Monoclonal antibody-beta-lactamase conjugates for the activation of a cephalosporin mustard prodrug.

Cephalosporin mustard (CM) was designed as an anticancer prodrug that could be activated in a site-specific manner by monoclonal antibody-beta-lactamase conjugates targeted to antigens present on tumor cell surfaces. Purified beta-lactamases from Bacillus cereus (BC beta L) and Escherichia coli (EC beta L) catalyzed the release of phenylenediamine mustard (PDM) from CM through a fragmentation reaction which occurs after the beta-lactam ring of CM is hydrolyzed. The Km and Vmax values were 5.7 microM and 201 mumol/min per mg for BC beta L and 43 microM and 29 mumol/min per mg for EC beta L, respectively. Conjugates of BC beta L were prepared by combining the F(ab')2 fragments of the maleimide-substituted monoclonal antibodies L6 and 1F5 with thiolated BC beta L. The conjugates showed little loss in enzymatic activity and bound nearly as well as the unmodified F(ab')2 monoclonal antibodies to antigens expressed on the H2981 human lung adenocarcinoma cell line (L6 positive, 1F5 negative). PDM was approximately 50-fold more cytotoxic than CM to H2981 cells. Treatment of the cells with L6-BC beta L followed by CM resulted in a level of cytotoxic activity that was comparable to that of PDM. This was most likely due to activation of CM by conjugate that bound to cell-surface antigens, since pretreatment of H2981 cells with BC beta L or 1F5-BC beta L enhanced the activity of CM to a lesser extent. Thus, we have shown that CM is a prodrug, and that it can be activated with immunological specificity by a monoclonal antibody-beta-lactamase conjugate.     

Uptake and concentration of bioactive macromolecules by K562 cells via the transferrin cycle utilizing an acid-labile transferrin conjugate.

The transferrin cycle was used to attempt the import of bioactive macromolecules into cells with the aid of an acid-labile cross-linking agent. Anti-tetanus F(ab')2 fragments were iodinated and then conjugated to transferrin with a newly developed acid-labile cleavable cross-linking reagent, bismaleimidoethoxy propane, following thiolation of both proteins. Noncleavable conjugates were also prepared. At saturating conjugate concentrations, the uptake rate for both conjugates averaged over the first 2 h is about 6.5 fmol/million cells/min. Incubation of loaded cells in fresh medium for 30 min and analysis of cell pellets and supernatants reveal that 1) of the previously cell-associated label, only intact conjugate (about 50% of the label) is returned to the medium; 2) most of the remaining cell-associated material for the cleavable conjugate is chromatographically coincident with free Fab with some contribution from free F(ab')2 fragments. In contrast, the cell pellets loaded with noncleavable conjugates contained intact transferrin-F(ab'), conjugates. These results are consistent with transferrin receptor-mediated uptake of acid-labile conjugate followed by hydrolysis in acidified endosomes and resulting in concentration of free F(ab')2 and Fab within a prelysosomal intracellular compartment. A protein shuttle such as transferrin may therefore be used with ketal based acid-labile cross-linkers to load foreign molecules into an intracellular compartment. In addition, these data provide independent confirmation of the low pH compartment within the transferrin cycle. This new methodology is applicable to other cases of receptor/ligand trafficking to report low pH compartments independent of morphological analysis. Since transferrin receptors are overexpressed in tumors, antineoplastic agents could be targeted to tumors as transferrin acid-labile conjugates. This import system might be particularly useful in combatting the tumor cell export of antitumor agents occurring in multidrug resistance.     

Multivalent conjugates of poly-gamma-D-glutamic acid from Bacillus licheniformis with antibody F(ab') and glycopeptide ligands

Poly-gamma-D-glutamic acid from Bacillus licheniformis is a water-soluble, nontoxic, nonimmunogenic exopolymer. Using synthetic linkers, the alpha-carboxylate side chains of PGA were conjugated to an exposed thiol side chain of an antibody F(ab') fragment, Mc109F4. Analysis of the PGA-Mc109F4 conjugate by gel filtration HPLC revealed a mixture of multivalent conjugates. The PGA-Mc109F4 conjugate retained biological activity, but showed a lower binding affinity to target BCL3B3 cells than free Mc109F4 F(ab')(2) by flow cytometry, and a lower efficacy for BCL3B3 growth inhibition than free Mc109F4 F(ab')(2). PGA was also conjugated with the free amino group of glycopeptide antibiotic vancomycin. The PGA-vancomycin conjugate showed slightly lower antibacterial activity than free vancomycin versus susceptible Bacillus subtilis, but slightly higher activity versus intrinsically resistant Leuconostoc mesenteroides.     

