performed and evidenced quite tolerable biocompatibility, thus suggesting potential use in biomedical applications. © 2015 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2016 , 54, 1479–1494