HeLa Cell Membranes Research Articles

Exploration of ZnO decorated g-C3N4 amphiphilic anticancer drugs for antiproliferative activity against human cervical cancer

Chemotherapeutic treatment of patients with cervical cancer cells often leads to severe side effects such as high drug toxicity to immune cells, poor penetration of chemotherapeutics across the blood, and lack of cancer selectivity. As a result, it is imperative to develop novel cancer treatment drugs that are free of side effects and toxicity. Hydrophobic-hydrophilic combinational anticancer medicines for chemotherapy have been recently introduced because they minimize multidrug resistance and side effects as a result of reduced dosage of each component in a drug and solubility in water increases. In this work, hydrophobic g-C3N4 (GCN) nanosheets decorated with hydrophilic ZnO (ZN) nanoparticles were fabricated via a two-step self-assembly process. The synthesized ZN@GCN nanocomposites were subsequently characterized by Powder X-ray diffraction analysis, Fourier Transform Infrared Spectroscopy (FTIR), High-resolution Transmission electron microscopy (HRTEM), selected area electron diffraction (SAED), Energy-Dispersive X-ray Analysis (EDAX), and Dynamic Light Scattering (DLS) for a crystal structure, functional group, surface morphology, elemental composition, particle size, polydispersity index, and zeta potential analysis. Furthermore, the antiproliferative effects of synthesized ZN@GCN nanocomposites in a human cervical cancer cell line (HeLa) were tested by MTT assay. The experiments revealed dose and time-dependent considerable cytotoxicity activity of the nanocomposites in the growing cells, with an IC50 value of 77.80 ± 2.75 μg/ml after 24 h incubation time. The apoptotic impact of ZN@GCN nanocomposite is achieved by the generation of free radicals; which potentially involves the disruption of mitochondrial membrane, cell damage, and DNA damage in HeLa cells. The amphiphilic ZN@GCN nanocomposite could be a promising anticancer drug, according to the observed properties and results obtained in our in vitro assay testing. Further research at the molecular level could point to its clinical promise in nanomedicine for the treatment of cervical cancer.

The AJ072 antibody against the human transferrin receptor labels HeLa cells by surface immunofluorescence

The AJ072 antibody against the human transferrin receptor labels the cell membrane of HeLa cells by surface immunofluorescence; AM236 does not.

Alkaline Phosphatase Enabled Fluorogenic Reaction and in situ Coassembly of Near-Infrared and Radioactive Nanoparticles for in vivo Imaging.

Smart near-infrared (NIR) fluorescence (FL) and positron emission tomography (PET) bimodal probes have shown promise for preoperative and intraoperative imaging of tumors. In this paper, we report an enzyme-activatable probe (P-CyFF-68Ga) and its cold probe (P-CyFF-Ga) using an enzyme-induced fluorogenic reaction and in situ coassembly strategy and demonstrate the utility for NIR FL/PET bimodality imaging of enzymatic activity. P-CyFF-68Ga and P-CyFF-Ga can be converted into dephosphorylated CyFF-68Ga and CyFF-Ga in response to alkaline phosphatase (ALP) and subsequently coassemble into fluorescent and radioactive nanoparticles (NP-68Ga). The ALP-triggered in situ formed NP-68Ga is prone to anchoring on the ALP-positive HeLa cell membrane, permitting the concurrent enrichment of NIR FL and radioactivity. The enhancements in NIR FL and radioactivity enables high sensitivity and deep-tissue imaging of ALP activity, consequently facilitating the delineation of HeLa tumor foci from the normal tissues in vivo.

Quantitative FRET Microscopy Reveals a Crucial Role of Cytoskeleton in Promoting PI(4,5)P2 Confinement.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an essential plasma membrane component involved in several cellular functions, including membrane trafficking and cytoskeleton organization. This function multiplicity is partially achieved through a dynamic spatiotemporal organization of PI(4,5)P2 within the membrane. Here, we use a Förster resonance energy transfer (FRET) approach to quantitatively assess the extent of PI(4,5)P2 confinement within the plasma membrane. This methodology relies on the rigorous evaluation of the dependence of absolute FRET efficiencies between pleckstrin homology domains (PHPLCδ) fused with fluorescent proteins and their average fluorescence intensity at the membrane. PI(4,5)P2 is found to be significantly compartmentalized at the plasma membrane of HeLa cells, and these clusters are not cholesterol-dependent, suggesting that membrane rafts are not involved in the formation of these nanodomains. On the other hand, upon inhibition of actin polymerization, compartmentalization of PI(4,5)P2 is almost entirely eliminated, showing that the cytoskeleton network is the critical component responsible for the formation of nanoscale PI(4,5)P2 domains in HeLa cells.

