12 research outputs found
Chitosan Modification of Adenovirus to Modify Transfection Efficiency in Bovine Corneal Epithelial Cells
BACKGROUND: The purpose of this study is to modulate the transfection efficiency of adenovirus (Ad) on the cornea by the covalent attachment of chitosan on adenoviral capsids via a thioether linkage between chitosan modified with 2-iminothiolane and Ad cross-linked with N-[gamma-maleimidobutyryloxy]succinimide ester (GMBS). METHODOLOGY/PRINCIPAL FINDINGS: Modified Ad was obtained by reaction with the heterobifunctional crosslinking reagent, GMBS, producing maleimide-modified Ad (Ad-GMBS). Then, the chitosan-SH was conjugated to Ad-GMBS via a thioether bond at different ratios of Ad to GMBS to chitosan-SH. The sizes and zeta potentials of unmodified Ad and chitosan-modified Ads were measured, and the morphologies of the virus particles were observed under transmission electron microscope. Primary cultures of bovine corneal epithelial cells were transfected with Ads and chitosan-modified Ads in the absence or presence of anti-adenovirus antibodies. Chitosan modification did not significantly change the particle size of Ad, but the surface charge of Ad increased significantly from -24.3 mV to nearly neutral. Furthermore, primary cultures of bovine corneal epithelial cells were transfected with Ad or chitosan-modified Ad in the absence or presence of anti-Ad antibodies. The transfection efficiency was attenuated gradually with increasing amounts of GMBS. However, incorporation of chitosan partly restored transfection activity and rendered the modified antibody resistant to antibody neutralization. CONCLUSIONS/SIGNIFICANCE: Chitosan can provide a platform for chemical modification of Ad, which offers potential for further in vivo applications
Chemical Modification of Adenoviral Capsid with Chitosan
本篇研究,我們利用一個正電性的天然高分子幾丁聚醣來對腺病毒外殼進行共價性的修飾。首先利用2-iminothiolane與幾丁聚醣反應,使幾丁聚醣硫醇化,此反應率達到46.9%。再利用N-[γ-maleimidobutyryloxy]succinimide ester (GMBS)與腺病毒反應,以作為與幾丁聚醣之間的架橋。將兩者混合以生成幾丁聚醣修飾的腺病毒。修飾前與修飾後的腺病毒進行粒徑與表面電位分析,病毒外觀形態改變則是利用穿透式電子顯微鏡(TEM)來觀察。本篇研究利用牛的角膜上皮細胞作為腺病毒感染力之測試,在有或無腺病毒抗體的培養基中評估修飾幾丁聚醣對腺病毒的影響。另外,我們也在幾丁聚醣上連接一些專一性的ligand,例如表皮生長因子(EGF)以及RGD等,再進而修飾腺病毒,嘗試改變腺病毒的細胞感染專一性。
由粒徑分析的結果可知幾丁聚醣的修飾對腺病毒的大小並無太大的變化,但卻明顯改變病毒表面電位,由原本的-24.3 mV轉變成幾乎不帶電。TEM的結果也指出幾丁聚醣修飾後的病毒外觀看來較為粗糙且不規則。另外我們發現腺病毒的感染能力隨著反應的GMBS的量增加而降低,而幾丁聚醣卻能使腺病毒恢復部分的感染能力。在低濃度抗體的環境下,幾丁聚醣修飾的腺病毒能有抵抗抗體中和的能力,但高濃度抗體的時候卻無此效果。此外,我們發現連接上ligand的腺病毒可能利用不同的路徑來感染細胞。
總結而言,幾丁聚醣此高分子提供了一個良好的平台讓我們加以利用。除了連接不同的專一性ligand以外,也可遮蔽抗體的辨認。未來將可利用此幾丁聚醣修飾的腺病毒來進行日後的in vivo研究。The purpose of this study is to covalently modify adenoviral capsid with chitosan, a cationic and natural polymer. To further expand applicability of chitosan, we have modified the amine group of chitosan with 2-iminothiolane to introduce thiol groups and obtained a 46.9% yield. Adenoviruses (Ads) were reacted with N-[γ-maleimidobutyryloxy]succinimide ester (GMBS), a heterobifuctional crosslinker, and maleimide-modified Ads (MalN-Ads) were obtained. Then, the chitosan-SH was reacted with MalN-Ads via thioether at different ratios of Ads to GMBS to chitosan-SH. The sizes and the zeta potentials of unmodified Ads and chitosan-modified Ads were measured, and morphology of the virus was observed under transmission electron microscope (TEM). Primary culture of bovine corneal epithelial cells was transduced with either adenoviruses or chitosan-modified Ads in the absence or presence of anti-adenovirus antibodies. Furthermore, we incorporated targeting ligands, such as epidermal growth factor (EGF) and RGD, on chitosan to further modify adenovirus.
Analysis of particle sizes showed modification with chitosan did not obviously affect the size of Ads, but zeta potential analysis revealed that the surface charge of Ads significantly changed form -24.3 mV to nearly neutral charge. The results of TEM also showed chitosan-modified Ads had a rough and irregular appearance. Depending on the increasing amounts of GMBS, the transduction efficiency was attenuated gradually. However, incorporation of chitosan could restore a part of transduction activity. Chitosan-modified Ads were resistant to antibody neutralization at a low antibody concentration, but could not work at a high concentration. We also speculate that ligand-linked chitosan-modified Ads could transducer cells via alternative pathway.
