Poly(2-oxazoline)-Based Polyplexes as a PEG-Free Plasmid DNA Delivery Platform

The present study expands the versatility of cationic poly(2-oxazoline) (POx) copolymers as a polyethylene glycol (PEG)-free platform for gene delivery to immune cells, such as monocytes and macrophages. Several block copolymers are developed by varying nonionic hydrophilic blocks (poly(2-methyl-2-oxazoline) (pMeOx) or poly(2-ethyl-2-oxazoline) (pEtOx), cationic blocks, and an optional hydrophobic block (poly(2-isopropyl-2-oxazoline) (iPrOx). The cationic blocks are produced by side chain modification of 2-methoxy-carboxyethyl-2-oxazoline (MestOx) block precursor with diethylenetriamine (DET) or tris(2-aminoethyl)amine (TREN). For the attachment of a targeting ligand, mannose, azide-alkyne cycloaddition click chemistry methods are employed. Of the two cationic side chains, polyplexes made with DET-containing copolymers transfect macrophages significantly better than those made with TREN-based copolymer. Likewise, nontargeted pEtOx-based diblock copolymer is more active in cell transfection than pMeOx-based copolymer. The triblock copolymer with hydrophobic block iPrOx performs poorly compared to the diblock copolymer which lacks this additional block. Surprisingly, attachment of a mannose ligand to either copolymer is inhibitory for transfection. Despite similarities in size and design, mannosylated polyplexes result in lower cell internalization compared to nonmannosylated polyplexes. Thus, PEG-free, nontargeted DET-, and pEtOx-based diblock copolymer outperforms other studied structures in the transfection of macrophages and displays transfection levels comparable to GeneJuice, a commercial nonlipid transfection reagent.     

Preparation of poly(glutamic acid) shielding micelles self-assembled from polylysine-b-polyphenylalanine for gene and drug codelivery

A novel amphiphilic cationic block copolymer polylysine-b-polyphenylalanine(PLL-b-PPhe) was synthesized and self-assembled into micelles in aqueous solution,then shielded with poly(glutamic acid)(marked as PG/PLL-b-PPhe) to codeliver gene and drug for combination cancer therapy.Here,doxorubicin(DOX) was selected to be loaded into PLL-b-PPhe micelles and the drug loading efficiency was 8.0%.The drug release studies revealed that the PLL-b-PPhe micelles were pH sensitive and the released DOX could reach to 53.0%,65.0%,72.0% at pH 7.4,6.8 and 5.0,respectively.In order to reduce positive charge and cytotoxicity of PLL-b-PPhe micelles,PG was used as shelding,simultaneously condensed with Bcl2 siRNA to form gene carrier system.Compared with PEI,PG/PLL-b-PPhe had excellent gene transfection efficiency,especially when the molar ratio of PLL to PPhe was 30:60 and the mixed mass ratio of PLL-b-PPhe to gene was 5:1.More importantly,DOX and Bcl2 siRNA gene codelivery system displayed remarkable cytotoxicity against B16 F10 cells.Confocal laser scanning microscopy(CLSM) and flow cytometry were used to characterize endocytosis of the codelivery system,and confirmed that both DOX and Bcl2 siRNA had been endocytosed into B16 F10 cells.The above results indicated that gene and drug codelivery was a promising strategy in future cancer therapy.     

Effects of chirality on gene delivery efficiency of polylysine

Chirality is a key factor in the biological activity of many biomolecules. Poly(L-lysine)(PLL), a polypeptide synthesized from L-lysine, is one of the mostly used cationic polymers for gene delivery. The effect of chirality of polylysine(PL) on its gene delivery remains unknown. Herein, we prepared three polylysines(PLs) with the similar molecular weight but different backbone chiralities including poly(L-lysine)(PLL), poly(D-lysine)(PDL) and poly(DL-lysine)(PDLL). The side chains of each PL were modified with propylene oxide(PO) of different chiralities including(R)PO,(S)PO and(R,S)PO. These PL-POs with distinct chirality in main and side chains could condense p DNA into polyplexes. The polyplexes had approximately the same size, zeta potential and binding ability, but showed distinct gene transfection efficiency. We found that the PLs of L-configuration in the main chain had higher transfection efficiency than that of D or DL configuration due to their faster cellular uptake, while the side chain chirality had no effect on transfection efficiency.     