Uptake of antitetanus F(ab')2 fragments into eukaryotic cells

1. In order to introduce antitetanus immunoglobulin fragments into eukaryotic cells, either antitetanus F(ab')2 or Fab' fragments have been linked to carrier molecules. Aciclovir, horseradish peroxidase, wheat germ agglutinin, and transferrin were tried as carriers. 2. F(ab')2-aciclovir and Fab'-horseradish peroxidase were not internalized by NG108-15 neurohybridoma cells. 3. [Fab']2-wheat germ agglutinin and F(ab')2-transferrin conjugates were internalized into various cells. 4. F(ab')2-transferrin conjugates were made with three different linkers: N-succinimidyl 3-(2-pyridyldithio) propionate, bis-maleimido hexane, and bis-maleimidoethoxy propane. All three conjugates were internalized but had a different fate inside the cells.     

Hormonotoxins. Preparation and characterization of ovine luteinizing hormone-gelonin conjugate

With the aim of targeting toxins to selected cells in the gonad, we have prepared conjugates of ovine luteinizing hormone (oLH) with a single chain ribosome-inactivating protein called gelonin. The two proteins were thiolated by using N-succinimidyl-3-(2-pyridyldithio)propionate and subsequently reacted under appropriate conditions to form oLH-S-S-gelonin complex. A complete biochemical analysis of thiolated oLH and oLH-gelonin conjugates has been performed. The linkage of the hormone to the toxin probably occurred through a single amino group in the alpha-subunit, with the beta-subunit remaining free. Modification of a single amino group on the alpha-subunit reduced receptor binding and immunological reactivity of the thiolated oLH, but subsequent complexing with the toxin-gelonin did not seriously compromise these activities. oLH and gelonin were calculated to be present in a 1:1 ratio in the hormonotoxin preparation. The conjugate retained significant steroidogenic activity in rat granulosa cells. Upon reaction with mouse tumor Leydig cells (MA-10 cells), the toxin component of the complex became internalized to a sufficient degree to effectively inhibit protein synthesis. The studies provide a rational basis for the design and study of large hormonotoxins.     

Polyethylene glycol modification of the monoclonal antibody A7 enhances its tumor localization

The F(ab')2 fragment of murine monoclonal antibody A7 was covalently bonded to polyethylene glycol (PEG, molecular weight: 5000) and the conjugate was compared to the parent F(ab')2 fragment by in vitro and in vivo studies. PEG-conjugated antibody fragment retained its antigen-binding activity in a competitive radioimmunoassay. The conjugate had a longer half-life and showed increased accumulation in tumors. Although the tumor: blood ratio for parent F(ab')2 fragment was higher than that for the conjugate, it showed higher value than whole MAb A7. The tissue: blood ratios were kept low with the conjugate, indicating that the conjugate was uptaken to normal organ with lesser extent, as compared with parent F(ab')2 fragment. Our findings indicate that this PEG-conjugated F(ab')2 fragment could be a promising carrier for use in targeting cancer chemotherapy.     

Tumor cell targeting of transferrin-PEG-TNF-alpha conjugate via a receptor-mediated delivery system: design, synthesis, and biological evaluation

