Utilization of a sol–gel method for encapsulation of doxorubicin

The sol-gel pre-doping method was used to encapsulate doxorubicin in silica gels and optimum conditions of preparation of drug-loaded gel were established, ensuring both reproducible and effective results of quantitative encapsulation of doxorubicin and its gradual but complete release. Doxorubicin was encapsulated in polysiloxane polymers using the method based on sol-gel encapsulation without a catalyst, with an acid catalyst (HCl) and a base catalyst (NH₃). The time of gelation of the gel loaded with doxorubicin, encapsulation efficiency of the drug and the degree of release of the drug from the gel are all affected by the kind of catalyst (acidic or basic) or its absence at the gel preparation stage, and the temperature of the gelation process. The time of sol gelation when using the NH₃ or HCl catalyst was 9 days at 21°C, 2 days at 30°C and 1.5 days at 37°C, while for the gel prepared without a catalyst it was 90 days at 21°C, 75 days at 30°C and 70 days at 37°C. The efficiency of doxorubicin encapsulation was 99.5 ± 0.5% (w/w) for acid-catalyzed gel, 98.9 ± 1.01% (w/w) for base-catalyzed gel and 86.4 ± 11.6% (w/w) for non-catalyzed gel. A 100% (w/w) release of doxorubicin by diffusion through pores was found only in the case of base-catalyzed gel after a 140-h incubation time. For acid-catalyzed gel and non-catalyzed gel, the total amounts of released doxorubicin after 140 h of incubation were 3-5% (w/w) and 9-11% (w/w), respectively. The stability of doxorubicin encapsulated in the three kinds of gel matrices was found to be improved compared to the stability of a free form of the drug in solution.     

Biocompatibility and gelation of chitosan-glycerol phosphate hydrogels

The aim of this study was to evaluate the cytotoxicity and gelation of thermosensitive chitosan-beta-glycerol phosphate (GP) solutions, which undergo sol-gel transition around body temperature. Chitosan 0.5-2% (w/v) mixed with GP 5-20% (w/v) solutions all gel at 37 degrees C and possess pH around the physiological range. High GP and chitosan concentrations result in faster gelation time. Extracts of all chitosan concentrations mixed with or without 5% (w/v) GP and 2% (w/v) chitosan combined with 10% (w/v) GP demonstrated up to 34% increase in proliferation rate of goat bone marrow derived mesenchymal stem cells when compared with control medium. Extracts from all other chitosan-GP combinations resulted in reduced cell proliferation relative to control medium. Increasing GP content in the gel resulted in a linear increase in the osmolality of the extracts in contact with the gels. The results of this study indicate that chitosan-GP is a biocompatible hydrogel, extracts of which can stimulate mesenchymal stem cell proliferation at certain concentrations. This material is therefore a promising vehicle for cell encapsulation and injectable tissue-engineering applications.     

Pluronic F127 as a cell encapsulation material: utilization of membrane-stabilizing agents

Thermoreversible gelation of the copolymer Pluronic F127 (generic name, poloxamer 407) in water makes it a unique candidate for cell encapsulation applications, either alone or to promote cell seeding and attachment in tissue scaffolds. At concentrations of 15-20% (w/w), aqueous Pluronic F127 (F127) solutions gel at physiological temperatures. The effect of F127 on viability and proliferation of human liver carcinoma cells (HepG2) was determined for both liquid and gel formulations. Cell concentration and viability over a 5-day period were measured by the trypan blue assay via hemocytometry and results were confirmed in both the MTT and LDH assays. With 0.1-5% (w/w) F127 (liquid), cells proliferated and maintained high viability over 5 days. However, at 10% (w/w) F127 (liquid), there was a significant decrease in cell viability and no cell proliferation was evident. HepG2 cell encapsulation in F127 concentrations ranging from 15 to 20% (w/w) (gel) resulted in complete cell death by 5 days. This was also true for the HMEC-1 (endothelial) and L6 (muscle) cell lines evaluated. Cell-seeding density did not affect cell survival or proliferation. Membrane-stabilizing agents (hydrocortisone, glucose, and glycerol) were added to the F127 gel formulations to improve cell viability. The steroid hydrocortisone demonstrated the most significant improvement in viability, from <2% (in F127 alone) to >70% (with 60 nM hydrocortisone added). These results suggest that F127 formulations supplemented with membrane-stabilizing agents can serve as viable cell encapsulation materials. In addition, hydrocortisone may be generally useful in the promotion of cell viability for a wide range of encapsulation materials.     

