Small-angle neutron scattering study of the n-decane effect on the bilayer thickness in extruded unilamellar dioleoylphosphatidylcholine liposomes

Dioleoylphosphatidylcholine (DOPC) and n-decane were mixed and hydrated afterwards in an excess of heavy water at 1 wt.% of DOPC. From this dispersion, unilamellar liposomes were prepared by extrusion through polycarbonate filter with 500-A pores. Small-angle neutron scattering (SANS) was conducted on these liposomes. From the Kratky-Porod plot ln[I(Q)Q2] vs. Q2 of SANS intensity I(Q) in the range of scattering vectors Q corresponding to the interval 0.001 A(-2) < or = Q2 < or = 0.006 A(-2), the liposome bilayer radius of gyration Rg and the bilayer thickness parameter d(g) = 12(0.5)Rg were obtained. The values of d(g) indicated that the bilayer thickness is within the experimental error constant up to n-decane/DOPC approximately 0.5 molar ratio, and then increases by 2.4 +/- 1.3 A up to n-decane/DOPC = 1.2 molar ratio.     

Small-angle neutron scattering study of the lipid bilayer thickness in unilamellar dioleoylphosphatidylcholine vesicles prepared by the cholate dilution method: n-decane effect

Previous X-ray diffraction studies on fully hydrated fluid lamellar egg phosphatidylcholine phases indicated a approximately 10 A increase of bilayer thickness in the presence of excess n-decane [Biochim. Biophys. Acta 597 (1980) 455], while the small-angle neutron scattering (SANS) on unilamellar extruded dioleoylphosphatidylcholine (DOPC) vesicles detected substantially smaller 2.4+/-1.3 A bilayer thickness increase at n-decane/DOPC molar ratio of 1.2 [Biophys. Chem. 88 (2000) 165]. The purpose of the present study is to investigate the n-decane effect on the bilayer thickness in unilamellar DOPC vesicles prepared by the sodium cholate (NaChol) dilution method. Mixed DOPC+NaChol micelles at DOPC and NaChol concentrations of 0.1 mol/l were prepared in 2H(2)O containing 0.135 mol/l NaCl. This micellar solution was diluted in 0.135 mol/l NaCl in 2H(2)O to reach the final DOPC and NaChol concentrations of 0.008 mol/l. Thirty microliters of n-decane solution in methanol was added to 1 ml of this dispersion. After methanol evaporation, SANS was conducted on the dispersions. From the Kratky-Porod plot ln[I(Q)Q(2)] vs. Q(2) of SANS intensity I(Q) in the range of scattering vector values Q corresponding to interval 0.001 A(-2)相似文献   

Conformational changes in single-strand DNA as a function of temperature by SANS

Small-angle neutron scattering (SANS) measurements were performed on a solution of single-strand DNA, 5'-ATGCTGATGC-3', in sodium phosphate buffer solution at 10 degrees C temperature increments from 25 degrees C to 80 degrees C. Cylindrical, helical, and random coil shape models were fitted to the SANS measurements at each temperature. All the shapes exhibited an expansion in the diameter direction causing a slightly shortened pitch from 25 degrees C to 43 degrees C, an expansion in the pitch direction with a slight decrease in the diameter from 43 degrees C to 53 degrees C, and finally a dramatic increase in the pitch and diameter from 53 degrees C to 80 degrees C. Differential scanning calorimeter scans of the sequence in solution exhibited a reversible two-state transition profile with a transition temperature of 47.5 +/- 0.5 degrees C, the midpoint of the conformational changes observed in the SANS measurements, and a calorimetric transition enthalpy of 60 +/- 3 kJ mol(-1) that indicates a broad transition as is observed in the SANS measurements. A transition temperature of 47 +/- 1 degrees C was also obtained from ultraviolet optical density measurements of strand melting scans of the single-strand DNA. This transition corresponds to unstacking of the bases of the sequence and is responsible for the thermodynamic discrepancy between its binding stability to its complementary sequence determined directly at ambient temperatures and determined from extrapolated values of the melting of the duplex at high temperature.     

