RESUMEN
Antibody drug conjugates (ADC) are an emerging class of pharmaceuticals consisting of cytotoxic agents covalently attached to an antibody designed to target a specific cancer cell surface molecule followed by internalization and intracellular release of payload to exhibit its anticancer activity. Targeted delivery of cytotoxic payload to a variety of specific cells has been demonstrated to have significant enhancement in clinical efficacy and dramatic reduction in off-target toxicity. Site-specific conjugation of payload to the antibody is highly desirable for development of ADC with well-defined antibody-to-drug ratio, enhanced internalization, reduced toxicity, improved stability, desired pharmacological profile and optimal therapeutic index. Here, we reported a site-specific conjugation strategy for evaluation of antibody internalization and efficacy of ADC designed to target SSEA4 on solid tumors. This strategy stems from the azido-fucose tag of a homogeneous antibody Fc-glycan generated via in vitro glycoengineering approach for site-specific conjugation and optimization of antibody-drug ratio to exhibit optimal efficacy. The ADC consisting of a chimeric anti-SSEA4 antibody chMC813-70, conjugated to the antineo-plastic agent monomethyl auristatin E via both cleavable and non-cleavable linkers showed excellent cytotoxicity profile towards SSEA4-bearing cancer cells. A clear distinction in cytotoxicity was observed among cancer cells with different SSEA4 expression levels.
RESUMEN
The chemoenzymatic remodeled monoclonal antidodies with well-defined glycan structure at the Fc domain display improved biological activities, such as ADCC and ADCP, and are more likely to yield a better safety profile by eliminating the non-human glycans derived from CHO cell culture. We covalently immobilize wild type endoglycosidase S (EndoS), fucosidase, and EndoS2 mutant on magnetic beads through a linker to efficiently generate homogeneous antibody glycoforms without additional purification step to remove endoglycosidase and fucosidase. We also used the biotinylated wild type EndoS2 and EndoS2 mutant in combination with covalently immobilized fucosidase on magnetic beads to allow the sequential removal of endoglycosidases and fucosidase for efficient glyco-engineering and isolation of antibodies without purifying deglycosylated antibody intermediate. Notably, the relatively expensive fucosidase can be recovered to reduce the cost, and the strong affinity of streptavidin to biotin would complete the isolation of biotinylated enzymes. We used Trastuzumab as a model to demonstrate both approaches were reliable for the large-scale production and isolation of antibodies without the residual contamination of endoglycosidase to avoid deglycosylation over storage time.
Asunto(s)
Antibacterianos/metabolismo , Desarrollo de Medicamentos , Glicósido Hidrolasas/metabolismo , Trastuzumab/metabolismo , alfa-L-Fucosidasa/metabolismo , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Biotinilación , Relación Dosis-Respuesta a Droga , Enzimas Inmovilizadas/genética , Enzimas Inmovilizadas/metabolismo , Glicósido Hidrolasas/genética , Fenómenos Magnéticos , Estructura Molecular , Mutación , Relación Estructura-Actividad , Trastuzumab/química , Trastuzumab/aislamiento & purificación , alfa-L-Fucosidasa/genéticaRESUMEN
INTRODUCTION: Several studies have identified a number of genes associated with Parkinson's disease (PD). Genomic rearrangements (exon dosage variations) in these genes have emerged as significant, causing mutations. However, exon dosage variations in several PD genes were rarely investigated in Chinese patients. OBJECTIVE: This study was aimed at determining the prevalence of PD-causing genes' exon rearrangements in Chinese sporadic early-onset PD (EOPD) patients. METHODS: A total of 150 Chinese sporadic EOPD patients and 100 healthy controls were enrolled. Multiplex ligation-dependent probe amplification (MLPA) was used to detect exon dosage in PD genes, including SNCA, PARKIN, UCHL1, PINK1, DJ1, LRRK2, and ATP13A2. Positive results were verified by real-time quantitative polymerase chain reaction. And exon sequencing was employed to screen for subtle mutations. Novel exon dosage variations were screened in families and controls. RESULTS: PARKIN exon rearrangements were detected in 10 (6.7%) patients, including a novel heterozygous duplication of PARKIN exons 1-4. Clinical investigation showed that the percentage of individuals with PARKIN exon rearrangements was higher in the younger patients. Notably, the MLPA screening detected a heterozygous deletion of UCHL1 exon 1 in a patient. MLPA analysis in the family detected the deletion in an asymptomatic sister, indicating incomplete penetrance. CONCLUSION: Exon copy number variations (CNVs) in the PARKIN gene are relatively common among Chinese sporadic EOPD patients, whereas exon CNVs in other known PD genes can also be detected. Our findings demonstrate that it is important to perform exon dosage analysis for several known PD genes to obtain a better mechanistic insight into PD pathogenesis.
