Dextrin-trypsin and ST-HPMA-trypsin conjugates: enzyme activity, autolysis and thermal stability.

Using monomethoxy poly(ethylene glycol) (mPEG)-trypsin conjugates we recently showed that both PEG molecular weight (1100-5000 g/mol) and linker chemistry affect the rate of protein autolysis and thermal stability. These important factors are often overlooked but they can guide the early choice of optimal polymer/chemistry for synthesis of a lead polymer therapeutic suitable for later formulation development. As we are currently developing dextrin- and semi-telechelic poly[N-(2-hydroxypropyl)methacrylamide] (ST-HPMA)-protein conjugates as new therapeutics, the aim of this study was to examine the effect of polymer on activity, autolysis and its thermal stability using trypsin conjugates as a model and compare to the data obtained for mPEG conjugates. Trypsin conjugates were first synthesized using succinoylated dextrin (Mw approximately 8000 g/mol, dextrin I; or approximately 61,000g/mol, dextrin II), and a ST-HPMA-COOH (Mw approximately 10,100g/mol). The conjugates had a trypsin content of approximately 54, 17 and 3 wt% respectively with <5% free protein. When amidase activity (K(M), V(max) and K(cat)) was determined by using N-benzoyl-L-arginine p-nitroanilide (BAPNA) as substrate, trypsin K(M) values were not altered by conjugation, but the V(max) was approximately 6-7-fold lower, and the substrate turnover rate (K(cat)) decreased by approximately 5-7-fold. The dextrin II-trypsin conjugate was more stable than the other conjugates and native trypsin at all temperatures between 30 and 70 degrees C, and also exhibited improved thermal stability in the autolysis assays at 40 degrees C.

Low concentration thresholds of plasma membranes for rapid energy-independent translocation of a cell-penetrating peptide.

The exact mechanisms by which cell-penetrating peptides such as oligo-arginines and penetratin cross biological membranes has yet to be elucidated, but this is required if they are to reach their full potential as cellular delivery vectors. In the present study, qualitative and quantitative analysis of the influence of temperature, peptide concentration and plasma membrane cholesterol on the uptake and subcellular distribution of the model cell-penetrating peptide octa-arginine was performed in a number of suspension and adherent cell lines. When experiments were performed on ice, the peptide at 2 microM extracellular concentration efficiently entered and uniformly labelled the cytoplasm of all the suspension cells studied, but a 10-fold higher concentration was required to observe similar results in adherent cells. At 37 degrees C and at higher peptide concentrations, time-lapse microscopy experiments showed that the peptide rapidly penetrated the entire plasma membrane of suspension cells, with no evidence of a requirement for nucleation zones to promote this effect. Cholesterol depletion with methyl-beta-cyclodextrin enhanced translocation of octa-arginine across the plasma membrane of suspension cells at 37 degrees C, but decreased overall peptide accumulation. Under the same conditions in adherent cells this agent had no effect on peptide uptake or distribution. Cholesterol depletion increased the overall accumulation of the peptide at 4 degrees C in KG1a cells, but this effect could be reversed by re-addition of cholesterol as methyl-beta-cyclodextrin-cholesterol complexes. The results highlight the relatively high porosity of the plasma membrane of suspension cells to this peptide, especially at low temperatures, suggesting that this feature could be exploited for delivering bioactive entities.

Thermo- and pH-responsive polymers in drug delivery.

Stimuli-responsive polymers show a sharp change in properties upon a small or modest change in environmental condition, e.g. temperature, light, salt concentration or pH. This behaviour can be utilised for the preparation of so-called 'smart' drug delivery systems, which mimic biological response behaviour to a certain extent. The possible environmental conditions to use for this purpose are limited due to the biomedical setting of drug delivery as application. Different organs, tissues and cellular compartments may have large differences in pH, which makes the pH a suitable stimulus. Therefore the majority of examples, discussed in this paper, deal with pH-responsive drug delivery system. Thermo-responsive polymer is also covered to a large extent, as well as double-responsive system. The physico-chemical behaviour underlying the phase transition will be discussed in brief. Then selected examples of applications are described.

In situ study of the thermoresponsive behavior of micropatterned hydrogel films by imaging ellipsometry.

A patterned hydrogel was immobilized on a polymer substrate by low-pressure argon plasma treatment using a masking technique. The polymer sample showed a thermoresponsive aggregation behavior in the region of 35-37 degrees C. The micropatterned, thermoresponsive hydrogel film has been characterized with imaging ellipsometry. The characterization was carried out on the dry film as well as on a swollen sample in water. The thermoresponsive behavior was studied in deionized water by temperature-dependent measurements in a solid-liquid cell. Through imaging ellipsometry, it was possible to distinguish the different regions of interest on a micrometer scale and to follow the swelling of the hydrogel part as a function of the temperature. It was possible to visualize the swelling as 3D profiles of Delta at various temperatures. Long-term changes of the sample could also be detected, which cannot be picked up by conventional ellipsometry.

Thermo-reversible swelling of thin hydrogel films immobilized by low-pressure plasma.

