PEG-doxorubicin conjugates: influence of polymer structure on drug release, in vitro cytotoxicity, biodistribution, and antitumor activity

Polymer-drug conjugates (polymer therapeutics) are finding increasing use as novel anticancer agents. Here a series of poly(ethylene glycol) PEG-doxorubicin (Dox) conjugates were synthesized using polymers of linear or branched architecture (molecular weight 5000-20000 g/mol) and with different peptidyl linkers (GFLG, GLFG, GLG, GGRR, and RGLG). The resultant conjugates had a drug loading of 2.7-8.0 wt % Dox and contained <2.0% free drug (% total drug). All conjugates containing a GFLG linker showed approximately 30% release of Dox at 5 h irrespective of PEG molecular weight or architecture. The GLFG linker was degraded more quickly (approximately 57% Dox release at 5 h), and the other linkers more slowly (<16% release at 5 h), by lysosomal enzymes in vitro. In vitro there was no clear relationship between cytotoxicity toward B16F10 cells and the observed Dox release rate. All PEG conjugates were more than 10-fold less toxic (IC50 values > 2 microg/mL) than free Dox (IC50 value = 0.24 microg/mL). Biodistribution in mice bearing sc B16F10 tumors was assessed after administration of PEGs (5000, 10000, or 20000 g/mol) radioiodinated using the Bolton and Hunter reagent or PEG-Dox conjugates by HPLC. The 125I-labeled PEGs showed a clear relationship between Mw and blood clearance and tumor accumulation. The highest Mw PEG had the longest plasma residence time and consequently the greatest tumor targeting. The PEG-Dox conjugates showed a markedly prolonged plasma clearance and greater tumor targeting compared to free Dox, but there was no clear molecular weight-dependence on biodistribution. This was consistent with the observation that the PEG-Dox conjugates formed micelles in aqueous solution comprising 2-20 PEG-Dox molecules depending on polymer Mw and architecture. Although PEG-Dox showed greater tumor targeting than free Dox, PEG conjugation led to significantly lower anthracycline levels in heart. Preliminary experiments to assess antitumor activity against sc B16F10 in vivo showed the PEG5000linear (L)-GFLG-Dox and PEG10000branched (B)-GLFG-Dox (both 5 mg/kg Dox-equiv) to be the most active with T/C values of 146 and 143%, respectively. Free Dox did not show significant activity in this model (T/C = 121%). Dose escalation of PEG5000(L)-GFLG-Dox to 10 mg/kg Dox-equiv prolonged further animal survival (T/C = 161%). Using the Dox-sensitive model ip L1210 (where Dox displayed a T/C = 150% after single ip dose), the PEG5000(L)-GFLG-Dox displayed a maximum T/C of 141% (10 mg/kg Dox-equiv) using a once a day (x3) schedule. Further studies are warranted with PEG5000(L)-GFLG-Dox to determine its spectrum of antitumor activity and also the optimum dosing schedule before clinical testing.     

Poly(ethylene glycol)-poly(ester-carbonate) block copolymers carrying PEG-peptidyl-doxorubicin pendant side chains: synthesis and evaluation as anticancer conjugates

Water soluble polymer anticancer conjugates can improve the pharmacokinetics of covalently bound drugs by limiting cellular uptake to the endocytic route, thus prolonging plasma circulation time and consequently facilitating tumor targeting by the enhanced permeability and retention (EPR) effect. Many of the first generation antitumor polymer conjugates used nonbiodegradable polymeric carriers which limits the molecular weight that can be safely used to <40,000 g/mol. The aim of this ambitious study was to synthesize and evaluate a novel, prototype biodegradable polymeric system based on high molecular weight, water-soluble functionalized polyesters. The main polymeric platform was prepared from bis(4-hydroxy)butyl maleate (DBM) and poly(ethylene glycol) (PEG4000) blocks to give the polymer DBM2-PEG4000 containing biodegradable carbonate bonds and having a M(w) of 100,000-190,000 g/mol; M(n) of 37,000-53,000 g/mol, and M(w)/M(n) of 3.0-3.7. Using thioether linkages, this polymer was then grafted with HS-PEG3000-Gly-Phe-Lue-Gly doxorubicin (HS-PEG3000-GFLG-Dox) pendant side chains ( approximately 30 per DBM2-PEG chain). The final construct, DBM2-PEG4000-S-PEG3000-GFLG-Dox had a total Dox content of 3-4 wt % and a free Dox content of < or = 0.7% total Dox. During incubation with isolated lysosomal enzymes, the rate of Dox release from the polymer backbone was relatively slow (<5% release over 5 h) compared to that seen for PEG5000-GFLG-Dox alone (>20% over 5 h). The in vitro cytotoxicity was assessed using B16F10 murine melanoma (MTT assay). DBM2-PEG4000-S-PEG3000-GFLG-Dox was 10-20-fold less toxic than free Dox. In vivo antitumor activity of the DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates was assessed using a subcutaneous (s.c.) B16F10 murine melanoma model, and an intraperitoneal (i.p.) L1210 leukaemia model. The increased toxicity (attributed to poor solubility) and low antitumor activity of DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates compared to PEG5000-GFLG-Dox and HPMA copolymer-Dox conjugates was attributed to the slow rate of Dox release. The DBM2-PEG4000-S-PEG3000-GFLG-Dox conjugates were considered unfavorable as candidates for further development. However, the successful scale-up synthesis of DBM2-PEG4000-S-PEG3000 constructs suggest that they are worthy of further investigation as carriers for controlled release and targeting of less hydrophobic agents.     