Asunto(s)
Enfermedad de Parkinson/genética , Ubiquitina-Proteína Ligasas/genética , Adulto , Edad de Inicio , Pueblo Asiatico/genética , Variaciones en el Número de Copia de ADN , Exones/genética , Femenino , Dosificación de Gen , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa Multiplex , MutaciónRESUMEN
Antibodies have been developed as therapeutic agents for the treatment of cancer, infection, and inflammation. In addition to binding activity toward the target, antibodies also exhibit effector-mediated activities through the interaction of the Fc glycan and the Fc receptors on immune cells. To identify the optimal glycan structures for individual antibodies with desired activity, we have developed an effective method to modify the Fc-glycan structures to a homogeneous glycoform. In this study, it was found that the biantennary N-glycan structure with two terminal alpha-2,6-linked sialic acids is a common and optimized structure for the enhancement of antibody-dependent cell-mediated cytotoxicity, complement-dependent cytotoxicity, and antiinflammatory activities.
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Fragmentos Fc de Inmunoglobulinas/química , Inmunoglobulina G/química , Polisacáridos/química , Rituximab/química , Acetilglucosamina/química , Acetilglucosamina/inmunología , Animales , Anticuerpos Antivirales/química , Anticuerpos Antivirales/inmunología , Anticuerpos Antivirales/uso terapéutico , Citotoxicidad Celular Dependiente de Anticuerpos , Proteínas Bacterianas/metabolismo , Bacteroides fragilis/enzimología , Línea Celular Tumoral , Femenino , Células HEK293 , Humanos , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Linfoma de Células B/patología , Ratones , Ratones Endogámicos BALB C , Neuraminidasa/metabolismo , Infecciones por Orthomyxoviridae/prevención & control , Ingeniería de Proteínas , Receptores de IgG/inmunología , Rituximab/inmunología , Ácidos Siálicos/química , Ácidos Siálicos/inmunología , Streptococcus pyogenes/enzimología , Relación Estructura-Actividad , Trastuzumab/química , Trastuzumab/inmunología , alfa-L-Fucosidasa/metabolismoRESUMEN
A comprehensive structure-activity relationship study on antibody Fc-glycosylation has been performed using the chimeric anti-SSEA4 antibody chMC813-70 as a model. The α-2,6 sialylated biantennary complex type glycan was identified as the optimal Fc-glycan with significant enhancement in antibody effector functions, including binding to different Fc receptors and ADCC.
Asunto(s)
Fragmentos Fc de Inmunoglobulinas , Inmunoglobulina G , Glicosilación , Relación Estructura-Actividad , Polisacáridos/metabolismoRESUMEN
Streptococcus pyogenes is a group A streptococcus (GAS) and an important human pathogen that causes a variety of diseases. Streptococcal pyrogenic exotoxin B (SPE B) and streptolysin S (SLS) are important virulence factors involved in GAS infection, but it is not clear which one is more virulent. Using an air pouch infection model, the wild-type strain NZ131, its isogenic mutants, and complementary mutants were used to examine the effects of SPE B and SLS on GAS infection. The results of the skin lesion and mouse mortality assays showed that although SPE B and SLS had a synergistic effect on GAS infection, SPE B played a more important role in local tissue damage while SLS had a more prominent effect on mouse mortality. Surveys of the exudates from the air pouch revealed that the expression of inflammatory cytokines was significantly inhibited in the sagB/speB-double-mutant JM4-infected mice. Furthermore, in vivo and in vitro studies showed that the isogenic mutant strains were more susceptible to the immune cell killing than the wild-type strain and that the sagB/speB-double-mutant JM4 was the most sensitive among these strains. Moreover, infection with the sagB/speB-double-mutant JM4 strain caused the least amount of macrophage apoptosis compared to infection with the wild-type NZ131 and the other complementary strains, which express only SPE B or SLS or both. Taken together, these results indicate that both SPE B and SLS contributed to GAS evasion from immune cell killing, local tissue damage, and mouse mortality.
