Search

This paper investigates the controlled release of hepatocyte growth factor (HGF) by biodegradable gelatin hydrogels and their HGF-induced angiogenic effect. Hydrogels of different degradabilities were prepared through chemical crosslinking gelatin with varied amounts of glutaraldehyde. When the gelatin hydrogels were radioiodinated and subcutaneously implanted into the back of mice, the remaining radioactivity of the hydrogels decreased with time. However, the remaining period became longer when the concentration of glutaraldehyde used for hydrogel preparation increased. Following implantation of gelatin hydrogels incorporating 125I-labeled HGF, the HGF radioactivity retained in the mouse subcutis for longer time periods as the glutaraldehyde concentration becomes higher. The time profile of HGF remaining in every gelatin hydrogel was in good accordance with that of hydrogel degradation, indicating HGF release as a result of hydrogel biodegradation. The gelatin hydrogel incorporating HGF histologically induced angiogenic change around the implanted hydrogel. Gelatin hydrogels incorporating 5 and 10 jig HGF significantly enhanced the number of capillaries newly formed around the implanted site. This was in marked contrast to free HGF of same dose form and HGF-free, empty gelatin hydrogel. The gelatin hydrogel incorporating HGF induced VEGF around the implanted site. In vitrobioassay revealed that HGF molecules interacting with gelatin, still exhibited the biological activity. The interacted HGF would be released from gelatin hydrogels only when they were degraded to generate water-soluble gelatin fragments. It is possible that the HGF associating gelatin fragments of bioactivating, results in induced angiogenic effect.

Bone is a dynamic tissue that undergoes significant turnover during the life cycle of an individual. Despite having a significant regenerative capability, trauma and other pathological scenarios commonly require therapeutic intervention to facilitate the healing process. Bone tissue engineering, where cellular and biological processes at a site are deliberately manipulated for a therapeutic outcome, offers a viable option for the treatment of skeletal diseases. In this review paper, we aim to provide a brief synopsis of cellular and molecular basis of bone formation that are pertinent to current efforts of bone healing. Different approaches for engineering bone tissue were presented with special emphasis on the use of soluble (diffusible) therapeutic agents to accelerate bone healing. The latter agents have been used for both local bone repair (i.e. introduction of agents directly to a site of repair) as well as systemic bone regeneration (i.e. delivery for regeneration throughout the skeletal system). Critical drug delivery and targeting issues pertinent for each mode of bone regeneration are provided. In addition, future challenges and opportunities in bone tissue engineering are proposed from the authors' perspective.

The goal of this paper was to investigate the fate of novel poly(methylidene malonate 2.1.2) microparticles with different surface properties, i.e. prepared with or without polyvinylalcohol (PVA), after oral administration, using in vitrocell culture and an in vivomice model. Incubation of particles with Caco-2 cells induced no cytotoxicity except for the microparticles prepared without PVA at high concentrations. At subtoxic concentrations, microparticles were highly associated to cells, independently of particles concentrations, particles surface properties (with or without PVA) or incubation time. Confocal microscopy analysis revealed that adsorption was the main phenomenon leading to the association of particles to cells. However, association was greater at 37°C than at 4°C, suggesting that an active process, such as endocytosis, could also occur. In vivo, radiolabeled particles were mainly found in luminal content and also adsorbed onto the epithelium. After 24 hours, more than 15 of PVA-free microparticles were still present in the gastrointestinal tract, compared to 5 for particles prepared with PVA. However, histological evaluation revealed low uptake of particles by Peyer's patches. As a conclusion, this study provided a good correlation between in vitroand in vivoevaluation. These particles could be useful for oral sustained release and delivery of drugs to intestinal and colon epithelium.

