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Biomedical subjects

Francis C Szoka

Publications and source records attributed to Francis C Szoka.

At least 19 recordsLinked to original sources
PubMed2006

Retro-convection enhanced delivery to increase blood to brain transfer of macromolecules.

A retro-convection enhanced delivery (R-CED) method has been developed to improve the entry of intravenously administered therapeutics within solid brain tumors. R-CED uses an osmotic gradient to withdraw brain interstitial fluid (ISF) in a controlled manner via an implanted microdialysis catheter. Withdrawal of ISF increases the local tissue specific gravity in normal brain and increases twofold the extravasation of intravenous Evans blue (EB) albumin in normal brain and in an orthotopic 9L tumor. R-CED also increases the extravasation of 70 nm fluorescent liposomes fivefold in the 9L tumor. Thus the transmembrane osmotic gradient induces movement of substances in the blood into the tissue parenchyma. Following probe removal, the magnitude of the R-CED effect on EB-albumin extravasation decreases to control values within 1.5 h in normal brain; however, the effect persists beyond 6 h in the tumor. There was no evidence of histologic damage to the neurons at either 6 h or 2 weeks after R-CED. These studies establish the feasibility of applying R-CED to increase the distribution of systemically administered drugs in both the normal tissue-tumor margin as well as in the central tumor core, holding forth the possibility of improved antitumor drug efficacy.

Albumins↗
PubMed2006

A single dose of doxorubicin-functionalized bow-tie dendrimer cures mice bearing C-26 colon carcinomas.

The antitumor effect of doxorubicin (DOX) conjugated to a biodegradable dendrimer was evaluated in mice bearing C-26 colon carcinomas. An asymmetric biodegradable polyester dendrimer containing 8-10 wt % DOX was prepared. The design of the dendrimer carrier optimized blood circulation time through size and molecular architecture, drug loading through multiple attachment sites, solubility through PEGylation, and drug release through the use of pH-sensitive hydrazone linkages. In culture, dendrimer-DOX was >10 times less toxic than free DOX toward C-26 colon carcinoma cells after exposure for 72 h. Upon i.v. administration to BALB/c mice with s.c. C-26 tumors, dendrimer-DOX was eliminated from the serum with a half-life of 16 +/- 1 h, and its tumor uptake was ninefold higher than i.v. administered free DOX at 48 h. In efficacy studies performed with BALB/c mice bearing s.c. C-26 tumors, a single i.v. injection of dendrimer-DOX at 20 mg/kg DOX equivalents 8 days after tumor implantation caused complete tumor regression and 100% survival of the mice over the 60-day experiment. No cures were achieved in tumor-implanted mice treated with free DOX at its maximum tolerated dose (6 mg/kg), drug-free dendrimer, or dendrimer-DOX in which the DOX was attached by means of a stable carbamate bond. The antitumor effect of dendrimer-DOX was similar to that of an equimolar dose of liposomal DOX (Doxil). The remarkable antitumor activity of dendrimer-DOX results from the ability of the dendrimer to favorably modulate the pharmacokinetics of attached DOX.

Animals↗
PubMed2006

Acid-triggered transformation of diortho ester phosphocholine liposome.

The use of pH-sensitive liposomes for acid-triggered site-specific drug delivery is one of the more promising approaches to improve the therapeutic index of drugs. Here, we report the synthesis, assembly, and hydrolysis of the first ortho ester phosphocholine (OEPC). The acid hydrolysis of OEPC liposomes consists of a lag phase and a burst phase. The lag time is pH-dependent-the lower the pH, the shorter the time. Upon acid hydrolysis, the OEPC liposomes were transformed into leaky metastable vesicles that rapidly collapsed in the presence of albumin. OEPC, when formulated with cationic lipid, significantly enhanced the transfection efficiency compared with that of the pH-insensitive formulation.

Esters↗
PubMed2005

Barriers to carrier mediated drug and gene delivery to brain tumors.

Brain tumor patients face a poor prognosis despite significant advances in tumor imaging, neurosurgery and radiation therapy. Potent chemotherapeutic drugs fail when used to treat brain tumors because biochemical and physiological barriers limit drug delivery into the brain. In the past decade a number of strategies have been introduced to increase drug delivery into the brain parenchyma. In particular, direct drug administration into the brain tumor has shown promising results in both animal models and clinical trials. This technique is well suited for the delivery of liposome and polymer drug carriers, which have the potential to provide a sustained level of drug and to reach cellular targets with improved specificity. We will discuss the current approaches that have been used to increase drug delivery into the brain parenchyma in the context of fluid and solute transport into, through and from the brain, with a focus on liposome and polymer drug carriers.

