Size- and Surface- Dual Engineered Small Polyplexes for Efficiently Targeting Delivery of siRNA

Though siRNA-based therapy has achieved great progress, efficient siRNA delivery remains a challenge. Here, we synthesized a copolymer PAsp(-N=C-PEG)-PCys-PAsp(DETA) consisting of a poly(aspartate) block grafted with comb-like PEG side chains via a pH-sensitive imine bond (PAsp(-N=C-PEG) block), a poly(l-cysteine) block with a thiol group (PCys block), and a cationic poly(aspartate) block grafted with diethylenetriamine (PAsp(DETA) block). The cationic polymers efficiently complexed siRNA into polyplexes, showing a sandwich-like structure with a PAsp(-N=C-PEG) out-layer, a crosslinked PCys interlayer, and a complexing core of siRNA and PAsp(DETA). Low pH-triggered breakage of pH-sensitive imine bonds caused PEG shedding. The disulfide bond-crosslinking and pH-triggered PEG shedding synergistically decreased the polyplexes’ size from 75 nm to 26 nm. To neutralize excessive positive charges and introduce the targeting ligand, the polyplexes without a PEG layer were coated with an anionic copolymer modified with the targeting ligand lauric acid. The resulting polyplexes exhibited high transfection efficiency and lysosomal escape capacity. This study provides a promising strategy to engineer the size and surface of polyplexes, allowing long blood circulation and targeted delivery of siRNA.     

Influence of Different Types of Block Copolymers on the Secondary Structure and Stability of the Staphylolytic Lysk Enzyme

A branched block copolymer of polyethyleneimine and polyethylene glycol (PEI–PEG) and a linear block copolymer of polylysine and polyethylene glycol (PLL–PEG) form complexes with the staphylolytic LysK enzyme. The complexation between LysK and cationic block-copolymers results in a 1.5–2.0-fold increase in the lytic activity of LysK and a 2–20-fold increase in its half-life. The stabilizing effects of the block copolymers depend on the temperature, the NaCl concentration, and the polymer/enzyme ratio. Being an effective stabilizer, linear PLL–PEG provides an increase in the LysK stability suitable for biomedical materials (LysK maintains 100% of its activity after four months at 4°C).     

Copolymerization of methacrylic acid with methyl methacrylate on a polyethylene glycol matrix

Copolymerization of methacrylic acid with [¹⁴C]-methyl methacrylate was carried out in the presence of polyethylene glycol (PEG) as a matrix. Composition and structure of complexes were determined using NMR techniques and radiometric measurements. It was found that the complexes consist of copolymer and PEG. Dependence of the composition of the copolymer on the composition of the initial mixture of monomers was examined. The reactivity ratios were calculated and compared with those for copolymerization without matrix. It was found that the influence of polyethylene glycol results in changing reactivity ratios of comonomers and kinetics of the process.     

Solvent-controlled organization of self-assembled polymeric monolayers on gold: an easy approach for the construction of protein resistant surfaces

Protein resistant surfaces based on poly(ethylene glycol) (PEG) coatings are extensively applied in the fields of biosensors, tissue engineering, fundamental cell-surface interaction research, and drug delivery systems. The structural organization of the PEG film on the surface has a significant effect on the performance of the film to resist protein adsorption. In this paper, we report an approach using solvent to control the organization of the polymeric monolayer on gold. A water soluble copolymer with grafted PEG side chains and alkyl disulfide side chains was synthesized. A polymeric monolayer was fabricated on a gold surface from different solutions (water- and toluene-based) of the copolymer. The organization of the polymeric monolayers was characterized by means of ellipsometry, cyclic voltammetry, contact angle, X-ray photoelectron spectroscopy, and atomic force microscopy. It was proven that the structural organization of the polymeric monolayer on a gold surface could be controlled by the solvent. A polymeric monolayer with PEG enriched at the outer level is obtained when water is used as the solvent. Various types of proteins, including fibrinogen, albumin, and normal human serum, were used to test the protein resistance of the gold surfaces modified by the polymeric monolayers. The polymeric monolayer formed from a water solution of the copolymer showed excellent protein resistance. In addition, by using water as the solvent, patterning of the polymeric monolayer could easily be achieved through a combination of lift-off and self-assembly. We believe that the approach reported here provides an easy, fast, and efficient way to fabricate a robust protein resistant surface.     