Macromolecular HPMA-Based Nanoparticles with Cholesterol for Solid-Tumor Targeting: Detailed Study of the Inner Structure of a Highly Efficient Drug Delivery System

We report a rigorous investigation into the detailed structure of nanoparticles already shown to be successful drug delivery nanocarriers. The basic structure of the drug conjugates consists of an N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer bearing the anticancer drug doxorubicin (Dox) bound via a pH-sensitive hydrazone bond and a defined amount of cholesterol moieties that vary in hydrophobicity. The results show that size, anisotropy, and aggregation number N(aggr) of the nanoparticles grows with increasing cholesterol content. From ab initio calculations, we conclude that the most probable structure of HPMA copolymer-cholesterol nanoparticles is a pearl necklace structure, where ellipsoidal pearls mainly composed of cholesterol are covered by a HPMA shell; pearls are connected by bridges composed of hydrophilic HPMA copolymer chains. Using a combination of techniques, we unambiguously show that the Dox moieties are not impregnated inside a cholesterol core but are instead uniformly distributed across the whole nanoparticle, including the hydrophilic HPMA shell surface.     

N-(2-hydroxypropyl)methacrylamide copolymer-6-(3-aminopropyl)-ellipticine conjugates. Synthesis, in vitro, and preliminary in vivo evaluation

Ellipticine derivatives have potential as anticancer drugs. Their clinical use has been limited, however, by poor solubility and host toxicity. As N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-anticancer conjugates are showing promise in early clinical trials, a series of novel HPMA copolymer conjugates have been prepared containing the 6-(3-aminopropyl)-ellipticine derivative (APE, NSC176328). Drug was linked to the polymer via GFLG or GG peptide side chains. To optimize biological behavior, HPMA copolymer-GFLG-APE conjugates with different drug loading (total APE: 2.3-7% w/w; free APE: <0.1% w/w) were synthesized. Conjugation of APE to HPMA copolymers considerably increased its aqueous solubility (>10-fold). HPMA copolymer-GG-APE did not liberate drug in the presence of isolated lysosomal enzymes (tritosomes), but HPMA copolymer-GFLG-APE released APE to a maximum of 60% after 5 h. The rate of drug release was influenced by drug loading; lower loading led to greater release. Whereas free APE (35 microg/mL) caused significant hemolysis (50% after 1 h), HPMA copolymer-APE conjugates were not hemolytic up to 300 microg/mL (APE-equiv). As would be expected from its cellular pharmacokinetics, HPMA copolymer-GFLG-APE was >75 times less cytotoxic than free drug (IC(50) approximately 0.4 microg/mL) against B16F10 melanoma in vitro. However, in vivo when tested in mice bearing s.c. B16F10 melanoma, HPMA copolymer-GFLG-APE (1-10 mg/kg single dose, APE-equiv) given i.p. was somewhat more active (highest T/C value of 143%) than free APE (1 mg/kg) (T/C =127%). HPMA copolymer-APE conjugates warrant further evaluation as potential anticancer agents.     