Animals↗
PubMed2005

Identification of a deoxyribonuclease I inhibitor from a phage-peptide library.

Deoxyribonuclease I (DNase I) is a divalent cation dependent endonuclease and thought to be a significant barrier to effective gene delivery. The only known DNase I-specific inhibitor is monomeric actin which acts by forming a 1:1 complex with DNase I. Its use, however, is restricted because of tendency to polymerize under certain conditions. We screened two random phage peptide libraries of complexity 10(8) and 10(9) for DNase I binders as candidates for DNase I inhibitors. A number of DNase I-binding peptide sequences were identified. When these peptides were expressed as fusion proteins with Escherichia coli maltose binding protein, they inhibited the actin-DNase I interaction (IC50 = 0.1-0.7 microM) and DNA degradation by DNase I (IC50 = 0.8-8 microM). Plasmid protection activity in the presence of DNase I was also observed with the fusion proteins. These peptides have the potential to be a useful adjuvant for gene therapy using naked DNA.

Actins↗
PubMed2005

Distribution in brain of liposomes after convection enhanced delivery; modulation by particle charge, particle diameter, and presence of steric coating.

We have investigated the role of diameter, charge, and steric shielding on the brain distribution of liposomes infused by convection enhanced delivery (CED) using both radiolabeled and fluorescent-labeled particles. Liposomes of 40 and 80-nm diameter traveled the same distance but penetrated significantly less than a 10-kDa dextran; whereas 200-nm-diameter liposomes penetrated less than 80 nm liposomes. A neutral liposome shielded by polyethylene glycol (PEG; 2 kDa; 10% by mole) penetrated significantly farther than an unshielded liposome. Even when shielded with PEG, positive surface charge (10% by mole) significantly reduced the penetration radius compared to a neutral or negative charged liposome (10% by mole). A mathematical CED model including a term for liposome cell binding was applied to analyze the radius of particle penetration. Neutral liposomes had a binding constant of k=0.0010+/-0.0002 min-1, whereas for positive charged liposomes k increased 50-fold. The binding constant was independently verified using a degradable lipid radiolabel that eliminated from the brain with a 9.9+/-2.0 h half-life, equivalent to the calculated elimination constant k=0.0012+/-0.0002 min-1. During CED, liposomes accumulated in a subpopulation of perivascular cells within the brain. A non-degradable lipid radiolabel showed that lipid components remained within these perivascular brain cells for at least 2 days. To reduce this uptake, 100-fold molar excess of non-labeled liposomes were co-infused with labeled liposomes, which significantly increased liposome penetration. These studies suggest that optimization of therapeutic CED using particles such as drug-loaded liposomes, polymeric nanoparticles, non-viral DNA complexes, and viruses will require a strategy to overcome particle binding and clearance by cells within the CNS.

Animals↗
PubMed2005

Low-pH-sensitive poly(ethylene glycol) (PEG)-stabilized plasmid nanolipoparticles: effects of PEG chain length, lipid composition and assembly conditions on gene delivery.

BACKGROUND: We have studied the effects of the poly(ethylene glycol) (PEG) chain length and acyl chain composition on the pH-sensitivity of acid-labile PEG-diorthoester (POD) lipids. The optimal conditions are described for preparation of DNA plasmid encapsulated POD nanolipoparticles (NLPs) which mediate high gene delivery activity in vitro with moderate cytotoxicity. METHODS AND RESULTS: A series of POD lipids with various PEG chain lengths (750, 2000, and 5000 Da) or acyl chains (distearoyl 18:0 or dioleoyl 18:1) were incorporated into DNA containing NLPs or model liposomes as a stealth and bioresponsive component. We investigated the collapse kinetics of the POD-stabilized liposomes when the PEG chain length was changed. We optimized a detergent dialysis method to encapsulate plasmid DNA into NLPs prepared from a mixture of the various POD lipids, cationic lipid and phosphatidylethanolamine lipid. A critical concentration (28 mM) of n-octyl-beta-D-glucopyranoside (OG) enabled high encapsulation of DNA plasmid into 100 nm particles with a neutral surface charge. The POD NLPs are stable at pH 8.5 but rapidly collapse (approximately 10 min) into aggregates at pH 5.0. In the detergent solution there is a metastable DNA-lipid intermediate that evolves into a stable NLP if the detergent is removed shortly after adding DNA to the lipid-detergent mixture. The rank order of transfection activity from NLPs containing PEG-lipid was POD 750 > POD 5000 = POD 2000 > non-pH-sensitive PEG-lipid. The particle size stability was in the reverse order. Binding of the NLPs to cells reached a maximum level by 12 hours. The POD NLPs had slightly less transfection activity but considerably lower cytotoxicity than the PEI-DNA polyplex. CONCLUSIONS: Of the PEG-orthoester lipids tested, POD 2000 is the better choice for the preparation of sterically stabilized NLPs with a small particle diameter, good stability, low cytotoxicity, and satisfactory transfection activity.