Development and characterization of biodegradable nanospheres as delivery systems of anti-ischemic adenosine derivatives

We report a preliminary study concerning the encapsulation modalities in nanoparticles of the anti-ischemic drug N6-cyclopentyladenosine (CPA) and its pro-drug 5'-octanoyl-CPA (Oct-CPA). The release of these compounds and the related pro-drug stability effects in human whole blood have been tested. Moreover, the influence of the delivery systems on CPA interaction toward human adenosine A1 receptor has been analysed. The nanospheres were prepared by nanoprecipitation or double emulsion solvent evaporation method using poly(lactic acid) and recovered by gel filtration or ultracentrifugation or dialysis. Free and encapsulated Oct-CPA was incubated in fresh blood and its stability was analysed with HPLC. Quite spherical nanoparticles with mean diameters ranging between 210+/-50 and 390+/-90 nm were obtained. No encapsulation occurred when CPA was used. Satisfactory results concerning drug content (0.1-1.1% w/w) and encapsulation efficiency (6-56%) were achieved when Oct-CPA was employed. The controlled release of the pro-drug was achieved, being released within a range of 1-4 h, or very slowly, depending on nanoparticle preparations. The hydrolysis rate of Oct-CPA in human whole blood appeared stabilized in human whole blood with modalities related to the release patterns. The presence of all nanoparticle preparations did not interfere with CPA interaction at its action site.     

Effect of binder additives on terbutaline hydrogels of alpha-PVA/NaCl/H(2)O system in drug delivery: I. Effect of gelatin and soluble starch

In order to prepare ecologically and biologically safety physical cross-linked hydrogel of a-PVA/NaCl/H(2)O system, we prepared blend hydrogel of natural polymeric binders like gelatin and starch in above system, and effect of these binder additives were evaluated on terbutaline release kinetics. Terbutaline (1%) hydrogels of a-PVA(7%)/NaCl(11%)/H(2)O and a-PVA(7%)/H(2)O system (Cyclic FT process) were prepared along with various concentrations of gelatin and soluble starch by feed-mixture dissolving method. In case of a-PVA/NaCl/H(2)O system only one cycle gelation was done at -20 degrees C for 24 h. On the other hand 3 cycles were done by freezing at -30 degrees C for 16 h followed by thawing at room temperature for 8 h. Drug release was done by paddle method (USP type II) with a rotation of 50 rpm at 37 degrees C in distilled water. Swelling of the gel was done at 37 degrees C for 45 h and melting temperatures of the gel were also studied using the upside-down test tube method. Comparatively lower values of release rate, diffusion coefficient and kinetic constant were found from the blend hydrogel of a-PVA/starch/NaCl/H(2)O system. When % cumulative release was plotted vs. square root of time it showed straight lines, which indicated Higuchi Matrix Dissolution Model. Inclusion of starch binder increased the degree of swelling compared with that of gelatin. 15 h was found as equilibrium swelling time. A Fickian swelling of this blend hydrogel system indicated the swelling controlled Fickian diffusion type of drug release. Melting temperatures of the blend hydrogels were characteristically higher (94-95 degrees C) than that of cyclic FT (72-76 degrees C), resulting a thermostable hydrogel for biological system. SEM morphological studies of gel surface indicated the well-developed interpenetrating macromolecular network structure like fish net in starch blend gel prevails over other hydrogel studied here. Gelatin has got characteristic tunnel (200 microm in diameter) inside the macromolecular network that contributes this system higher release kinetics than that of starch.     