Neutron and light-scattering studies of DNA gyrase and its complex with DNA

The solution structure of Escherichia coli DNA gyrase, an enzyme that catalyzes the ATP-dependent supercoiling of DNA, has been characterized by small-angle neutron scattering (SANS) and dynamic light-scattering (DLS). The enzyme and its complex with a 172 base-pair fragment of duplex DNA, in H2O or 2H2O solvent, were studied by contrast variation and the measurement of hydrodynamic parameters as a function of scattering angle. The complex was also measured in the presence of 5'-adenylyl-beta,gamma-imidodiphosphate (ADPNP), a non-hydrolyzable ATP analog that is known to support limited supercoiling. The values of the radius of gyration, Rg = 67 A, from SANS and the hydrodynamic radius, Rh = 64 A, from DLS predict a larger than expected volume for the enzyme, supporting the notion of channels or cavities within the molecule. In addition, several classes of models were rejected based on SANS data obtained in 2H2O at larger scattering angles. The best fit to both the SANS and DLS data is obtained for oblate, inhomogeneous particles approximately 175 A wide and 52 A thick. Such particles provide a large surface area for DNA interaction. Both Rg and Rh values change very little upon addition of DNA, suggesting that DNA binds in a manner that does not significantly change the shape of the protein. No appreciable change in structure is found with the addition of ADPNP. However, the higher-angle SANS data indicate a slight rearrangement of the enzyme in the presence of nucleotide.     

Small angle neutron scattering as sensitive tool to detect ligand-dependent shape changes in a plant lectin with β-trefoil folding and their dependence on the nature of the solvent

The galactoside-specific Viscum album L. agglutinin (VAA) is a potent biohazard akin to ricin and a mitogen for immune and tumor cells. These activities depend on cell surface binding to glycans. It is an open question whether the process of ligand binding alters the lectin's shape. Small angle neutron scattering (SANS) experiments revealed that the carbohydrate ligand lactose induced a decrease of the radius of gyration of dimeric VAA from 54.5 +/- 1 to 49.5 +/- 1 A in water. Apparently, VAA in aqueous solution and at the concentrations tested at 3.6 mg/ml and above adopts a compacted structure as response to ligand binding. In contrast to the behavior in aqueous solution, lactose binding in DMSO resulted in an increase of the lectin's radius of gyration from 49 +/- 1 to 55.5 +/- 1 A. Because shape changes may be reflected in the thermostability of the protein, this parameter was examined by activity assays of protein exposed to 60 degrees C and 70 degrees C and by differential scanning calorimetry (DSC). In line with the lactose-induced conformational alterations revealed by the SANS experiments, lactose presence enhanced the thermostability of VAA in water. Thus, binding of the carbohydrate ligand in solution can entail changes in shape and thermostability in the case of the tested plant lectin.     

Structural studies of agar-gelatin complex coacervates by small angle neutron scattering, rheology and differential scanning calorimetry

Agar-gelatin complex coacervates are studied by small angle neutron scattering (SANS), rheology (in both flow and temperature scan modes) and differential scanning calorimetry (DSC) in order to probe the microscopic structure of this dense protein-polysaccharide-rich phase. DSC and isochronal temperature sweep (rheology) experiments yielded a characteristic temperature at approximately 35+/-2 degrees C. Rheology data revealed a second characteristic temperature at approximately 75+/-5 degrees C which was absent in DSC thermograms. In the flow mode, shear viscosity (eta) was found to scale with (Carreau model) applied shear rate (gamma ) as: eta(gamma ) approximately (gamma )(-k) with k=1.2+/-0.2 indicating non-Newtonian and shear-thinning features independent of ionic strength. The static structure factor S(q) deduced from SANS data in the low wave vector (0.018 A(-1)相似文献   