In vitrostudies have shown that conjugation of a model compound [p-di(hydroxye-thyl)-amino-D-phenylalanine (D-MOD)] with L-Glu can improve D-MOD permeation through the bovine brain microvessel endothelial cell monolayers (Sakaeda et al., 2000). The transport of this D-MOD-L-Glu conjugate is facilitated by the L-Glu transport system. In this paper, we evaluate the in vivobrain delivery of model compounds (i.e. D-MOD, p-nitro-D-phenylalanine (p-nitro-D-Phe), 5, 7-dichlorokynurenic acid (DCKA) and D-kyotor-phin) and their L-Glu conjugates. DCKA was also conjugated with L-Asp and L-Gln amino acids. The analgesic activities of D-kyotorphin and its L-Glu conjugate were also evaluated. The results showed that the brain-to-plasma concentration ratio of D-MOD-L-Glu was higher than the D-MOD alone; however, the plasma concentration of both compounds were the same. The plasma concentration of p-nitro-D-Phe-L-Glu conjugate was higher than the parent p-nitro-D-Phe; however, the brain-to-plasma concentration ratio of p-nitro-D-Phe was higher than its conjugate. On the other hand, both DCKA and DCKA conjugates have a low brain-to-plasma concentration ratio due to their inability to cross the blood-brain barrier (BBB). The L-Asp and L-Glu conjugates of DCKA have elevated plasma concentrations relative to DCKA; however, the DCKA-L-Gln conjugate has the same plasma concentration as DCKA. For D-kyotorphin, both the parent and the L-Glu conjugate showed similar analgesic activity. In conclusion, conjugation of a non-permeable drug with L-Glu may improve the drug's brain delivery; however, this improvement may depend on the physicochemical and receptor binding properties of the conjugate.

Recent synchrotron-based X-ray diffraction studies have enabled us to comprehensively solve the self-assembled structures in mixtures of cationic liposomes (CLs) complexed with linear λ-DNA. In one case the CL-DNA complexes were found to consist of a higher ordered multilamellar structure (labeled LCα with DNA sandwiched between cationic bilayer membranes. The membrane charge density is found to control the DNA interaxial spacing with high densities leading to high DNA compaction between lipid bilayers. A second self-assembled structure (labeled HCII) consists of linear DNA strands coated by cationic lipid monolayers and arranged on a 2D hexagonal lattice. In this paper we report on a combined X-ray diffraction and optical microscopy study of CLs complexed with functional supercoiled plasmid DNA. We describe the self-assembled structures in cell culture medium for both a high transfectant complex (DOTAP/DOPE, ΦDOPC = 0.72) and a low transfectant complex (DOTAP/DOPC, ΦDOPC = 0.72). Fluorescence optica microscopy shows two distinct interactions between these two types of complexes and mouse fibroblast L-cells, demonstrating the existence of a correlation between structure and transfection efficiency.

In this paper, the L-glutamate (L-Glu) transport system was targeted to improve the delivery of a model compound, p-di(hydroxyethyl)-amino-D-phenylalanine (D-MOD), through the blood-brain barrier (BBB) in vitro cell culture model. D-MOD is an analogue of an antitumor agent D-melphalan. To target the L-Glu transport system, D-MOD was conjugated to L-Glu to give D-MOD-L-Glu conjugate. D-MOD and D-MOD-L-Glu transport properties were evaluated using the bovine brain microvessel endothelial cell (BBMEC) monolayers. The results suggest that D-MOD-L-Glu conjugate permeates through the BBMEC monolayers more readily than the parent D-MOD. The improvement of transport may be due to the recognition of D-MOD-L-Glu by the L-Glu transport system. The transport mechanism was evaluated using several different experiments including: (a) concentration-dependent studies; (b) temperature-dependent studies; (c) substrate inhibition studies; and (d) metabolic inhibitor studies. The D-MOD-L-Glu transport was inhibited by the change of temperature from 37°C to 4°C. At higher concentrations, the transport of D-MOD-L-Glu reached plateau due to saturation. Furthermore, some amino acids (i.e., L-Glu, L-Asp, D-Asp, and L-Gln) inhibited the transport of D-MOD-L-Glu; presumably the conjugate was competing with these amino acids for the same transport system. Metabolic inhibitors (i.e., 2,4-dinitrophenol and sodium azide) suppressed the transport of the conjugate. However, the conjugate was not transported by monocarboxylic acid, dipeptide and neutral amino acid transporters. In conclusion, the L-Glu transport system can be utilized to facilitate a non-permeable drug across the BBB by conjugating the drug with L-Glu amino acid.