PEGylation is a procedure of growing interest for enhancing the therapeutic and biotechnological potential of peptides and proteins. Transferrin (Tf) has been proposed to be useful for targeting cancer cells. The aim of this study was to modify PEGylated recombinant human tumor necrosis factor alpha (PEG-TNF-alpha) with Tf to form Tf-PEG-TNF-alpha conjugates, which would maintain the advantages of PEGylation and also achieve the function of active targeting to tumor cells. In PEGylation reactions with 5-, 20-, 40-, and 60-fold molar excess of 3.4 kDa N-hydroxysuccinimide-PEG-maleimide (PT1, PT2, PT3, and PT4, respectively), PEG-TNF-alpha conjugates with different PEG chains were synthesized. A perfusion chromatography technique using a cation-exchange column was introduced to purify PEG-TNF-alpha conjugates. PT4 with about five PEG chains was selected as a lead candidate due to highest extent of PEGylation and maximum reaction yield. Thiolated Tf was conjugated to the maleimide group at the distal end of the PEG chains on the PEG-TNF-alpha conjugates, with the resulting Tf-PEG-TNF-alpha conjugates after purification containing approximately one Tf ligand on one TNF-alpha molecule. The conjugate of Tf and PT4 (TPT4) was selected to assess the specificity and affinity to transferrin receptor (TfR) on two kinds of tumor cells, K562 and KB. Both the receptor binding assays and the competition experiments were performed using radioligand binding analysis. The results demonstrated that TPT4 as well as Tf bound specifically to the TfR on the tumor cell surface and the affinity of the conjugate to TfR was similar to that of native Tf. In contrast, PEG-TNF-alpha demonstrated no specificity. The biodistribution and antitumor effects were investigated in S-180 tumor-bearing mice. It was found that TPT4 could markedly alter in vivo behavioral characteristics of TNF-alpha. Compared with TNF-alpha and PT4, extravasated TPT4 in tumor tissues exhibited a significantly delayed blood clearance and the highest intratumoral TNF-alpha levels. Furthermore, the inhibitory rate of tumor of TPT4 enhanced 5.3- and 1.8-fold over that of TNF-alpha and PT4, indicating that TPT4 exhibited the highest antitumor activity. These results suggested that Tf-PEG-TNF-alpha was a useful long circulating conjugate with the capabilities of specific receptor binding resulting in enhanced antitumor activity of TNF-alpha.     

A micro-scale method for the conjugation of affinity-purified Fab' to beta-D-galactosidase from Escherichia coli

A micro-scale method for the conjugation of affinity-purified Fab' to beta-D-galactosidase from Escherichia coli is described. Rabbit anti-human chorionic gonadotropin serum (0.2 ml) was digested with pepsin to convert IgG to F(ab')2 and applied to a column of human chorionic gonadotropin-Sepharose 4B, followed by elution at pH 2.5. The affinity-purified anti-human chorionic gonadotropin F(ab')2 was mixed with non-specific goat F(ab')2 (0.5 mg) as a carrier, reduced with 2-mercaptoethylamine to split F(ab')2 to Fab' and conjugated to beta-D-galactosidase using N,N'-o-phenylenedimaleimide. The affinity-purified rabbit anti-human chorionic gonadotropin Fab'-beta-D-galactosidase conjugate was separated from non-specific goat Fab'-beta-D-galactosidase conjugate and unconjugated beta-D-galactosidase by affinity chromatography on a column of goat (anti-rabbit IgG) IgG-Sepharose 4B using 4 M urea. The amount of the affinity-purified conjugate obtained was 56-69 micrograms. The detection limit of human chorionic gonadotropin by a sandwich enzyme immunoassay technique was improved 30-fold by using the affinity-purified conjugate as compared with that before affinity-purification. This method is applicable to the conjugation with alkaline phosphatase from calf intestine and probably also other enzymes which are stable in 4 M urea.     

In vivo gene delivery to tumor cells by transferrin-streptavidin-DNA conjugate.