Sonication-induced gelation of silk fibroin for cell encapsulation

Purified native silk fibroin forms beta-sheet-rich, physically cross-linked, hydrogels from aqueous solution, in a process influenced by environmental parameters. Previously we reported gelation times of days to weeks for aqueous native silk protein solutions, with high ionic strength and temperature and low pH responsible for increasing gelation kinetics. Here we report a novel method to accelerate the process and control silk fibroin gelation through ultrasonication. Depending on the sonication parameters, including power output and time, along with silk fibroin concentration, gelation could be controlled from minutes to hours, allowing the post-sonication addition of cells prior to final gel setting. Mechanistically, ultrasonication initiated the formation of beta-sheets by alteration in hydrophobic hydration, thus accelerating the formation of physical cross-links responsible for gel stabilization. K(+) at physiological concentrations and low pH promoted gelation, which was not observed in the presence of Ca(2+). The hydrogels were assessed for mechanical properties and proteolytic degradation; reported values matched or exceeded other cell-encapsulating gel material systems. Human bone marrow derived mesenchymal stem cells (hMSCs) were successfully incorporated into these silk fibroin hydrogels after sonication, followed by rapid gelation and sustained cell function. Sonicated silk fibroin solutions at 4%, 8%, and 12% (w/v), followed by mixing in hMSCs, gelled within 0.5-2 h. The cells grew and proliferated in the 4% gels over 21 days, while survival was lower in the gels with higher protein content. Thus, sonication provides a useful new tool with which to initiate rapid sol-gel transitions, such as for cell encapsulation.     

Poly(N-isopropylacrylamide) copolymer films as vehicles for the sustained delivery of proteins to vascular endothelial cells

The aim of this study was to establish the capacity of thermoresponsive poly(N-isopropylacrylamide) copolymer films to deliver bioactive concentrations of vascular endothelial growth factor (VEGF165) to human aortic endothelial cells (HAEC) over an extended time period. Films were prepared using a 50:50 (w/w) mixture of non-crosslinkable and crosslinkable copolymers of the following monomer compositions (w/w): 85:15, N-isopropylacrylamide (NiPAAm):N-tert-butylacrylamide (NtBAAm); and 85:13:2 NiPAAm:NtBAAm:acrylamidobenzophenone (ABzPh, crosslinking agent), respectively. After crosslinking by UV irradiation, the ability of films to incorporate a fluorescently labeled carrier protein (FITC-labeled BSA, 1 mg loaded per film), at 4 degrees C, was first established. Incorporation into the matrix was confirmed by the observation that increasing film thickness from 5 to 10 microm increased release from collapsed films at 37 degrees C (1.76 +/- 0.15 and 10.98 +/- 3.38 microg/mL, respectively, at 24 h postloading) and that this difference was maintained at 5 days postloading (1.81 +/- 0.25 and 13.8 +/- 2.3 microg/mL, respectively). Incorporation was also confirmed by visualization using confocal microscopy. When 10-microm films were loaded with a BSA solution (1 mg/mL) containing VEGF165 (3 microg/mL), sustained release of VEGF165 was observed (10.75 +/- 3.11 ng at 24 h; a total of 31.32 +/- 8.50 ng over 7 days). Furthermore, eluted VEGF165 increased HAEC proliferation by 18.2% over control. The absence of cytotoxic species in medium released from the copolymer films was confirmed by the lack of effect of medium (incubated with copolymer films for 3 days) on HAEC viability. In conclusion this study has shown that NiPAAm:NtBAAm copolymers can be loaded with a therapeutic protein and can deliver bioactive concentrations to human vascular endothelial cells over an extended time period.     