聚己内酯－聚乙二醇嵌段共聚物Ⅱ．组成、结晶性同降解性间关系

采用ＤＳＣ法测定了新嵌段共聚物聚己内酯（ＰＣＬ）－聚乙二醇（ＰＥＧ）嵌段共聚物（ＰＣＥ）的结晶性，研究了共聚物的结晶性同组成及降解性的关系．结果表明随着共聚物中ＰＥＧ组分的含量和分子量增加，共聚物的结晶性下降，亲水性提高，降解速度加快．     

聚己内酯－聚乙二醇嵌段共聚物Ⅱ．组成、结晶性同降解性间关系

采用ＤＣＳ法测定了新嵌段共聚物聚己内酯－聚乙二醇嵌段共聚物的结晶性，研究了共聚物的结晶性同组成及降解性的关系。结果表明随着共聚物中ＰＥＧ组分的含量和分子量增加，共聚物的结晶性下降，亲水性提高，降解速度加快。     

Water-soluble complexes from random copolymer and oppositely charged surfactant. 2. Complexes of poly(ethylene glycol)-based cationic random copolymer and bile salts

The complexes formed between the positively charged random copolymers (RCPs) of methoxy-poly(ethylene glycol) monomethacrylate (MePEGMA) and (3-(methacryloylamino)propyl)trimethylammonium chloride (MAPTAC) with oppositely charged biosurfactants (bile salts) were studied using turbidimetric titration, steady-state fluorescence, dynamic light scattering, and electron microscopy. Studies showed that the complexes of the RCPs of MAPTAC and MePEGMA with less than 68 mol % of PEG content precipitate in water, whereas the complexes of the copolymer with 89 and 94 mol % of PEG content do not precipitate in the entire range of composition of the mixture including stoichiometric compositions when the electroneutral complexes are formed. The complexes with true hydrophobic domains, which are a prerequisite characteristic to serve as a carrier, can be obtained at much lower concentration than the critical micelle concentration of the corresponding surfactant. For a particular surfactant, hydrophobic domains are obtained at lower Z-/+ for the random copolymer with lower PEG content. The hydrodynamic radii of these complexes vary over a range of 20-35 nm. Overall results reveal that these complexes are qualitatively similar to the polyion complex micelles or block ionomer complexes obtained from the block copolymers and oppositely charged surfactants. As the surfactants used in this study are biocompatible, we hope that these soluble particles will be promising vectors in the field of drug delivery.     

Polycaprolactone–poly(ethylene glycol) block copolymer,I: synthesis and degradability in vitro

A new type of biodegradable polymer material, poly(caprolactone)–poly(ethylene glycol) block copolymer (PCL-b-PEG), was synthesized by means of direct copolycondensation of ε-caprolactone with poly(ethylene glycol) in the presence of a Ti(OBu)₄ catalyst. The degradability of the polycaprolactone was improved by introducing a PEG component into it. The degradation of PCL-b-PEG copolymer increase with a decreasing crystallinity of the copolymer, and can be controlled by adjusting the component ratio of the copolymer.     

Self-assembly of a diblock copolymer with pendant disulfide bonds and chromophore groups: a new platform for fast release

An amphiphilic block copolymer comprising poly(ethylene glycol) (PEG) and poly(2-(methacryloyl)oxyethyl-2'-hydroxyethyl disulfide) (PMAOHD) blocks was synthesized by atom transfer radical polymerization (ATRP). Pyrenebutyric acid was conjugated to the block copolymer by esterification, and a block copolymer with pendant disulfide bonds and pyrenyl groups (PEG-b-P(MAOHD-g-Py)) was obtained. (1)H NMR and gel permeation chromatography (GPC) results demonstrated the successful synthesis of the block copolymer. The cleavage of the disulfide bonds and the degrafting of the pyrenyl groups were investigated in THF and a THF/methanol mixture. Fluorescence spectroscopy, GPC, and (1)H NMR results demonstrated fast cleavage of the disulfide bonds by Bu(3)P in THF. Fluorescence results showed the ratio of the intensity of the excimer peak to the monomer peak decreased rapidly within 20 min. GPC traces of the block copolymer moved to a long retention time region after addition of Bu(3)P, indicating the cleavage of the disulfide bonds and the degrafting of the pyrenyl groups. PEG-b-P(MAOHD-g-Py) can self-assemble into micelles with poly(MAOHD-g-Py) cores and PEG coronae in a mixture of methanol and THF (9:1 by volume). The dissociation of the micelles in the presence of Bu(3)P was investigated. After cleavage of the disulfide bonds in the micellar cores, a pyrene-containing small molecular compound and a block copolymer with pendant thiol groups were produced. Transmission electron microscopy (TEM), dynamic light scattering (DLS), and (1)H NMR were employed to track the dissociation of the polymeric micelles. All the techniques demonstrated the dissociation of the micelles and the fast release of pyrenyl groups from the micelles.     

