Polypeptide Shell Research Articles

Tumor-Associated Enzyme-Activatable Spherical Nucleic Acids.

Maximizing the tissue-targeting efficiency of nanomaterials while also protecting them from rapid clearance from the bloodstream and limiting their immunogenicity remains a central problem in the field of systemic-administered nanomedicine. Herein, we introduce a generalizable strategy to simultaneously increase tumor accumulation, prolong blood circulation, and limit nonspecific immune activation of nanomaterials via peptide-based, tumor-responsive, "sheddable" coatings. Spherical nucleic acids (SNAs) were designed and synthesized to contain an exterior coating composed of zwitterionic polypeptides with recognition sequences for tumor-associated proteases. In the presence of matrix metalloproteinases (MMPs), the polypetide coating is rapidly cleaved, leading to increased cellular uptake of these SNAs, relative to SNAs containing nonsheddable shells. Moreover, the zwitterionic nature of the polypeptide shell shields the SNAs from immune system recognition, which extends their blood circulation time and improves tumor accumulation and in vivo cellular uptake relative to control SNAs with no protective coating. Taken together, these results indicate that this strategy is a viable method for increasing nanoparticle tumor accumulation and can have utility for the systemic delivery of oligonucleotides and nanomaterials to target cells in vivo with low immunogenicity.

Development of an Integrated High Serum Stability Zwitterionic Polypeptide-Based Nanodrug with Both Rapid Internalization and Endocellular Drug Releasing for Efficient Targeted Chemotherapy

Chemotherapeutic nanodrugs have to penetrate through many biological barriers before reaching the tumor cells. Thus, high stability of the nanocarrier before reaching tumor cells and fast release of the carried drugs in targeted tumor cells are required. In this work, inspired by the intrinsic zwitterionic surface property, mainly formed by glutamic acid and lysine residues, of the plasma protein surface, the zwitterionic poly(glutamyl lysine-co-aspartic acid-co-cysteine) peptide (P(EK-D-C)) was synthesized for conjugating n-mercaptoalkanoic acid (MA) with different chain lengths on cysteine residues through a disulfide linkage to load hydrophobic doxorubicin (DOX). The results showed that the slightly negative-biased zwitterionic nanodrugs were very stable in both resistance to nonspecific plasma protein adsorption and prevention of premature DOX release at physiological pH 7.4 due to the zwitterionic polypeptide shell and the sharp contrast in polarity between the shell and DOX-loaded core, while they can quickly release the loaded DOX through responding to both low pH values in the endosome/lysosome and high glutathione concentrations in the tumor cell cytoplasm. Furthermore, the enhanced internalization of these nanodrugs led to about 60% higher in vitro cytotoxicity against MCF-7 cells at pH 6.7 than at pH 7.4, whereas the in vitro cytotoxicity of DOX·HCl at pH 6.7 was only 75% of the value at pH 7.4. In vivo results revealed that the stable nanodrugs conjugated with the long hydrophobic 12-mercaptododecanoic acid had higher tumor inhibition rate and lower systematic toxicity on MCF-7 tumor-bearing mice than the less stable nanodrugs conjugated with the short 8-mercaptooctaoic acid and were significantly superior to DOX·HCl. These results indicate that the combination of high stability in circulation and fast release in tumor cells of nanodrugs can enhance high efficacy targeted chemotherapy. This pH/redox-sensitive zwitterionic polypeptide nanocarrier might provide an excellent vehicle for solid tumor treatment.

Biodegradable and Dual-Responsive Polypeptide-Shelled Cyclodextrin-Containers for Intracellular Delivery of Membrane-Impermeable Cargo.