In vitro evaluation of in situ gels as short term vitreous substitutes

Dysfunction of the vitreous humor, present in the posterior cavity of the eye, leads to its detachment from the retina and vision loss. In this study, biopolymers were evaluated as in situ gels for short term vitreous substitution. Biophysical characterization revealed that the viscosity of the vitreous was >4000 cP at a shear rate of 0.15/s and it formed a gel with elastic modulus G' greater than the viscous modulus G'. Biopolymers of gellan and hyaluronic acid (8:2 w/w, 1% concentration) were low viscosity liquids at 37 degrees C and gelation was triggered both by the addition of 0.18 mM CaCl(2) as well as ocular temperature, thus making them feasible as in situ gels. Gelation was confirmed by viscoelastic moduli where G' was greater than G', similar to the vitreous and unlike that of silicone oil, a common vitreous substitute. The gels had a viscosity >5000 cP at a shear rate of 0.512/s, excellent light transmittance and absence of syneresis. Contact angle studies with water and simulated ocular fluids showed that gellan hyaluronic acid gels had similar wetting properties to that of vitreous with contact angles of 27 degrees +/- 1 degrees , 36.7 degrees +/- 1.6 degrees , and 33.7 degrees +/- 0.5 degrees for water, simulated tear fluid, and simulated aqueous humor, respectively. The results of this study suggest that biopolymers of gellan and hylauronic acid are suitable as in situ gels, have biophysical properties similar to that of the vitreous, and may be promising as alternatives to silicone oil as short-term vitreous substitutes.     

Physical matrices stabilized by enzymatically sensitive covalent crosslinks

This work describes a unique system of gel and gel-like materials formed from physical bonds between heparin and heparin binding peptides (dG-PBD) coupled to multivalent poly(ethylene glycol) vinyl sulfone star polymers (PEGVS) and formed from covalent bonds between an enzymatically sensitive crosslinker and PEGVS. Dynamic mechanical testing revealed that the viscoelastic behavior and gelation kinetics of 10% (w/v) gels formed from 2:1 dG-PBD:PEGVS, heparin, and crosslinker (2:1 gels) and from 3:1 dG-PBD:PEGVS, heparin, and crosslinker (3:1 materials) were significantly influenced by the presence of both physical and covalent bonds. Furthermore, the mechanical properties of both compositions were thermally responsive and reversible. At 4 degrees C, the storage modulus, G', for 2:1 gels (5005.3+/-592.0Pa) and 3:1 materials (5512.0+/-272.7Pa) were statistically similar; however, at 45 degrees C, G' of 2:1 gels decreased to 477.9+/-150.4Pa, and the viscoelastic behavior of 3:1 materials was dominated by viscous behavior. In addition, the mechanical properties of 2:1 gels and 3:1 materials were sensitive to the frequency of the applied stress at 4 degrees C, 20 degrees C, and at 37 degrees C. Although the covalent bonds within the materials provided mechanical stability, the overall viscoelastic response of this system could be dominated by physical crosslinks under certain conditions. Results from degradation studies indicated that the crosslinker was sensitive to collagenase type I but not to thrombin or heparinase I, and a hemolysis assay suggested that the 2:1 gels and 3:1 materials might be biocompatible. These materials could be useful to study the role of physical interactions within networks that mimic the extracellular matrix.     

Triggered release of calcium from lipid vesicles: a bioinspired strategy for rapid gelation of polysaccharide and protein hydrogels