Antisense oligonucleotides delivered to the lysosome escape and actively inhibit the hepatitis B virus

The subcellular fate and activity in inhibiting the hepatitis B virus of free and N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-phosphorothioate oligonucleotides were studied. Their internalization and subcellular fate were monitored with confocal microscopy. A fraction of the internalized free oligonucleotides escaped into the cytoplasm and nucleus of Hep G2 cells but were not active antiviral agents. Covalently attaching the oligonucleotides to the HPMA copolymers via nondegradable dipeptide GG spacers resulted in sequestering the oligonucleotides in vesicles after internalization. Conjugation of the oligonucleotides to an HPMA copolymer via a lysosomally cleavable tetrapeptide GFLG spacer resulted in release of the oligonucleotide in the lysosome and subsequent translocation into the cytoplasm and nucleus of the cells. The HPMA copolymer-oligonucleotide conjugate possessed antiviral activity, indicating that phosphorothioate oligonucleotides released from the carrier in the lysosome were able to escape into the cytoplasm and nucleus and remain active. The Hep G2 cells appeared to actively internalize the phosphorothioate oligonucleotides as oligonucleotide-HPMA copolymer conjugates were internalized to a greater extent than unconjugated polymers.     

Small-angle neutron scattering study of the structure of protein/detergent complexes

Small-angle neutron scattering (SANS) was used to study the structure of protein/sodium dodecylsulfate complexes. Two water soluble proteins, bovine serum albumin (BSA) and ovalbumin (OVA), were used. The protein concentration was kept constant at 1 wt %, and protein/detergent wt ratio varied between 1/1, 1/1.5, 1/2 and 1/3. Absolute intensities of SANS distributions were analyzed by a fractal model. Analyses of large Q portions of SANS distributions established that sodium dodecylsulfate (SDS) molecules bound to a protein/SDS complex form micelle-like clusters. On the other hand, analyses of small Q portions of SANS distributions clearly showed that the arrangement of micelle-like clusters resembles a fractal packing of spheres. We showed that a protein/SDS complex can be characterized by four parameters extracted from the scattering experiment, namely, the average micelle size and its aggregation number, the fractal dimension characterizing the conformation of the micellar chains, the correlation length giving the extent of the unfolded polypeptide chains, and the numbers of micelle-like clusters in the complex.     

Enhanced biorecognition and internalization of HPMA copolymers containing multiple or multivalent carbohydrate side-chains by human hepatocarcinoma cells.

N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers containing pendant saccharide moieties (galactosamine, lactose, and triantennary galactose) were synthesized. The relationship between the content of saccharide moieties and three-dimensional arrangement of galactose residues and their biorecognition and internalization by human hepatocarcinoma HepG2 cells was investigated. The results obtained clearly indicated preferential binding of the trivalent galactose and the lactose-containing copolymers to these cells. The higher the saccharide moieties content in HPMA copolymers, the higher the levels of binding. The biorecognition of the glycosylated HPMA copolymers by HepG2 cells was inhibited by free lactose. The data on the internalization and subcellular trafficking of HPMA copolymer conjugates obtained by confocal fluorescence microscopy correlated well with the flow cytometric analysis of their biorecognition by target cells. Structural features of the glycosides responsible for the specific recognition of the HPMA copolymers have been identified. The results underline the potential of glycosylated HPMA copolymers for delivery of pharmaceutical agents to hepatocarcinoma cells.     

PDEPT: polymer-directed enzyme prodrug therapy. 2. HPMA copolymer-beta-lactamase and HPMA copolymer-C-Dox as a model combination

Polymer-directed enzyme prodrug therapy (PDEPT) is a novel two-step antitumor approach that uses a combination of a polymeric prodrug and polymer-enzyme conjugate to generate a cytotoxic drug rapidly and selectively at the tumor site. Previously we have shown that N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound cathepsin B can release doxorubicin intratumorally from an HPMA copolymer conjugate PK1. Here we describe for the first time the synthesis and biological characterization of a PDEPT model combination that uses an HPMA-copolymer-methacryloyl-glycine-glycine-cephalosporin-doxorubicin (HPMA-co-MA-GG-C-Dox) as the macromolecular prodrug and an HPMA copolymer conjugate containing the nonmammalian enzyme beta-lactamase (HPMA-co-MA-GG-beta-L) as the activating component. HPMA-co-MA-GG-C-Dox had a molecular weight of approximately 31 600 Da and a C-Dox content of 5.85 wt %. Whereas free beta-L has a molecular weight of 45 kDa, the HPMA-co-MA-GG-beta-L conjugate had a molecular weight in the range of 75-150 kDa, and following purification no free enzyme was detectable. Against the cephalosporin C or HPMA-co-MA-GG-C-Dox substrates, the HPMA-co-MA-GG-beta-L conjugate retained 70% and 80% of its activity, respectively. In vivo (125)I-labeled HPMA-co-MA-GG-beta-L showed prolonged plasma concentration and greater tumor targeting than (125)I-labeled beta-L due to the enhanced permeability and retention (EPR) effect. Moreover, administration of HPMA-co-MA-GG-C-Dox iv to mice bearing sc B16F10 melanoma followed after 5 h by HPMA-co-MA-GG-beta-L led to release of free Dox. The PDEPT combination caused a significant decrease in tumor growth (T/C = 132%) whereas neither free Dox nor HPMA-co-MA-GG-C-Dox alone displayed activity. The PDEPT combination displayed no toxicity at the doses used, so further evaluation of this approach to establish the maximum tolerated dose (MTD) is recommended.     