Bilayer thickness and lipid interface area in unilamellar extruded 1,2-diacylphosphatidylcholine liposomes: a small-angle neutron scattering study

Small-angle neutron scattering (SANS) experiments have been performed on large unilamellar liposomes prepared from 1,2-dilauroylphosphatidylcholine (DLPC), 1,2-dimyristoyl-phosphatidylcholine (DMPC) and 1,2-distearoylphosphatidylcholine (DSPC) in heavy water by extrusion through polycarbonate filters with 500 A pores. The neutron scattering intensity I(Q) in the region of scattering vectors Q corresponding to 0.0015 A(-2) < or = Q(2) < or = 0.0115 A(-2) was fitted using a step function model of bilayer neutron scattering length density and supposing that the liposomes are spherical and have a Gaussian distribution of radii. Using the lipid volumetric data, and supposing that the thickness of bilayer polar region equals to d(H) = 9+/-1 A and the water molecular volume intercalated in the bilayer polar region is the same as in the aqueous bulk aqueous phase, the steric bilayer thickness d(L), the lipid surface area A(L) and the number of water molecules per lipid molecule N intercalated in the bilayer polar region were obtained: d(L) = 41.58+/-1.93 A, A(L) = 57.18+/-1.00 A(2) and N = 6.53+/-1.93 in DLPC at 20 degrees C, d(L) = 44.26+/-1.42 A, A(L) = 60.01+/-0.75 A(2) and N = 7.37+/-1.94 in DMPC at 36 degrees C, and d(L) = 49.77+/-1.52 A, A(L) = 64.78+/-0.46 A(2) and N = 8.67+/-1.97 in DSPC at 60 degrees C. After correcting for area thermal expansivity alpha approximately 0.00417 K(-1), the lipid surface area shows a decrease with the lipid acyl chain length at 60 degrees C: A(L) = 67.56+/-1.18 A(2) in DLPC, A(L) = 66.33+/-0.83 A(2) in DMPC and A(L) = 64.78+/-0.46 A(2) in DSPC. It is also shown that a joint evaluation of SANS and small-angle X-ray scattering on unilamellar liposomes can be used to obtain the value of d(H) and the distance of the lipid phosphate group from the bilayer hydrocarbon region d(H1).     

Small angle neutron scattering studies of C8 and C9 and their interactions in solution.

Small angle neutron scattering (SANS) results revealed that contrary to most reports C9 is not a globular protein. Its radius of gyration (Rg) at pH 8 and an ionic strength of 0.5 is 32.2 +/- 1.4 A increasing to 35 A at physiologic ionic strength. In contrast, C8, which has a 2.2-fold larger mass, has a similar Rg value [34.6 +/- 1.6 A]. Calibration plots of Rg vs. M(r) indicate that native C8 is a spherical protein whereas native C9 is elongated. From previous reports it was known that native C8 and C9 associate in solutions of low ionic strength. SANS results confirmed this observation but also demonstrated that C8-C9 heterodimers are already formed at physiologic ionic strength. The dimeric complex is globular [Rg = 40 +/- 0.8 A] indicating that the proteins associate side-by-side rather than end-to-end. In contrast, in presence of the drug Suramin, a potent inhibitor of the assembly of the C5b-9 complex, C9 forms a complex with twice the molecular mass that is still elongated (Rg = 48.8 +/- 0.8 A), suggesting that in this case the protein dimerizes end-to-end via a bridging Suramin molecule.     

Assembly of bacteriophage T7. Dimensions of the bacteriophage and its capsids.