Engineering and development of model lipid membranes mimicking the HeLa cell membrane

Cells are complex systems whose interaction with nanocarriers, i.e., liposomes, are continuously under investigation to improve drug uptake. Model membranes can facilitate the understanding of the processes involved in fusion or endocytosis. In this work, we engineered two different lipid model membranes, vesicles and supported lipid bilayers (SLBs), mimicking the lipid composition of the HeLa cell plasma membrane. We characterized the model using atomic force microscopy (AFM) and fluorescence. We found that liposomes formed with four lipid components mimicking the HeLa cell bilayer show a liquid ordered fluid nature between 13 °C and 34 °C and yield featureless SLBs onto mica. We evaluated the fusion between the model and liposomes positively charged with and without cholesterol by AFM-based force spectroscopy and fluorescence techniques, such as Förster resonance energy transfer, fluorescence lifetime decay and fluorescence anisotropy. The results indicated a primary electrostatic interaction between the HeLa bilayer model and the liposomes. It was also confirmed the well-known fact that cholesterol enhances the fusion process with the engineered HeLa bilayer. All results support the usefulness of the engineered model in the rationale design of liposomes for drug delivery.

Variants of human CLDN9 cause mild to profound hearing loss

Hereditary deafness is clinically and genetically heterogeneous. We investigated deafness segregating as a recessive trait in two families. Audiological examinations revealed an asymmetric mild to profound hearing loss with childhood or adolescent onset. Exome sequencing of probands identified a homozygous c.475G>A;p.(Glu159Lys) variant of CLDN9 (NM_020982.4) in one family and a homozygous c.370_372dupATC;p.(Ile124dup) CLDN9 variant in an affected individual of a second family. Claudin 9 (CLDN9) is an integral membrane protein and constituent of epithelial bicellular tight junctions (TJs) that form semipermeable, paracellular barriers between inner ear perilymphatic and endolymphatic compartments. Computational structural modeling predicts that substitution of a lysine for glutamic acid p.(Glu159Lys) alters one of two cis-interactions between CLDN9 protomers. The p.(Ile124dup) variant is predicted to locally misfold CLDN9 and mCherry tagged p.(Ile124dup) CLDN9 is not targeted to the HeLa cell membrane. In situ hybridization shows that mouse Cldn9 expression increases from embryonic to postnatal development and persists in adult inner ears coinciding with prominent CLDN9 immunoreactivity in TJs of epithelia outlining the scala media. Together with the Cldn9 deaf mouse and a homozygous frameshift of CLDN9 previously associated with deafness, the two bi-allelic variants of CLDN9 described here point to CLDN9 as a bona fide human deafness gene.

Preparation of carbon dots-based nanoparticles and their research of bioimaging and targeted antitumor therapy.

Carbon dots (CDs) are nanomaterials with excellent photoluminescence property, usually used in the field of bioimaging tumor cells. However, its practical applicability in cancer therapeutics is limited by CDs' insensitive surface properties to complicated tumor microenvironment in vivo. Herein, a new type of innovative biomimetic nanoparticles has been formed with HeLa cell membranes (CM) and multifunctional CDs containing antitumor and bioimaging activities. The CDs are prepared by a facile one-step microwave-assisted procedure. Gallic acid is used as carbon resource and antitumor active molecule. Gelatin is treated as the nitrogen resource. Citric acid monohydrate is used as the auxiliary carbon source and the Hela CM is used for tumor targeting. A series of fluorescence analyses has proved its homotypic targeting and ability of diagnosis. Besides, in vitro and in vivo antitumor experiments further indicate their better antitumor efficiency. The findings show the totally new nanoparticles' feasibilities of dealing with the clinical therapy problems as well as applying for the integration of diagnosis and targeting therapy.

