Balb/c 마우스에서 Keyhole limpet hemocyanine (KLH)의 항원성에 대한 PAMAM dendrimer 의 면역증강 효과

이가영(Ga-Young Lee),김민지(Min Jee Kim),김소연(So Yeon Kim),이경복(Kyung Bok Lee),오동현(Dong Hyun Oh),조영호(Young Ho Cho),유영춘(Yung Choon Yoo) 한국생명과학회 2020 생명과학회지 Vol.30 No.10

본 연구에서는 Keyhole limpet hemocyanin (KLH)에 대한 체액성 및 세포성 면역반응 유도에 대한 PAMAM dendrimer G4 (PAMAM)의 증강 효과를 조사하였다. PAMAM을 KLH와 2주 간격으로 2회 피하주사로 면역한 후, KLH에 대한 특이항체를 측정한 결과, KLH+PAMAM 면역 그룹은 KLH만을 단독으로 면역한 그룹에 약 30배 이상 높은 유의한 항체가(IgG+IgA+IgM) 상승을 나타냈다. ELISA 분석에 의해 KLH 특이적인 면역글로부린의 isotype을 측정한 결과, PAMAM를 혼합하여 면역함으로서 IgG1, IgG2a, IgG2b, IgG3 및 IgM 항체의 역가가 유의하게 증가하는 것으로 확인되었다. 또한 면역 개시 7주째에 면역동물에 KLH 항원을 피하주사하고 swelling reaction을 통해 세포성 면역반응인 지연형 과민반응(DTH)을 측정한 결과, KLH+PAMAM으로 면역한 그룹에서 KLH만을 단독으로 면역한 그룹에 비해 높은 DTH 유도활성이 관찰되었다. 한편 면역동물의 비장세포를 취하여 in vitro에서 KLH로 재자극한 후 림프구 증식반응과 사이토카인 유도활성을 측정한 결과, PAMAM을 혼합하여 면역한 그룹에서 KLH 단독 면역 그룹에 비해 림프구의 증식반응에 유의하게 증가하였으며, Th1 type (IFN-γ)과 Th2 type (IL-4) 사이토카인의 생성도 모두 상승하는 것으로 확인되었다. 이상의 결과로부터 PAMAM dendrimer는 함께 투여된 항원물질에 의해 유도되는 세포성 면역과 체액성 면역을 상승시키는 활성이 있는 것으로 확인되었으며, 이는 PAMAM dendrimer가 면역 adjuvant로서 응용 가능한 소재임을 입증하는 것이다. The adjuvant effect of PAMAM dendrimer G4 (PAMAM) on the induction of humoral and cellular immune responses against keyhole limpet hemocyanin (KLH) was examined. Mice were immunized subcutaneously twice at two-week intervals with KLH, with or without PAMAM dendrimer (100 μg/mouse), and the mice immunized with KLH+PAMAM showed significantly higher antibody titers against KLH than those immunized with KLH alone. The assay for determining the isotypes of the antibodies showed that PAMAM augmented the KLH-specific antibody titers of IgG1, IgG2a, IgG2b, IgG3, and IgM. In addition, mice immunized twice with KLH+PAMAM followed by a subcutaneous injection of KLH (20 μg/site) 7 weeks after the primary immunization exhibited a higher delayed-type hypersensitivity (DTH) reaction than those treated with KLH alone. In an in vitro analysis of T lymphocyte proliferation in response to KLH in week 8, the splenocytes of mice treated with KLH+ PAMAM showed significantly higher proliferating activity than those treated with KLH alone, and the culture supernatants of cell cultures from mice immunized with added PAMAM dendrimer showed higher levels of KLH-specific cytokine (IL-4 and IFN-r) production. These results suggest that PAMAM dendrimer G4 possesses a potent immune-adjuvant activity for enhancing both humoral and cell-mediated immunity specific to foreign antigens.

Basic Amino Acid-Conjugated Polyamidoamine Dendrimers with Enhanced Gene Transfection Efficiency