Bioreducible cationic random copolymer for gene delivery

Cationic polymers have been widely investigated for gene delivery, although their low transfection efficiency and high cytotoxicity limit their application. We synthesized a bioreducible cationic random copolymer, poly(cystamine bisacylamide‐aminoethyl piperazine)‐co‐poly(cystamine bisacylamide‐histamine) (denoted as CBA‐AEP‐His) from N,N′‐cystamine bis acrylamide (CBA) with aminoethyl piperazine (AEP) and histamine (His). CBA‐AEP‐His copolymer possesses disulfide linkages that endow it with redox‐responsivity to the intracellular environment. This polymer efficiently condenses pZNF580 into complexes with the size of 160 ± 4 nm to 280 ± 5 nm and positive zeta potential of 20 ± 0.3 mV to 30 ± 0.4 mV. The gel‐retardation assay shows that CBA‐AEP‐His can retard pZNF580 even at a low mass ratio of 1/1. The gene complexes were triggered to release pZNF580 when exposed to the reducing environment of dithiothreitol (DTT). CBA‐AEP‐His random copolymer presented higher buffer capacity owing to its His moieties, which protected pZNF580 from DNase degradation. The gene transfection results reveal that CBA‐AEP‐His can efficiently deliver pZNF580 and transfect EA. Hy926 cells. The MTT assay indicates that CBA‐AEP‐His and its complexes exhibit lower cytotoxicity than PEI25KDa. These results illustrate that CBA‐AEP‐His had promising properties for gene delivery, which may provide a suitable platform for the development of a non‐viral gene carrier.     

Solubilisation of [60]fullerenes using block copolymers and evaluation of their photodynamic activities

Unmodified [60]fullerenes (C60) were solubilised with high stability using various type of poly(ethylene glycol) (PEG) based block copolymer micelles. Block copolymer micelle-incorporated C60 fullerenes were studied in cultures for biological activities using human cervical cancer HeLa cells. As a result, the cationic block copolymer micelles delivered C60 into the cells depending on their surface densities and showed cytotoxicity under photoirradiation.     

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.     

Hydrogen bonds in polycation improve the gene delivery efficiency in the serum-containing environment

Cationic polymers with high charge density could effectively condense the DNA and achieve gene transfection; however, it often brings non-negligible cytotoxicity. Notably, the high charge density gene vector fails in the serum environment, limiting further application in vivo. In this paper, an efficient and reliable non-viral gene vector of poly (amidoamine) (PAA) was designed by introducing diacryolyl-2,6-diaminopyridine (DADAP) onto the PAA backbone through Michael-addition polymerization, which provides high transfection efficiency in a serum-containing environment. Diacryolyl-2,6-diaminopyridine and cationic parts provided multiple interactions between gene vectors and DNA, including hydrogen bond and electrostatic interactions. The introduction of hydrogen bonding can effectively reduce the charge density of polyplexes without reducing the DNA condensing ability, incorporating the diaminopyridine group and cationic part in PAA chains successfully consolidated cellular uptake, endosome destabilization, and transfection efficiency for the PAA/DNA complexes with low cytotoxicity. The constructed vector with multiple interactions presented 6 times higher transfection efficiency in serum-free and 9 times in serum-containing environment than that of branched polyethyleneimine (PEI 25K) in 293T cells in vitro. Therefore, introducing the hydrogen band to form low charge density polyplexes with high transfection efficiency and low cytotoxicity has a great potential in gene delivery.     

