Clickable functionalization of liposomes with the gH625 peptide from Herpes simplex virus type I for intracellular drug delivery

Liposomes externally modified with the nineteen residues gH625 peptide, previously identified as a membrane‐perturbing domain in the gH glycoprotein of Herpes simplex virus type I, have been prepared in order to improve the intracellular uptake of an encapsulated drug. An easy and versatile synthetic strategy, based on click chemistry, has been used to bind, in a controlled way, several copies of the hydrophobic gH625 peptide on the external surface of 1,2‐dioleoyl‐sn‐glycero‐3‐phosphocholine (DOPG)‐based liposomes. Electron paramagnetic resonance studies, on liposomes derivatized with gH625 peptides, which are modified with the 2,2,6,6‐tetramethylpiperidine‐1‐oxyl‐4‐amino‐4‐carboxylic acid (TOAC) spin label in several peptide positions, confirm the positioning of the coupled peptides on the liposome external surface, whereas dynamic light scattering measurements indicate an increase of the diameter of the liposomes of approximately 30 % after peptide introduction. Liposomes have been loaded with the cytotoxic drug doxorubicin and their ability to penetrate inside cells has been evaluated by confocal microscopy experiments. Results suggest that liposomes functionalized with gH625 may act as promising intracellular targeting carriers for efficient delivery of drugs, such as chemotherapeutic agents, into tumor cells.     

Sequential functionalization of janus‐type dendrimer‐like poly(ethylene oxide)s with camptothecin and folic acid

Janus‐type dendrimer‐like poly(ethylene oxide)s (PEOs) of 1st, 2nd, and 3rd generation carrying terminal hydroxyl functions on one side and cleavable ketal groups on the other were used as substrates to conjugate folic acid as a folate receptor and camptothecin (CPT) as a therapeutic drug in a sequential fashion. The conjugation of both FA and CPT was accomplished by “click chemistry” based on the 1,3 dipolar cycloaddition coupling reaction. First, the hydroxyl functions present at one face of Janus‐type dendrimer‐like PEOs were transformed into alkyne groups through a simple Williamson‐type etherification reaction. Next, the ketals carried by the other face of the dendrimer‐like PEOs were hydrolyzed, yielding twice as many hydroxyls which were subsequently subjected to an esterification reaction using 2‐bromopropionic bromide. Before substituting azides for the bromide of 2‐bromopropionate esters just generated in the presence of NaN₃, an azido‐containing amidified FA derivative was reacted through click chemistry with alkyne functions introduced on the other face of the dendrimer‐like PEOs. A purposely designed alkyne‐functionalized biomolecule derived from CPT was conjugated to the azido functions carried by the dendritic PEOs by a second “click reaction.” In this case, twice as many CPT as FA moieties were finally conjugated to the two faces of the Janus‐type dendrimer‐like PEOs, the numbers of folate and CPT introduced being 2 and 4, 4 and 8, and 8 and 16 for samples of 1st, 2nd, and 3rd generation, respectively (route A). An alternate route for functionalizing the dendrimer‐like PEO of 1st generation consisted, first, in conjugating the azido‐containing CPT onto the alkyne groups present on one face of the dendritic PEO scaffold. The alkyne‐functionalized FA was further introduced by click chemistry after the bromides of 2‐bromopropionate esters were chemically transformed into azido groups. The corresponding prodrug thus contains 2 CPT and 4 FA external moieties (route B). Every reaction step product was thoroughly characterized by ¹H NMR spectroscopy. A preliminary investigation into the water solution properties of these functionalized dendritic PEOs is also presented. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

X‐Ray Crystal Structure of a Second‐Generation Peptide Dendrimer in Complex with Pseudomonas aeruginosa Lectin LecB

Dendrimers are regularly branched molecular trees which are notoriously difficult to crystallize. Herein we report the crystal structure of a C‐fucosylated second generation peptide dendrimer as complex with lectin LecB in which the only dendrimer‐lectin contact is the LecB bound glycoside (PDB 6S5S). In contrast to a previously reported crystal structure of a first‐generation peptide dendrimer as LecB complex in which the dendrimer formed trimers connected by intermolecular β‐sheets (PDB 5D2A), the present structure features a globular monomeric state held together by intramolecular backbone hydrogen bonds and assembled into a non‐covalent dimer stabilized by hydrophobic contacts between leucine side‐chains and proline‐phenylalanine CH‐π stacking interactions. Molecular dynamics and circular dichroism studies suggest that this crystal structure resembles the structure of the peptide dendrimer in solution. Structures of a partially resolved dendrimer (PDB 6S5R) and of C‐fucosylated disulfide bridged peptide dimers connecting different LecB tetramers are also reported (PDB 6S7G, PDB 6S5P).     