박중현,최준식,박종상 한국고분자학회 2014 Macromolecular Research Vol.22 No.5

In this study, we characterized the polyamidoamine (PAMAM) dendrimer derivatives conjugated with basicamino acids as effective nonviral vector systems for gene delivery. Using PAMAM dendrimer (generation 4) as a corepolymer, we further synthesized PAMAM G4-Histidine-Lysine (PAMAM G4-H-K) and PAMAM G4-Histidine-Ornithine (PAMAM G4-H-O). Lysine and ornithine have cationic charged groups that can contribute to the condensationof DNA and interaction with cellular membranes. Histidine has an imidazole ring group that can induce a protonbuffering effect. In this report, we performed experiment to evaluate the basic amino acid-PAMAM conjugates asefficient and safe gene carriers. The mean diameter and zeta potential value of the PAMAM conjugates/DNA complexwere measured to be around 100 nm and 30 mV, respectively. It was observed that the PAMAM derivatives and plasmidDNA can form polyplexes at weight ratio 1.5 by agarose gel retardation and PicoGreen reagent assay. Furthermore,the PAMAM derivatives have shown high buffering capacity compared to the native PAMAM dendrimer. We performedthe cytotoxicity and transfection assay in the HeLa, HepG2, HEK 293, and NIH3T3 cell lines. While the transfectionefficiency was remarkable in all cell lines tested, the cytotoxicity level was very low. Based on these characteristics,it is suggested that the basic amino acid-conjugated PAMAM dendrimers could be utilized as promising gene deliverypolymeric vectors for effective gene therapy.

Dipeptide-functionalized polyamidoamine dendrimer-mediated apoptin gene delivery facilitates apoptosis of human primary glioma cells

Bae, Yoonhee,Green, Eric S.,Kim, Goo-Young,Song, Su Jeong,Mun, Ji Young,Lee, Sunray,Park, Jong-Il,Park, Jong-sang,Ko, Kyung Soo,Han, Jin,Choi, Joon Sig Elsevier 2016 International journal of pharmaceutics Vol.515 No.1

<P><B>Abstract</B></P> <P>Glioblastoma multiform (GBM) is the most frequent and aggressive form of brain tumors in adults. However, the development of more efficient and safe nonviral vector gene therapy represents a promising therapeutic approach, using a tumor-specific killer gene, named apoptin. In this study, we describe the efficacy of non-viral gene delivery vectors, the amino acid-conjugated PAMAM derivatives (PAMAM-H-R and PAMAM-H-K) in delivering a therapeutic gene, displaying affinity toward human primary glioma cells (GBL-14 cells) and dermal fibroblasts. We analyzed transfection efficiency, using luciferase (Luci) and a pDNA encoding for enhanced fluorescent protein (EGFP), and cytotoxicity in both cells. The results show that transfection efficiency of PAMAM-H-R improved compared to native PAMAM dendrimer, but cytotoxicity of PAMAM-H-R and PAMAM-H-K were very low. We treated both cells with a polyplex formation of PAMAM-H-R or PAMAM-H-K/apoptin, and analyzed their cellular uptake and localization by flow cytometry and confocal microscopy. Furthermore, we analyzed the endosomal escape effect using TEM images, and found that PAMAM-H-R showed very fast escape from endosome to the cytosol. Caspase 3 activity assay, cell cycle distribution, and JC-1 analysis showed apoptosis induced by apoptin in GBL-14 cells. This indicates that PAMAM-H-R can be a potential nonviral vector gene delivery carrier for brain tumor therapy. The present study demonstrates that PAMAM-H-R/apoptin gene polyplex can be used as an effective therapeutic candidate for GBM due to its selective induction of apoptosis in primary glioma cells as a potential nonviral gene delivery carrier for brain tumor therapy.</P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>

Comparison between arginine conjugated PAMAM dendrimers with structural diversity for gene delivery systems

Kim, T.i.,Bai, C.Z.,Nam, K.,Park, J.s. Elsevier Science Publishers 2009 Journal of controlled release Vol.136 No.2

Mono- and di-arginine conjugated PAMAM dendrimers (G=3 or 4) were synthesized to examine the structure-activity relationships of arginine conjugation for gene delivery systems. Number of conjugated arginines was examined by <SUP>1</SUP>H NMR (PAMAM3-R: 31, PAMAM4-R: 60, PAMAM3-R2: 59, PAMAM4-R2: 116). They could retard pDNA at a charge ratio of 2 and form polyplexes with sizes less than 250 nm from a charge ratio of 4. Di-arginine conjugated dendrimers showed higher Zeta-potential values and polyplex stabilities than mono-arginine conjugates. PAMAM3-R and PAMAM4-R showed low cytotoxicities even at high concentration but PAMAM3-R2 and PAMAM4-R2 exhibited significant cytotoxicities at high concentration. PAMAM3-R2 displayed greater transfection efficiency than PAMAM3-R, although the transfection efficiency of PAMAM4-R2 was not higher than that of PAMAM4-R in all condition. PAMAM3-R2 and PAMAM4-R2 polyplexes were observed to show the good intra-nuclear localization in comparison with PAMAM3-R and PAMAM4-R. It is concluded that di-arginine conjugation to PAMAM dendrimers can improve polyplex stability, intra-nuclear localization, and transfection efficiency but also induce charge density- and generation-dependent cytotoxicity. Therefore, a novel strategy for highly densed arginine conjugation maintaining low cytotoxicity will be needed for the development of efficient gene delivery carriers.