To target disseminated tumors in vivo, transgenes [beta-galactosidase gene, green fluorescence protein (GFP) gene, herpes simplex virus thymidine kinase (HSV-TK)] were conjugated to transferrin (Tf) by a biotin-streptavidin bridging, which is stoichiometrically controllable, and Tf receptor (Tf-R) affinity chromatography, which selects Tf conjugates with intact receptor bindings sites from reacting with the linker. Tf-beta-galactosidase plasmid conjugate thus constructed was specifically transfected to human erythroleukemia cells (K562) via Tf-R without the aid of any lysosomotropic agents. The transfection efficiency of the conjugate was superior to those of lipofection (1% staining) and retroviral vector (5%) and slightly lower than that of adenovirus (70%). The high level of expression with our conjugate was confirmed using other tumor cells (M7609, TMK-1) whereas in normal diploid cells (HEL), which express low levels of Tf-R, expression was negligible. When GFP gene conjugates were systemically administered through the tail vein to nude mice subcutaneously inoculated with tumor, expression of GFP mRNA was found almost exclusively in tumors and to a much lesser extent in muscles, whereas GFP revealed by fluorescence microscopy was detected only in the former. To exploit a therapeutic applicability of this method, suicide gene therapy using Tf-HSV-TK gene conjugate for massively metastasized k562 tumors in severe combined immune-deficient mice was conducted, and a marked prolongation of survival and significant reduction of tumor burden were confirmed. Thus, this method could also be used for gene therapy to disseminated tumors.     

Pretargeting for cancer radioimmunotherapy with bispecific antibodies: role of the bispecific antibody's valency for the tumor target antigen

The use of a divalent effector molecule improves bispecific antibody (bsMAb) pretargeting by enabling the cross-linking of monovalently bound bsMAb on the cell surface, thereby increasing the functional affinity of a bsMAb. In this work, it was determined if a bsMAb with divalency for the primary target antigen would improve bsMAb pretargeting of a divalent hapten. The pretargeting of a (99m)Tc-labeled divalent DTPA-peptide, IMP-192, using a bsMAb prepared by chemically coupling two Fab' fragments, one with monovalent specificity to the primary target antigen, carcinoembryonic antigen (CEA), and to indium-loaded DTPA [DTPA(In)], was compared to two other bsMAbs, both with divalency to CEA. One conjugate used the whole anti-CEA IgG, while the other used the anti-CEA F(ab')(2) fragment to make bsMAbs that had divalency to CEA, but with different molecular weights to affect their pharmacokinetic behavior. The rate of bsMAb blood clearance was a function of molecular weight (IgG x Fab' < F(ab')(2) x Fab' < Fab' x Fab' conjugate). The IgG x Fab' bsMAb conjugate had the highest uptake and longest retention in the tumor. However, when used for pretargeting, the F(ab')(2) x Fab' conjugate allowed for superior tumor accretion of the (99m)Tc-IMP-192 peptide, because its more rapid clearance from the blood enabled early intervention with the radiolabeled peptide when tumor uptake of the bsMAb was at its peak. Excellent peptide targeting was also seen with the Fab' x Fab' conjugate, albeit tumor uptake was lower than with the F(ab')(2) x Fab' conjugate. Because the IgG x Fab' bsMAb cleared from the blood so slowly, when the peptide was given at the time of its maximum tumor accretion, the peptide was captured predominantly by the bsMAb in the blood. Several strategies were explored to reduce the IgG x Fab' bsMAb remaining in the blood to take advantage of its 3-4-fold higher tumor accretion than the other bsMAb conjugates. A number of agents were tested, including those that could clear the bsMAb from the blood (e.g., galactosylated or nongalactosylated anti-id antibody) and those that could block the anti-DTPA(In) binding arm [e.g., DTPA(In), divalent-DTPA(In) peptide, and DTPA coupled to bovine serum albumin (BSA) or IgG]. When clearing agents were given 65 h after the IgG x Fab' conjugate (time of maximum tumor accretion for this bsMAb), (99m)Tc-IMP-192 levels in the blood were significantly reduced, but a majority of the peptide localized in the liver. Increasing the interval between the clearing agent and the time the peptide was given to allow for further processing of the bsMAb-clearing agent complex did not improve targeting. At the dose and level of substitution tested, galacosylated BSA-DTPA(In) was cleared too quickly to be an effective blocking agent, but BSA- and IgG-DTPA(In) conjugates were able to reduce the uptake of the (99m)Tc-IMP-192 in the blood and liver. Tumor/nontumor ratios compared favorably for the radiolabeled peptide using the IgG x Fab'/blocking agent combination and the F(ab')(2) x Fab' (no clearing/blocking agent), and peptide uptake 3 h after the blocking agent even exceeded that of the F(ab')(2) x Fab'. However, this higher level of peptide in the tumor was not sustained over 24 h, and actually decreased to levels lower than that seen with the F(ab')(2) x Fab' by this time. These results demonstrate that divalency of a bsMAb to its primary target antigen can lead to higher tumor accretion by a pretargeted divalent peptide, but that the pharmacokinetic behavior of the bsMAb also needs to be optimized to allow for its clearance from the blood. Otherwise, blocking agents will need to be developed to reduce unwanted peptide uptake in normal tissues.     