A Novel Pentaerythritol-based Carbosilane Liquid Crystalline Dendrimer Containing 12 Nitroazobenzene Groups on the Periphery

A novel liquid crystalline dendrimer with peripheral mesogenic units was successfully prepared. Azo-reaction and Williamson synthesis were employed in the preparation of the mesogenic unit 4-[4-(6-hydroxyhexyloxy)-phenylazo]nitrobenzene (M-NO2). A terminal Si-Cl functional carbosilane dendrimer based on pentaerythritol was used as dendritic scaffold and subsequently functionalized with the aforementioned groups. Investigation of the liquid crystalline properties of the mesogen-functionalized dendrimer PCSi-IG-NO2 by polarizing optical microscopy, DSC, and X-ray diffraction showed that it exhibits smectic E (SE) phase, different from the corresponding mesogenic unit, which shows nematic phase. Furthermore, the temperatures of both the melting point and the clearing point of the mesogen-functionalized dendrimer decrease, and the temperature region of the SE phase is wider than that of the nematic phase.     

基于碘乙酸功能化树枝状聚合物的硫巯化肽段选择性富集新方法

蛋白质的硫巯化是一种重要的氧化翻译后修饰，在细胞衰老、内质网应激、血管舒张、细胞凋亡等过程中扮演重要角色。利用蛋白质组学技术表征硫巯化修饰具有十分重要的意义。该文发展了一种基于碘乙酸功能化的聚酰胺-胺树枝状聚合物（PAMAM-INS），并结合滤膜辅助的样品预处理技术用于硫巯化肽段的富集（简称FADE策略），利用FADE策略能从100倍质量干扰的牛血清白蛋白酶解产物中选择性地富集出硫巯化标准肽段。将细胞培养稳定同位素标记技术引入到FADE策略中，并将其应用于不同浓度梯度硫氢化钠刺激的SHSY5Y细胞硫巯化蛋白质组分析，共鉴定到163条硫巯化肽段。生物信息学的结果表明，硫巯化修饰可能在中枢神经系统中扮演着重要角色。     

Isolation and Characterization of Precise Dye/Dendrimer Ratios

Fluorescent dyes are commonly conjugated to nanomaterials for imaging applications using stochastic synthesis conditions that result in a Poisson distribution of dye/particle ratios and therefore a broad range of photophysical and biodistribution properties. We report the isolation and characterization of generation 5 poly(amidoamine) (G5 PAMAM) dendrimer samples containing 1, 2, 3, and 4 fluorescein (FC) or 6‐carboxytetramethylrhodamine succinimidyl ester (TAMRA) dyes per polymer particle. For the fluorescein case, this was achieved by stochastically functionalizing dendrimer with a cyclooctyne “click” ligand, separation into sample containing precisely defined “click” ligand/particle ratios using reverse‐phase high performance liquid chromatography (RP‐HPLC), followed by reaction with excess azide‐functionalized fluorescein dye. For the TAMRA samples, stochastically functionalized dendrimer was directly separated into precise dye/particle ratios using RP‐HPLC. These materials were characterized using ¹H and ¹⁹F NMR spectroscopy, RP‐HPLC, UV/Vis and fluorescence spectroscopy, lifetime measurements, and MALDI.     

Multivalency in the Gas Phase: H/D Exchange Reactions Unravel the Dynamic “Rock ’n’ Roll” Motion in Dendrimer–Dendrimer Complexes

Noncovalent dendrimer–dendrimer complexes were successfully ionized by electrospray ionization of partly protonated amino‐terminated polypropylene amine (POPAM) and POPAM dendrimers fully functionalized with benzo[21]crown‐7 on all branches. Hydrogen/deuterium exchange (HDX) experiments conducted on dendrimer–dendrimer complexes in the high vacuum of a mass spectrometer give rise to a complete exchange of all labile NH hydrogen atoms. As crown ethers represent noncovalent protective groups against HDX reactions on the ammonium group to which they are coordinated, this result provides evidence for a very dynamic binding situation: each crown is mobile enough to move from one ammonium binding site to another. Schematically, one might compare this motion with two rock ’n’ roll dancers that swirl around each other without completely losing all contact at any time. Although the multivalent attachment certainly increases the overall affinity, the “microdynamics” of individual site binding and dissociation remains fast.     