The dimensions of bacteriophage T7 and T7 capsids have been investigated by small-angle x-ray scattering. Phage T7 behaves like a sphere of uniform density with an outer radius of 301 +/- 2 A (excluding the phage tail) and a calculated volume for protein plus nucleic acid of 1.14 +/- 0.05 x 10(-16) ml. The outer radius determined for T7 phage in solution is approximately 30% greater than the radius measured from electron micrographs, which indicates that considerable shrinkage occurs during preparation for electron microscopy. Capsids that have a phagelike envelope and do not contain DNA were obtained from lysates of T7-infected Escherichia coli (capsid II) and by separating the capsid component of T7 phage from the phage DNA by means of temperature shock (capsid IV). In both cases the peak protein density is at a radius of 275 A; the outer radius is 286 +/- 4 A, approximately 5% smaller than the envelope of T7 phage. The thickness of the envelope of capsid II is 22 +/- 4 A, consistent with the thickness of protein estimated to be 23 +/- 5 A in whole T7 phage, as seen on electron micrographs in which the internal DNA is positively stained. The volume in T7 phage available to package DNA is estimated to be 9.2 +/- 0.4 x 10(-17) ml. The packaged DNA adopts a regular packing with 23.6 A interplanar spacing between, DNA strands. The angular width of the 23.6 A reflection shows that the mean DNA-DNA spacing throughout the phage head is 27.5 +/- less than 2.2 A. A T7 precursor capsid (capsid I) expands when pelleted for x-ray scattering in the ultracentrifuge to essentially the same outer dimensions as for capsids II and IV. This expansion of capsid I can be prevented by fixing with glutaraldehyde; fixed capsid I has peak density at a radius of 247 A, 10% less than capsid II or IV.     

Prosomes (proteasomes) of higher plants

From different plant tissues such as tobacco (Nicotiana rustica), potato (Solanum tuberosum), and mung bean (Phaseolus radiatus), ring- or cylinder-shaped particles called prosomes were isolated by either sucrose gradient centrifugation or fast protein liquid chromatography (FPLC). These particles have a diameter of 12 to 14 nm and a length of 16 to 18 nm. They migrate under conditions of nondenaturing gel electrophoresis as one distinct band. Sedimentation coefficient and buoyant density in Cs2SO4 of the plant prosomes were determined by analytical ultracentrifugation to be approximately 23S and 1.23 g/cm3, respectively. The total molecular mass was estimated by gel filtration to be 650 kDa. Plant prosomes are composed of 12 to 15 proteins with molecular masses in the range of 24 to 35 kDa with isoelectric points of pH 5 to 7 as revealed by two-dimensional gel electrophoresis. The protein patterns of prosomes from the three different plant species are very similar. Polyclonal antisera against potato prosomes reacted in Western blots with prosomal proteins of all three plant species. They also bind to some prosomal proteins of animal species. Antisera against animal prosomes react with some proteins of plant prosomes. As shown by lectin blotting, plant prosomes are glycosylated carrying glucosyl- or mannosyl, and N-acetylgalactosaminyl residues. Prosomal preparations contain non-stoichiometric amounts of small RNA of about 80 kDa. These results suggest that plant prosomes are structurally and functionally homologous to prosomes of other eukaryotic cells.     

An analysis by low-angle neutron scattering of the structure of the acetylcholine receptor from Torpedo californica in detergent solution.