Synthesis of bioadhesive lectin-HPMA copolymer-cyclosporin conjugates

An amino group containing cyclosporin A (CsA) derivative has been synthesized and conjugated to N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer via an aromatic azo bond, which can be specifically cleaved by azoreductase activity in colon to release the drug for the treatment of colon diseases. Lectins, peanut (Arachis hypogea) agglutinin (PNA) and wheat germ agglutinin (WGA), have been conjugated to HPMA copolymer-CsA derivative conjugates (PCsA), respectively, to give bioadhesive conjugates. The PNA and WGA are the targeting proteins that can bind to diseased colon tissue and healthy tissue, respectively. There were on average four P(CsA) copolymer chains attached on one WGA molecule with a drug content of 16.0 wt % and five P(CsA) copolymer chains attached on one PNA molecule with a drug content of 11.5 wt %. The incubation of a P(CsA) copolymer with the rat cecal contents resulted in the cleavage of the azo bond and release of the cyclosporin derivative. The biological evaluation of the conjugates is under way.     

The vitelline envelope of eggs from the giant keyhole limpet Megathura crenulata. I. Chemical composition and structural studies.

The egg vitelline envelope of the marine invertebrate Megathura crenulata is a glycoprotein composed of 37.3 mol % protein and 62.7 mol % carbohydrate. Of the total amino acid content, 61 mol % consists of a single amino acid, threonine. The carbohydrate content includes galactosamine, galactose, and fucose. The molar ratio of threonine to galactosamine is about 1:1. Most of the threonine residues are linked to galactosamine residues via O-glycosidic bonds. A single peptide that was purified following alkaline borohydride treatment of the vitelline envelope had the structure: Abu-Pro-Abu-(Abu6, Pro1, Thr1), where Abu is 2-aminobutyric acid. Several sugar residues have been isolated following the alkaline hydrolysis of the vitelline envelope that include an octasaccharide Gal4Fu4, an hexasaccharide Gal3Fu3, a trisaccharide Gal3, fucose, and galactose. It is proposed that the vitelline envelope of Megathura crenulata eggs is composed of polypeptide chains built to a large extent of closely spaced threonine residues. Almost every threonine residue is linked to a saccharide moiety.     

Polyacetal-doxorubicin conjugates designed for pH-dependent degradation

Terpolymerization of poly(ethylene glycol) (PEG), divinyl ethers, and serinol can be used to synthesize water soluble, hydrolytically labile, amino-pendent polyacetals (APEGs) suitable for drug conjugation. As these polyacetals display pH-dependent degradation (with faster rates of hydrolysis at acidic pH) and they are not inherently hepatotropic after intravenous (iv) injection, they have potential for development as biodegradable carriers to facilitate improved tumor targeting of anticancer agents. The aim of this study was to synthesize a polyacetal-doxorubicin (APEG-DOX) conjugate, determine its cytotoxicity in vitro and evaluate its potential for improved tumor targeting in vivo compared to an HPMA copolymer-DOX conjugate in clinical development. Amino-pendent polyacetals were prepared, and following succinoylation (APEG-succ), the polymeric intermediate conjugated to DOX via one of three methods using carbodiimide mediated coupling (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) in aqueous solution was the most successful). The resultant APEG-DOX conjugates had a DOX content of 3.0-8.5 wt %, contained <1.2% free DOX (relative to total DOX content) and had a M(w) = 60000-100000 g/mol and M(w)/M(n) = 1.7-2.6. In vitro cytotoxicity studies showed APEG-DOX to be 10-fold less toxic toward B16F10 cells than free DOX (IC(50) = 6 microg/mL and 0.6 microg/mL respectively), but confirmed the serinol-succinoyl-DOX liberated during main-chain degradation to be biologically active. When administered iv to C57 black mice bearing subcutaneous (sc) B16F10 melanoma, APEG-DOX of M(w) = 86000 g/mol, and 5.0 wt % DOX content exhibited significantly (p < 0.05) prolonged blood half-life and enhanced tumor accumulation compared to an HPMA copolymer-GFLG-DOX conjugate of M(w) = 30000 g/mol and 6.2 wt % DOX content. Moreover, APEG-DOX exhibited lower uptake by liver and spleen. These observations suggest that APEG anticancer conjugates warrant further development as novel polymer therapeutics for improved tumor targeting.     