In Vitro Synthesis of the E. coli Sec Translocon from DNA

Difficulties in constructing complex lipid/protein membranes have severely limited the development of functional artificial cells endowed with vital membrane‐related functions. The Sec translocon membrane channel, which mediates the insertion of membrane proteins into the plasma membrane, was constructed in the membrane of lipid vesicles through in vitro expression of its component proteins. The components of the Sec translocon were synthesized from their respective genes in the presence of liposomes, thereby bringing about a functional complex. The synthesized E. coli Sec translocon mediated the membrane translocation of single‐ and multi‐span membrane proteins. The successful translocation of a functional peptidase into the liposome lumen further confirmed the proper insertion of the translocon complex. Our results demonstrate the feasible construction of artificial cells, the membranes of which can be functionalized by directly decoding genetic information into membrane functions.     

苯丙氨酸改性树枝状聚赖氨酸的合成及表征

以双芴甲氧羰基赖氨酸(Di- Fmoc -Lysine)为支化单元采用固相多肽合成技术制备了一种树枝状聚赖氨酸.该聚合物表面活性氨基通过与芴甲氧羰基苯丙氨酸(Fmoc- Phe)反应获得表面完全替代的苯丙氨酸改性树枝状聚赖氨酸.产物的分子量和结构经MS和NMR进行了表征.研究结果表明,利用该方法得到的产物分子量单一,是一种潜在的理想非病毒治疗载体.     

Click‐Conjugation of Nanogold‐Functionalized PAMAM Dendrimer: Toward a Novel Electrochemical Detection Platform

This work reports a new electrochemical monitoring platform for sensitive detection of Cu²⁺ coupling click chemistry with nanogold‐functionalized PAMAM dendrimer (AuNP‐PAMAM). The system involved an alkyne‐modified carbon electrode and an azide‐functionalized AuNP‐PAMAM. Initially, the added Cu²⁺ was reduced to Cu⁺ by the ascorbate, and then the azide‐modified AuNP‐PAMAM was covalently conjugated to the electrode via Cu⁺‐catalyzed azide‐alkyne click reaction. The carried AuNPs accompanying PAMAM dendrimer could be directly monitored by stripping voltammetry after acidic pretreatment. By introduction of high‐loading PAMAM dendrimer with gold nanoparticles, as low as 2.8 pM Cu²⁺ (ppt) could be detected, which was 125‐fold lower than that of gold nanoparticle‐based labeling strategy. The method exhibited high specificity toward target Cu²⁺ against other potentially interfering ions, and was applicable for monitoring Cu²⁺ in drinking water with satisfactory results.     

Synthesis of carbohydrate‐linked poly(polyoxometalate) poly(amido)amine dendrimers

Mannose‐functionalized and ethoxyethanol‐functionalized poly(amido)amine dendrimers bound multiple vanadate‐substituted polyoxotungstate Wells–Dawson‐type polyoxometalates (POMs). Dendrimers incorporating 10–30 POMs were characterized with NMR, transmission electron microscopy, and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry techniques. The number of metal clusters per dendrimer molecule varied according to the dendrimer generation and the nature of the surface functional groups. Efforts aimed at using the poly(polyoxometalate) dendrimers as oxidation catalysts are also described. © 2005 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 43: 3059–3066, 2005     

Direct Cytosolic Delivery of siRNA Using Nanoparticle‐Stabilized Nanocapsules

The use of nanoparticle‐stabilized nanocapsules (NPSCs) for the direct cytosolic delivery of siRNA is reported. In this approach, siRNA is complexed with cationic arginine‐functionalized gold nanoparticles by electrostatic interactions, with the resulting ensemble self‐assembled onto the surface of fatty acid nanodroplets to form a NPSC/siRNA nanocomplex. The complex rapidly delivers siRNA into the cytosol through membrane fusion, a mechanism supported by cellular uptake studies. Using destabilized green fluorescent protein (deGFP) as a target, 90 % knockdown was observed in HEK293 cells. Moreover, the delivery of siRNA targeting polo‐like kinase 1 (siPLK1) efficiently silenced PLK1 expression in cancer cells with concomitant cytotoxicity.     

TERS Detection of αVβ3 Integrins in Intact Cell Membranes

Integrins are important membrane receptors that form focal adhesions with the extracellular matrix and are transmembrane signaling proteins. We demonstrate that nanoparticles functionalized with c‐RGDfC ligands bind to intact cell membranes and selectively enhance the amino acid signals of the integrin receptor when coupled with tip‐enhanced Raman scattering (TERS) detection. Controlling the plasmonic interaction between the functionalized nanoparticle and the TERS tip provides a clear Raman signal from α_Vβ₃ integrins in the cell membrane that matches the signal of the purified integrin receptor. Random aggregation of nanoparticles on the cell does not provide the same spectral information. Chemical characterization of membrane receptors in intact cellular membranes is important for understanding membrane signaling and drug targeting. These results provide a new method to investigate the chemical interactions associated with ligand binding to membrane receptors in cells.     