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Proteínas Bacterianas/metabolismo , Exotoxinas/metabolismo , Infecciones Estreptocócicas/mortalidad , Infecciones Estreptocócicas/patología , Streptococcus pyogenes/patogenicidad , Estreptolisinas/metabolismo , Factores de Virulencia/metabolismo , Animales , Proteínas Bacterianas/genética , Modelos Animales de Enfermedad , Sinergismo Farmacológico , Exotoxinas/genética , Humanos , Evasión Inmune , Masculino , Ratones , Ratones Endogámicos BALB C , Infecciones Estreptocócicas/inmunología , Infecciones Estreptocócicas/microbiología , Streptococcus pyogenes/genética , Estreptolisinas/genética , Virulencia , Factores de Virulencia/genéticaRESUMEN
A cDNA encoding a bifunctional acetylxylan esterase/xylanase, XynS20E, was cloned from the ruminal fungus Neocallimastix patriciarum. A putative conserved domain of carbohydrate esterase family 1 was observed at the N-terminus and a putative conserved domain of glycosyl hydrolase family 11 was detected at the C-terminus of XynS20E. To examine the enzyme activities, XynS20E was expressed in Escherichia coli as a recombinant His(6) fusion protein and purified by immobilized metal ion-affinity chromatography. Response surface modeling combined with central composite design and regression analysis was then applied to determine the optimal temperature and pH conditions of the recombinant XynS20E. The optimal conditions for the highest xylanase activity of the recombinant XynS20E were observed at a temperature of 49 degrees C and a pH of 5.8, while those for the highest carbohydrate esterase activity were observed at a temperature of 58 degrees C and a pH of 8.2. Under the optimal conditions for the enzyme activity, the xylanase and acetylxylan esterase specific activities of the recombinant XynS20E toward birchwood xylan were 128.7 and 873.1 U mg(-1), respectively. To our knowledge, this is the first report of a bifunctional xylanolytic enzyme with acetylxylan esterase and xylanase activities from rumen fungus.
Asunto(s)
Acetilesterasa/metabolismo , Clonación Molecular , Neocallimastix/enzimología , Neocallimastix/genética , Xilanos/metabolismo , Xilosidasas/metabolismo , Acetilesterasa/química , Acetilesterasa/genética , Acetilesterasa/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión , Búfalos/microbiología , Cromatografía de Afinidad , ADN Complementario , ADN de Hongos/genética , Escherichia coli/genética , Cinética , Datos de Secuencia Molecular , Neocallimastix/metabolismo , Proteínas Recombinantes de Fusión/química , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes de Fusión/metabolismo , Rumen/microbiología , Alineación de Secuencia , Especificidad por Sustrato , Temperatura , Xilosidasas/química , Xilosidasas/genética , Xilosidasas/aislamiento & purificaciónRESUMEN
The focus of our investigation was to determine the feasibility of using six visual rating scales as whole-brain imaging markers for monitoring atrophied brain volume in Parkinson's disease (PD). This was a prospective cross-sectional single-center observational study. A total of 98 PD patients were enrolled and underwent an MRI scan and a battery of neuropsychological evaluations. The brain volume was calculated using the online resource MRICloud. Brain atrophy was rated based on six visual rating scales. Correlation analysis was performed between visual rating scores and brain volume and clinical features. We found a significant negative correlation between the total scores of visual rating scores and quantitative brain volume, indicating that six visual rating scales reliably reflect whole brain atrophy in PD. Multiple linear regression-based analyses indicated severer non-motor symptoms were significantly associated with higher scores on the visual rating scales. Furthermore, we performed sample size calculations to evaluate the superiority of visual rating scales; the result show that using total scores of visual rating scales as an outcome measure, sample sizes for differentiating cognition injury require significantly fewer subjects (n = 177) compared with using total brain volume (n = 2524). Our data support the use of the total visual rating scores rather than quantitative brain volume as a biomarker for monitoring cerebral atrophy.