Novel Biodegradable Block Copolymers of Poly(ethylene glycol) (PEG) and Cationic Polycarbonate: Effects of PEG Configuration on Gene Delivery

A novel amine‐functionalized polycarbonate was synthesized and its excellent gene transfection ability in vitro is demonstrated. In the framework of adapting the cationic polycarbonate for in vivo gene delivery applications, here the design and synthesis of biodegradable block copolymers of poly(ethylene glycol) (PEG) and amine‐functionalized polycarbonate with a well‐defined molecular architecture and molecular weight is achieved by metal‐free organocatalytic ring‐opening polymerization. Copolymers in triblock cationic polycarbonate‐block‐PEG‐block‐cationic polycarbonate and diblock PEG‐block‐cationic polycarbonate configurations, in comparison with a non‐PEGylated cationic polycarbonate control, are investigated for their influence on key aspects of gene delivery. Among the polymers with similar molecular weights and N content, the triblock copolymer exhibit more favorable physicochemical (i.e., DNA binding, size, zeta‐potential, and in vitro stability) and biological (i.e., cellular uptake and luciferase reporter gene expression) properties. Importantly, the various cationic polycarbonate/DNA complexes are biocompatible, inducing minimal cytotoxicities and hemolysis. These results suggest that the triblock copolymer is a more useful architecture in future cationic polymer designs for successful systemic therapeutic applications.     

Dendronised block copolymers as potential vectors for gene transfection

A series of block copolymers containing a dendronised cationic block for efficient DNA binding and a poly(ethylene glycol) block for encapsulation of the complex were synthesised in a modular fashion using a combination of click chemistry and ring-opening metathesis polymerisation. DNA binding experiments, investigated using gel electrophoresis, dynamic light scattering and transmission electron microscopy, showed that all polymers prepared in this study strongly complex DNA and self-assemble into polyion complex micelles with apparent hydrodynamic radii ranging from 20-120 nm at physiological pH (7.4). The in vitro transfection efficiency and toxicity of these potential non-viral vectors were also evaluated in HeLadouble dagger cells using plasmid DNA encoding for green fluorescent protein as the reporter gene.     

Novel cationic 6-lauroxyhexyl lysinate modified poly(lactic acid)-poly(ethylene glycol) nanoparticles enhance gene transfection

The leading principle of non-viral delivery systems for gene therapy is to mediate high levels of gene expression with low cytotoxicity. Nowadays, biodegradable nanoparticles formulated with poly(lactic acid)-poly(ethylene glycol) (PLA-PEG) were wildly developed. However, the relative lower gene transfection efficiency and higher cytotoxicity still remained critical problems. To address these limitations, PLA-PEG nanoparticles have been composited with other components in their formulation. Here, a novel cationic lipid, 6-lauroxyhexyl lysinate (LHLN), was fabricated onto PLA-PEG nanoparticles as a charge modifier to improve the transfection efficiency and cytotoxicity. The obtained cationic LHLN modified PLA-PEG nanoparticles (LHLN-PLA-PEG NPs) could condense pDNA thoroughly via electrostatic force, leading to the formation of the LHLN-PLA-PEG NPs/pDNA complexes (NPs/DNA complexes). The nanoparticles obtained have been characterized in relation to their physicochemical and biological properties, and the results are extremely promising in terms of low cell toxicity and high transfection efficiency. These results indicated that the novel cationic LHLN modified PLA-PEG nanoparticles could enhance gene transfection in vitro and hold the potential to be a promising non-viral nanodevice.     

Nonionic Block Copolypeptide Micelles Containing a Hydrophobic racemic-Leucine Core

Block copolymer micelles have been used extensively as carriers for therapeutic drugs and diagnostic molecules. Here, we report the synthesis of nonionic, block copolypeptides, K(P) (x)(rac-L)(y), which have a "reversed" rod-coil structure composed of a hydrophilic, rod-like, α-helical segment attached to a disordered, racemic hydrophobic segment. The self assembly of these block copolypeptides in water was studied, and their compositions were optimized to identify a sample, K(P) (100)(rac-L)(10), which is able to form well defined micelles that are very stable against dilution, high temperatures, and various media. Micelle structure was determined using a combination of electron microscopy and dynamic light scattering measurements. The potential of these micelles as drug delivery carriers was evaluated by encapsulation of the anticancer drug camptothecin. The drug containing micelles were found to be stable.     

