PAMAM activated

Keywords. Polyamidoamine dendrimers. Activation of PAMAM dendrimers. Transfection, Gene transfer, DNA-dendrimer complex... 

Structure of PAMAM Dendrimers and Activated PAMAM Dendrimers 230... 

Theory of Gene Transfer with Activated PAMAM Dendrimers.232... 

Newly synthesized PAMAM dendrimers have a defined size and shape and can be used in gene transfer experiments, but the efficiency of transfection can be greatly increased by a process called activation of the dendrimer. In activation, some of the tertiary amines are removed, resulting in a molecule with a higher... 

Fig.1. Non-activated and activated PAMAM-dendrimers. Schematic diagram of a non-acti-vated (left) and activated dendrimer (middle). The right panel shows a magnification of the dendrimer branches...

Activated PAMAM dendrimers interact with DNA to form a DNA-dendrimer complex with a toroid-like structure (Fig. 2). Such DNA-dendrimer complexes have diameters of 50-100 nm [10],which means that the DNA molecules are highly condensed in these complexes. A 6-kb plasmid alone, for example, has an extended structure several hundred nanometers in diameter. In transfection experiments, typically an 8- to 12-fold excess of positive amino groups over negatively... 

Since mild activation conditions appear to be important, a number of solution activation conditions were tested. PAMAM dendrimers are comprised of amide bonds, so the favorable conditions for refro-Michael addition reactions, (low pH, high temperature and the presence of water) may be able to cleave these bonds. Table 1 shows a series of reaction tests using various acid/solvent combinations to activate the dendrimer amide bonds. Characterization of the solution-activated catalysts with Atomic Absorption spectroscopy, FTIR spectroscopy and FTIR spectroscopy of adsorbed CO indicated that the solution activation generally resulted in Pt loss. Appropriate choice of solvent and acid, particularly EtOH/HOAc, minimized the leaching. FTIR spectra of these samples indicate that a substantial portion of the dendrimer amide bonds was removed by solution activation (note the small y-axis value in Figure 4 relative... 

The N-linked pentasaccharide core Man3(GlcNAc)2 glycopeptide bearing the extracellular MMP inducer sequence 517 (emmprin 34—58) has been successfully linked to G(l) PAMAM aminodendrimer by thioester activation (Fig. 63). The resulting octameric 30 kDa construct was obtained in low yield, but was purified by preparative electrophoresis and fully characterized by MALDI-TOF mass spectrometry. The multivalent architecture was built to evaluate the requirement of emmprin multimerization for inducing MMP expression.384... 

Other than the epoxy groups available on one Priostar dendrimer type and a methyl ester available on a PAMAM dendrimer, the commercial suppliers generally don t offer a selection of spontaneously reactive dendrimers for bioconjugation purposes. For this reason, most of the applications published for coupling biomolecules to dendrimers have used various modification or activation steps to create the appropriate reactive groups for conjugation (e.g., Leon etal., 1996). 

Use of sulfo-NHS-LC-SPDP or other heterobifunctional crosslinkers to modify PAMAM dendrimers may be done along with the use of a secondary conjugation reaction to couple a detectable label or another protein to the dendrimer surface. Patri et al. (2004) used the SPDP activation method along with amine-reactive fluorescent labels (FITC or 6-carboxytetramethylrhodamine succinimidyl ester) to create an antibody conjugate, which also was detectable by fluorescent imaging. Thomas et al. (2004) used a similar procedure and the same crosslinker to thiolate dendrimers for conjugation with sulfo-SMCC-activated antibodies. In this case, the dendrimers were labeled with FITC at a level of 5 fluorescent molecules per G-5 PAMAM molecule. 

Hong et al. (2004) also found that modification of PAMAM dendrimers with a short PEG linker arm could act to reduce nonspecificity caused by the amines on the dendrimer-modified surface. An azido-PEGj-aininc spacer was activated with nitrophenyl carbamate to yield an activated intermediate that could be used to modify the amines on the dendrimer (Figure 7.24). Reaction at high molar ratio resulted in about 61 PEG-azido spacers on the dendrimer. Reduction of the azido group to an amine using triphenylphosphine in THF provided the dendrimer-PEG-amine derivative for surface modification. The added presence of the PEG spacer arm reduced... 

Jacobsen et al. reported enhanced catalytic activity by cooperative effects in the asymmetric ring opening (ARO) of epoxides.[38] Chiral Co-salen complexes (Figure 4.27) were used, which were bound to different generations of commercial PAMAM dendrimers. As a direct consequence of the second-order kinetic dependence on the [Co(salen)] complex concentration of the hydrolytic kinetic resolution (HKR), reduction of the catalyst loading using monomeric catalyst leads to a sharp decrease in overall reaction rate. 

To investigate this dendritic effect, a dimeric model compound was synthesized which mimics the tethered relationship of two catalytic units within one branch of the PAMAM dendrimer. All dendritic catalysts were more active in the HKR than the parent complex. Furthermore, the dendritic catalysts also displayed significantly higher activity than the dimeric model compound. The authors proposed that this positive dendritic effect arises from restricted conformation imposed by the dendrimer structure, thereby creating a bigger effective molarity of [Co(salen)] units. Alternatively, the multimeric nature of the dendrimer, may lead to higher order in productive cooperative interactions between the catalytic units. 

Within the temperature range from 10 to 40°C, both PAMAM dendrimers in EDA [5] and PPI dendrimers in water [22] showed a linear relationship between In r and /T, in good agreement with the kinetic rate theory of flow [46]. The apparent activation energies of flow (En) were constant and independent of temperature, and it was shown for PAMAM/EDA systems that the dependence of En on solution concentration was linear for all generations examined [5]. This was considerably different from the typical relationships for the solutions of linear and/or randomly branched chain polymers, where a break in the slope of... 

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