Factors influencing the ability of nuclear localization sequence peptides to enhance nonviral gene delivery

Nonviral gene delivery is limited by inefficient transfer of DNA from the cytoplasm to the nucleus. Nuclear localization sequence (NLS) peptides have been widely used to exploit intracellular transport mechanisms and promote nuclear uptake of DNA. However, the exact conditions to successfully utilize the properties of NLS peptides are still unclear. In the present study a panel of NLS peptides that bind different transport receptors were compared for their ability to enhance nonviral gene transfer. Several factors such as method of incorporating the NLS peptide, type of NLS peptide, DNA morphology, and proper characterization of NLS peptide/DNA conjugates were identified as important considerations in utilizing NLS peptides to enhance gene transfer. In particular, it was shown that a peptide derived from human T cell leukaemia virus type 1 (HTLV) was able to effectively condense DNA into discrete particles and mediate levels of transgene expression up to 32-fold greater than polylysine-based polyplexes. This is the first study to demonstrate efficient transfection mediated by an importin beta-binding peptide based on the HTLV sequence. Promising results were also achieved with a 7-fold increase in gene expression using a NLS peptide/DNA conjugate formed by site-specific linkage of an extended SV40 peptide via a peptide nucleic acid (PNA) clamp. Altogether, the results from this study should help to define the requirements for successful NLS-enhanced transfection.     

Magnetic resonance imaging of inducible E-selectin expression in human endothelial cell culture.

Covalent conjugates of the cross-linked iron oxide nanoparticles (CLIO) and high-affinity (K(d)(app) = 8.5 nM) anti-human E-selectin (CD62E) F(ab')(2) fragments were prepared and tested in vitro to establish feasibility of endothelial proinflammatory marker magnetic resonance (MR) imaging. The conjugates were obtained by using thiol-disulfide exchange reaction between 3-(2-pyridyl)propionyl-CLIO and S-acetylthioacetate-modified F(ab')(2) fragments. The purified CLIO-F(ab')(2) conjugates (average hydrodynamic diameter 40.6 nm) were used in experiments with the live human endothelial umbilical vein cells (HUVEC). Cells treated with IL-1 beta expressed E-selectin and showed a 100-200 times higher binding of CLIO particles (83-104 ng iron/million cells) than control cells. The binding resulted in a high superparamagnetism of HUVEC with the transverse water proton relaxation time (T2) decrease to 30-40 ms in cell precipitates. Cells did not bind/internalize CLIO-F(ab')(2) conjugates prepared using a control fragment or nonconjugated iron oxide particles before or after treatment with IL-1 beta. MR imaging of cells showed a highly specific T2-weighted signal darkening associated with cells treated with IL-1 beta and incubated with anti-E selectin. Demonstration of MR imaging of E-selectin expression justifies further development of MR-targeted agents for monitoring tumor vascular endothelial proliferation, angiogenesis, and atherosclerosis.     

Preparation and performance of bispecific F(ab' gamma)2 antibody containing thioether-linked Fab' gamma fragments