The bioinspired strategy of triggered release of Ca2+ from liposomal compartments was used to induce rapid gelation of polysaccharide and protein-based hydrogels. Thermally triggerable liposomes were designed by entrapping CaCl2 within liposomes constructed of 90% dipalmitoylphosphatidylcholine and 10% dimyristoylphosphatidylcholine. These liposomes released greater than 90% of entrapped Ca2+ when heated to 37 degrees C. A precursor fluid containing liposomes suspended in aqueous sodium alginate remained fluid for several days at room temperature but gelled rapidly when heated to 37 degrees C, as a result of Ca2+ release and formation of crosslinked Ca-alginate. Alternatively, thermally triggered Ca2+ release from liposomes was used to activate enzyme-catalyzed crosslinking of proteins to form hydrogels. A mixture of Ca-loaded liposomes, fibrinogen, and a Ca2+-dependent transglutaminase enzyme (either human recombinant FXIII or guinea pig liver transglutaminase) remained fluid indefinitely when stored at room temperature, but gelled rapidly when heated to 37 degrees C. SDS-PAGE of the reaction mixture revealed that gelation was due to enzymatic crosslinking of the alpha and gamma chains of fibrinogen, and oscillating rheometry revealed gel formation within 10 min of heating to 37 degrees C. This new approach may be useful for developing rapidly gelling injectable biomaterials that can be stored at room temperature and injected in a minimally invasive manner into a body tissue or cavity, upon which rapid solidification would occur. This versatile bioinspired strategy could be utilized for the delivery of biomaterials for tissue repair and reconstruction, and local site-directed drug delivery.     

Kinetics of poly(propylene fumarate) synthesis by step polymerization of diethyl fumarate and propylene glycol using zinc chloride as a catalyst

Diethyl fumarate and propylene glycol were reacted in the presence of a zinc chloride catalyst to synthesize poly(propylene fumarate) (PPF) over a period of 12 hours. The kinetics of the transesterification polymerization at 130 degrees C, 150 degrees C, and 200 degrees C were determined by gel permeation chromatography (GPC) analysis. The initial rate of polymerization at each temperature was quantified by calculating the rate of change of the number average molecular weight (Mn). At 200 degrees C, gelation of the PPF occurred after 4 h. GPC analysis of the reaction showed that PPF synthesized at 150 degrees C had a higher final Mn of 4600 (+/- 190) and a higher weight average molecular weight of 10500 (+/- 760) than at 130 degrees C (n = 3). The chemical structure of the PPF was verified by NMR and FT-IR analysis. This study demonstrated that the maximum Mn of PPF by a transesterification reaction is limited due to gelation of PPF at high temperature.     

Preparation of 125I-labeled human growth hormone of high quality binding properties endowed with long-term stability

125I-labeled human growth hormone (125I-labeled.hGH) was prepared by using two variants of the chloramine T labelling procedure and purified by polyacrylamide gel electrophoresis (PAGE) of the reaction mixture. Variant A produced a tracer with high specific activity (100 +/- 10 microCi/microgram), high maximal binding capacity to antibodies (93%) and long-term stability (at least 150 days at -20 degrees C). No diiodinated tyrosil residues could be detected in this tracer. Variant B was devised to obtain higher yields of labeled hormone. The electrophoresis of the iodination mixture revealed two radioactive components with Rm values of 0.49 and 0.55 which result from the iodination of hGH variants preexisting in the starting material. Both tracers had similar specific activities (70 +/- 10 microCi/microgram), high maximal binding capacity to antibodies or receptors (80-100%, after 80 days of their obtention) and high stability (at least 100 days at -20 degrees C). It is concluded that the iododerivatives of hGH obtained by either method are adequate to perform radioimmunoassay and receptor studies and have long-term stability.     

Increased positivity of skin test and allergenic stability of glycerinated soybean hull extracts