Human Cancer Cell Membrane-Cloaked Fe3O4 Nanocubes for Homologous Targeting Improvement.

Surface modification of nanoparticles with cellular protein components is a new biomimetic modification strategy, which utilizes the inherent affinity between homologous cells to introduce the same surface molecules into nanoparticles to improve the targeting performance. In this study, oleic acid (OA)-coated Fe3O4 nanocubes were prepared by a high-temperature thermal decomposition method and modified by 3, 4-dihydroxyphenylpropionic acid (DHCA); then, HeLa cell membranes were introduced onto the surface of the nanocubes through mixed coextrusion to try to endow them with the targeting function of natural cells. The results show that the prepared Fe3O4 nanocubes have high monodispersity, excellent water solubility, and biocompatibility. Moreover, the Fe3O4 nanocubes encapsulated by cellular protein show an obvious core-shell structure and the specific targeting property to HeLa cells is improved significantly, which is expected to be used in clinical targeted diagnosis and treatment of cancer.

Volumetric Semantic Instance Segmentation of the Plasma Membrane of HeLa Cells.

In this work, an unsupervised volumetric semantic instance segmentation of the plasma membrane of HeLa cells as observed with serial block face scanning electron microscopy is described. The resin background of the images was segmented at different slices of a 3D stack of 518 slices with 8192 × 8192 pixels each. The background was used to create a distance map, which helped identify and rank the cells by their size at each slice. The centroids of the cells detected at different slices were linked to identify them as a single cell that spanned a number of slices. A subset of these cells, i.e., the largest ones and those not close to the edges were selected for further processing. The selected cells were then automatically cropped to smaller regions of interest of 2000 × 2000 × 300 voxels that were treated as cell instances. Then, for each of these volumes, the nucleus was segmented, and the cell was separated from any neighbouring cells through a series of traditional image processing steps that followed the plasma membrane. The segmentation process was repeated for all the regions of interest previously selected. For one cell for which the ground truth was available, the algorithm provided excellent results in Accuracy (AC) and the Jaccard similarity Index (JI): nucleus: JI , AC , cell including nucleus JI , AC , cell excluding nucleus JI , AC . A limitation of the algorithm for the plasma membrane segmentation was the presence of background. In samples with tightly packed cells, this may not be available. When tested for these conditions, the segmentation of the nuclear envelope was still possible. All the code and data were released openly through GitHub, Zenodo and EMPIAR.

Accurate construction of cell membrane biomimetic graphene nanodecoys via purposeful surface engineering to improve screening efficiency of active components of traditional Chinese medicine

Biomimetic nanoengineering presents great potential in biomedical research by integrating cell membrane (CM) with functional nanoparticles. However, preparation of CM biomimetic nanomaterials for custom applications that can avoid the aggregation of nanocarriers while maintaining the biological activity of CM remains a challenge. Herein, a high-performance CM biomimetic graphene nanodecoy was fabricated via purposeful surface engineering, where polyethylene glycol (PEG) was used to modifying magnetic graphene oxide (MGO) to improve its stability in physiological solution, so as to improve the screening efficiency to active components of traditional Chinese medicine (TCM). With this strategy, the constructed PEGylated MGO (PMGO) could keep stable at least 10 days, thus improving the CM coating efficiency. Meanwhile, by taking advantage of the inherent ability of HeLa cell membrane (HM) to interact with specific ligands, HM-camouflaged PMGO showed satisfied adsorption capacity (116.2 mg/g) and selectivity. Finally, three potential active components, byakangelicol, imperatorin, and isoimperatorin, were screened from Angelica dahurica, whose potential antiproliferative activity were further validated by pharmacological studies. These results demonstrated that the purposeful surface engineering is a promising strategy for the design of efficient CM biomimetic nanomaterials, which will promote the development of active components screening in TCM.

Disordered Nanocomposite Islands for Nanospeckle Illumination Microscopy in Wide‐Field Super‐Resolution Imaging

AbstractNanospeckle illumination microscopy (NanoSIM) based on disordered metallic nanocomposite island substrates is described. The nanoisland substrates can be fabricated without lithographic techniques. Azimuthal scanning illumination of nanoislands creates near‐field distribution that excites an arbitrary number of basis images to produce super‐resolution. Experimental studies of point‐spread images using NanoSIM with 360 basis images obtained by azimuthal scanning find spatial resolution improved by more than three times with an achievable full‐width‐at‐half‐maximum at 81.1 nm on average. The approach is confirmed by imaging aggregated nanobeads and nanoscale distribution of gangliosides on the HeLa cell membrane.