A simple and efficient method is described for the production of pure bispecific F(ab' gamma)2 heterodimers, in which the individual antibody Fab' gamma fragments are joined via a stable thioether linkage. Hybrid molecules were constructed from both mouse monoclonal and rabbit polyclonal antibodies with equal efficiency, in the combinations mouse-rabbit and mouse-mouse. Peptic F(ab' gamma)2 fragments from the two chosen antibodies were first reduced to provide Fab' gamma SH. The SH groups on one of the Fab' gamma SH partners were then fully alkylated with o-phenylenedi-maleimide to provide free maleimide groups. Finally the two preparations, Fab' gamma mal and Fab' gamma SH, combined under conditions which allowed cross-linking of the maleimide and SH groups and avoided reoxidation of SH groups. The major product isolated from the reaction mixture after chromatography was always the F(ab' gamma)2 heterodimer (50 to 70%), other products being unreacted Fab' gamma and trace amounts of putative F(ab' gamma)3. Immunochemical analysis revealed that the thioether-linked F(ab' gamma)2 molecules were essentially all heterodimers, most of which had been joined via their Fd chains. The dual specificity of F(ab' gamma)2 heterodimers was tested functionally in three systems: 1) the combination (anti-idiotype + anti-phycoerythrin) linked L2C cells to the fluorochrome phycoerythrin, allowing fluorescence analysis; 2) the combination (anti-idiotype + anti-saporin) linked L2C cells to the ribosome-inactivating protein saporin, and transformed a subtoxic dose of saporin into a highly toxic mixture which prevented further protein synthesis by L2C cells; and 3) the combination of anti-idiotype with 3G8 (antibody to the Fc gamma receptor CD16) subjected L2C cells to cytotoxic attack by human mononuclear effectors.     

Evaluation of cell-penetrating peptides (CPPs) as vehicles for intracellular delivery of antisense peptide nucleic acid (PNA)

Cell-penetrating peptides (CPPs) are characterized by their ability to be internalized in mammalian cells. To investigate the relative potency of CPPs as carriers of medicinally relevant cargo, a positive read-out assay based on the ability of a peptide nucleic acid (PNA) oligomer to promote correct expression of a recombinant luciferase gene was employed. Seven different CPPs were included in the study: Transportan, oligo-arginine (R7-9), pTat, Penetratin, KFF, SynB3, and NLS. The CPP-PNA conjugates were synthesized by different conjugation chemistries: continuous synthesis, maleimide coupling, and ester or disulfide linkage. Under serum-free conditions PNA-SS-Transportan-amide (ortho)-PNA was found to be the most potent conjugate, resulting in maximum luciferase signal at a concentration of 1-2 microM. (D-Arg)9-PNA showed optimal efficacy at 5 microM but gave rise to only one-third of the luciferase signal obtained with the Transportan conjugate. The pTat- and KFF-PNA conjugates showed significantly lower efficacy. The penetratin-, SynB3-. and NLS-PNA conjugates showed only minimal or no activity. Serum was found to have a drastic negative impact on CPP-driven cellular uptake. PNA-SS-Transportan-acid (ortho) and (D-Arg)9-PNA were least sensitive to the presence of serum. Both the chemical nature and, in the case of Transportan, the position of the peptide PNA coupling were found to have a major impact on the transport capacity of the peptides. However, no simple relationship between linker type and antisense activity of the conjugates could be deduced from the data.     

Evaluation of a high-affinity QCM immunosensor using antibody fragmentation and 2-methacryloyloxyethyl phosphorylcholine (MPC) polymer

This study evaluated construction of a highly affinitive quartz crystal microbalance (QCM) immunosensor using anti-C-reactive protein (CRP) antibody and its fragments for CRP detection. Three types of antibody were immobilized on the surface of a QCM via covalent-bounding. Then affinity was evaluated through antigen-antibody binding between CRP and its antibody. Affinity between antigen-antibody was shown to be highest when anti-CRP F(ab')2-IgG antibody (70 microg/mL) was immobilized on the QCM. In case of anti-CRP F(ab')2-IgG antibody, affinity which was attributable to antigen-antibody binding was almost twice that of anti-CRP IgG antibody, which is used conventionally for QCM immunosensors. In addition, when it was treated with 2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate, so-called MPC polymer, highly affinitive and selective immunosensing for CRP was achieved without non-specific binding from plasma proteins in human serum. When anti-CRP F(ab')2-IgG antibody was immobilized on the QCM, the detection limit and the linearity of CRP calibration curve were achieved at concentrations from 0.001 to 100 microg/dL even during investigation in serum samples. Experimental results verified the successful construction of a highly affinitive and selective QCM-immunosensor which was modified with anti-CRP F(ab')2-IgG antibody and MPC polymer.     