聚肽接枝共聚物的自组装行为研究

Polymeric micelles of poly(γ-benzyl L-glutamate)(PBLG)-poly(ethylene oxide)(PEO) graft copolymer were prepared by the dialysis method in deionized water. Fluorescence spectroscopy, nuclear magnetic resonance(NMR) and transmission electron microscope(TEM) were used for the investigation of the self-assembly of PBLG-PEO graft copolymer. Fluorescence spectrosco0y measurements suggest that PBLG-PEO graft copolymer associates to form polymeric micelles in water. ^1H NMR measurements further prove that in aqueous medium PBLG-PEO graft copolymer could assemble into polymeric micelles with PBLG segments as the hydrophobic inner core and PEO segments as the hydrophilic shell. The results of the TEM observations show that the polymeric micelles of PBLG-PEO graft copolymer are almost spindly shaped, which are different from the morphology of the spherical micelles formed by PBLG-PEO block copolymer. Polymeric micelles formed by polypeptide copolymer have potential application as drug carrier in controlled-release delivery system.     

A reactive oxygen species-responsive dendrimer with low cytotoxicity for efficient and targeted gene delivery

Nonviral vectors have been attracting more attention for several advantages in gene delivery and the development of nonviral gene ca rriers with high delivery efficiency and low cytotoxicity has long been a key project.Starburst polyamidoamine dendrimers are a class of synthetic polymers with unique structural and physical characteristics.However,when they are used as gene carrier,the gene transfection efficiency is not satisfactory.Herein,a novel thioketal-core polyamidoamine dendrimer(i.e.,ROS...     

PEG-detachable polyplex micelles based on disulfide-linked block catiomers as bioresponsive nonviral gene vectors

PEG-based polyplex micelles, which can detach the surrounding PEG chains responsive to the intracellular reducing environment, were developed as nonviral gene vectors. A novel block catiomer, PEG-SS-P[Asp(DET)], was designed as follows: (i) insertion of biocleavable disulfide linkage between PEG and polycation segment to trigger PEG detachment and (ii) a cationic segment based on poly(aspartamide) with a flanking N-(2-aminoethyl)-2-aminoethyl group, P[Asp(DET)], in which the Asp(DET) unit acts as a buffering moiety inducing endosomal escape with minimal cytotoxicity. The polyplex micelles from PEG-SS-P[Asp(DET)] and plasmid DNA (pDNA) stably dispersed in an aqueous medium with a narrowly distributed size range of approximately 80 nm due to the formation of hydrophilic PEG palisades while undergoing aggregation by the addition of 10 mM dithiothreitol (DTT) at the stoichiometric charge ratio, indicating the PEG detachment from the micelles through the disulfide cleavage. The PEG-SS-P[Asp(DET)] micelles showed both a 1-3 orders of magnitude higher gene transfection efficiency and a more rapid onset of gene expression than PEG-P[Asp(DET)] micelles without disulfide linkages, due to much more effective endosomal escape based on the PEG detachment in endosome. These findings suggest that the PEG-SS-P[Asp(DET)] micelle may have promising potential as a nonviral gene vector exerting high transfection with regulated timing and minimal cytotoxicity.     

Novel biodegradable polymers as gene carriers

This study investigated two new biodegradable polymers as gene controlled-released coatings for gene transfer. Poly(ethylene glycol)-co-poly(D,L-lactic acid) (PELA) and poly(ethylene glycol)-co-poly(lactic acid)-co-poly(glycolic acid) random copolymer (PELGA) were synthesized and used as microspheres matrices with encapsulated plasmid pCH110. The plasmid loading efficiency, cytotoxicity, transfection efficiency and in vitro degradation and release profiles of microsphere complexes were evaluated in details. The biodegradable polymers showed high DNA loading efficiency and low cytotoxicity as gene controlled-released coatings, and the poly(ethylene glycol) (PEG) contents of polymer matrices influenced the diameter, loading efficiency and transfection efficiency of plasmid DNA within the microspheres. The average diameters of PELA and PELGA microspheres were between 0.5 and 1.5 microm, and the plasmid loading efficiency was 62 and 73% for PELA and PELGA microspheres with 10% PEG content, respectively. In vitro testing showed a gradual release profile of DNA from polymeric matrices. The polymers/DNA microspheres had high transfection efficiency and early gene expression and maintenance of gene expression level for up to 96 h, although transfection efficiency were slightly lower than that of liposome in the initial 24 h. The biodegradable polymeric materials possess potential superiority as gene carriers.     