Animals↗
PubMed2005

Toroid formation in charge neutralized flexible or semi-flexible biopolymers: potential pathway for assembly of DNA carriers.

BACKGROUND: The theoretical state diagram for semi-flexible macromolecules such as DNA predicts that a tightly wound toroid can be a stable structure. Experimentally, toroids roughly 100 nm in diameter are routinely observed for DNA in the presence of multivalent cations at low DNA concentration. Theory also predicts toroids can form between non-DNA semi-flexible polymers and multivalent counterions. This phenomenon provides a means to co-package DNA with functionalized anionic polymers to create gene delivery systems. METHODS AND RESULTS: We show using electron microscopy that non-DNA polymers (polylysine, polyglutamic acid, and dextran sulfate) form toroids when mixed with multi- or polyvalent ions of opposite charge. The non-DNA toroids are similar in diameter to ones made with DNA. The results using dextran sulfate, a semi-flexible polymer, are explained by current theory. However, theory predicts that high flexibility in polypeptides should discourage their incorporation into stable toroids. To explain these latter observations we propose that charge neutralization facilitates secondary structure formation, which confers stiffness, thereby allowing stable toroids for the polypeptides studied. We measured the secondary structure of the toroid-forming polypeptides using circular dichroism (CD). The CD spectrum indicates the polypeptides undergo transitions from non-ordered structures (random coil) to ordered secondary structures (either alpha-helix or beta-sheet) upon charge neutralization which supports the hypothesis. The type of secondary structure is dependent on the type of multivalent counterion used to form the toroids. Formation of the polypeptide toroids confers resistance to heat denaturation of the resulting polypeptide secondary structure. The CD spectrum of DNA in a toroid also is changed from that of uncomplexed DNA, but all of the counterions used to form DNA toroids created structures with similar CD spectra in the DNA region (250-290 nm). CONCLUSIONS: The toroid structure obtained using DNA is observed in other semi-flexible non-DNA polymers such as dextran sulfate, and also in flexible polymers such as polylysine and polyglutamic acid upon charge neutralization with multivalent counterions. In the flexible polymers we propose that this phenomenon is due to induction of secondary structure upon charge neutralization, which decreases polymer flexibility, i.e. increases polymer stiffness, to enable toroid formation. These results have significant implications for the co-assembly of non-DNA anionic polymers with DNA to create nanoscopic gene carriers.

Biopolymers↗
PubMed2005

Thiocholesterol-based lipids for ordered assembly of bioresponsive gene carriers.

A series of thiocholesterol-based cationic lipids (TCL) has been designed and synthesized by the attachment of thiocholesterol to a cationic amine via a disulfide bond. TCL can be incorporated into liposomes and used to package DNA into a lipoplex, thereby protecting it from DNase digestion. DNA is rapidly released from the complex in the presence of low concentrations of reducing agents. The lipoplex mediated efficient transfection activity and had low cytotoxicity. To improve the biocompatibility of the cationic lipoplex, TCL were used as a component in the assembly of a nanolipoparticle (NLP). The particle surface was subsequently modified by disulfide exchange to replace the cationic group with a negatively charged (glutathione) or zwitterionic (cysteine) reducing agent. A cell-binding ligand (TAT peptide, sequence GRKKRRQRRRGYG) was then incorporated onto the particle surface to enhance the particle-cell recognition. The sequentially assembled cell-binding NLP with a zwitterionic surface gave a larger transfection yield than the cationic NLP at all concentrations tested. At low DNA concentrations, the enhancement was 80-fold. The disulfide cationic lipids and the sequential assembly strategy enable one to tailor the surface charge, hydrophilicity, and recognition elements of a nanosized gene carrier. This results in increased gene transfer activity in a biocompatible particle.

Cholesterol↗
PubMed2005

Designing dendrimers for biological applications.

Dendrimers are branched, synthetic polymers with layered architectures that show promise in several biomedical applications. By regulating dendrimer synthesis, it is possible to precisely manipulate both their molecular weight and chemical composition, thereby allowing predictable tuning of their biocompatibility and pharmacokinetics. Advances in our understanding of the role of molecular weight and architecture on the in vivo behavior of dendrimers, together with recent progress in the design of biodegradable chemistries, has enabled the application of these branched polymers as anti-viral drugs, tissue repair scaffolds, targeted carriers of chemotherapeutics and optical oxygen sensors. Before such products can reach the market, however, the field must not only address the cost of manufacture and quality control of pharmaceutical-grade materials, but also assess the long-term human and environmental health consequences of dendrimer exposure in vivo.