Use of maleimide-thiol coupling chemistry for efficient syntheses of oligonucleotide-enzyme conjugate hybridization probes

Two general methods which exploit the reactivity of sulfhydryl groups toward maleimides are described for the synthesis of oligonucleotide-enzyme conjugates for use as nonradioisotopic hybridization probes. In the first approach, 6-maleimidohexanoic acid succinimido ester was used to couple 5'-thiolated oligonucleotide to calf intestine alkaline phosphatase to provide a 1:1 conjugate in 80-85% yield. The second strategy employed N,N'-1,2-phenylenedimaleimide to cross-link thiolated horseradish peroxidase or beta-galactosidase with a 5'-thiolated oligonucleotide in 58% and 65% yields, respectively. The oligonucleotide-alkaline phosphatase conjugate was able to detect 6 amol of target DNA in 4 h, while the horseradish peroxidase conjugate was found to be 40-fold lower in its sensitivity of detection by using dye precipitation assays.     

Structure-activity relationships of water-soluble cationic methacrylate/methacrylamide polymers for nonviral gene delivery.

A number of water-soluble cationic carriers was evaluated as transfectant. Almost all studied cationic methacrylate/methacrylamide polymers were able to condense the structure of plasmid DNA, yielding polymer/plasmid complexes (polyplexes) with a size of 0.1-0.3 micron and a slightly positive zeta-potential, which can be taken up by cells, e.g., via endocytosis. However, the transfection efficiency and the cytotoxicity of the polymers differed widely: the highest transfection efficiency and cytotoxicity were observed for poly[2-(dimethylamino)ethyl methacrylate], p(DMAEMA). Assuming that polyplexes enter cells via endocytosis, p(DMAEMA) apparently has advantageous properties to escape the endosome. A possible explanation is that, due to its average pK(a) value of 7.5, p(DMAEMA) is partially protonated at physiological pH and might behave as a proton sponge. This might cause a disruption of the endosome, which results in the release of both the polyplexes and cytotoxic endosomal/lysosomal enzymes into the cytosol. On the other hand, the analogues of p(DMAEMA) studied here have a higher average pKa value and have, consequently, a higher degree of protonation and a lower buffering capacity. This might be associated with a lower tendency to destabilize the endosome, resulting in both a lower transfection efficiency and a lower cytotoxicity. Furthermore, molecular modeling showed that, of all studied polymers, p(DMAEMA) has the lowest number of interactions with DNA. We therefore hypothesized that the superior transfection efficiency of p(DMAEMA) containing polyplexes can be ascribed to an intrinsic property of p(DMAEMA) to destabilize endosomes combined with an easy dissociation of the polyplex once present in the cytosol and/or the nucleus.     

Release characteristics of novel pH-sensitive p(HEMA-DMAEMA) hydrogels containing 3-(trimethoxy-silyl) propyl methacrylate

An amphiphilic hydrogel of poly(2-hydroxyethyl methacrylate) cross-linked with tetraethyleneglycol diacrylate (TEGDA) was synthesized to contain the hydrophobic monomer 3-(trimethoxy-silyl) propyl methacrylate (PMA) and the pH-responsive, hydrophilic monomer N',N'-dimethylaminoethyl methacrylate (DMAEMA). The gels were separately loaded with two biomolecular probes, insulin and protamine, via both physical entrapment and equilibrium imbibition methods. The release profiles for these biomolecular probes, possessing similar MW (5.7 and 4-6 kDa, respectively) but different pI's (5.3 and 10.0, respectively), were investigated with respect to variation in the pH of the bathing medium as well as the DMAEMA content, and the cross-link density of the hydrogel. Gels exhibited classical Fickian diffusion release profiles. For a typical gel composition 66:15:10:09 mol % (HEMA:DMAEMA:PMA:TEGDA), as the pH of the release media decreased from 7.3 to 4.0, the rate of release of both biomolecular probes increased. When loaded via entrapment, the insulin release rate increased ca. 4-fold (1.0-3.7 x 10(-7) cm(2) s(-1)), whereas that of protamine increased 10-fold (0.3-3.3 x 10(-7) cm(2) s(-1)). When loaded by imbibition, the insulin diffusion coefficient increased 2-fold (3.8-7.2 x 10(-7) cm(2) s(-1)), whereas that of protamine increased 3-fold (1.9-5.5 x 10(-7) cm(2) s(-1)). The reduction of pH, through its protonation of the gel network, has a more dramatic influence on protamine release, the result of its higher pI (10.0) compared to that of insulin (5.3). As the DMAEMA content of the hydrogel was increased from 0 to 20 mol %, the diffusion coefficient of protamine increased by ca. 7-fold (1.7-12.2 x 10(-7) cm(2) s(-1)), whereas that of insulin increased only ca. 2-fold (1.7-4.0 x 10(-7) cm(2) s(-1)). This differential release confirms the role of internal protonation in effecting the greater release of the protonated drug molecule. Increasing the TEGDA content from 3 to 15 mol % reduced the diffusion coefficient ca. 3-fold for insulin (1.6-0.5 x 10(-7) cm(2) s(-1)) and 5-fold for protamine (4.0-0.8 x 10(-7) cm(2) s(-1)). The final D(ip) at 15 mol % TEGDA suggests that the smaller mesh size offsets any differential release that arises from protonation. The presence of PMA in the hydrogel formulation, which contributes additional cross-links by reason of the formation of siloxane macromers, did not change the usually observed Fickian diffusion mechanism.     