The transport of membrane impermeable compounds into cells is a prerequisite for the efficient cellular delivery of hydrophilic and amphiphilic compounds and drugs. Transport into the cell's cytosolic compartment should ideally be controllable and it should involve biologically compatible and degradable vehicles. Addressing these challenges, nanocontainers based on cyclodextrin amphiphiles that are stabilized by a biodegradable peptide shell are developed and their potential to deliver fluorescently labeled cargo into human cells is analyzed. Host–guest mediated self‐assembly of a thiol‐containing short peptide or a cystamine‐cross‐linked polypeptide shell on cyclodextrin vesicles produce short peptide‐shelled (SPSVss) or polypeptide‐shelled vesicles (PPSVss), respectively, with redox‐responsive and biodegradable features. Whereas SPSVss are permeable and less stable, PPSVss effectively encapsulate cargo and show a strictly regulated release of membrane impermeable cargo triggered by either reducing conditions or peptidase treatment. Live cell experiments reveal that the novel PPSVSS are readily internalized by primary human endothelial cells (human umbilical vein endothelial cells) and cervical cancer cells and that the reductive microenvironment of the cells’ endosomes trigger release of the hydrophilic cargo into the cytosol. Thus, PPSVSS represent a highly efficient, biodegradable, and tunable system for overcoming the plasma membrane as a natural barrier for membrane‐impermeable cargo.

Organophilic, Superparamagnetic, and Reversibly Thermoresponsive Silica-Polypeptide Core-Shell Particles.

Particles with a superparamagnetic cobalt inner core, silica outer core, and covalently bound homopolypeptide shell were investigated under thermal and magnetic stimuli. The homopolypeptide was poly(ε-carbobenzyloxy-l-lysine), PCBL, which is known to exhibit a thermoreversible coil ⇔ helix transition when dissolved as a pure polymer in m-cresol. Tethering to a core particle did not prevent PCBL from undergoing this conformational transition, as confirmed by dynamic light scattering and optical rotation, but the transition was broadened compared to that of the untethered polymer. The Co@SiO2-PCBL hybrid particles retained the superparamagnetic properties of the cobalt inner nougat. Indeed, some response remains even after aging for >5 years. The aged PCBL shell also preserved its responsiveness to temperature, although differences in the shape of the size vs temperature transition curve were observed compared to the freshly made particles. A reversible coil ⇔ helix transition for a particle-bound polypeptide in a pure organic solvent is rare. In addition to providing a convenient tool for characterizing coil ⇔ helix transitions for surface-bound polypeptides without interference from pH or the strong ionic forces that dominate behavior in aqueous systems, the Co@SiO2-PCBL/m-cresol system may prove useful in studies of the effect of shell polymer conformation on colloid interactions. The stability of the magnetic core and polypeptide shell suggest a long shelf life for Co@SiO2-PCBL, which can, in principle, be deprotected to yield positively charged Co@SiO2-poly(l-lysine) particles for possible transfection or antimicrobial applications or chained magnetically to produce responsive poly(colloids).

Inlaying Radiosensitizer onto the Polypeptide Shell of Drug-Loaded Ferritin for Imaging and Combinational Chemo-Radiotherapy.

Rationale: Ferritin with unique hollow cavity is an emerging protein-based nanoplatform for anticancer-drug delivery, but the in vivo chemotherapeutic effectiveness is still unsatisfactory with such a monotherapy modality, which is urgently in need of improvement.Methods: Here a novel ferritin nanotheranostic with anticancer-drug doxorubicin encapsulated into its hollow interior and nanoradiosensitizer bismuth sulfide nanocrystals inlayed onto its polypeptide shell was synthesized for combinational therapeutic benefits. The formation mechanism of bismuth sulfide nanocrystals based on ferritin has been analyzed. The in vitro and in vivo treatment effects were carried out on HeLa cancer cells and tumor-bearing mice, respectively. The biocompatibility and excretion of the ferritin nanotheranostic have also been evaluated to guarantee their biosafety.Results: The polypeptide shell of ferritin provides nucleation sites for the bismuth sulfide nanocrystals through coordination interaction, and simultaneously inhibits the further growth of bismuth sulfide nanocrystals, rendering the bismuth sulfide nanocrystals like rivets inlaying onto the polypeptide firmly, which can not only strengthen the architectural stability of ferritin to prevent drug burst leakage during systemic circulation, but also act as excellent computed tomography contrast agents and nanoradiosensitizers for in vivo imaging-guided cancer combinational treatments.Conclusions: The design concept of inlaying bismuth sulfide nanocrystals onto the polypeptide shell of doxorubicin-encapsulated ferritin significantly inhibits the tumor growth and simultaneously further broadens the application of ferritin in nanomedicine.