The acetylcholine receptor from the electric tissue of Torpedo californica is a large, integral membrane protein containing four different types of polypeptide chains. The structure of the purified receptor in detergent solution has previously been investigated by sedimentation analysis and gel filtration. Sedimentation analysis yielded a molecular weight of 250,000 for the protein moiety of the receptor monomer-detergent complex; hydrodynamic characteristics such as the Stokes radius, however, refer to the receptor-detergent complex. In this paper we report the results of our use of low-angle neutron scattering to investigate the shape of the receptor-detergent (Triton X-100 from Rohm & Haas Co., Philadelphia, Pa.) complex and separately of its protein and detergent moieties. By adjustment of the neutron-scattering density of the solvent with D2O to match that of one or the other of the moieties, its contribution to the scattering can be nearly, if not completely, eliminated. Neutron scattering from Triton X-100 micelles established that this detergent is contrast matched in approximately 18% D2O. Scattering measurements on the receptor-detergent complex in this solvent yielded a radius of gyration of the acetylcholine receptor monomer of 46 +/- 1A. The radius of gyration and molecular volume (305,000 A3) of the receptor are inconsistent with a compact spherical shape. These parameters are consistent with, for example, a prolate cylinder of dimensions (length x diameter) approximately 150 x approximately 50 A or an oblate cylinder, approximately 25 x approximately 130 A. More complex shapes are possible and in fact seem to be required to reconcile the present results with previous electron microscopic and x-ray analyses of receptor in membrane and with considerations of the function of the receptor in controlling ion permeability. The neutron-scattering data yield, in addition, an independent determination of the molecular weight of the receptor protein (240,000 +/- 40,000), the extent of Triton X-100 binding in the complex (approximately 0.4 g/g protein), and from the extended scattering curve, an approximation to the shape of the receptor-Triton X-100 complex, namely an oblate ellipsoid of axial ratio 1:4.     

Using small-angle neutron scattering to study the solution conformation of N-(2-hydroxypropyl)methacrylamide copolymer-doxorubicin conjugates

Our past research developed two N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-doxorubicin (Dox) conjugates that became the first synthetic polymer-anticancer conjugates to be evaluated clinically. The first, FCE28068, contained Dox bound to the polymeric carrier via a tetrapeptidic linker (glycine-phenylalanine-leucine-glycine (GFLG)) (Mw approximately 30,000 g/mol; approximately 8 wt % drug), and the second, FCE28069, contained additionally galactosamine (Gal) (Mw approximately 30,000 g/mol; approximately 7.5 wt % Dox) again bound by a GFLG linker. Galactosamine was included to promote hepatocyte/hepatoma targeting via the asialoglycoprotein receptor. Both conjugates showed antitumor activity and were clinically less toxic than free Dox (2-5 fold). However, despite their similar chemical characteristics, the conjugates displayed a significantly different maximum-tolerated dose (MTD) in patients. The aim of this study, therefore, was to use small-angle neutron scattering (SANS) to explore the solution behavior of a small library of HPMA polymer conjugates including FCE28068, FCE28069, and their pharmaceutical formulations, plus as reference compounds HPMA copolymer-GFLG conjugates containing aminopropanol (Ap) or galactosamine (Gal) alone (i.e., without Dox). The SANS data obtained showed that HPMA copolymer-GFLG-Ap conjugates (containing 5 and 10 mol % side chains) showed evidence of polymer aggregation, however, no indication of aggregation was observed for FCE28068 and FCE28069 over the concentration range studied (2.5-50 mg/mL). Clear differences in the scattering behavior for the two conjugates were observed at equivalent concentration. Data were best fitted by a model for polydisperse Gaussian coils, and the HPMA copolymer-Dox conjugate with Gal (FCE28069) exhibited a larger radius of gyration (Rg) (by approximately 2.5 nm) compared to FCE28068. In conclusion, we have shown that SANS will be a valuable tool to elucidate conformation-performance relationships for polymer-drug conjugates.     

Solution structure and dynamics of cartilage aggrecan

We studied the structure and dynamics of porcine laryngeal aggrecan in solution using a range of noninvasive techniques: dynamic light scattering (DLS), small-angle neutron scattering (SANS), video particle tracking (VPT) microrheology, and diffusing wave spectroscopy (DWS). The data are analyzed within the framework of a combined static and dynamic scaling model, and evidence is found for reptation of the comb backbones with unentangled side-chain dynamics. Small-angle neutron scattering indicated standard polyelectrolyte scaling of the mesh size (xi) with concentration (c) in semidilute solutions for the whole aggrecan aggregate, xi = Ac(-0.47+/-0.04), with the prefactor (A) implying there is on average 60 nm between the aggrecan subunits along the backbone. VPT demonstrated large exponents for the power law dependence of the intrinsic viscosity (eta) on the polymer concentration in the semidilute concentration regime, eta approximately c(alpha); with alpha equal to 2.04 +/- 0.06 and 1.95 +/- 0.08 for the assembled and disassembled aggrecan aggregates, respectively. DWS at high frequencies (10(4)-10(5) Hz) gave evidence for internal Rouse modes of the aggrecan monomers, independent of the degree of self-assembly of the molecules.     