BACKGROUND: Results obtained from previous experiences with soybean hull antigens suggest that the addition of glycerine to the extract increases positivity of the skin test. OBJECTIVE: The purpose of this study was to investigate the effect of this glycerine addition and the influence of different storage temperatures on the potency of soybean hull extracts. METHODS: Twenty-two asthmatic patients admitted to emergency rooms during one of the soybean-related Barcelona asthma epidemics were evaluated 2 years after the last epidemic for: (1) sensitivity to prick test with glycerinated and non-glycerinated extracts, both fresh and stored at 4 degrees C, - 20 degrees C and - 70 degrees C for 30 days, and (2) specific IgE to a fresh hull extract. All extracts were also studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: No differences in positivities were detected for prick tests performed the same day of preparation between non-glycerinated (5/22; 22.7%) and glycerinated (4/22; 18.2%) extracts. After 30 days storage, positivities of prick test with non-glycerinated extracts stored at 4 degrees C did not significantly differ from the results obtained on the first day. However, prick tests performed with glycerinated extracts stored for 30 days showed increased positivity for the extracts stored at 4 degrees C (7/22; 31%) (P < 0.05) as well as for those stored at - 20 degrees C and - 70 degrees C (9/22; 40.9%) (P < 0.001). This latter percentage is similar to that of specific immunoglobulin E detected by radioimmunoassay (10/22; 45. 4%). Also, for the glycerinated extracts only, the quantitative response showed an increase in mean diameter of the induration at 30 days at the three temperatures, even though owing to the great standard deviation and limited number of patients, it was only significant for the extract stored at - 20 degrees C (P < 0.01). SDS-PAGE gel densitometry at 30 days demonstrated a loss of protein in the bands with a molecular weight higher than 66.2 kDa for non-glycerinated extracts stored at 4 degrees C. CONCLUSIONS: The results of this study confirm previous observations with soybean hull allergen extracts and indicate that (1) storage of soybean hull extracts with glycerine addition for 30 days at the concentration studied (1:100 w/v) determines a significant increase in the positivity of skin tests, and (2) glycerine addition stabilizes soybean hull extracts at any of the temperatures studied.     

Hydrogen-bonded polymer gel and its application as a temperature-sensitive drug delivery system

The mixture of poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide) triblock copolymer (F-68) and poly vinyl alcohol (PVA) forms a polymer complex gel by intra/intermolecular interaction via hydrogen bonding in water, which is verified by differential scanning calorimetry. With 30 wt% F-68 aqueous solution and 15 wt% PVA aqueous solution, F-68/PVA complex gel was prepared and its swelling transition was observed at approximately 37 degrees C. Based on the temperature-sensitivity of hydrogen bondings in F-68/PVA complex gel, temperature sensitive drug delivery system has been designed and characterized. For the stability in the aqueous media, F-68/PVA complex gel was prepared with a form of polymeric bead, followed by the encapsulation with poly(lactide-co-glycolide) membrane. With changing the ratio of F-68/PVA, the swelling transition of polymer complex gel was manipulated and pulsatile release of acetoaminophen, used as a model drug, was demonstrated in response to pulsatile change of temperature between 35 degrees C and 40 degrees C.     

Thermal assembly of a biomimetic mineral/collagen composite

A strategy is described for exploiting temperature driven self-assembly of collagen and thermally triggered liposome mineralization to form a mineralized collagen composite from an injectable precursor fluid. Optical density and rheological experiments demonstrated the formation of a collagen gel when acid-soluble type I collagen solutions (1-7 mg/ml) were heated to 24-30 degrees C. Scanning calorimetry experiments demonstrated that mixtures of calcium- and phosphate-loaded liposomes composed of dipalmitoylphosphatidylcholine (90 mol%) and dimyristoylphosphatidylcholine (10 mol%) were stable at room temperature but formed calcium phosphate mineral when heated above 35 degrees C, a consequence of the release of entrapped salts at the lipid chain melting transition. The formation of calcium phosphate mineral induced by triggered release of calcium and phosphate was detected as an endothermic transition (deltaH=6.2+/-1.1 kcal/mol lipid) near the lipid chain melting transition (Tm=37 degrees C). Combining an acid-soluble collagen solution with calcium- and phosphate-loaded liposomes resulted in a liposome/collagen precursor fluid, which when heated from room temperature to 37 degrees C formed a mineralized collagen gel. The dynamic storage modulus of the collagen scaffold increased upon mineralization, and direct nucleation of mineral from the collagen scaffold was detected by electron microscopy.     