Curcumin loaded iron functionalized biopolymeric nanofibre reinforced edible nanocoatings for improved shelf life of cut pineapples

The current research investigates the development of curcumin (Cur) loaded chitosan (CS)/iron functionalized cellulose nanofibers (mgCNF) based edible coating for perishable cut fruit, which is a non-toxic eatable packaging with improved anti-cancer, physicochemical, thermal, and food properties. The modification in cellulose nanofibres (CNF) through fabricating mgCNF improved the surface properties of Cur loaded CS materials (suggested by FESEM). Further, the weight percentage of various elements such as carbon, oxygen, iron, and nitrogen was found to be as 54.4, 38.4, 5.2, and 1.9 % (w/w), respectively, in the developed Cur loaded CS/mgCNF (1.5 % (w/w)) based materials (obtained by EDX analysis). Interestingly, the work provides value addition to the perishable cut pineapple fruit in form of eatable packaging materials with the iron content of ∼3.6 ppm, which is within permissible limit according to WHO guidelines. It is worth mentioning that the presence of Cur with CS based biocomposites exhibited anti-cancer activity by disrupting the cell membrane of HeLa (cervical cancer) cells with cell viability of ∼17 % and ∼98 % for CS/mgCNF (1.5 % (w/w)) with and without Cur, respectively. The thermal properties in terms of onset degradation temperatures (10 % weight reduction) for Cur loaded CS, CS/CNF (1 % (w/w)) and CS/mgCNF (1 % (w/w)) were 78.6, 90.8, and 82.3 °C, respectively, which provides an additional approach to use these materials for heat unstable food items. Therefore, the present investigation provides a novel approach towards supplementing the iron functionalized food products with anti-cancer activity and improved performance to reduce food waste.

Identification of Novel FNIN2 and FNIN3 Fibronectin-Derived Peptides That Promote Cell Adhesion, Proliferation and Differentiation in Primary Cells and Stem Cells.

In recent years, a major rise in the demand for biotherapeutic drugs has centered on enhancing the quality and efficacy of cell culture and developing new cell culture techniques. Here, we report fibronectin (FN) derived, novel peptides fibronectin-based intergrin binding peptide (FNIN)2 (18-mer) and FNIN3 (20-mer) which promote cell adhesion proliferation, and the differentiation of primary cells and stem cells. FNIN2 and 3 were designed based on the in silico interaction studies between FN and its receptors (integrin α5β1, αvβ3, and αIIbβ3). Analysis of the proliferation of seventeen-cell types showed that the effects of FNINs depend on their concentration and the existence of expressed integrins. Significant rhodamine-labeled FNIN2 fluorescence on the membranes of HeLa, HepG2, A498, and Du145 cells confirmed physical binding. Double coating with FNIN2 or 3 after polymerized dopamine (pDa) or polymerized tannic acid (pTA) precoating increased HBEpIC cell proliferation by 30–40 percent, suggesting FNINs potently affect primary cells. Furthermore, the proliferation of C2C12 myoblasts and human mesenchymal stem cells (MSCs) treated with FNINs was significantly increased in 2D/3D culture. FNINs also promoted MSC differentiation into osteoblasts. The results of this study offer a new approach to the production of core materials (e.g., cell culture medium components, scaffolds) for cell culture.

Nuclear Stiffness Decreases with Disruption of the Extracellular Matrix in Living Tissues.