Isolation and physical characterization of an exocellular polysaccharide

The physical properties of a polysaccharide produced by the lactic acid bacterium Lactococcus lactis subsp. cremoris strain NIZO B40 were investigated. Separation of the polysaccharide from most low molar mass compounds in the culture broth was performed by filtration processes. Residual proteins and peptides were removed by washing with a mixture of formic acid, ethanol, and water. Gel permeation chromatography (GPC) was used to size fractionate the polysaccharide. Fractions were analyzed by multiangle static light scattering in aqueous 0.10 M NaNO3 solutions from which a number- (Mn) and weight-averaged (Mw) molar mass of (1.47 +/- 0.06).10(3) and (1.62 +/- 0.07).10(3) kg/mol, respectively, were calculated so that Mw/Mn approximately 1.13. The number-averaged radius of gyration was found to be 86 +/- 2 nm. From dynamic light scattering an apparent z-averaged diffusion coefficient was obtained. Upon correcting for the contributions from intramolecular modes by extrapolating to zero wave vector a hydrodynamic radius of 86 +/- 4 nm was calculated. Theoretical models for random coil polymers show that this z-averaged hydrodynamic radius is consistent with the z-averaged radius of gyration, 97 +/- 3 nm, as found with GPC.     

An elongated model of the Xenopus laevis transcription factor IIIA-5S ribosomal RNA complex derived from neutron scattering and hydrodynamic measurements.

The precise molecular composition of the Xenopus laevis TFIIIA-5S ribosomal RNA complex (7S particle) has been established from small angle neutron and dynamic light scattering. The molecular weight of the particle was found to be 95,700 +/- 10,000 and 86,700 +/- 9000 daltons from these two methods respectively. The observed match point of 54.4% D2O obtained from contrast variation experiments indicates a 1:1 molar ratio. It is concluded that only a single molecule of TFIIIA, a zinc-finger protein, and of 5S RNA are present in this complex. At high neutron scattering contrast radius of gyration of 42.3 +/- 2 A was found for the 7S particle. In addition a diffusion coefficient of 4.4 x 10(-11) [m2 s-1] and a sedimentation coefficient of 6.2S were determined. The hydrodynamic radius obtained for the 7S particle is 48 +/- 5 A. A simple elongated cylindrical model with dimensions of 140 A length and 59 A diameter is compatible with the neutron results. A globular model can be excluded by the shallow nature of the neutron scattering curves. It is proposed that the observed difference of 15 A in length between the 7S particle and isolated 5S RNA most likely indicates that part(s) of the protein protrudes from the end(s) of the RNA molecule. There is no biochemical evidence for any gross alteration in 5S RNA conformation upon binding to TFIIIA.     

Structure of the myosin head in solution and the effect of light chain 2 removal.

Structural properties of rabbit skeletal myosin head (S1) and the influence of the DTNB light chain (LC2) on the size and shape of myosin heads in solution were investigated by small angle x-ray scattering. The LC2 deficient myosin head, S1 (-LC2), and the S1 containing LC2 light chain, S1 (+LC2) were studied in parallel. The respective values of the radius of gyration were found to be (40.2 +/- 0.5) A and (46.7 +/- 1) A, while the maximum dimension was (190 +/- 15) A for both species. The large difference between the two Rg values suggest that LC2 is located close to one extremity of the myosin head, in agreement with most electron microscopy observations. All models derived from the x-ray scattering pattern of the native myosin head share a common overall morphology, showing two main regions, an asymmetric globular portion which tapers smoothly into a thinner domain of roughly equivalent length making an angle of approximately 60 degrees, with a contour length of approximately 210 A.     