Complexation of the Zn, Co, Cd, and Pb ions by metallothioneins: A QM/MM simulation

Complexation of divalent metal ions (Zn, Co, Cd and Pb) with metallothioneins (MT), typical low-molecular proteins of diverse higher organisms, has been theoretically studied within the all-electron approximation B3LYP/DZP. The active site of MT (metal ions and cysteine residues) adopts the structure, in which cadmium, cobalt and zinc ions reveal a tetrahedral location of the sulfur atoms. A pyramidal structure is typical for the coordination environment of the lead complexes in the MT active site. The Gibbs energies of the active site formation were calculated accounting the influence of water medium in terms of the IEFPCM approximation. For all studied metallothioneins, the metal–active site complexes demonstrate close Gibbs formation energies per one metal ion. The influence of the amino acid environment of the MT active sites has been studied with the two-shell ONIOM model and B3LYP/DZP:UFF approximation. A simple scheme for calculation of relative metal–MT binding energies has been developed. Even under the pronounced destabilization of the lead complexes by a polypeptide shell, the total energetic effect is sufficient to make accumulation of lead ions by metallothioneins more efficient than Cd and, especially, Zn and Co. The following general trend of stability of the MT complexes has been found: Co≈Zn≪Cd≪Pb. These results compared with the experimental regularities of bioaccumulation of heavy metal salts by bacteria Bacillus are briefly discussed.

SiRNA-Loaded Polyion Complex Micelle Decorated with Charge-Conversional Polymer Tuned to Undergo Stepwise Response to Intra-Tumoral and Intra-Endosomal pHs for Exerting Enhanced RNAi Efficacy.

Small interfering RNA (siRNA) needs an efficient delivery vehicle to reach the cytoplasm of target cells for successful RNA interference (RNAi) therapy. This study aimed to develop an siRNA-loaded polyion complex (PIC) micelle equipped with a smart polymeric shell featuring tumor targetability and endosome escapability for enhanced RNAi activity in cancer cells. To this end, an acidic pH-responsive polypeptide was designed to exert a stepwise change in its charged state from negative to modestly positive and highly positive in response to slightly acidic environment of tumor (pH ∼6.7) and further lowered-pH condition of late endosomal compartments (pH ∼5.0), respectively, for selective binding to cancer cell surface and subsequent endosome disruption. This polypeptide, termed PAsp(DET-CDM/DBCO), was synthesized by introducing acid-labile carboxydimethyl maleate (CDM) and dibenzylcyclooctyne (DBCO) moieties into a polyaspartamide derivative bearing two-repeated aminoethylene side chains (PAsp(DET)). Then, PAsp(DET-CDM/DBCO) was installed on the surface of disulfide cross-linked PIC micelles prepared from cholesterol-modified siRNA (Chol-siRNA) and azide-poly(ethylene glycol)-b-poly[(3-mercaptopropylamidine)-L-lysine] (N3-PEG-b-PLys(MPA)) through the copper-free click reaction. Successful PAsp(DET-CDM/DBCO) coverage of PIC micelles was confirmed by a significant decrease in ζ-potential as well as a narrowly distributed size of 40 nm. The PAsp(DET-CDM/DBCO)-installed micelles significantly improved the gene-silencing efficiency in cultured lung cancer cells, compared with nonmodified control micelles, especially after incubation at pH 6.7. This improved silencing activity was nicely correlated with the facilitated cellular uptake of siRNA payloads at the acidic pH and the efficient endosomal escape. These results demonstrate that the acidic pH-responsive polypeptide shell is a promising design strategy for tumor-targeted siRNA delivery.