含氟高分子基因载体

阳离子高分子被广泛应用为非病毒类基因载体,但这类高分子材料的转染效率与细胞毒性之间通常存在"恶性"关联,即获得高转染效率时往往会伴随严重的细胞毒性.如何制备兼具高效、低毒特点的高分子载体是成功实施基因治疗的关键.含氟高分子是一类具有独特理化性质的高分子,能够在低电荷密度条件下与核酸形成稳定的复合物,从而实现高效、低毒的基因转染.含氟功能基团可帮助阳离子高分子改善复合物稳定性、细胞内吞、内涵体逃逸、胞内核酸释放等多个环节,从而赋予了含氟高分子在基因递送过程中的氟效应.该专论系统地总结了含氟高分子基因载体的研究,介绍了含氟高分子的基因递送性能、作用机理以及在基因治疗、基因编辑中的应用,并对含氟高分子载体的未来发展进行了展望.     

Synthesis and Characterization of Cationic Glycidyl-Based Poly(aminoester)-Folic Acid Targeting Conjugates and Study on Gene Delivery

A new poly(aminoester) (EPAE-FA) containing folic acid and amino groups in the backbone and side chain was synthesized. EPAE-FA self-assembled readily with the plasmid DNA (pCMV-βgal) in HEPES buffer and was characterized by dynamic light scattering, zeta potential, fluorescence images, and XTT cell viability assays. To evaluate the transfection effect of graft ratio of FA on the EPAE system, EPAE-FA polymers with two different graft ratios (EPAE-FA12k and EPAE-FA14k) were also prepared. This study found that all EPAE-FA polymers were able to bind plasmid DNA and yielded positively charged complexes with nano-sized particles ( < 200 nm). To assess the transfection efficiency mediated by EPAE and EPAE-FA polymers, we performed in vitro transfection activity assays using FR-negative (COS-7) and FR-positive (HeLa) cells. The EPAE-FA12k/DNA and EPAE-FA14k/DNA complexes were able to transfect HeLa cell in vitro with higher transfection efficiency than PEI25k/DNA at the similar weight ratio. These results demonstrated that the introduction of FA into EPAE system had a significant effect on transferring ability for FR-positive cells (HeLa). Examination of the cytotoxicity of PEI25k and EPAE-FA system revealed that EPAE-FA system had lower cytotoxicity. In this paper, EPAE-FA seemed to be a novel cationic poly(aminoester) for gene delivery and an interesting candidate for further study.     

Folic Acid Modified Polymeric Micelles for Intravesical Instilled Chemotherapy

In this study,a targeting micellar drug delivery system was developed for intravesical instilled chemotherapy of bladder cancer.The amphiphilic diblock copolymer poly(ε-caprolactone)-block-poly(ethylene glycol) (PCL-b-PEO) with functional amino group (NH2) at the end of PEO block was synthesized.Then the copolymer was conjugated with folic acid (FA) and fluorescein isothiocyannate (FITC) via the PEO-NH2 terminus,and then assembled into micelles with the target moiety and fluorescence labeling.In addition,drug loaded micelles were also fabricated with anticancer drug doxorubicin (DOX) encapsulated in the hydrophobic core.The micelles were characterized in terms of size,drug loaded efficiency and critical micellization concentration (CMC) by means ofDLS,UV and fluorescence spectra.In vitro cellular uptake and cytotoxicity studies showed that FA modified PCL-b-PEO-FA micelles have a greater targeting efficiency to human bladder cancer cell (T-24 cell) compared to PCL-b-PEO-NH2 micelles due to the conjugation of FA on the surface,while no targeting effect to normal tissue originated human embryonic kidney 293 (HEK-293) cells was observed,enabling the micelles a promising drug carrier for intravesical instilled chemotherapy of bladder cancer.     