PEG-PLA block copolymer as potential drug carrier: preparation and characterization

Diblock and multiblock copolymers composed of a poly(D,L-lactide) (PLA) or poly(trimethylene carbonate) (PTMC) core with a hydrophilic chain of poly(ethylene glycol) (PEG) were prepared. These copolymers, in which the core is connected to PEG through a polyfunctional molecule such as citric, mucic, or tartaric acid, may be used to form nanoparticles for drug delivery applications. Branched copolymers were prepared by direct amidation between the polyfunctional acid and methoxy PEGamine, followed by ring-opening polymerization of lactide or trimethyl carbonate to form the PLA and PTMC block copolymers. In addition, a complex multiblock copolymer of biotin-PEG-poly[lactic-co-(glycolic acid)] (PLGA) for application in an avidin-biotin system was prepared for possible design of nanospheres with targeting properties. Studies of drug release from polymeric systems containing multiblock copolymers and studies of polymer degradation were also performed.     

Tailoring of block copolymers based on the stoichiometric control of the end‐functionality of telechelic oligomers and the utilization of large‐scale fractionation by phase fluctuation chromatography: A synthetic strategy for the preparation of end‐functionalized poly(L‐lactide)‐block‐poly(oxyethylene)

An AB diblock copolymer of poly(L ‐lactide) (PLLA) and poly(oxyethylene) (PEG) with a cinnamate terminal in the PEG block was prepared by the copolymerization of L ‐lactide and partially end‐modified PEG followed by fractionation. The first step was the terminal modification of PEG with cinnamoyl chloride (CC), in which the degree of cinnamoylation of the hydroxyl terminals of PEG was roughly controlled by the feed ratio of both reactants. The resultant PEG cinnamate was subjected to copolymerization with L ‐lactide to produce a mixture of unreacted PEG dicinnamate (C‐PEG‐C), the diblock copolymer (PLLA‐PEG‐C), and the triblock copolymer (PLLA‐PEG‐PLLA) corresponding to the three components of the PEG cinnamate. This mixture was separated by phase fluctuation chromatography (PFC) to obtain PLLA‐PEG‐C in sufficient purity. This process, involving the stoichiometric control of the terminal reaction of telechelic oligomers and the utilization of PFC for fractionation, can be an efficient method for synthesizing end‐functionalized diblock copolymers from readily available telechelic oligomers. © 2000 John Wiley & Sons, Inc. J Polym Sci A: Polym Chem 38: 2405–2414, 2000     

可降解PET/PEG共聚物的合成

利用聚合物法通过熔融缩聚直接合成了可降解的PET／PEG共聚物，通过红外光谱、色质联用分析对聚合反应机理进行了研究，讨论了聚合条件对聚合物分子量的影响，测定了聚合物的体外降解。     

Di- and triblock siRNA-PEG copolymers: PEG density effect of polyelectrolyte complexes on cellular uptake and gene silencing efficiency

Di- and triblock siRNA/PEG copolymers were synthesized and complexed with cationic SLN for assessing their gene silencing efficiency as a function of PEG density. A sssiRNA and a sassiRNA were separately conjugated with PEG via a disulfide linkage. AB-type diblock and ABA-type triblock copolymers were successfully prepared by stoichiometric hybridization of sssiRNA-PEG conjugate with sassiRNA and sassiRNA-PEG conjugate, respectively. The resultant di- and triblock copolymers were characterized by means of GPC and gel electrophoresis. The serum stability of siRNA in the copolymers was enhanced as compared to that of naked siRNA. Using cationic SLN as a model carrier, the PEGylation density effect of the siRNA-PEG/SLN complexes on gene silencing and cellular uptake was analyzed.     