PAMAM Dendrimers Conjugated with L-Arginine and γ-Aminobutyric Acid as Novel Polymeric Gene Delivery Carriers

Son, Sang Jae,Yu, Gwang Sig,Choe, Yun Hui,Kim, Youn-Joong,Lee, Eunji,Park, Jong-Sang,Choi, Joon Sig Korean Chemical Society 2013 Bulletin of the Korean Chemical Society Vol.34 No.2

In this study, we synthesized functional dendrimer derivatives as nonviral gene delivery vectors. Poly(amidoamine) dendrimer (PAMAM, generation 4) was modified to possess functional amino acids to enhance gene transfection efficiency. PAMAM G4 derivatives conjugated with L-arginine (Arg) and ${\gamma}$-aminobutyric acid (GABA) showed higher transfection efficiency and lower cytotoxicity compared to the native PAMAM G4 dendrimer. The polyplex of the PAMAM G4 derivative/pDNA was evaluated using an agarose gel retardation assay and Picogreen reagent assay. Additionally, the MTT assay was performed to examine the cytotoxicity of synthesized polymers. All PAMAM G4 derivatives showed lower cytotoxicity than PEI25kD. Particularly, PAMAM G4-GABA-Arg displayed enhanced transfection efficiency compared to the native PAMAM G4 dendrimer.

Enhanced transfection of primary cortical cultures using arginine-grafted PAMAM dendrimer, PAMAM-Arg

Kim, Jung-Bin,Choi, Joon Sig,Nam, Kihoon,Lee, Minhyung,Park, Jong-Sang,Lee, Ja-Kyeong Elsevier 2006 Journal of controlled release Vol.114 No.1

<P><B>Abstract</B></P><P>PAMAM-Arg is a cationic arginine-grafted polyamidoamine (PAMAM) dendrimer. In the previous study, we reported that PAMAM-Arg facilitates transfection in a range of mammalian cell types. In the present study, we investigated the transfection efficiency of PAMAM-Arg in primary cortical cultures, which are known to be extremely vulnerable to exogenous gene transfection. PAMAM-Arg/DNA complexes showed particularly high transfection efficiencies and low cytotoxicity in primary cortical cells, as compared to other gene carriers such as, native PAMAM, polyethylenimine (BPEI), and Lipofectamine. Efficient transfection was not limited to neurons but extended to all three glial cells, astrocytes, microglia, and oligodendrocytes, present in these primary cortical cultures. The potential use of PAMAM-Arg was demonstrated by efficient gene knock-down by transfecting HMGB1 shRNA-expressing plasmid. The numbers of green fluorescent protein (GFP)-positive and HMGB1-negative cells indicated that PAMAM-Arg/shRNA-expressing plasmid complex suppressed target gene expression in over 40% of cells, which is the highest level achieved to date in primary cortical culture by any gene carrier. Here, we present evidence of the successful delivery and expression of both a reporter gene and of a shRNA-expressing plasmid in primary cortical cells, which demonstrates the potential of PAMAM-Arg for mediating gene delivery to primary neuronal cells.</P>

Modulation of Electroosmotic Flow through Skin: Effect of Poly(Amidoamine) Dendrimers

김혜지,오승열 한국응용약물학회 2018 Biomolecules & Therapeutics(구 응용약물학회지) Vol.26 No.2

The objective of this work is to evaluate the effect of polyamidoamine (PAMAM) dendrimers on electroosmotic flow (EOF) through skin. The effect of size and concentration of dendrimer was studied, using generation 1, 4 and 7 dendrimer (G1, G4 and G7, respectively). As a marker molecule for the direction and magnitude of EOF, a neutral molecule, acetoaminophen (AAP) was used. The visualization of dendrimer permeation into the current conducting pore (CCP) of skin was made using G4–fluorescein isothiocyanate (FITC) conjugate and confocal microscopy. Without dendrimer, anodal flux of AAP was much higher than cathodal or passive flux. When G1 dendrimer was added, anodal flux decreased, presumably due to the decrease in EOF by the association of G1 dendrimer with net negative charge in CCP. As the generation increased, larger decrease in anodal flux was observed, and the direction of EOF was reversed. Small amount of methanol used for the preparation of dendrimer solution also contributed to the decrease in anodal flux of AAP. Cross-sectional view perpendicular to the skin surface by confocal laser scanning microscope (CLSM) study showed that G4 dendrimer-FITC conjugate (G4-FITC) can penetrate into the viable epidermis and dermis under anodal current. The permeation route seemed to be localized on hair follicle region. These results suggest that PAMAM dendrimers can permeate into CCP and change the magnitude and direction of EOF. Overall, we obtained a better understanding on the mechanistic insights into the electroosmosis phenomena and its role on flux during iontophoresis.