AbstractPurpose: In this paper, the authors attempt to construct a mathematical model to correlate the biological activities of 63 polyamine transport inhibitors in LI 210 cells with their physicochemical parameters.Method: The inhibitory constants (Kiwere obtained from the published work of Bergeron et al.Non-weighted least square method was used in deriving the regression equations with a BMDP program. An AM1 subroutine of the HyperChem program was used to optimize the geometry and calculate the molecular dipole moments and the distance between two terminal amino groups. A CQSAR program was used to calculate Clog P(oct./w.).Results: A good correlation (r2= 0.81) was obtained by using a five-parameter equation including the distance between two terminal amino groups (d), the number of cationic charge (Charge), molecular weight (μ), dipole moment (μ), and hydrogen bond forming ability (Hb).Conclusion: This model accounts for 81 of the variance in the data and can be used to estimate transport-inhibitory activity of many other polyamine analogues. It gives some quantitative information about the relationship between the polyamine analogues' function as transport inhibitors and their molecular structures.

AbstractWe have previously demonstrated the development of acoustically reflective liposomes as a novel ultrasound contrast agent, that can be conjugated to antibodies for site specific acoustic enhancement of pathologically altered vascular tissue. The liposomes are echogenic due to the lipid composition, without gas entrapment, and have a size of less than one micron (Alkan-Onyuksel et al., 1996). When conjugated to anti-fibrinogen antibodies, the liposomes have the ability to attach to fibrin coated surfaces and thrombi in vitro as demonstrated by scanning electron microscopy and ultrasound imaging (Demos et al., 1997a). Anti-fibrinogen liposomes were shown to attach to fibrous atheroma and thrombi in a Yucatan miniswine model of induced atherosclerosis whereas liposomes conjugated to anti-intercellular adhesion molecule-1 (anti-ICAM-1) were demonstrated to target early stage atherosclerotic plaques (Demos et al., 1997b).The purpose of this study is to evaluate the binding characteristics of anti-fibrinogen liposomes in vitro under a variety of flow conditions in order to optimize the targeting ability of the immunoliposomes. Radiolabeled anti-fibrinogen liposomes were applied to fibrin coated filter paper and placed in a flow circuit under controlled flow conditions. Row conditions were altered to study the effects of different shear stresses, temperature, plasma flow and pulsatile flow on the retention of liposomes to fibrin after set time periods. The retention of liposomes conjugated to polyclonal and monoclonal antibodies as well as Fab fragments made from monoclonal antibodies were compared. The binding characteristics of liposomes conjugated to different quantities of polyclonal antibodies were analyzed. At physiological shear stress of 1.5 N/m2(15 dynes/cm2) over 70 of the liposomes remained attached to fibrin after two hours. A smaller and greater portion of the liposomes remained attached at higher and lower shear stresses respectively. Plasma components and temperature had no effect on liposomal retention whereas pulsatile flow resulted in a slight reduction in binding. Monoclonal antibodies showed a slight trend of reduced retention to fibrin over time as compared with polyclonal antibodies and Fab fragments. The quantity of antibody conjugated to the liposomes plays a role in liposome retention as demonstrated by the reduction in liposome retention caused by reducing the quantity of antibody conjugated to the liposomes. Anti-fibrinogen liposomes were retained to the fibrin surface to a large extent under all flow conditions likely to occur in vivo and therefore can provide site specific ultrasound contrast for a long enough time period to allow for imaging after injection.