RESUMEN
The first systematic investigation of the effect of high mannose, hybrid, and bi- and tri-antennary complex type glycans on the effector functions of antibodies was achieved by the discovery of novel Endo-S2 mutants generated by site-directed mutagenesis as glycosynthases with broad substrate specificity.
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Anticuerpos/química , Glicosiltransferasas/química , Polisacáridos/química , Anticuerpos/metabolismo , Glicósido Hidrolasas/genética , Glicosilación , Glicosiltransferasas/genética , Mutagénesis Sitio-Dirigida , Ingeniería de Proteínas , Receptores de IgG/metabolismo , Streptococcus pyogenes/enzimología , Relación Estructura-Actividad , Especificidad por SustratoRESUMEN
Correction for 'Development of glycosynthases with broad glycan specificity for the efficient glyco-remodeling of antibodies' by Sachin S. Shivatare et al., Chem. Commun., 2018, 54, 6161-6164.
RESUMEN
A new class of broadly neutralizing antibodies (bNAbs) from HIV donors has been reported to target the glycans on gp120--a glycoprotein found on the surface of the virus envelope--thus renewing hope of developing carbohydrate-based HIV vaccines. However, the version of gp120 used in previous studies was not from human T cells and so the glycosylation pattern could be somewhat different to that found in the native system. Moreover, some antibodies recognized two different glycans simultaneously and this cannot be detected with the commonly used glycan microarrays on glass slides. Here, we have developed a glycan microarray on an aluminium-oxide-coated glass slide containing a diverse set of glycans, including homo- and mixed N-glycans (high-mannose, hybrid and complex types) that were prepared by modular chemo-enzymatic methods to detect the presence of hetero-glycan binding behaviours. This new approach allows rapid screening and identification of optimal glycans recognized by neutralizing antibodies, and could speed up the development of HIV-1 vaccines targeting cell surface glycans.
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Anticuerpos Neutralizantes/inmunología , Anticuerpos Anti-VIH/inmunología , VIH-1/inmunología , Polisacáridos/síntesis química , Vacunas contra el SIDA/inmunología , Proteína gp120 de Envoltorio del VIH/inmunología , Humanos , Ligandos , Polisacáridos/química , Polisacáridos/inmunologíaRESUMEN
Anticancer therapies are often compromised by nonspecific effects and challenged by tumour environments' inherent physicochemical and biological characteristics. Often, therapeutic effect can be increased by addressing multiple parameters simultaneously. Here we report on exploiting extravasation due to inherent vascular leakiness for the delivery of a pH-sensitive polymer carrier. Tumours' acidic microenvironment instigates a charge reversal that promotes cellular internalization where endosomes destabilize and gene delivery is achieved. We assess our carrier with an aggressive non-small cell lung carcinoma (NSCLC) in vivo model and achieve >30% transfection efficiency via systemic delivery. Rejuvenation of the p53 apoptotic pathway as well as expression of KillerRed protein for sensitization in photodynamic therapy (PDT) is accomplished. A single administration greatly suppresses tumour growth and extends median animal survival from 28 days in control subjects to 68 days. The carrier has capacity for multiple payloads for greater therapeutic response where inter-individual variability can compromise efficacy.