Biocompatible Materials↗
PubMed2004

GALA: a designed synthetic pH-responsive amphipathic peptide with applications in drug and gene delivery.

GALA is a 30 amino acid synthetic peptide with a glutamic acid-alanine-leucine-alanine (EALA) repeat that also contains a histidine and tryptophan residue as spectroscopic probes. It was designed to explore how viral fusion protein sequences interact with membranes. The sequence selected was long enough to span a bilayer in the alpha-helix, the glutamic acids (Glu) were selected to provide a pH-dependent negatively charged side-chain and the EALA repeat was adjusted so that the peptide would have a hydrophobic face of sufficient hydrophobicity to interact with the bilayer when the peptide was in an alpha-helix. GALA converts from a random coil to an amphipathic alpha-helix when the pH is reduced from 7.0 to 5.0. At neutral pH, GALA is water soluble while at acid pH, GALA binds to bilayer membranes. The nature of the association and the type of peptide-peptide interactions in the membrane depend upon the physico-chemical properties of the bilayer such as the acyl chain composition of the phospholipids and the presence of cholesterol. Neutral and negatively charged bilayers composed of saturated phospholipids of 14-16 acyl chain length are solubilized into peptide-lipid discs by GALA. GALA can induce fusion between small unilamellar vesicles (SUV) composed of unsaturated phospholipids. Most importantly GALA forms a transmembrane peptide pore comprised of approximately 10 GALA alpha-helical monomers that are arrayed in an alpha-helix perpendicular to the plane of the membrane. Membrane leakage from neutral or negatively charged vesicles at pH 5.0 can be adequately explained by a mathematical model assuming that GALA becomes incorporated into the vesicle bilayer and aggregates to form a transbilayer pore consisting of 10 (+/-2) peptides. The lipid compositions of model bilayer have important effects on the GALA transbilayer insertion mechanism and peptide orientation. Insertion of the pore into the membrane dramatically accelerates transmembrane phospholipid flip-flop. Cationic peptides designed based upon GALA but containing a lysine-alanine-leucine-alanine (KALA) motif can interact with nucleic acids and perturb biomembranes. The pH-controlled membrane permealization induced by GALA and related peptides serve as a paradigm for the design of environmentally responsive peptidic delivery vehicles for drugs and genes.

Animals↗
PubMed2004

Determination and modeling of kinetics of cancer cell killing by doxorubicin and doxorubicin encapsulated in targeted liposomes.

Various mathematical approaches have been devised to relate the cytotoxic effect of drugs in cell culture to the drug concentration added to the cell culture medium. Such approaches can satisfactorily account for drug response when the drugs are free in solution, but the approach becomes problematic when the drug is delivered in a drug delivery system, such as a liposome. To address this problem, we have developed a simple model that assumes that the cytotoxic potency of a drug is a function of the intracellular drug level in a critical compartment. Upon exposure to drug, cell death commences after a lag time, and the cell kill rate is dependent on the amount of drug in the critical intracellular compartment. The computed number of cells in culture, at any time after exposure to the drug, takes into account the cell proliferation rate, the cell kill rate, the average intracellular drug concentration, and a lag time for cell killing. We have applied this model to compare the cytotoxic effect of doxorubicin (DOX), or DOX encapsulated in a liposome that is targeted to CD44 on B16F10 melanoma cells in culture. CD44 is the surface receptor that binds to hyaluronan and is overexpressed on various cancer cells, including B16F10. We have shown previously that the drug encapsulated in hyaluronan-targeted liposomes was more potent than was the free drug. The model required the determination of the cell-associated DOX after the cells were incubated with various concentrations of the free or the encapsulated drug for 3 h, and the quantification of cell number at various times after exposure to the drug. The uptake of encapsulated drug was greater than that of the free drug, and the ratio of cell association of encapsulated:free drug was 1.3 at 0.5 micro g/ml and increased to 3.3 at 20 micro g/ml DOX. The results demonstrate that the enhanced potency of the encapsulated drug could stem from its enhanced uptake. However, in certain cases, where larger amounts of the free drug were added, such that the intracellular amounts of drug exceeded those obtained from the encapsulated drug, the numbers of viable cells were still significantly smaller for the encapsulated drug. This finding demonstrates that for given amounts of intracellular DOX, the encapsulated form was more efficient in killing B16F10 cells than the free drug. The outcome was expressed in the kinetic model as a 5-6-fold larger rate constant of cell killing potency for the encapsulated drug versus the free drug. The model provides a quantitative framework for comparing the cytotoxic effect in cultured cells when applying the drug in the free form or in a delivery system.