PAMAM Dendrimers Conjugated with L-Arginine and γ-Aminobutyric Acid as Novel Polymeric Gene Delivery Carriers

Sang Jae Son,유광식,Yun Hui Choe,김윤중,이은지,박종상,최준식 대한화학회 2013 Bulletin of the Korean Chemical Society Vol.34 No.2

In this study, we synthesized functional dendrimer derivatives as nonviral gene delivery vectors. Poly(amidoamine) dendrimer (PAMAM, generation 4) was modified to possess functional amino acids to enhance gene transfection efficiency. PAMAM G4 derivatives conjugated with L-arginine (Arg) and γ-aminobutyric acid (GABA) showed higher transfection efficiency and lower cytotoxicity compared to the native PAMAM G4 dendrimer. The polyplex of the PAMAM G4 derivative/pDNA was evaluated using an agarose gel retardation assay and Picogreen reagent assay. Additionally, the MTT assay was performed to examine the cytotoxicity of synthesized polymers. All PAMAM G4 derivatives showed lower cytotoxicity than PEI25kD. Particularly, PAMAM G4-GABA-Arg displayed enhanced transfection efficiency compared to the native PAMAM G4 dendrimer.

Modulation of Electroosmotic Flow through Skin: Effect of Poly(Amidoamine) Dendrimers

Kim, Hye Ji,Oh, Seaung Youl The Korean Society of Applied Pharmacology 2018 Biomolecules & Therapeutics(구 응용약물학회지) Vol.26 No.2

The objective of this work is to evaluate the effect of polyamidoamine (PAMAM) dendrimers on electroosmotic flow (EOF) through skin. The effect of size and concentration of dendrimer was studied, using generation 1, 4 and 7 dendrimer (G1, G4 and G7, respectively). As a marker molecule for the direction and magnitude of EOF, a neutral molecule, acetoaminophen (AAP) was used. The visualization of dendrimer permeation into the current conducting pore (CCP) of skin was made using G4-fluorescein isothiocyanate (FITC) conjugate and confocal microscopy. Without dendrimer, anodal flux of AAP was much higher than cathodal or passive flux. When G1 dendrimer was added, anodal flux decreased, presumably due to the decrease in EOF by the association of G1 dendrimer with net negative charge in CCP. As the generation increased, larger decrease in anodal flux was observed, and the direction of EOF was reversed. Small amount of methanol used for the preparation of dendrimer solution also contributed to the decrease in anodal flux of AAP. Cross-sectional view perpendicular to the skin surface by confocal laser scanning microscope (CLSM) study showed that G4 dendrimer-FITC conjugate (G4-FITC) can penetrate into the viable epidermis and dermis under anodal current. The permeation route seemed to be localized on hair follicle region. These results suggest that PAMAM dendrimers can permeate into CCP and change the magnitude and direction of EOF. Overall, we obtained a better understanding on the mechanistic insights into the electroosmosis phenomena and its role on flux during iontophoresis.

Modulation of Electroosmotic Flow through Skin: Effect of Poly(Amidoamine) Dendrimers

( Hye Ji Kim ),( Seaung Youl Oh ) 한국응용약물학회 2018 Biomolecules & Therapeutics(구 응용약물학회지) Vol.26 No.2

The objective of this work is to evaluate the effect of polyamidoamine (PAMAM) dendrimers on electroosmotic flow (EOF) through skin. The effect of size and concentration of dendrimer was studied, using generation 1, 4 and 7 dendrimer (G1, G4 and G7, respectively). As a marker molecule for the direction and magnitude of EOF, a neutral molecule, acetoaminophen (AAP) was used. The visualization of dendrimer permeation into the current conducting pore (CCP) of skin was made using G4-fluorescein isothiocyanate (FITC) conjugate and confocal microscopy. Without dendrimer, anodal flux of AAP was much higher than cathodal or passive flux. When G1 dendrimer was added, anodal flux decreased, presumably due to the decrease in EOF by the association of G1 dendrimer with net negative charge in CCP. As the generation increased, larger decrease in anodal flux was observed, and the direction of EOF was reversed. Small amount of methanol used for the preparation of dendrimer solution also contributed to the decrease in anodal flux of AAP. Cross-sectional view perpendicular to the skin surface by confocal laser scanning microscope (CLSM) study showed that G4 dendrimer-FITC conjugate (G4-FITC) can penetrate into the viable epidermis and dermis under anodal current. The permeation route seemed to be localized on hair follicle region. These results suggest that PAMAM dendrimers can permeate into CCP and change the magnitude and direction of EOF. Overall, we obtained a better understanding on the mechanistic insights into the electroosmosis phenomena and its role on flux during iontophoresis.