Photochemically cross-linked collagen gels as three-dimensional scaffolds for tissue engineering

Collagen gels have many favorable attributes for tissue engineering, but the gels undergo dramatic contraction when cells are added because of the weak noncovalent bonds that form during spontaneous gelation. We hypothesized that photochemically cross-linking collagen gels would make suitable scaffolds for tissue engineering with favorable cell viability and minimal gel contraction. Rose Bengal and riboflavin were chosen as candidate photo-initiators for gel cross-linking using 532- and 458-nm-light wavelengths, respectively. Chondrocyte viability was measured after initial gelation for several concentrations of initiators. Cell viability and gel contraction were then measured using chondrocytes and fibroblasts over 7 days of culture. Rose Bengal used at concentrations necessary for gelation resulted in little or no cell viability. Short-term viability results showed that 0.25- or 0.5-mM concentrations of riboflavin, and 40 s of illumination permitted more than 90% cell viability. Using riboflavin concentrations of 0.25 or 0.5 mM, long-term chondrocyte viability was 113.1 +/- 11.6% and 25.4 +/- 2.7%, respectively, at day 7. Although non-cross-linked chondrocyte constructs contracted to 59.9 +/- 11.8% of their original diameter and fibroblasts contracted to 24.9 +/- 5.0% of their original diameter by day 7, the cross-linked constructs retained 88.8 +/- 7.4% and 85.5 +/- 5.0% of the original diameter, respectively. In conclusion, by photochemically cross-linking collagen gels using riboflavin and visible light, stable gel scaffolds with favorable cell survival can be produced.     

Encapsulation of brewers yeast in chitosan coated carrageenan microspheres by emulsification/thermal gelation

Brewers yeast was encapsulated in kappa-carrageenan microspheres using an emulsification-thermal gelation approach. Due to heat sensitivity of the yeast at temperatures in excess of 36 degrees C, mixtures of low and high gelation temperature carrageenans were tested to obtain a blend yielding a gelation temperature under 40 degrees C. A 20:80 dispersion of 2% carrageenan sol containing cells, in warm canola oil, produced microspheres upon cooling, with a mean diameter of 450 microm and narrow size dispersion (span of 1.2). Application of a chitosan membrane coat to minimize cell release, increased the mean microsphere diameter to 700 microm, due to the coat thickness and swelling of the microspheres. This diameter was designed so as to minimize mass transfer limitations. Batch fermentations were carried out in a 3 L reactor on a commercial wort medium. Cell loading was 10(7) cells mL(-1) microspheres, and cell "burst" release was observed upon inoculation into fresh medium, whether microspheres were coated or not. The kinetics of intra- and extracapsular cell growth were determined. Increased concentrations of extracapsular free cells could be accounted for by growth in the wort medium, and by ongoing release from the gel microspheres, whether coated or not. Cell release from chitosan-coated carrageenan microspheres was less than that from uncoated microspheres, likely due to retention by the membrane coat. Growth kinetics and alpha-amino nitrogen consumption of encapsulated yeast were higher than that of free cells, and differences in alcohol and ester profiles were also observed, likely due to modified metabolism of the encapsulated yeast.     

A simple one-step protocol for preparing small-sized doxorubicin-loaded liposomes.

A simple, single-step, extrusion-free protocol for preparing doxorubicin-loaded liposomes (100150 nm), based on the ethanol injection method (EIM), is described. Efficient encapsulation of doxorubicin (up to 98%) was obtained concomitantly with liposome preparation avoiding the need for an additional loading step. Parameters such as stock concentration of phospholipid, injection ratio, lipid composition, and drug-to-phospholipid ratio affected the resultant liposome size and magnitude of doxorubicin encapsulation. A lipid stock concentration (50 mM) and injection ratio (1:10) resulted in 96.0 +/- 2.92% encapsulation efficiency of doxorubicin (drug-to-lipid mole ratio: 0.192) and mean diameter of 135 +/- 2.32 nm for SCOL-2 formulation (DSPC/ cholesterol /oleic acid: 2/2/1; molar ratios). Replacement of phospholipid DSPC with DMPC or DPPC did not affect the mean liposome size and doxorubicin-encapsulation efficiency. These findings offer promise for scale-up and development on a large-scale production of doxorubicin-loaded liposomes for effective cancer therapy.     