Carbon monoxide-releasing micelles for immunotherapy

With the discovery of important biological roles of carbon monoxide (CO), the use of this gas as a therapeutic agent has attracted attention. However, the medical application of this gas has been hampered by the complexity of the administration method. To overcome this problem, several transition-metal carbonyl complexes, such as Ru(CO)(3)Cl(glycinate), [Ru(CO)(3)Cl(2)](2), and Fe(η(4)-2-pyrone)(CO)(3), have been used as CO-releasing molecules both in vitro and in vivo. We sought to develop micellar forms of metal carbonyl complexes that would display slowed diffusion in tissues and thus better ability to target distal tissue drainage sites. Specifically, we aimed to develop a new CO-delivery system using a polymeric micelle having a Ru(CO)(3)Cl(amino acidate) structure as a CO-releasing segment. The CO-releasing micelles were prepared from triblock copolymers composed of a hydrophilic poly(ethylene glycol) block, a poly(ornithine acrylamide) block bearing Ru(CO)(3)Cl(ornithinate) moieties, and a hydrophobic poly(n-butylacrylamide) block. The polymers formed spherical micelles in the range of 30-40 nm in hydrodynamic diameter. Further characterization revealed the high CO-loading capacity of the micelles. CO-release studies showed that the micelles were stable in physiological buffer and serum and released CO in response to thiol-containing compounds such as cysteine. The CO release of the micelles was slower than that of Ru(CO)(3)Cl(glycinate). In addition, the CO-releasing micelles efficiently attenuated the lipopolysaccharide-induced NF-κB activation of human monocytes, while Ru(CO)(3)Cl(glycinate) did not show any beneficial effects. Moreover, cell viability assays revealed that the micelles significantly reduced the cytotoxicity of the Ru(CO)(3)Cl(amino acidate) moiety. This novel CO-delivery system based on CO-releasing micelles may be useful for therapeutic applications of CO.     

Structure and interactions of charged triblock copolymers studied by small-angle X-ray scattering: dependence on temperature and charge screening

A series of thermo-responsive cationic triblock copolymers composed of methoxy-poly(ethylene glycol) (MPEG, hydrophilic), poly(N-isopropylacrylamide) (PNIPAAM, temperature sensitive), and poly((3-acrylamidopropyl) trimethyl ammonium chloride) (PN(+), cationic) has been investigated as a function of temperature and ionic strength. In the MPEG-b-PNIPAAM-b-PN(+) copolymers, the MPEG block length is constant, and the lengths of the PNIPAAM and PN(+) blocks are varied. The solubility of the PNIPAAM block decreases with increasing temperature, and the triblock copolymer thus provides the possibilities of studying micelles with both neutral and charged blocks in the micelle corona as well as the interplay between these two blocks as the electrostatic interactions are varied by addition of salt. Investigation of the systems by densitometry and small-angle X-ray scattering (SAXS) in a temperature range from 20 to 70 °C gave detailed information on the behavior both below and above the critical micelle temperature (CMT). A clear effect of the addition of salt is observed in both the apparent partial specific volume, obtained from the densitometry measurements, and the SAXS data. Below the CMT, the single polymers can be described as Gaussian chains, for which the repulsive interchain interactions, originating from the charged PN(+) block, have to be taken into account in salt-free aqueous solution. Increasing the salt concentration of the solution to 30 mM NaCl leads to an increase in the apparent partial specific volume, and the electrostatic repulsive interchain interactions between the single polymers vanish. Raising the temperature results in micelle formation, except for the copolymer with only 20 NIPAAM units. The SAXS data show that the polymer with the medium PNIPAAM block length forms spherical micelles, whereas the polymer with the longest PNIPAAM block forms cylindrical micelles. Increasing the temperature further above the CMT results in an increase in the micellar aggregation number for both of the polymers forming spherical and cylindrical micelles. The addition of salt to the solution also influences the aggregates formed above the CMT. Overall, the micelles formed in the salt solution have a smaller cross-section radius than those in aqueous solution without added salt.