Cationic poly(amidoamine) promotes cytosolic delivery of bovine RNase A in melanoma cells, while maintaining its cellular toxicity

Ribonucleases are known to cleave ribonucleic acids, inducing cell death. RNase A, a member of the ribonuclease family, generally displayed poor in vitro activity. This has been attributed to factors such as low intracellular delivery. Poly(amidoamine)s have been used to promote the translocation of non-permeant proteins to the cytosol. Our objective was to demonstrate that poly(amidoamine)s could potentially promote the delivery of RNase A to selected cell line. Interactions of three cationic poly(amidoamine)s (P1, P2 and ISA1) with wild-type bovine RNase A were investigated using gel retardation assays, DLS and microcalorimetry. Although the polymers and the protein are essentially cationic at physiological pH, complexation between the PAAs and RNase A was observed. The high sensitivity differential scanning calorimetry (HSDSC) thermograms demonstrated that the thermal stability of the protein was reduced when complexed with ISA1 (Tmax decreased by 6.5 °C) but was not affected by P1 and P2. All the polymers displayed low cytotoxicity towards non-cancerous cells (IC50 > 3.5 mg mL?1). While RNase A alone was not toxic to mouse melanoma cells (B16F1), P1 was able to promote cytosolic delivery of biologically active RNase A, increasing cell death (IC50 = 0.09 mg mL?1)

Multiple Scale Reorganization of Electrostatic Complexes of PolyStyrene Sulfonate and Lysozyme

We report on a SANS investigation into the potential for these structural reorganization of complexes composed of lysozyme and small PSS chains of opposite charge if the physicochemical conditions of the solutions are changed after their formation. Mixtures of solutions of lysozyme and PSS with high matter content and with an introduced charge ratio [-]/[+]intro close to the electrostatic stoichiometry, lead to suspensions that are macroscopically stable. They are composed at local scale of dense globular primary complexes of radius ~ 100 {\AA}; at a higher scale they are organized fractally with a dimension 2.1. We first show that the dilution of the solution of complexes, all other physicochemical parameters remaining constant, induces a macroscopic destabilization of the solutions but does not modify the structure of the complexes at submicronic scales. This suggests that the colloidal stability of the complexes can be explained by the interlocking of the fractal aggregates in a network at high concentration: dilution does not break the local aggregate structure but it does destroy the network. We show, secondly, that the addition of salt does not change the almost frozen inner structure of the cores of the primary complexes, although it does encourage growth of the complexes; these coalesce into larger complexes as salt has partially screened the electrostatic repulsions between two primary complexes. These larger primary complexes remain aggregated with a fractal dimension of 2.1. Thirdly, we show that the addition of PSS chains up to [-]/[+]intro ~ 20, after the formation of the primary complex with a [-]/[+]intro close to 1, only slightly changes the inner structure of the primary complexes. Moreover, in contrast to the synthesis achieved in the one-step mixing procedure where the proteins are unfolded for a range of [-]/[+]intro, the native conformation of the proteins is preserved inside the frozen core

Influence of complexing polyanions on the thermostability of basic protiens.

Lysozyme (Lyz), chymotrypsinogen (Cht), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as model proteins capable of forming water-soluble polyelectrolyte complexes with linear synthetic polyanions. The complex formation with sodium poly(methacrylate) (PMA), sodium poly(acrylate) (PAA), sodium poly(anetholsulfonate) (PAS), and potassium poly(vinylsulfate) (PVS) markedly reduced the temperature of protein denaturation, Tmax, as determined by differential scanning calorimetry (DSC). The effect of sodium poly(styrenesulfonate) (PSS) on Lyz was so drastic that the protein melting peak was not observed at all during DSC measurements. The temperature shift, most pronounced for Lyz, increased upon substitution of the polyanions according to the following series: PMA < PVS < PAA < PAS < PSS. Decomposition of the complexes by addition of either sodium chloride or poly(N-ethyl-4-vinylpyridinium) cation completely restored the initial Tmax of the protein (except for PSS and PAS). The complex formation slightly affected the enzyme activity up to temperatures close to Tmax of the polyanion-protein complex. On further heating, the activity of the complex decreased steeply, whereas the free enzyme maintained a high activity. The data obtained strongly suggest that the protein-polyelectrolyte interactions in solution, while leaving the thermostability and activity of the proteins practically unaffected over a rather wide temperature range, result in the effective denaturation of proteins once a certain critical temperature is achieved. This finding appears to be crucial for further development of immobilized enzymes in biotechnology and essential for understanding mechanisms and principles of the functioning of proteins immobilized on charged matrices in vivo

Influence of Complexing Polyanions on the Thermostability of Basic Proteins

Lysozyme (Lyz), chymotrypsinogen (Cht), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used as model proteins capable of forming water-soluble polyelectrolyte complexes with linear synthetic polyanions. The complex formation with sodium poly(methacrylate) (PMA), sodium poly(acrylate) (PAA), sodium poly(anetholsulfonate) (PAS), and potassium poly(vinylsulfate) (PVS) markedly reduced the temperature of protein denaturation, Tmax, as determined by differential scanning calorimetry (DSC). The effect of sodium poly(styrenesulfonate) (PSS) on Lyz was so drastic that the protein melting peak was not observed at all during DSC measurements. The temperature shift, most pronounced for Lyz, increased upon substitution of the polyanions according to the following series: PMA < PVS < PAA < PAS < PSS. Decomposition of the complexes by addition of either sodium chloride or poly(N-ethyl-4-vinylpyridinium) cation completely restored the initial Tmax of the protein (except for PSS and PAS). The complex formation slightly affected the enzyme activity up to temperatures close to Tmax of the polyanion-protein complex. On further heating, the activity of the complex decreased steeply, whereas the free enzyme maintained a high activity. The data obtained strongly suggest that the protein-polyelectrolyte interactions in solution, while leaving the thermostability and activity of the proteins practically unaffected over a rather wide temperature range, result in the effective denaturation of proteins once a certain critical temperature is achieved. This finding appears to be crucial for further development of immobilized enzymes in biotechnology and essential for understanding mechanisms and principles of the functioning of proteins immobilized on charged matrices in vivo