Reciprocal interactions between the cell nucleus and the extracellular matrix lead to macroscale tissue phenotype changes. However, little is known about how the extracellular matrix environment affects gene expression and cellular phenotype in the native tissue environment. Here, it is hypothesized that enzymatic disruption of the tissue matrix results in a softer tissue, affecting the stiffness of embedded cell and nuclear structures. The aim is to directly measure nuclear mechanics without perturbing the native tissue structure to better understand nuclear interplay with the cell and tissue microenvironments. To accomplish this, an atomic force microscopy needle-tip probe technique that probes nuclear stiffness in cultured cells to measure the nuclear envelope and cell membrane stiffness within native tissue is expanded. This technique is validated by imaging needle penetration and subsequent repair of the plasma and nuclear membranes of HeLa cells stably expressing the membrane repair protein CHMP4B-GFP. In the native tissue environment ex vivo, it is found that while enzymatic degradation of viable cartilage tissues with collagenase 3 (MMP-13) and aggrecanase-1 (ADAMTS-4) decreased tissue matrix stiffness, cell and nuclear membrane stiffness is also decreased. Finally, the capability for cell and nucleus elastography using the AFM needle-tip technique is demonstrated. These results demonstrate disruption of the native tissue environment that propagates to the plasma membrane and interior nuclear envelope structures of viable cells.

Effects of PCB70 and PCB75 on HeLa cell proliferation, membrane integrity and cell signaling pathway

Polychlorinated biphenyls (PCBs) are persistent pollutants and pose toxic effects to humans and environment. In the current study, we focused to assess the effects of PCBs congeners (PCB70 and PCB75) upon HeLa cells using different concentrations (10-3, 10-2, 1, 10 and 15 µg/mL) and time (12, 24 and 48 h). The 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay revealed that PCB70 and PCB75 induces cell proliferation at low doses (10-3, 10-2 and 1 µg/g) and inhibit the proliferation at high doses (10 and 15 µg/g) with different time suggesting cytotoxicity. The HeLa cells were analyzed for quantification of membrane integrity and it is found that PCB70 did not cause deformity in membrane integrity. However, PCB75 showed to cause deformity in membrane integrity in dose and time dependent manners. The results from western blot assay demonstrated that PCB70 and PCB75 exposure induced the expression of extracellular signal regulated-kinases (ERK1/2) and Jun N-terminal kinases (JNK) in dose- and time-dependent manners in the HeLa cells. The findings might provide mechanistic insight of PCB congeners induced cytotoxicity to HeLa cells.

Lipid membranes exposed to dispersions of phytantriol and monoolein cubosomes: Langmuir monolayer and HeLa cell membrane studies

The interactions of liquid-crystalline nanoparticles based on lipid-like surfactants, glyceryl monooleate, monoolein (GMO) and 1,2,3-trihydroxy-3,7,11,15-tetramethylhexadecane, phytantriol (PT) with selected model lipid membranes prepared by Langmuir technique were compared. Monolayers of DPPC, DMPS and their mixture DPPC:DMPS 87:13 mol% were used as simple models of one leaflet of a cell membrane. The incorporation of cubosomes into the lipid layers spread at the air-water interface was followed by surface-pressure measurements and Brewster angle microscopy. The cubosome - membrane interactions lead to the fluidization of the model membranes but this effect depended on the composition of the model membrane and on the type of cubosomes. The interactions of PT cubosomes with lipid layers, especially DMPS-based monolayer were stronger compared with those of GMO-based nanoparticles. The kinetics of incorporation of cubosomal material into the lipid layer was influenced by the extent of hydration of the polar headgroups of the lipid: faster in the case of smaller, less hydrated polar groups of DMPS than for strongly hydrated uncharged choline of DPPC. The membrane disrupting effect of cubosomes increased at longer times of the lipid membrane exposure to the cubosome solution and at larger carrier concentrations. Langmuir monolayer observations correspond well to results of studies of HeLa cells exposed to cubosomes. The larger toxicity of PT cubosomes was confirmed by MTS. Their ability to disrupt lipid membranes was imaged by confocal microscopy. On the other hand, PT cubosomes easily penetrated cellular membranes and released cargo into various cellular compartments more effectively than GMO-based nanocarriers. Therefore, at low concentrations, they may be further investigated as a promising drug delivery tool.