Determination of asymmetric structure of ganglioside-DPPC mixed vesicle using SANS,SAXS, and DLS

Functions of mammalian cell membrane microdomains being rich in glycosphingolipids, so-called rafts, are now one of the current hot topics in cell biology from the intimate relation to cell adhesion and signaling. However, little is known about the role of glycosphingolipids in the formation and stability of the domains. By the use of the inverse contrast variation method in small-angle neutron scattering (SANS), combined with small-angle x-ray scattering (SAXS) and dynamic light scattering (DLS), we have determined an asymmetric internal structure of the bilayer of the small unilamellar vesicle (SUV) of monosialoganglioside (G(M1))-dipalmitoylphosphatidylcholine (DPPC) mixture ([G(M1)]:[DPPC] = 0.1:1). A direct method using a shell-model fitting with a size distribution function describes consistently all experimental results of SANS, SAXS, and DLS. We have found that G(M1) molecules predominantly localize at SUV outer surface to form a highly hydrophilic layer which is dehydrated with the rise of temperature from 25 degrees C to 55 degrees C accompanied by the conformational change of the oligosaccharide chains. The average SUV size determined is approximately 200 A, which is comparable to the reported value 260 +/- 130 A of glycosphingolipids microdomains. The present results suggest that the preferential asymmetric distribution of gangliosides is essential to define the size and stability of the domains.     

Hydration of NaDNA by neutron quasi-elastic scattering.

Preliminary results of neutron quasi-elastic scattering experiments are reported for hydrated paracrystals of sodium deoxyribonucleic acid (NaDNA). The samples were investigated at two water contents: 3.5 +/- 1.0 and 9.5 +/- 1.5 mol H2O per mole nucleotide. The results of the scattering experiments were almost independent of whether the NaDNA fibers were oriented parallel or perpendicular to the momentum transfer. The data indicate that at the lower hydration the water molecules do not diffuse appreciably on the time scale of the neutron measurements (approximately 3 X 10(-10) s). At the higher hydration the water molecules diffuse isotropically in a sphere of 9 A in diameter with a diffusion coefficient of (5 +/- 2) X 10(-6) cm2 s-1.     

Understanding the mechanism of action of poly(amidoamine)s as endosomolytic polymers: correlation of physicochemical and biological properties

Bioresponsive poly(amidoamine)s (PAA)s are currently under development as endosomolytic polymers for intracellular delivery of proteins and genes. Here for the first time, small-angle neutron scattering (SANS) is used to systematically investigate the pH-dependent conformational change of an endosomolytic polymer, the PAA ISA 23. The radius of gyration of the ISA23 was determined as a function of pH and counterion, the aim being to correlate changes in polymer conformation with membrane activity assessed using a rat red blood cell haemolysis assay. With decreasing pH, the ISA23 radius of gyration increased to a maximum (R(g) approximately 80 A) around pH = 3, before subsequently decreasing once more. At high pH and therefore high ionic strengths, the polymer is negatively charged and adopts a rather compact structure (R(g) approximately 20 A), presumably with the dissociated carboxylic groups on the exterior of the polymer coil. At low pH, the coil again collapses (R(g) < 20 A), presumably due to the effects of the high ionic strength. It is concluded that the nature of the salt form has no direct bearing on the size of the polymer coil, but it does indirectly determine the prevailing pH and, hence, polymer conformation. Pulsed-gradient spin-echo NMR measurements were in good agreement with the SANS estimates of the radius of gyration, although ISA23 polydispersity does complicate the data interpretation/comparison. These results support the proposed mode of action of PAAs, namely a coil expansion on passing from a neutral pH (extracellular) to an acidic pH (endosomal and lysosomal) environments. The results do, however, suggest that the charge on the polymer shows a closer correlation with the haemolysis activity rather than the polymer conformation.