Encapsulation of EV71-specific IgY antibodies by multilayer polypeptide microcapsules and its sustained release for inhibiting enterovirus 71 replication

To save egg yolk immunoglobulin (IgY) from damage by protease digestion in oral delivery, IgY specific to enterovirus 71 (EV71) was loaded into polypeptide microcapsules by the method of layer-by-layer encapsulation. Porous CaCO3 particles doped with IgY were used as templates which were coated with multilayer poly (L-lysine) (PLL) and poly (L-glutamic acid) (PGA). Then, IgY-loaded polypeptide microcapsules were fabricated after the removal of CaCO3 templates. The alternating adsorption was confirmed by the zeta potential and the successful formation of a multilayer polypeptide (PLL/PGA) shell was visualized by SEM. The influence of the PLL/PGA layer numbers on the IgY sustained release performance was studied in simulated gastric fluid. IgY release could be accurately controlled by adjusting the layer number of the polypeptide shells and only 45% of the IgY was released from 5-bilayer microcapsules in the initial 2 hours. The biological activity of the encapsulated IgY was investigated both in vitro and in vivo. Encapsulated EV71-specific IgY remained highly active and capable of neutralizing EV71 after multilayer polypeptide encapsulation, which provided a promising method to protect EV71-specific IgY in the gastric environment and to specifically release IgY at the target intestinal site for the prevention and control of EV71 infectious diseases.

Hybrid materials achieved by polypeptide grafted magnetite nanoparticles through a dopamine biomimetic surface anchored initiator

Herein we report the preparation of well-defined hybrid materials based on magnetite nanoparticles coated with a polypeptide shell attached covalently through dopamine. Highly crystalline and hydrophobic magnetite nanoparticles have been modified at the surface using a biomimetic adhesive, dopamine, which provides the magnetite particles with primary amine groups. Hybrid magnetite–polypeptide systems have been, then, obtained by ring opening polymerization (ROP) of γ-benzyl-L-glutamate N-carboxyanhydride. These core–shell nanoparticles, magnetite–polypeptide, constitute an interesting alternative to other magnetite–polymer structures (PEG or dextran) because they present the following advantages: (1) the stability provided by a polypeptide shell covalently bonded to the magnetite surface through a dopamine anchor molecule instead of other non-covalent interactions; (2) the posterior chain deprotection of a poly(γ-benzyl-L-glutamate) (PBLG) shell transforms the system into magnetite–poly(glutamic acid) nanoparticles (PLGA), with high stability in aqueous solutions and a large amount of carboxylic acid groups; and (3) the easiness to incorporate compounds with biological activity into the PBLG structure, for instance, by loading procaine by aminolysis reaction. Therefore, the magnetite–PBLG nanoparticles depicted herein can be potentially employed as magnetic drug carriers or as scaffolds to engineer more complex hybrid materials. The structure and composition of these systems have been extensively characterized.

Magnetite–Polypeptide Hybrid Materials Decorated with Gold Nanoparticles: Study of Their Catalytic Activity in 4-Nitrophenol Reduction

The preparation and physical–chemical characterization of gold decorated hybrid materials based on individual magnetite nanoparticles coated with a poly(γ-benzyl-l-glutamate) shell is here presented. First, the synthesis of hybrid magnetite–polypeptide is achieved using a mimetic adhesive molecule, dopamine, which is responsible of both, anchoring the magnetite and initiating the ring-opening polymerization of γ-benzyl-l-glutamate N-carboxyanhydride. Besides, and in a second step, new molecules of dopamine are introduced to provide functionality to the poly(γ-benzyl-l-glutamate) by simple aminolysis reaction, which allow the growth and the stabilization of gold nanoparticles inside the polypeptide shell. Finally, the potential of these nanoparticles as a magnetic catalyst is proved by the reduction reaction of nitrophenol to aminophenol. The 100% efficiency with the magnetic recovery after eight catalytic cycles is also demonstrated.

Polypeptide core–shell silica nanoparticles with high grafting density by N-carboxyanhydride (NCA) ring opening polymerization as responsive materials and for bioconjugation