Micellization of lithium perfluoroheptanoate and its aggregation on poly(ethylene glycol) oligomers in water

The interaction of lithium perfluoroheptanoate (LiPFHep) with poly(ethylene glycol) (PEG) of different molecular weights (300 < MW < 20 000 Da) was investigated in water at 298.15 and 308.15 K by the isothermal titration calorimetry (ITC). Density and sound velocity measurements were also performed at 288.15, 298.15, and 308.15 K, while viscosity and conductivity data were only collected at 298.15 K. The aggregation process of this surfactant on the PEG polymeric chain was found to be very similar to the process exhibited by the two homologous perfluorooctanoate and perfluorononanoate. Viscosity and ITC data indicated that the formation of polymer-surfactant complexes between PEG and LiPFHep also leads to a conformational change in the polymer. The aggregation of micelles of the lithium perfluoro surfactants on the PEG polymeric chain is characterized by a comparable thermodynamic stability, which results from a balance of enthalpy and entropy contributions, which both increase with the length of the surfactant hydrophobic chain.     

PEGylated polyethyleneimine grafted silica nanoparticles: enhanced cellular uptake and efficient siRNA delivery

The present paper reports the utilization of hybrid nanocomposite particles consisting of PEI25k-PEG5k copolymer grafted silica nanoparticles (SiO₂NPs) for enhanced cellular uptake and siRNA delivery. High-resolution transmission electron microscopy and dynamic light scattering measurements ensured the average particle size of the final hybrid component as 45 nm (core SiO₂, 28–30 nm and shell PEI25k-PEG5k, 12–15 nm). Surface morphology from atomic force microscopy analysis showed the significant relationship between the particle size and shape. ²⁹Si and ¹³C cross-polarization–magic angle spinning solid state nuclear magnetic resonance (NMR), ¹H-NMR, and Fourier transform infrared spectroscopy were used to obtain the relevant structural information (such as Q3, silanol; Q4, siloxane functional groups of SiO₂NPs; resonance shifts and bending vibrations of PEI25k, –CH₂–CH₂–NH–; and PEG5k, –CH₂–CH₂–O–) from copolymer nanoparticle. Stable complexation of siRNA and nanocomposite particle (wt.%:wt.%) was achieved from 1:5 to 1:15 ratio. Nanocomposite particle (N/P) ratio and siRNA concentration determine the stability and knockdown efficiency of the PEI25k-PEG5k-graft-SiO₂NPs–siRNA complexes. It was shown that highly positively charged (zeta potential, +66 mV) PEI25k-PEG5k-graft-SiO₂NPs result in strong affinity with negatively charged siRNA. Confocal microscopy showed intensified cellular uptake of siRNA into cytoplasm of A549 cancer cell utilized for in vitro study. In conclusion, the coherence, graft density of copolymer-SiO₂NPs, and siRNA concentration were found to strongly influence the stability, and hence determine the knockdown efficiency, of PEI25k-PEG5k-graft-SiO₂NPs–siRNA complexes.     

Preparation, characterization and biocompatibility of poly(ethylene glycol)-poly(n-butyl cyanoacrylate) nanocapsules with oil core via miniemulsion polymerization

A new type of nanocapsules with an oil core, coated by poly(ethylene glycol) (PEG) was designed. The loading efficiency and the biocompatibility of the polymeric nanocapsules were evaluated when it was used as a carrier for hydrophobic agent paclitaxel. The nanocapsules were synthesized through miniemulsion polymerization of butylcyanoacrylate (BCA) with PEG as initiator. The particle size and zeta potential of nanocapsules were influenced by the PEG content in the polymerization system. Fourier transform infrared (FTIR) spectra and ¹H NMR demonstrated the chemical coupling between PEG and poly(butylcyanoacrylate) (PBCA). Thermal characteristics of the copolymer were investigated by differential scanning calorimetry (DSC). The encapsulation efficiency increased concurrently with the increase of the PEG content in the system. The hemolytic assay and the cytotoxicity measurement showed that the PEG coating could significantly reduce the hemolytic potential and cytotoxicity of the nanocapsules. The results showed that the PEG-PBCA nanocapsules could be an effective carrier for hydrophobic agents.     

Fabrication of polymeric micelles with core–shell–corona structure for applications in controlled drug release

Thermo-responsive polymeric micelles of poly (ethylene glycol)-b-poly(2-hydroxyethyl methacrylate-g-lactide)-b-poly(N-isopropylacrylamide) (PEG-P(HEMA-PLA)-PNIPAM) with core–shell–corona structure were fabricated for applications in controlled drug release. The graft copolymer of PEG-P(HEMA-PLA)-PNIPAM was self-assembled into core–shell micelles with a densely PLA core and mixed PEG/PNIPAM shells at 25 °C in aqueous media. By increasing the temperature above the lower critical solution temperature of PNIPAM, these core–shell micelles could be converted into core–shell–corona micelles because of the collapse of PNIPAM block on the PLA core as the inner shell and the soluble PEG block stretching outside as the outer corona. Anticancer drug doxorubicin (DOX) was loaded in the polymeric micelles as a model drug. Compared with polymeric micelles formed by liner PEG-b-PLA-b-PNIPAM triblock copolymer, these polymeric micelles exhibited higher loading capacity, and release of DOX from the polymeric micelles with core–shell–corona structure was well-controlled.     

