Leriche Emma-Dune, Hubert-Roux Marie, Afonso Carlos, Lange Catherine M, Grossel Martin C, Maire Florian, Loutelier-Bourhis Corinne
Normandie Université, COBRA, UMR6014 and FR3038, Université de Rouen, CNRS, IRCOF, 1 rue Tesnière, 76821 Mont-Saint-Aignan Cedex, France.
School of Chemistry, University of Southampton, Highfield, Hants SO17 1BJ Southampton, UK.
Molecules. 2014 Dec 12;19(12):20731-50. doi: 10.3390/molecules191220731.
Highly branched polyamidoamine (PAMAM) dendrimers presenting biological activities have been envisaged as non-viral gene delivery vectors. They are known to associate with nucleic acid (DNA) in non-covalent complexes via electrostatic interactions. Although their transfection efficiency has been proved, PAMAMs present a significant cytotoxicity due to their cationic surface. To overcome such a drawback, different chemical modifications of the PAMAM surface have been reported such as the attachment of hydrophobic residues. In the present work, we studied the complexation of DNA duplexes with different low-generation PAMAM; ammonia-cored G0(N) and G1(N) PAMAM, native or chemically modified with aromatic residues, i.e., phenyl-modified-PAMAM G0(N) and phenylalanine-modified-PAMAM G1(N). To investigate the interactions involved in the PAMAM/DNA complexes, also called dendriplexes, we used electrospray ionization (ESI) coupled to ion mobility spectrometry-mass-spectrometry (IM-MS). ESI is known to allow the study of non-covalent complexes in native conditions while IM-MS is a bidimensional separation technique particularly useful for the characterization of complex mixtures. IM-MS allows the separation of the expected complexes, possible additional non-specific complexes and the free ligands. Tandem mass spectrometry (MS/MS) was also used for the structural characterization. This work highlights the contribution of IM-MS and MS/MS for the study of small dendriplexes. The stoichiometries of the complexes and the equilibrium dissociation constants were determined. The [DNA/native PAMAM] and [DNA/modified-PAMAM] dendriplexes were compared.
具有生物活性的高度支化聚酰胺-胺（PAMAM）树枝状大分子已被设想为非病毒基因传递载体。已知它们通过静电相互作用与核酸（DNA）形成非共价复合物。尽管已证明其转染效率，但由于其阳离子表面，PAMAM具有显著的细胞毒性。为克服这一缺点，已报道了对PAMAM表面进行的不同化学修饰，例如连接疏水残基。在本研究中，我们研究了不同低代PAMAM（氨核G0(N)和G1(N) PAMAM）与DNA双链体的络合情况，这些PAMAM为天然的或用芳香族残基进行化学修饰的，即苯基修饰的PAMAM G0(N)和苯丙氨酸修饰的PAMAM G1(N)。为了研究PAMAM/DNA复合物（也称为树枝状复合物）中涉及的相互作用，我们使用了与离子淌度光谱-质谱联用（IM-MS）的电喷雾电离（ESI）。已知ESI可用于在天然条件下研究非共价复合物，而IM-MS是一种二维分离技术，对复杂混合物的表征特别有用。IM-MS可以分离预期的复合物、可能的额外非特异性复合物和游离配体。串联质谱（MS/MS）也用于结构表征。这项工作突出了IM-MS和MS/MS在研究小型树枝状复合物方面的作用。测定了复合物的化学计量比和平衡解离常数。比较了[DNA/天然PAMAM]和[DNA/修饰PAMAM]树枝状复合物。