Silica nanoparticles were furnished with a functional polypeptide shell to create a pH-responsive inorganic–organic hybrid material. Free amine groups present on silica nanoparticles initiated the N-carboxyanhydride (NCA) polymerization of particular amino acid NCAs directly onto the inorganic support, offering a convenient method to functionalize silica core nanoparticles with a uniformly dense polypeptide shell. Poly(γ-benzyl-L-glutamate) (PBLG), poly(e-carbobenzyoxy-L-lysine) (PZLL), and S-tert-butyl protected polycysteine (PtBLC) were grafted from the silica core both independently as homopolypeptides and simultaneously to form a copolypeptide shell, highlighting the versatility that the grafting mechanism possesses. The grafting of PBLG was investigated in detail at 0 °C and 20 °C to determine any differences in the size and uniformity of the polypeptide shell formed. Dynamic light scattering (DLS) analysis revealed a correlation between the thickness of the uniform organic shell and the amount of amino acid in the monomer feed, with higher linearity at the lower polymerization temperature. Size Exclusion Chromatography (SEC) analyses of degrafted PBLG confirmed the DLS results. A high grafting density of around 0.4 PBLG chains per nm2 was calculated highlighting the control afforded in this approach to polypeptide grafting. Subsequent deprotection of the PBLG homopolypeptide shell afforded pH-sensitive poly(glutamic acid) (PGA) coupled silica nanoparticles. The selective release of a model rhodamine B dye was demonstrated to emphasize the potential that these hybrid nanomaterials have for the on-demand release of payload molecules in response to a targeted pH trigger. Moreover, covalent bioconjugation was successfully shown by attachment of green fluorescent protein.

Preparation of Organosoluble Silica−Polypeptide Particles by “Click” Chemistry

The synthesis of organo-soluble hybrid particles with a hydrophobic, α-helical polypeptide shell and silica core has been achieved by combining ring-opening polymerization and click chemistry. Alkyne end-terminated poly(γ-stearyl-α-l-glutamate) (alkyne-PSLG) was prepared by the ring-opening polymerization of the N-carboxyanhydride of γ-stearyl-α-l-glutamate using propargylamine as initiator. The molecular weight and structure of this polypeptide were characterized by GPC and MALDI, and the α-helical nature was established by 1H NMR and FTIR. Azide-functionalized silica particles (azido-silica) were prepared by the functionalization of silica particles with 3-bromopropyltrichlorosilane followed by nucleophilic substitution with sodium azide. The azide functionalization was confirmed by FTIR and XPS. Alkyne-PSLG was coupled to azido-silica by click reaction in tetrahydrofuran or toluene in the presence of pentamethyldiethylenetriamine and copper(I) bromide. Further characterization of the product using XPS, FTIR, and 1H NMR revealed that the grafted polypeptide retained its α-helical nature and formed colloidal particles that readily dispersed in organic solvents. These hydrophobic, polypeptide-functionalized particles can serve as model systems in studies of colloid dynamics and/or crystallization. They may also find use in investigations designed to model enzyme activation or the properties of hydrophobic proteins in cell membranes.

Polypeptide Modification of Multiwalled Carbon Nanotubes by a Graft‐From Approach

AbstractSummary: Covalent surface functionalization of carbon nanotubes with polypeptides is promising for possible medical applications. This work presents a graft‐from approach to perform the polypeptide modification of multiwalled carbon nanotubes (MWNTs). The raw MWNTs are first amine‐functionalized. The amine‐functionalized MWNTs are then used as the initiator to initiate the ring‐opening polymerization of γ‐benzyl‐L‐glutamate N‐carboxyanhydride (BLG‐NCA), to result in the polypeptide‐grafted MWNTs. FT‐IR, XPS, and TGA data demonstrate that the functionalization is successful. The TEM images of the products show that the thickness of the polypeptide shell of the PBLG‐MWNT is about 4.5–22 nm. Using the facile route developed here, carbon nanotubes functionalized with other types of polypeptides can be easily fabricated using the corresponding NCAs.Strategy for the polypeptide modification of multiwalled carbon nanotubes by a graft‐from approach.magnified imageStrategy for the polypeptide modification of multiwalled carbon nanotubes by a graft‐from approach.

α‐Helical Polypeptide Microcapsules Formed by Emulsion‐Templated Self‐Assembly

alpha-Helical peptide microcapsules were prepared by the emulsion-templated self-assembly of amphiphilic poly(gamma-benzyl L-glutamate)s (PBLG) 1. By mixing solutions of 1 in dichloromethane (in the form of a sodium salt) with water, oil-in-water emulsions were obtained. Spontaneous stripping of the dichloromethane phase caused a decrease in the diameter of the microdroplets and finally stable microcapsules formed. The microcapsules contain an inner aqueous phase as observed by confocal laser scanning microscopy (CLSM). Binding of hydrophobic pyrene molecules to the polypeptide shell was also demonstrated. The present polypeptide microcapsules are stable even after drying in air and they would serve as supramolecular vehicles for both hydrophobic and water-soluble molecules.