Optimizing Antimicrobial Peptide Dendrimers in Chemical Space

We used nearest‐neighbor searches in chemical space to improve the activity of the antimicrobial peptide dendrimer (AMPD) G3KL and identified dendrimer T7 , which has an expanded activity range against Gram‐negative pathogenic bacteria including Klebsiellae pneumoniae, increased serum stability, and promising activity in an in vivo infection model against a multidrug‐resistant strain of Acinetobacter baumannii. Imaging, spectroscopic studies, and a structural model from molecular dynamics simulations suggest that T7 acts through membrane disruption. These experiments provide the first example of using virtual screening in the field of dendrimers and show that dendrimer size does not limit the activity of AMPDs.     

De novo Design of Selective Membrane‐Active Peptides by Enzymatic Control of Their Conformational Bias on the Cell Surface

Selectively targeting the membrane‐perturbing potential of peptides towards a distinct cellular phenotype allows one to target distinct populations of cells. We report the de novo design of a new class of peptide whose ability to perturb cellular membranes is coupled to an enzyme‐mediated shift in the folding potential of the peptide into its bioactive conformation. Cells rich in negatively charged surface components that also highly express alkaline phosphatase, for example many cancers, are susceptible to the action of the peptide. The unfolded, inactive peptide is dephosphorylated, shifting its conformational bias towards cell‐surface‐induced folding to form a facially amphiphilic membrane‐active conformer. The fate of the peptide can be further tuned by peptide concentration to affect either lytic or cell‐penetrating properties, which are useful for selective drug delivery. This is a new design strategy to afford peptides that are selective in their membrane‐perturbing activity.     

Double‐Layer Nanogold and Poly(amidoamine) Dendrimer‐ Functionalized PVC Membrane Electrode for Enhanced Electrochemical Immunoassay of Total Prostate Specific Antigen

A simple and portable electrochemical immunosensor for the detection of total prostate specific antigen (t‐PSA) in human serum was developed using a double‐layer nanogold particles and dendrimer‐functionalized polyvinyl chloride (PVC) membrane as immunosensing interface. To fabricate such a multifunctional PVC electrode, an o‐phenylenediamine‐doped PVC membrane was initially constructed, then nanogold particles and poly(amidoamine) G4‐dendrimer with a sandwich‐type format were assembled onto the PVC membrane surface, and then t‐PSA antibodies (anti‐PSA) were adsorbed on the nanogold surface. The detection principle of the immunosensor is based on the change in the electric potential before and after the antigen‐antibody interaction. The experimental conditions and the factors influencing the performance of the immunosensor were investigated. Under optimal conditions, the proposed immunosensor exhibits good electrochemical behavior in the dynamic range of 0.5–18 ng/mL relative to t‐PSA concentration with a relative low detection limit of 0.1 ng/mL (S/N=3). The precision, reproducibility, and stability of the immunosensor are acceptable. In addition, 43 serum specimens were assayed by the as‐prepared immunosensor, and consistent results were obtained in comparison with those obtained by the standard enzyme‐linked immunosorbent assay (ELISA). Compared with the conventional ELISAs, the developed immunoassay system was simple and rapid without labeling and separation steps. Importantly, the immobilization and detection methodologies could be extended for the immobilization and detection of other biomarkers.     

Synthesis of spherical and hemispherical sugar‐containing poly(ornithine) dendrimers

A novel sugar‐containing poly(ornithine) dendrimer is synthesized for possible antigen delivery and related applications. The dendrimer contains an ornithine dendron as interior scaffolding and oligosaccharides on the periphery, which provide an attachment site for a peptide antigen. Maltose or lactose is bound to both hemispherical and spherical poly(ornithine) dendrimer generation 3 (G3) by reductive amination between its reducing end and the peripheral amino group of the dendrimer using a borane‐pyridine complex in a buffer solution at 50 °C. The degree of substitution of sugar is changed by varying the molar ratio of sugar to dendrimer. When the surface of spherical poly(ornithine) dendrimer G3 is modified by binding β‐alanine to the 16 amino groups, highly substituted maltose‐ or lactose‐β‐alanine‐poly(ornithine) dendrimer G3 is obtained in high yield after 7 days of reaction. The structures of these sugar‐containing dendrimers are characterized by NMR and matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry analyses. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 1400–1414, 2004     