In conclusion, chitosan can provide a great platform of chemical modification on adenoviruses. It permits incorporation of a range of targeting molecules, but also of other biological effectors. Hence, chitosan-modified Ads are potential for further in vivo studies.中文摘要 I
Abstract II
Contents IV
List of Figures VI
List of Tables X
Chapter 1. Introduction 1
Chapter 2. Literature Review 3
2-1. Introduction to Chitosan4 3
2-2. The Application of Chitosan in Ophthalmology4 3
2-3. Routes for the Ocular Delivery33 4
2-4. Introduction to Adenoviruses3 5
2-5. The Application of Adenoviruses in Cornea3 6
2-6. Chemical Modification of Adenoviruses with Polymers47 6
Chapter 3. Materials and Methods 9
3-1. Experimental Skeleton 9
3-2. Materials 9
3-3. Primary Culture of Bovine Corneal Epithelial Cells 12
3-4. Amplification and Purification of Adenoviruses 13
3-5. Modification of Adenoviruses with Chitosan 14
3-6. Characterization of Chitosan-Modified Ads 15
3-7. In Vitro Transduction 17
3-8. Retargeting of Ligand-Linked Chitosan-Modified Adenoviruses 18
Chapter 4. Results 20
4-1. Modification of Chitosan 20
4-2. Characterization of Chitosan-Modified Ads 20
4-3. Reaction of Adenoviruses with GMBS Substantially Attenuates Transduction Efficiency 21
4-4. Incorporation of Chitosan Restores Part of Transduction Activity 22
4-5. Transduction in the Presence of Neutralizing Antibody 23
4-6. Retargeting of Ligand-Linked Chitosan-Modified Adenoviruses to Alternative Receptors 24
Chapter 5. Discussion 25
Chapter 6. Conclusion 28
References 29
Figures 38
Tables 5
Infection efficiency (A) and relative fluorescent intensity (B) from flow-cytometric analysis of corneal epithelial cells infected with unmodified Ad or chitosan-modified Ad at different ratios at 1000 Ad particles/cell in the absence or presence of neutralizing antibody.
<p>Each point represents the mean ± S.D. (n = 3). *P<0.05 (Student's t-test).</p
Comparison of different reaction amounts of GMBS and chitosan-SH to yield different Ad/GMBS/ChiSH ratios.
<p>Comparison of different reaction amounts of GMBS and chitosan-SH to yield different Ad/GMBS/ChiSH ratios.</p
Variations of chitosan-modified Ad at different Ad∶GMBS and Ad∶GMBS∶ChiSH ratios.
<p>For size, each point represents the particle number mean ± S.E.M. For zeta potential, each point represents the mean of zeta potential ± S.E.M (n = 100).</p
Phase contrast and fluorescence photomicrographs, respectively, of primary cultured corneal epithelial cells infected with Ad-GMBS at a ratio of 1∶100 (A), (E); Ad-GMBS-ChiSH at a ratio of 1∶100∶7 (B), (F); Ad-GMBS-ChiSH at a ratio of 1∶100∶70 (C), (G); and Ad-GMBS-ChiSH at a ratio of 1∶100∶700 (D), (H) at 1000 Ad particles/cell.
<p>Magnification = 100×. Infection efficiency (I) and relative fluorescence intensity (J) from flow-cytometric analysis of corneal epithelial cells infected with Ad, Ad-GMBS at a ratio of 1∶100, Ad-GMBS-ChiSH at a ratio of 1∶100∶7, Ad-GMBS-ChiSH at a ratio of 1∶100∶70 and Ad-GMBS-ChiSH at a ratio of 1∶100∶700 at 1000 Ad particles/cell. Each point represents the mean ± S.D. (n = 3). **P<0.001 (Student's t-test).</p
Infection efficiencies from flow-cytometric analysis of corneal epithelial cells infected at a MOI of 200, 1000 or 5000 Ad particles/cell with unmodified Ad or chitosan-modified Ad at varying ratios.
<p>Each point represents the mean ± S.D. (n = 3).</p
Fluorescence photomicrographs of primary cultured corneal epithelial cells infected with unmodified Ad (A)–(D), Ad-GMBS-ChiSH at a ratio of 1∶1∶7 (E)–(H), Ad-GMBS-ChiSH at a ratio of 1∶1∶70 (I)–(L), Ad-GMBS-ChiSH at a ratio of 1∶1∶700 (M)–(P), Ad-GMBS-ChiSH at a ratio of 1∶10∶7 (Q)–(T), Ad-GMBS-ChiSH at a ratio of 1∶10∶70 (U)–(X) and Ad-GMBS-ChiSH at a ratio of 1∶10∶700 (Y)–(AB) at 1000 Ad particles/cell in the absence or presence of neutralizing antibody.
<p>Magnification = 100×.</p
Fluorescence photomicrographs of primary cultured corneal epithelial cells.
<p>Primary cultured corneal epithelial cells infected by unmodified Ad (A)–(C) or chitosan-modified Ad at Ad∶GMBS∶ChiSH ratios of 1∶1∶7 (D)–(F), 1∶10∶7 (G)–(I), and 1∶100∶7 (J)–(L). Magnification = 100×.</p