AbstractIn antibody-targeted enzyme prodrug therapy, a monoclonal antibody (mAb) covalently linked to an enzyme is commonly exploited to concentrate the enzyme on the tumor cell surface prior to administration of a relatively nontoxic prodrug. The tumor-localized enzyme then converts the prodrug into a cytotoxic agent, which in turn diffuses into the tumor causing localized cell death. In this paper, we have substituted folic acid for the mAb as a mean of delivering an attached enzyme, penicillin-V amidase (PVA), to folate receptor (FR)-positive tumor cells. The enzyme PVA is capable of converting a doxorubicin-N-p-hydroxyphenoxyacetamide prodrug (DPO) into its potent parent drug, doxorubicin. For PVA targeting, each PVA molecule was covalently labeled with three molecules of folic acid via the formation of amide bonds. In vitrobinding assays showed that folate-PVA-125I conjugates bind specifically to KB cells (FR-positive tumor cells) but not to A549 cells (FR-negative tumor cells). Moreover, in a series of in vitrocytotoxicity tests, folate-PVA conjugates were found to kill folate receptor positive but not receptor negative cells, and when bound to FR-positive cells, folate-PVA conjugates rendered the DPO prodrug as toxic as free doxorubicin (IC⊂50, 0.6 μM). Finally, preliminary in vivoplasma clearance studies in normal mice revealed that i.v. administered folate-PVA-125I and PVA-125I are both cleared from the blood within a 24 h time period, removing concern that nonspecifically trapped folate-PVA might activate prodrug in nontargeted tissues. In view of the fact that only a small number of folate-PVA molecules are required to mediate killing of target cells in vitro, these data argue that folate-targeted enzyme prodrug therapy should be considered for tumor eradication in vivo.

Quantitative and qualitative evidence from our laboratories on the absorption and translocation of polystyrene latex nanoparticles both by histological (qualitative) and analytical measurement of levels of polystyrene (quantitative) is briefly reviewed in this paper. We have previously compared the uptake of nonionized polystyrene latex ranging in size from 50nm to 3μm, and made some comparisons of uptake between carboxylated microspheres and nonionic systems, showing the lower uptake of the former through the lymphoid tissue of the gastrointestinal tract. Size is a key parameter, uptake increasing with decreasing particle diameter. Early evidence suggested that uptake is by way of the Peyer's patches and other elements of the gut associated lymphoid tissue (GALT). Adsorption of hydrophilic block-copolymers onto polystyrene markedly reduces the uptake by intestinal GALT. Modification of the surface with specific ligands such as by covalent attachment of tomato lectin molecules has indicated widespread uptake by non-GALT tissues, following their binding to and internalisation by enterocytes. The ability to decrease and increase uptake is clear evidence of a phenomenon which has the potential for further control to allow it to be exploited fully for drug or vaccine delivery. The evidence to date with nanoparticles as carriers systems for labile drugs such as proteins by the oral route remains to be substantiated.

An aqueous dispersion of niosomes (non-ionic surfactant vesicles) emulsified in an external oil phase forms the vesicle-in-water-in-oil (v/w/o) system described in this paper. The properties of the surfactant used to form the vesicles, the surfactant or surfactant mixture used to stabilize the emulsion and the nature of the oil phase can be changed to provide systems of different capacities for drug or antigen and different release characteristics. The same nonionic surfactant is used as the principle amphipile to form the niosomes and to stabilize the w/o emulsion, thus promoting stability by decreasing transfer of surfactant between the stabilizing monolayers and the vesicle bilayers. The in vitro release of carboxyfluoroscein and 5-fluorouracil encapsulated within the niosomes of the v/w/o system has been investigated, the nature of the oil phase and surfactant-oil interactions being important in determining the rate of solute release. Initial studies of the system in vivo, as an adjuvant for tetanus toxoid, using cottonseed oil as the external oil phase, showed enhanced immunological activity over the free antigen or vesicles.

The charge density of Poly(D-lysine) was reduced by the carbamylation of the lysyl residues with potassium cyanate. A decrease in the charge density of poly(D-lysine) by 25% and 50% reduced the cytotoxicity of the ligand to cultured L929 cells by a 5-, and a 20 to 25-fold level, respectively, as estimated by using either the viability or the protein assay. The uptake of cyanate-modified poly(D-lysine) ligands in cultured L929 cells was not reduced, while the uptake of poly(D-lysine)/Heparin complex was reduced by 80%, as compared to that of unmodified poly(D-lysine). The in vivo biodistribution of cyanate-modified poly(D-lysine) ligands in the lungs and the liver of mice was not altered in comparison to that of unmodified poly(D-lysine), whereas the poly(D-lysine)/Heparin complex was only accumulated in the liver but not in the lungs. The data in this paper indicate that a 50% decrease in the positive charge density of poly(D-lysine) reduces the toxicity, but not the carrier potential of this poly-cationic ligand.