The haemocyanin of the whelk, Busycon contrarium (Conrad): aggregation states and subunit structure

1. The haemocyanin of the left-handed whelk Busycon contrarium (Conrad) exists largely as six or more multi-decameric aggregates characterized by sedimentation coefficients of approximately 105S, 132S, 155S, 170S, 185S and about 200-220S. 2. These aggregates represent di- to hepta- or octa-decameric assemblies of the basic haemocyanin decamer having a mol. wt of 4.3 x 10(6)-4.5 x 10(6). 3. The fully dissociated subunits in 8.0 M urea (pH 8.5) and at pH 11.1, 0.01 M EDTA have mol. wts of 4.78 x 10(5) and 4.62 x 10(5), close to one-tenth of the mol. wt of the basic decameric unit of most gastropod haemocyanins. 4. The pH dependence of the mol. wts (Mw), studied by light-scattering at the constant protein concentration of 0.010%, exhibit bell-shaped pH transition profiles with mol. wt values of about 16 x 10(6) in the presence of 0.01 M Mg2+, in the pH region from about pH 4.5-8.0; in the absence of stabilizing divalent ions the observed mol. wt is about 10 x 10(6) at pH 4.5-7.0. Below pH 4.5 and above 7.0-8.0 there is a sharp drop in mol. wt to about 4 x 10(5)-4.5 x 10(5). 5. The transition profiles observed with both the urea and salt series of probes investigated at concentration = 0.010% are found to produce aggregation at low reagent concentrations with mol. wt changes from about 9 x 10(6)-12 x 10(6)-14 x 10(6), followed by a decrease in mol. wt below 4.3 x 10(6)-4.5 x 10(6) of the haemocyanin decamers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Star structure of antibody-targeted HPMA copolymer-bound doxorubicin: a novel type of polymeric conjugate for targeted drug delivery with potent antitumor effect

The aim of this study was to compare the properties and antitumor potential of a novel type of antibody-targeted N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer-bound doxorubicin conjugates with star structure with those of previously described classic antibody-targeted or lectin-targeted HPMA copolymer-bound doxorubicin conjugates. Classic antibody-targeted conjugates were prepared by aminolytic reaction of the multivalent HPMA copolymer containing side-chains ending in 4-nitrophenyl ester (ONp) reactive groups with primary NH(2) groups of the antibodies. The star structure of antibody-targeted conjugates was prepared using semitelechelic HPMA copolymer chains containing only one reactive N-hydroxysuccinimide group at the end of the backbone chain. In both types of conjugates, B1 monoclonal antibody (mAb) was used as a targeting moiety. B1 mAb recognizes the idiotype of surface IgM on BCL1 cells. The star structure of the targeted conjugate had a narrower molecular mass distribution than the classic structure. The peak in the star structure was around 300-350 kDa, while the classic structure conjugate had a peak around 1300 kDa. Doxorubicin was bound to the HPMA copolymer via Gly-Phe(D,L)-Leu-Gly spacer to ensure the controlled intracellular delivery. The release of doxorubicin from polymer conjugates incubated in the presence of cathepsin B was almost twice faster from the star structure of targeted conjugate than from the classic one. The star structure of the targeted conjugate showed a lower binding activity to BCL1 cells in vitro, but the cytostatic activity measured by [(3)H]thymidine incorporation was three times higher than that seen with the classic conjugate. Cytostatic activity of nontargeted and anti-Thy 1.2 mAb (irrelevant mAb) modified HPMA copolymer-bound doxorubicin was more than hundred times lower as compared to the star structure of B1 mAb targeted conjugate. In vivo, both types of conjugates targeted with B1 mAb bound to BCL1 cells in the spleen with approximately the same intensity. The classic structure of the targeted conjugate bound to BCL1 cells in the blood with a slightly higher intensity than the star structure. Both types of targeted conjugates had a much stronger antitumor effect than nontargeted HPMA copolymer-bound doxorubicin and free doxorubicin. The star structure of targeted conjugate had a remarkably higher antitumor effect than the classic structure: a single intravenous dose of 100 microg of doxorubicin given on day 11 completely cured five out of nine experimental animals whereas the classic structure of targeted conjugate given in the same schedule only prolonged the survival of experimental mice to 138% of control mice. These results show that the star structure of antibody-targeted HPMA copolymer-bound doxorubicin is a suitable conjugate for targeted drug delivery with better characterization, higher cytostatic activity in vitro, and stronger antitumor potential in vivo than classic conjugates.     