Conjugation of siRNA with Comb‐Type PEG Enhances Serum Stability and Gene Silencing Efficiency

A thiol‐modified siRNA targeting the enhanced green fluorescence protein (eGFP) gene was conjugated with RAFT‐synthesized, pyridyl disulfide‐functional poly(PEG methyl ether acrylate)s (p(PEGA)s). siRNA‐p(PEGA) conjugates demonstrated significantly enhanced in vitro serum stability and nuclease resistance compared to the unmodified and thiol‐modified siRNA. The complexes of siRNA‐p(PEGA) conjugates with a fusogenic peptide, KALA ((+)/(–) = 2) inhibited the protein expression approximately 28‐fold more than the KALA complex of the unmodified siRNA. The protein inhibition caused by siRNA‐p(PEGA)‐KALA complexes (56 ± 5%–58 ± 3% of the fluorescence expressed in non‐treated cells) was comparable to the effect of the unmodified siRNA‐lipofectamine complex (77 ± 7%).

     

Efficient intracellular siRNA delivery by ethyleneimine-modified amphiphilic macromolecules

New materials that can bind and deliver oligonucleotides such as short interfering RNA (siRNA) without toxicity are greatly needed to fulfill the promise of therapeutic gene silencing. Amphiphilic macromolecules (AMs) were functionalized with linear ethyleneimines to create cationic AMs capable of complexing with siRNA. Structurally, the parent AM is formed from a mucic acid backbone whose tetra-hydroxy groups are alkylated with 12-carbon aliphatic chains to form the hydrophobic component of the macromolecule. This alkylated mucic acid is then mono-functionalized with poly(ethylene glycol) (PEG) as a hydrophilic component. The resulting AM contains a free carboxylic acid within the hydrophobic domain. In this work, linear ethyleneimines were conjugated to the free carboxylic acid to produce an AM with one primary amine (1N) or one primary amine and four secondary amines (5N). Further, an AM with amine substitution both to the free carboxylic acid in the hydrophobic domain and also to the adjacent PEG was synthesized to produce a polymer with one primary amine and eight secondary amines (9N), four located on each side of the AM hydrophobic domain. All amine-functionalized AMs formed nanoscale micelles but only the 5N and 9N AMs had cationic zeta potentials, which increased with increasing number of amines. All AMs exhibited less inherent cytotoxicity than linear polyethyleneimine (L-PEI) at concentrations of 10 μM and above. By increasing the length of the cationic ethyleneimine chain and the total number of amines, successful siRNA complexation and cellular siRNA delivery was achieved in a malignant glioma cell line. In addition, siRNA-induced silencing of firefly luciferase was observed using complexes of siRNA with the 9N AM and comparable to L-PEI, yet showed better cell viability at higher concentrations (above 10 μM). This work highlights the promise of cationic AMs as safe and efficient synthetic vectors for siRNA delivery. Specifically, a novel polymer (9N) was identified for efficient siRNA delivery to cancer cells and will be further evaluated.     

含有二个聚醚软链段的聚醚聚酯嵌段共聚物的合成及其血液相容性的研究

本文报道了一类新的具有二种聚醚软链段(PTMGT和PEGT)和一种聚酯硬链段(PET)的混合聚醚-聚酯嵌段共聚物(MPEE)的合成和它的血液相容性,并与具有相同软、硬链段比及相同软链段组成比(PTMGT/PEGT)的二种聚醚聚酯嵌段共聚物(PTMGT-PET和PEGT-PET)的共混物(BPEE)的性质进行了比较,结果表明:(1)聚醚聚酯嵌段共聚物的血液相容性可以通过引入亲水性好的PEGT组分而得到提高;(2)在多数的组成比下,共聚型的MPEE具有比共混型的BPEE优良的血液相容性;(3)特定的组成比:PTMGT/PEGT=60/40(mol),共混型的BPEE:(60/40)呈现最好的血液相容性以及最佳的力学性质。研究中发现材料的微相分离结构同血液相容性有关,细微的相分离结构可导致优良的血液相容性。