Organophilic Colloidal Particles with a Synthetic Polypeptide Coating

Composite colloidal particles with a silica core and a synthetic, homopolypeptide shell have been produced by initiation of benzyl-l-glutamate N-carboxyanhydride monomer from primary amine functionalized silica particles. The resulting poly(γ-benzyl-α,l-glutamate)-coated spheres were characterized by electron microscopy, dynamic light scattering, infrared spectroscopy, and thermogravimetry. The polypeptide shell accounts for about 20% of the total mass of the particles, which are reasonably uniform in size. Infrared spectra show an α-helical secondary structure, but other conformations are not excluded. A geometrical analysis is applied to calculate the maximum number of amino groups that might realistically participate in initiation. The actual shell thickness is smaller than expected on the basis of these geometrical considerations, which reflects undesired termination steps or the conversion of some monomer to unattached polymer by trace initiator impurities.

Direct visualization of polypeptide shell of ferritin molecule by atomic force microscopy

The polypeptide shell of the ferritin molecule has been imaged in water by atomic force microscopy (AFM). The central dip and the quaternary structure could be observed on the surface of the ferritin molecule anchored inhomogeneously in two dimensions. These structures observed in the AFM images are quite similar to the electron density map near the top of the apoferritin viewed down from a 4-fold axis structure reported previously (S. H. Banyard, D. K. Stammers, and P. M. Harrison, 1978. Nature (Lond.). 271:282-284). It has been achieved by introducing a "self-screening effect" of the surface charges of the AFM sample (S. Ohnishi, M. Hara, T. Furuno, and H. Sasabe. 1992. Biophys. J. 63:1425-1431) and the specially sharpened stylus of AFM cantilever.

Magnetoferritin: in vitro synthesis of a novel magnetic protein.

The iron storage protein ferritin consists of a spherical polypeptide shell (apoferritin) surrounding a 6-nanometer inorganic core of the hydrated iron oxide ferrihydrite (5Fe2O3.9H2O). Previous studies have shown that the in vitro reconstitution of apoferritin yields mineral cores essentially identical to those of the native proteins. A magnetic mineral was synthesized within the nanodimensional cavity of horse spleen ferritin by the use of controlled reconstitution conditions. Transmission electron microscopy and electron diffraction analysis indicate that the entrapped mineral particles are discrete 6-nanometer spherical single crystals of the ferrimagnetic iron oxide magnetite (Fe3O4). The resulting magnetic protein, "magnetoferritin," could have uses in biomedical imaging, cell labeling, and separation procedures.

Isolation and characterization of ferritin from the hepatopancreas of the musselMytilus edulis

The main iron-binding protein in the hepatopancreas of the mussel Mytilus edulis, which had been previously iron-loaded by exposure to carbonyl iron (spheres of elemental iron less than 5 μm diameter), has been isolated to electrophoretic purity and identified as ferritin. This ferritin has Mr, of 480000, pI of 4.7-5.0 and is composed of two subunits, Mr 18500 and Mr 24600. Under the electron microscope, it appears as electron-dense iron cores of average diameter 5 nm surrounded by a polypeptide shell to a final average overall diameter of 11 nm. The purified protein contains, on average, 200 iron atoms/molecule protein. On immunodiffusion, M. edulis hepatopancreas ferritin gives a partial cross-reaction with antiserum to horse spleen ferritin and lamprey (Geotria australis) liver ferritin but does not react with antiserum to chiton (Acanthopleura hirtosa) haemolymph ferritin.

Biomineralization of iron: Isolation of ferritin from the hemolymph of the limpet Patella vulgata

Ferritin has been isolated from the hemolymph of the limpet Patella vulgata. The protein was homogeneous by electrophoretic criteria and appeared under the electron microscope as a central electron dense core surrounded by a polypeptide shell. The iron content of the protein was 22.5%, present in the central core which had a median diameter of 70–75 Å and was poorly crystalline. The polypeptide shell consisted of a single subunit of apparent M r 25 000.