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   

Separation and identification of Drosophila myosin light chains

Myosin was extracted from the larvae and adult flies of Drosophila melanogaster, and purified by column chromatography in the presence of KI. Myosin light chains were separated from heavy chains by column chromatography after treatment of the myosin with urea, and they were identified by 2D-gel electrophoresis. Tubular muscles and fibrillar muscles have different light chains. Lt1 (Mw = 31,000), Lt2 (Mw = 30,000), Lt2' (Mw = 30,000), and Lt3 (Mw = 20,000) exist in the tubular myosin of both larvae and adult flies; Lf1 (Mw = 34,000), Lf2 (Mw = 30,000), Lf2' (Mw = 30,000), and Lf3 (Mw = 20,000) exist in the fibrillar myosin. Polyacrylamide gel electrophoresis of myosin under nondissociating conditions revealed that there was one major myosin isozyme in each type of adult muscle, and the re-electrophoresis of each isozyme on SDS gel confirmed our identification of the light chains.     

The Targeted Transduction of MMP-Overexpressing Tumor Cells by ACPP-HPMA Copolymer-Coated Adenovirus Conjugates

We have designed and tested a new way to selectively deliver HPMA polymer-coated adenovirus type 5 (Ad5) particles into matrix metalloproteinase (MMP)-overexpressing tumor cells. An activatable cell penetrating peptide (ACPP) was designed and attached to the reactive 4-nitrophenoxy groups of HPMA polymers by the C-terminal amino acid (asparagine, N). ACPPs are activatable cell penetrating peptides (CPPs) with a linker between polycationic and polyanionic domains, and MMP-mediated cleavage releases the CPP portion and its attached cargo to enable cell entry. Our data indicate that the transport of these HPMA polymer conjugates by a single ACPP molecule to the cytoplasm occurs via a nonendocytotic and concentration-independent process. The uptake was observed to finish within 20 minutes by inverted fluorescence microscopy. In contrast, HPMA polymer-coated Ad5 without ACPPs was internalized solely by endocytosis. The optimal formulation was not affected by the presence of Ad5 neutralizing antibodies during transduction, and ACPP/polymer-coated Ad5 also retained high targeting capability to several MMP-overexpressing tumor cell types. For the first time, ACPP-mediated cytoplasmic delivery of polymer-bound Ad5 to MMP-overexpressing tumor cells was demonstrated. These findings are significant, as they demonstrate the use of a polymer-based system for the targeted delivery into MMP-overexpressing solid tumors and highlight how to overcome major cellular obstacles to achieve intracellular macromolecular delivery.     

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.     

In situ gellable oxidized citrus pectin for localized delivery of anticancer drugs and prevention of homotypic cancer cell aggregation

The aim of this study was to develop in situ gellable hydrogels composed of periodate oxidized citrus pectin (OP) for localized anticancer drug delivery and evaluate the potential of OP to inhibit cancer metastasis. Doxorubicin (Dox) was coupled to OP by imine bonds. Adipic dihydrazide (ADH) was used for cross-linking of the Dox-OP conjugates. The Dox-OP conjugate solution gelled within 2 min after addition of ADH. The release rate of Dox from the hydrogels was controllable by an additive amount of ADH. The released Dox retained anticancer activity. OP was shown to have a potency to prevent homotypic cancer cell aggregation compared to unmodified citrus pectin, strongly suggesting that OP released from hydrogels in vivo will inhibit cancer metastasis. These results indicate that OP hydrogels have the potential to prevent progression of primary cancer by the released Dox and generation of metastatic cancer by the released OP.     