Insulin release from islets of Langerhans entrapped in a poly(N-isopropylacrylamide-co-acrylic acid) polymer gel

A copolymer of N-isopropylacrylamide (98 mol% in feed) and acrylic acid, poly(N-isopropylacrylamide-co-acrylic acid) (P(NIPAAm-co-AAc)), was prepared by free radical polymerization for development of a thermally reversible polymer to entrap islets of Langerhans for a refillable biohybrid artificial pancreas. A 5 wt% solution of the polymer in Hanks' balanced salt solution forms a gel at 37 degrees C that exhibits no syneresis. Diffusion of fluorescein isothiocyanate (FITC) dextrans having molecular weights of 4400 and 70000 were used to evaluate mass transport in the gel at 37 degrees C. Insulin secretion from islets in the polymer gel was also investigated in both static and dynamic systems. The polymer gel exhibited excellent diffusion of FITC dextran 4400 and FITC dextran 70000 with diffusion ratios, D/D0 (ratio of diffusion in the gel to diffusion in water), of 0.20+/-0.04 and 0.35+/-0.17, respectively. Human islets entrapped in the polymer gel showed prolonged insulin secretion in response to basal (5.5 mM) glucose concentration compared to free human islets. Rat islets showed prolonged insulin secretion in response to high (16.5 mM) glucose concentrations compared to free rat islets. Rat islets in the polymer gel maintained insulin secretion in response to the higher glucose concentration for over 26 days. Rat islets entrapped by the polymer also released higher quantities of insulin more rapidly in response to changes in concentrations of glucose and other stimulants than rat islets entrapped in an alginate control. These results suggest that this material would provide adequate diffusion for rapid insulin release in an application as a synthetic extracellular matrix for a biohybrid artificial pancreas.     

Poly(N-isopropylacrylamide) co-polymer films as potential vehicles for delivery of an antimitotic agent to vascular smooth muscle cells.

INTRODUCTION: Local delivery of antimitotic agents is a potential therapeutic strategy for protection of injured coronary vasculature against intimal hyperplasia and restenosis. This study sought to establish the principle that thermoresponsive poly(N-isopropylacrylamide) co-polymer films can be used to deliver, in a controlled manner, an antimitotic agent to vascular smooth muscle cells (VSMC). METHODS: A series of co-polymer films was prepared, using varying ratios (w/w) of N-isopropylacrylamide (NiPAAm) monomer to N-tert-butylacrylamide (NtBAAm) and loaded with the antimitotic agent colchicine (100 nmol/film) at room temperature. RESULTS: The extent of colchicine release at 37 degrees C was inversely proportional to the amount of NtBAAm in co-polymer films: release after 48 h from 85:15, 65:35 and 50:50 (NiPAAm:NtBAAm) films was 26, 17 and 0.5 nmol, respectively. In cytotoxicity studies, when medium incubated with co-polymers for 24 h (in the absence of colchicine) was further incubated with target bovine aortic smooth muscle cells (BASMC), no loss of cell viability occurred. Colchicine released from all three co-polymer films significantly inhibited proliferation and random migration of BASMC: 100 nM colchicine (released from 65:35 NiPAAm:NtBAAm) reduced cell proliferation to 25.7+/-1.7% of levels seen in the absence of colchicine (control) and random cell migration to 37.7+/-5.7% of control (mean+/-S.E.M., n = 3, P < .01 and P < .05, respectively). The magnitudes of these effects were comparable to those seen in separate experiments with native colchicine and were observed in samples of released colchicine which had been stored at -20 degrees C for up to 6 months. CONCLUSIONS: This study has shown that the release of the antimitotic agent colchicine, from NiPAAm/NtBAAm co-polymer films can be manipulated by changes in co-polymer composition. Furthermore, such drug released at 37 degrees C retains comparable bioactivity to that of native colchicine.