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Apoptosis/fisiología , Carcinoma de Pulmón de Células no Pequeñas/terapia , Técnicas de Transferencia de Gen , Proteínas Fluorescentes Verdes/metabolismo , Fotoquimioterapia/métodos , Microambiente Tumoral/fisiología , Proteína p53 Supresora de Tumor/metabolismo , Animales , Línea Celular Tumoral , Dimetilsulfóxido , Endosomas/metabolismo , Glutamatos , Humanos , Concentración de Iones de Hidrógeno , Etiquetado Corte-Fin in Situ , Indoles , Espectroscopía de Resonancia Magnética , Ratones , Ratones Endogámicos BALB CRESUMEN
The ß-L-arabinofuranosidase (HypBA1) from Bifidobacterium longum JCM 1217 hydrolyzes the ß-1,2-linked arabinofuranose disaccharide to release L-arabinoses. HypBA1 was classified into glycoside hydrolase family 127 (GH127) by the CAZy website (http://www.cazy.org/). The enzyme was expressed in Escherichia coli and the purified recombinant protein was crystallized. Crystals belonging to the primitive hexagonal space group P3x21, with unit-cell parameters a = b = 75.9, c = 254.0Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.78Å resolution. A BLASTP search (http://blast.ncbi.nlm.nih.gov/) of the Protein Data Bank did not reveal any similar crystal structures. Structural determination by using SeMet MAD and MIR methods is in progress.
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Bifidobacterium/enzimología , Glicósido Hidrolasas/química , Glicósido Hidrolasas/aislamiento & purificación , Cristalización , Difracción de Rayos XRESUMEN
A small interfering RNA (siRNA)-loaded polyelectrolyte constructed with branched polyethylenimine (bPEI) and copolymers, consisting of polyethylene glycol (PEG), histidine (His), and glutamic acid (Glu), was developed in order to provide a tumor acidosis-triggered delivery system with low cytotoxicity.
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Antineoplásicos/farmacología , Sistemas de Liberación de Medicamentos , Ácido Glutámico/química , Histidina/química , Polietilenglicoles/farmacología , Polietileneimina/farmacología , ARN Interferente Pequeño/farmacología , Antineoplásicos/química , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Electrólitos/química , Electrólitos/farmacología , Humanos , Concentración de Iones de Hidrógeno , Modelos Moleculares , Estructura Molecular , Polietilenglicoles/química , Polietileneimina/química , ARN Interferente Pequeño/química , Relación Estructura-ActividadRESUMEN
Loading of viral vectors in synthetic polymers is a promising strategy for overcoming hurdles associated with viral gene delivery. For enhanced gene expression at a specific site, gene transfer by using hydrogels represents a versatile approach. In this study, adeno-associated virus serotype 2 containing the green fluorescent protein gene (rAAV2-GFP) were loaded into poly(ethylene glycol) (PEG) hydrogels, with and without incorporation of poly-L-hisditine (polyHis). Inclusion of polyHis created pH responsive hydrogels in a physiological range of tissues, containing the damaged vasculature and activated phagocytosis. The fraction of polyHis used controlled the degree of swelling, water uptake and subsequent degradation of the hydrogels and release rate of rAAV2-GFP. The swelling ratio of the PEG-polyHis hydrogels increased inversely with environment pH. As pH declined from 7.4 to 6.0, PEG-polyHis hydrogel swelling ratio and degradation rate increased 875% and 135%, respectively. As a result, release and transduction efficiency of the rAAV2-GFP from PEG-polyHis hydrogel in human HT-1080 fibrosarcoma cells increased significantly compared to a PEG hydrogel. Transduction rate can be controlled by the hydrogels' polyHis concentration and is sensitive to localized decreases in pH consistent with inflammation. This is relevant to optimizing parameters for wound care and regenerative medicine applications.