Morphological characterization of the pathway of endocytosis and intracellular processing of transferrin and alpha-fetoprotein in human T lymphocytes stimulated with phytohemagglutinin (PHA)

Covalent conjugates of transferrin (Tf) and alpha-fetoprotein (AFP) with horseradish peroxidase (HRP) have been used to follow, at the ultrastructural level, the uptake and the intracellular pathway of these proteins in peripheral blood human lymphocytes stimulated by phytohemagglutinin (PHA) to blast formation. Both proteins enter specifically the cells via vesicles (60-70 nm in diameter) and endosomes. They are then observed in multivesicular bodies and tubular vesicular elements in the Golgi region. AFP is thus found in the same subcellular compartments as Tf and is probably also recycled, as most of the 125I-labeled protein leaves the cells undegraded. Unstimulated lymphocytes do not internalize significantly AFP-HRP. The uptake of a noncovalent conjugate of AFP-HRP and [3H]-arachidonic acid [3H-(20:4)] is usually poor, at 37 degrees C, in unstimulated lymphocytes as well as, at 4 degrees C, in lymphocytes stimulated for 72 h. Stimulated lymphocytes incubated at 37 degrees C with the radioactive conjugate show a heavy labeling of cell organelles and more particularly of lipid droplets. AFP could regulate the intracellular delivery of fatty acid molecules.     

Antimicrobial surfaces of viologen-quaternized poly((2-dimethyl amino)ethyl methacrylate)-Si(100) hybrids from surface-initiated atom transfer radical polymerization

To improve the antimicrobial ability of silicon-based bioelectronics and to tailor the silicon surfaces for inhibiting biofilm formation, well-defined functional polymer-Si(100) hybrids, consisting of nearly monodispersed poly((2-dimethylamino)ethyl methacrylate) (P(DMAEMA)) covalently tethered on the silicon surface and functionalized by viologen moieties, were prepared. P(DMAEMA)-Si hybrids were prepared via surface-initiated atom transfer radical polymerization (ATRP) of (2-dimethylamino)ethyl methacrylate (DMAEMA) on the hydrogen-terminated Si(100) surfaces (Si−H surfaces). The tertiary amino groups of the covalently immobilized (Si−C bonded) P(DMAEMA) brushes on the silicon substrates were quaternized by an alkyl halide to produce a high concentration of quaternary ammonium groups with biocidal functionality. Alternatively, covalent coupling of viologen moieties to the tertiary amino groups of P(DMAEMA) brushes produced the quaternized P(DMAEMA)-Si(100) hybrids with substantially enhanced antimicrobial capability, as well as capability to effectively inhibit biofilm formation. Thus, the viologen-quaternized P(DMAEMA)-Si(100) hybrids possess good antibacterial surface properties and are potentially useful to the silicon-based bioelectronics to ensure their efficiency, durability and reliability.