Animals↗
PubMed2003

Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes.

The "proton sponge hypothesis" postulates enhanced transgene delivery by cationic polymer-DNA complexes (polyplexes) containing H+ buffering polyamines by enhanced endosomal Cl- accumulation and osmotic swelling/lysis. To test this hypothesis, we measured endosomal Cl- concentration, pH, and volume after internalization of polyplexes composed of plasmid DNA and polylysine (POL), a non-buffering polyamine, or the strongly buffering polyamines polyethylenimine (PEI) or polyamidoamine (PAM). [Cl-] and pH were measured by ratio imaging of fluorescently labeled polyplexes containing Cl- or pH indicators. [Cl-] increased from 41 to 80 mM over 60 min in endosomes-contained POL-polyplexes, whereas pH decreased from 6.8 to 5.3. Endosomal Cl- accumulation was enhanced (115 mM at 60 min) and acidification was slowed (pH 5.9 at 60 min) for PEI and PAM-polyplexes. Relative endosome volume increased 20% over 75 min for POL-polyplexes versus 140% for PEI-polyplexes. Endosome lysis was seen at >45 min for PEI but not POL-containing endosomes, and PEI-containing endosomes showed increased osmotic fragility in vitro. The slowed endosomal acidification and enhanced Cl- accumulation and swelling/lysis were accounted for by the greater H+ buffering capacity of endosomes containing PEI or PAM versus POL (>90 mM versus 46 H+/pH unit). Our results provide direct support for the proton sponge hypothesis and thus a rational basis for the design of improved non-viral vectors for gene delivery.

Animals↗
PubMed2003

Mechanism of pH-triggered collapse of phosphatidylethanolamine liposomes stabilized by an ortho ester polyethyleneglycol lipid.

The mechanism of pH-triggered destabilization of liposomes composed of a polyethyleneglycol-orthoester-distearoylglycerol lipid (POD) and phosphatidyl ethanolamine (PE) has been studied using an ANTS/DPX leakage and a lipid-mixing assay. We developed a kinetic model that relates POD hydrolysis to liposome collapse. This minimum-surface-shielding model describes the kinetics of the pH-triggered release of POD/PE liposomes. In the model, when acid-catalyzed hydrolysis lowers the mole percentage of POD on the liposome surface to a critical level, intervesicular lipid mixing is initiated, resulting in a burst of contents release. Two phases of content leakage are observed: a lag phase and a burst phase. During the lag phase, less than 20% of liposomal contents are released and the leakage begins to accelerate when approaching to the transition point. During the burst phase, the leakage rate is dependent on interbilayer contact. The burst phase occurs when the surface density of the PEG lipid is 2.3 +/- 0.6 mol%, regardless of the pH. Vesicles containing 4 mol% of a pH-insensitive PEG-lipid conjugate and 10% POD did not leak contents or collapse at any pH. These data are consistent with the stalk theory to describe the lamellar-to-inverted hexagonal phase transition and set a lower bound of approximately 16 PE lipids on the external monolayer as the contact site required for lipid mixing between two bilayers.

Computer Simulation↗
PubMed2003

Chemical approaches to triggerable lipid vesicles for drug and gene delivery.

Effective drug delivery requires the precise spatial and temporal delivery of therapeutic agents to the target site. To this end, a variety of chemical and physicochemical approaches have been devised to create lipid vesicles (liposomes) that can be triggered to release their contents in a controlled fashion. The triggers include changes in pH, redox potential, temperature, or the level of specific enzymes. We review the chemistries that have recently been applied to exploit the pH and redox potential triggers so as to release vesicle contents in the appropriate biological location.

Genetic Therapy↗
PubMed2002

Synthesis of vesicles on polymer template.

Reactive single-tail cationic surfactants self-assemble on the anionic block copolymer templates. These systems spontaneously arrange in small vesicles of nanoscale size. The vesicles are further stabilized by dimerization of the assembled surfactant monomers forming double-tail surfactants bound to the block copolymer. The resulting systems are resistant to changes in environmental characteristics such as pH, ionic strength, and temperature variations. Hydrophilic macromolecules can be encapsulated in the internal aqueous volume of these vesicles. The simplicity of the preparation makes these systems promising as drug and gene delivery carriers.

Isothiuronium↗