The trend to deliver drugs to defined areas of the body involves sophisticated carriers systems. In addition to the in vitro drug release profile one must be aware of the in vivo behaviour of the dosage form and the drug. Gamma scintigraphy is an elegant way to gain insights of the actual in vivo distribution pattern of dosage forms. This technique relies on the use of radioactive tracers included into the medicament and selected so as to enable an optimum detection by a gamma ray camera. The choice of a convenient label enables the in vivo determination of the targeting of the formulation administered through a large number of routes. The present paper reviews applications of gamma scintigraphy for the evaluation of dosage forms administered by the parenteral, rectal, buccal, nasal, pulmonary, and ophthalmic routes.

In the present paper effects of i.p. treatment with free or liposome-entrapped doxorubicin (DXR) on rat peritoneal exudate cell (PEC) populations were examined. Two types of DXR-liposomes were used: one type consisting of egg-phosphatidylcholine/phosphatidylserine/cholesterol (molar ratio 10/1/4, mean size approx. 0.3 μm) and the other type of distearoylphosphatidylcholine/dipalmitoylglycerol/cholesterol (molar ratio 10/1/10, mean size approx. 0.8 μm).Dramatically fewer PEC could be recovered from rats given free DXR or DXR-liposomes i.p. (10 mg DXR/kg body weight) 24 h before sacrifice than from control rats. Also the ratio of leukocyte species was modified by treatment with DXR, in free or liposomal form; in both cases the relative number of macrophages tended to increase. HPLC determination of the amount of DXR associated with monolayers obtained by seeding PEC harvested after a single i.p. dose of free DXR or liposomal DXR suggests that peritoneal macrophages are not particularly active in endocytosing DXR-liposomes. It was observed that the peritoneal macrophages phagocytosed DXR-containing granules from degranulating mast cells after both i.p. treatment with free DXR and DXR-liposomes. Decreased yields of PEC following i.p. treatment with free or liposomal DXR as well as an involvement of mast cells in the processing of both free DXR and liposome-encapsulated DXR in the peritoneal cavity may have important consequences for approaches to i.p. chemotherapy.

Present paper proposes a new system to administer triamterene, a sparing potassium diuretic which presents absorption problems when administered as a free powder, due to its low solubility in water.To increase the dissolution rate and subsequent oral bioavailability of this drug, it has been formulated as solid dispersion. This method involves preparation by the melting carrier method using D-mannitol as matrix. These systems were subjected to USP XXII dissolution rate determination. The results revealed a marked dissolution rate increase of included triamterene in solid dispersions when compared with micronized drug. Further in vivo assays have demonstrated the absorption efficiency for the proposed systems when referred to pure drug. In vitro-in vivo correlation between the parameter T80% (from dissolution rate studies) and the pharmacokinetic one Ke, has found to be acceptable. All the results obtained make this system suited for further formulation in the pharmaceutical industry.

Adenosine triphosphate (ATP) was proposed in various medical applications, as a possible bioenergetic substrate. Unfortunately, ATP is very difficult to use at a therapeutic level because of its high sensitivity to enzymatic hydrolysis making this molecule unstable in biological fluids. ATP is also a highly hydrophilic molecule that is unable to cross biological membranes. To try to develop a system able to protect ATP against degradation and to efficiently deliver this bioenergetic substrate, its liposomal encapsulation in multilamellar vesicles was carried out. One of the studies described in this paper deals with the efficiency of liposomal ATP in the treatment of cerebral ischemia. Our results show that encapsulation was able to protect ATP from its degradation by ectonucleotidases and that liposomal ATP was active against experimental brain ischemia. The other study deals with the effect of ATP on the motility and the acrosomal reaction of human spermatozoa. The results show that co-incubating ATP-loaded liposomes with sperm cells was able to induce the process of capacitation in vitro and might therefore be a useful tool in the procedure of in vitro fertilization.