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Dependovirus/química , Dependovirus/metabolismo , Histidina/química , Hidrogeles/química , Polietilenglicoles/química , Polímeros/química , Línea Celular Tumoral , Humanos , Concentración de Iones de HidrógenoRESUMEN
Alkaline phytases from Bacillus species, which hydrolyze phytate to less phosphorylated myo-inositols and inorganic phosphate, have great potential as additives to animal feed. The thermostability and neutral optimum pH of Bacillus phytase are attributed largely to the presence of calcium ions. Nonetheless, no report has demonstrated directly how the metal ions coordinate phytase and its substrate to facilitate the catalytic reaction. In this study, the interactions between a phytate analog (myo-inositol hexasulfate) and divalent metal ions in Bacillus subtilis phytase were revealed by the crystal structure at 1.25 Å resolution. We found all, except the first, sulfates on the substrate analog have direct or indirect interactions with amino acid residues in the enzyme active site. The structures also unraveled two active site-associated metal ions that were not explored in earlier studies. Significantly, one metal ion could be crucial to substrate binding. In addition, binding of the fourth sulfate of the substrate analog to the active site appears to be stronger than that of the others. These results indicate that alkaline phytase starts by cleaving the fourth phosphate, instead of the third or the sixth that were proposed earlier. Our high-resolution, structural representation of Bacillus phytase in complex with a substrate analog and divalent metal ions provides new insight into the catalytic mechanism of alkaline phytases in general.
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6-Fitasa/química , 6-Fitasa/metabolismo , Bacillus subtilis/enzimología , Cationes Bivalentes/metabolismo , Inositol/análogos & derivados , 6-Fitasa/genética , Catálisis , Cristalografía por Rayos X , Inositol/metabolismo , Modelos Moleculares , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Especificidad por SustratoRESUMEN
An insecticidal toxin gene, tccC, was cloned from the recently discovered novel species Pseudomonas taiwanensis using degenerate PCR and genomic walking. The DNA sequence of the tccC gene (2,940 bp) has an open reading frame encoding 980 amino acids with a calculated molecular weight of 107.93 kDa. The amino acid sequence alignment showed the highest sequence identity (41.2%) with the insecticidal toxin from Pseudomonas entomophila. To examine the insecticidal functionality of the tccC gene product, TccC was heterologously expressed in Escherichia coli as a recombinant His6 fusion protein and purified by immobilized metal ion-affinity chromatography. The recombinant TccC was fed to Drosophila larvae at a concentration of 350 ppm, which induced about 60% mortality within 72 h. The recombinant TccC was stable at pH 7.0 and at 37 °C. When the pH was less than 5.0 or greater than 9.0, or temperature was greater than 55 °C, less than 20% Drosophila larvae mortality was observed. These results prove that Pseudomonas taiwanensis could be used as a source for developing novel biopesticides.
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Toxinas Bacterianas/genética , Toxinas Bacterianas/farmacología , Clonación Molecular , Insecticidas/farmacología , Pseudomonas/metabolismo , Secuencia de Aminoácidos , Animales , Toxinas Bacterianas/química , Toxinas Bacterianas/metabolismo , Secuencia de Bases , Drosophila/efectos de los fármacos , Drosophila/fisiología , Insecticidas/química , Insecticidas/metabolismo , Larva/efectos de los fármacos , Larva/fisiología , Datos de Secuencia Molecular , Filogenia , Pseudomonas/química , Pseudomonas/clasificación , Pseudomonas/genéticaRESUMEN
Inefficient release of polymer/DNA complexes from endocytic vesicles into the cytoplasm and the cytotoxic nature of cationic polymers are two of the primary causes of poor gene delivery. EG-polyurethane [poly(ethylene glycol)-PU, Poly 1], EGDM-polyurethane [poly(ethylene glycol), 2-(dimethylamino)ethylamine-PU, Poly 2], and MDEADM-polyurethane [N-methyldiethanolamine, 2-(dimethylamino)ethylamine-PU, Poly 3] were designed in this study to overcome these obstacles. The structural characteristics of polyurethanes and physicochemical properties of their formed complexes with DNA were determined to correlate their transfection efficiency. The results revealed that Poly 2 and Poly 3 could bind with plasmid DNA and yield positively charged complexes with a size required for transfection. Poly 3 showed the best in buffering capacity and its formed complexes with DNA could transfect COS-7 cells better than those of Poly 2 and Poly 1. This study reveals that the amine groups in the polymeric structure and the buffer capacity of a polymeric transfectant would affect its potential in DNA delivery. Also the size and binding properties of DNA and polymeric transfectants can be in correlation to the transfection efficiency of resulting DNA/polymer complexes.