Synthesis,Redox Activity of Rigid Ferrocenyl Dendrimers,and Isolation of Robust Ferricinium and Class‐II Mixed‐Valence Dendrimers

The coupling reactions of ethynylferrocene with trihalogenoarenes do not lead to ethynylferrocenyl arenes that are soluble enough to form the basis of a suitable construction of stiff ferrocenylethynyl arene‐cored dendrimers, which explains the previous lack of reports on stiff ferrocenyl dendrimers. However, rigid ferrocenyl‐terminated dendrimers have been synthesized from 1,3,5‐tribromo‐ and triiodobenzene through Sonogashira and Negishi reactions with 1,2,3,4,5‐pentamethyl‐1′‐ethylnylferrocene ( 1 a ), according to 1→2 connectivity. With compound 1 a , the construction of a soluble dendrimer ( 10 a ) that contained 12 ethynylpentamethylferrocenyl termini was achieved. Stiff dendrimer 10 a shows a single, reversible cyclic voltammetry (CV) wave (with adsorption), which disfavors the hopping heterogeneous electron‐transfer mechanism that is postulated for redox‐terminated dendrimers that contain flexible tethers. The selectivity of these Sonogashira reactions allows the synthesis of an arene‐cored dendron ( 2 c ) that contains both ethynylferrocenyl and 1,2,3,4,5‐pentamethyl‐ferrocenylethynyl redox groups, thus leading to the construction of a dendrimer ( 7 c ) that contains both types of differently substituted ferrocenyl groups with two well‐separated reversible CV waves. Upon selective oxidation, this mixed dendrimer ( 7 c ) leads to a class‐II mixed‐valence dendrimer, 7 c [PF₆]₃, as shown by Mössbauer spectroscopy, whereas oxidation of the related fully pentamethylferrocenylated dendrimer ( 7 a ) leads to the all‐ferricinium dendrimer, 7 a [PF₆]₆.     

Design of peptide–dendrimer conjugates with tumor homing and antitumor effects

Development of drug delivery systems for cancer therapy is a crucial issue. Previously, some peptides were designed as tumor homing cell-penetrating peptides with antitumor activities. In this study, dual function dendrimers with tumor targeting activities and antitumor effects were designed using the tumor targeting CPP44 peptide for acute myelogenous leukemia (AML) and the antitumor p16^INK4a peptide. Two types of peptide–dendrimer conjugates were synthesized. One was a CPP44-linked p16^INK4a peptide-conjugated dendrimer (tandem linked dendrimer) and the other was a dendrimer conjugated with separate CPP44 and p16^INK4a peptides (parallel linked dendrimer). In addition, a peptide cathepsin B substrate was linked to the antitumor p16^INK4a peptide to release it from the carriers. These peptide–dendrimer conjugates produced more effective antitumor effects than a CPP44-linked p16^INK4a peptide. The parallel linked dendrimer showed less association with AML cells than the tandem linked dendrimer, but had greater antitumor effects. This suggested that both cellular uptake and antitumor peptide cleavage affected the antitumor activities of dual functional peptide-conjugated dendrimers.     

Synthesis,characterization, and thermal properties of dendrimer‐star,block‐comb copolymers by ring‐opening polymerization and atom transfer radical polymerization

Novel and well‐defined dendrimer‐star, block‐comb polymers were successfully achieved by the combination of living ring‐opening polymerization and atom transfer radical polymerization on the basis of a dendrimer polyester. Star‐shaped dendrimer poly(?‐caprolactone)s were synthesized by the bulk polymerization of ?‐caprolactone with a dendrimer initiator and tin 2‐ethylhexanoate as a catalyst. The molecular weights of the dendrimer poly(?‐caprolactone)s increased linearly with an increase in the monomer. The dendrimer poly(?‐caprolactone)s were converted into macroinitiators via esterification with 2‐bromopropionyl bromide. The star‐block copolymer dendrimer poly(?‐caprolactone)‐block‐poly(2‐hydroxyethyl methacrylate) was obtained by the atom transfer radical polymerization of 2‐hydroxyethyl methacrylate. The molecular weights of these copolymers were adjusted by the variation of the monomer conversion. Then, dendrimer‐star, block‐comb copolymers were prepared with poly(L ‐lactide) blocks grafted from poly(2‐hydroxyethyl methacrylate) blocks by the ring‐opening polymerization of L ‐lactide. The unique and well‐defined structure of these copolymers presented thermal properties that were different from those of linear poly(?‐caprolactone). © 2006 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 44: 6575–6586, 2006