Thin films of graft copolymers consisting of poly(N-isopropylacrylamide) (PNiPAAm) or poly(N,N-diethylacrylamide) (PDEAAm) as polymer backbone and poly(ethyleneglycol) as side chains were cross-linked on fluoropolymer substrates by low-pressure plasma treatment. All immobilized polymers exhibit a lower critical solution temperature between 34 and 40 degrees C. The swelling and collapsing of the hydrogels was examined with temperature-dependent spectroscopic ellipsometry. Two time ranges of swelling were observed: a fast 'dynamic' and a slow 'equilibrium' swelling. The dynamic swelling occurs within minutes or less, whereas the equilibrium swelling needs several days to complete. The surface-bound hydrogels show a shift in the transition temperature toward lower temperatures compared with the behavior in solution. Full reversibility of the dynamic swelling/collapsing was found, but the temperature scan exhibits a hysteresis between heating and cooling cycles. The PNiPAAm-containing hydrogels show a sharper transition compared to the PDEAAm-containing hydrogels, which is almost linear over a wide temperature range.

Thermo-responsive PNiPAAm-g-PEG films for controlled cell detachment.

A series of graft copolymers consisting of either poly(N-isopropylacrylamide) (PNiPAAm) or poly(N,N-diethylacrylamide) (PDEAAm) as a thermo-responsive component in the polymer backbone and poly(ethyleneglycol) (PEG) were immobilized as thin films and cross-linked on a fluoropolymer substrate using low-pressure argon plasma treatment. The surface-immobilized hydrogels exhibit a transition from partially collapsed to completely swollen, which is in the range of 32-35 degrees C and corresponds to the lower critical solution temperature of the soluble polymers. The hydrogels were used as cell carriers in culture experiments with L929 mouse fibroblast cells to probe for cell adhesion, proliferation, and temperature-dependent detachment of cell layers. The fibroblast cells adhere, spread, and proliferate on the hydrogel layers at 37 degrees C and become completely detached after reducing the temperature by 3 K. The cell release characteristics were further correlated to the swelling and collapsing behavior of the hydrogel films and the polymer solutions as measured in PBS solution and RPMI cell cultivation medium. It could be shown that, long before the swelling has completed upon temperature reduction, the cells detach. This can be attributed to the large content of PEG present in the hydrogel, which weaken the cell adhesion strength to the hydrogel layers.

Template Synthesis and Reactions of Tricarbonylmolybdenum Phosphadithiamacrocycle Complexes.

Treatment of [Me(4)N](2)[PhP(CH(2)CH(2)S)(2)] with [Mo(CO)(3)(NCMe)(3)] affords the reactive intermediate [Me(4)N](2)[Mo(CO)(3){PhP(CH(2)CH(2)S)(2)}] (1), which undergoes oxidation to afford [Mo{PhP(CH(2)CH(2)S)(2)}(2)] (2). Reaction of 1 with a variety of dichloroalkanes produces [Mo(CO)(3){c-PhP(CH(2)CH(2)S)(2)X}] (X = CH(2)CH(2), CH(2)CH(2)CH(2), CH(2)CHMe or CH(2)CH(OH)CH(2)). The structure of [Mo(CO)(3){c-PhP(CH(2)CH(2)S)(2)CH(2)CH(2)}] (3) has been established by X-ray crystallography and consists of a Mo(CO)(3) fragment facially coordinated by the tridentate c-PhP(CH(2)CH(2)S)(2)CH(2)CH(2) ligand. Reaction of 3 with bromine affords seven-coordinate [Mo(CO)(2){c-PhP(CH(2)CH(2)S)(2)CH(2)CH(2)}Br(2)] (7), the X-ray crystal structure of which reveals a carbonyl-capped octahedral geometry. Treatment of 3 with sulfur results in loss of the Mo(CO)(3) fragment and isolation of c-PhPS(CH(2)CH(2)S)(2)CH(2)CH(2) (8), the X-ray structure of which shows a nine-membered ring with the phosphorus center bearing phenyl and sulfide substituents. Reduction of 8 with sodium naphthalenide affords the parent ligand c-PhP(CH(2)CH(2)S)(2)CH(2)CH(2). Crystal data: 2, C(20)H(26)MoP(2)S(4), triclinic P&onemacr;, a = 8.105(3) Å, b = 8.263(3) Å, c = 17.663(4) Å, alpha = 100.29(2) degrees, beta = 99.78(2) degrees, gamma = 98.81(2) degrees, Z = 2; 3, C(15)H(17)MoO(3)PS(2), monoclinic P2(1)/n, a = 9.600(3) Å, b = 15.594(5) Å, c = 11.335(3) Å, beta = 93.01(2) degrees, Z = 4; 7, C(14)H(17)Br(2)MoO(2)PS(2), monoclinic P2(1)/c, a = 17.039(3) Å, b = 8.686(2) Å, c = 12.466(3) Å, beta = 100.52(2) degrees, Z = 4; 8, C(12)H(17)PS(3), monoclinic P2(1), a = 6.651(4) Å, b = 7.313(2) Å, c = 14.687(9) Å, beta = 101.62(3) degrees, Z = 2.