Characterization of monolayers and liposomes that mimic lipid composition of HeLa cells

In this work, based on several studies, we develop an artificial lipid membrane to mimic the HeLa cell membrane using 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (POPE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-l-serine (POPS) and cholesterol (CHOL). This is then a means to further study the fusion process of specific engineered liposomes. To characterize the mimicked HeLa cell membrane, we determined a series of surface pressure–area (π-A) isotherms and the isothermal compression modulus was calculated together with the dipole moment normal to the plane of the monolayer. The existence of laterally segregated domains was assessed using a fluorescence technique (Laurdan) and two microscopy techniques: Brewster angle microscopy (BAM) and atomic force microscopy (AFM) of Langmuir-Blodgett films (LBs) extracted at 30 mN m−1. To examine the nature and composition of the observed domains, force spectroscopy (FS) based on AFM was applied to the LBs. Finally, two engineered liposome formulations were tested in a fusion assay against mimicked HeLa cell membrane LBs, showing good results and thereby opening the door to further assays and uses.

Metamaterial-Assisted Photobleaching Microscopy with Nanometer Scale Axial Resolution.

The past two decades have witnessed a dramatic progress in the development of novel super-resolution fluorescence microscopy technologies. Here, we report a new fluorescence imaging method, called metamaterial-assisted photobleaching microscopy (MAPM), which possesses a nanometer-scale axial resolution and is suitable for broadband operation across the entire visible spectrum. The photobleaching kinetics of fluorophores can be greatly modified via a separation-dependent energy transfer process to a nearby metamaterial. The corresponding photobleaching rate is thus linked to the distance between the fluorophores and the metamaterial layer, leading to a reconstructed image with exceptionally high axial resolution. We apply the MAPM technology to image the HeLa cell membranes tagged with fluorescent proteins and demonstrate an axial resolution of ∼2.4 nm with multiple colors. MAPM utilizes a metamaterial-coated substrate to achieve super-resolution without altering anything else in a conventional microscope, representing a simple solution for fluorescence imaging at nanometer axial resolution.

Lattice complex assembled by noncompetitive anti-EGFR antibodies regulates actin cytoskeletal reorganization

BackgroundRecent evidence of clinical trials highlights that the combination of two noncompetitive anti-EGFR antibodies can benefit patients with several cancers. Previous studies propose that a lattice complex assembled by antibodies and EGFR down-regulates surface EGFR by rapid internalization of the complex. However, there remains a paucity of evidence and understanding on the existence of a lattice complex on cell surface and its cellular processes of internalization.MethodsHerein, we used three dimensions structured illumination microscopy to directly observe the actual morphology of the lattice complex formed on Hela cell membrane after noncompetitive anti-EGFR antibody combinations, and we explored the internalized mechanism of noncompetitive antibody combinations by constructing a PIP2 consumption system.ResultWe observed the lattice complex (length > 1 μm) on the surface of living cell after preincubation with Cetuximab and H11, but combination of Cetuximab and single domain antibody 7D12 fails to assemble the lattice, these results demonstrates the importance of symmetrical structure of conventional antibody for lattice formation. Interestingly, the lattice complex assembles along with cytoskeletal fibers, and its internalization recruits a large amount of PIP2 and triggers the rearrangement of F-actin.ConclusionsThe above data suggests that large-size lattice complex affects membrane fluidity and dynamic reorganization of cytoskeletal, which may be responsible for its rapid internalization. These new insight will aid in current rational combination design of anti-EGFR antibodies.

Selective Control of Cell Activity with Hydrophilic Polymer-Covered Cationic Nanoparticles.

Cationic polymers exhibit high cytotoxicity via strong interaction with cell membranes. To reduce cell membrane damage, a hydrophilic polymer is introduced to the cationic nanoparticle surface. The hydrophilic polymer coating of cationic nanoparticles resulted in a nearly neutral nanoparticle. These particles are applied to mouse fibroblast (3T3) and human cervical adenocarcinoma (Hela) cells. Interestingly, nanoparticles with a long cationic segment decrease cell activity regardless of cell type, while those with a short segment only affect 3T3 cell activity at lower concentrations less than 500 µg mL-1 . Most nanoparticles are located inside 3T3 cells but on the cell membrane of Hela cells. The short cationic nanoparticle shows negligible cell membrane damage despite its high accumulation on Hela cell membranes. Cell activity changed by hydrophilic polymer-coated cationic nanoparticles is caused by incorporated nanoparticle accumulation in the cells, not cell membrane damage. To suppress the cytotoxicity from the cationic polymer, cationic nanoparticle needs to completely cover with hydrophilic polymer so as not to exhibit the cationic effect and applies to cell with low concentrations to reduce the nonselective cytotoxicity from the cationic polymer.