Reduction-Responsive Disassemblable Core-Cross-Linked Micelles Based on Poly(ethylene glycol)-b-poly(N-2-hydroxypropyl methacrylamide)-Lipoic Acid Conjugates for Triggered Intracellular Anticancer Drug Release

Reduction-sensitive reversibly core-cross-linked micelles were developed based on poly(ethylene glycol)-b-poly(N-2-hydroxypropyl methacrylamide)-lipoic acid (PEG-b-PHPMA-LA) conjugates and investigated for triggered doxorubicin (DOX) release. Water-soluble PEG-b-PHPMA block copolymers were obtained with M(n,PEG) of 5.0 kg/mol and M(n,HPMA) varying from 1.7 and 4.1 to 7.0 kg/mol by reversible addition-fragmentation chain transfer (RAFT) polymerization. The esterification of the hydroxyl groups in the PEG-b-PHPMA copolymers with lipoic acid (LA) gave amphiphilic PEG-b-PHPMA-LA conjugates with degrees of substitution (DS) of 71-86%, which formed monodispersed micelles with average sizes ranging from 85.3 to 142.5 nm, depending on PHPMA molecular weights, in phosphate buffer (PB, 10 mM, pH 7.4). These micelles were readily cross-linked with a catalytic amount of dithiothreitol (DTT). Notably, PEG-b-PHPMA(7.0k)-LA micelles displayed superior DOX loading content (21.3 wt %) and loading efficiency (90%). The in vitro release studies showed that only about 23.0% of DOX was released in 12 h from cross-linked micelles at 37 °C at a low micelle concentration of 40 μg/mL, whereas about 87.0% of DOX was released in the presence of 10 mM DTT under otherwise the same conditions. MTT assays showed that DOX-loaded core-cross-linked PEG-b-PHPMA-LA micelles exhibited high antitumor activity in HeLa and HepG2 cells with low IC(50) (half inhibitory concentration) of 6.7 and 12.8 μg DOX equiv/mL, respectively, following 48 h incubation, while blank micelles were practically nontoxic up to a tested concentration of 1.0 mg/mL. Confocal laser scanning microscope (CLSM) studies showed that DOX-loaded core-cross-linked micelles released DOX into the cell nuclei of HeLa cells in 12 h. These reduction-sensitive disassemblable core-cross-linked micelles with excellent biocompatibility, superior drug loading, high extracellular stability, and triggered intracellular drug release are promising for tumor-targeted anticancer drug delivery.     

Fate of N-(2-hydroxypropyl)methacrylamide copolymers with pendent galactosamine residues after intravenous administration to rats

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymers bearing galactosamine residues accumulate in the liver after intravenous administration to rats (Duncan, R., Kopec̆ek, J., Rejmanová, P. and Lloyd, J.B. (1983) Biochim. Biophys. Acta 755, 518–521). In this study HPMA copolymers bearing pendent galactosamine residues (1.0–11.6 mol%) were injected intravenously intor rats and their rates of blood clearance and liver accumulation were measured. A level of substitution of 4 mol% was found to be sufficient to cause substantial deposition in the liver 30 min after administration. The most highly substituted polymer (11.6 mol%) was directed rapidly to the liver, 80–90% being recovered there less than 10 min after administration. Separation of liver into hepatocytes and non-parenchymal cells indicated that polymer was largely associated with the hepatocytes, and density-gradient subcellular fractionation of liver at various times after administration confirmed that polymer was internalized by liver cells and transported, with time, into the secondary lysosomes. Experiments using isolated rat hepatocytes indicated that HPMA copolymers with high galactosamine content have higher affinity for the hepatocyte plasma membrane. HPMA copolymers containing galactosamine and in addition glycylglycyltyrosianmide side-chains were used to demonstrate release of a drug analogue across the lysosomal membrane. These polymers were radioiodinated and, following intravenous administration to rats, the liver lysosomes were isolated and incubated at 37°C in 0.25 M sucrose. Radioactivity was released from the lysosomes faster than the lysosomal enzyme arylsulphatase, an observation that indicates intralysosomal hydrolysis of the copolymer side-chain with subsequent passage of low molecular weight degradation product across the lysosomal membrane.