Orwik

Molecular implications in the nanoencapsulation of the anti-tuberculosis drug rifampicin within flower-like polymeric micelles.

Tuberculosis (TB) is the second most deadly infectious disease behind the Human Immunodeficiency Virus (HIV). An effective pharmacotherapy has been available for more than 5 decades. However, the length of the treatment and the pill burden result in ... expand abstractlow patient compliance and adherence to the regimens. Nanotechnologies can overcome these basic technological drawbacks. The present work explored the molecular implications governing the encapsulation and water solubilization of RIF within flower-like micelles of poly(epsilon-caprolactone)-b-poly(ethylene glycol)-poly(epsilon-caprolactone) (PCL-PEG-PCL) block copolymers. Ten derivatives of different molecular weight and hydrophobic/hydrophilic caprolactone/ethylene oxide ratio (CL/EO) were synthesized by a fast and high-yield Microwave-Assisted Polymer Synthesis (MAPS) technique; CL/EO values are determined by taking the ratios of the number of repeating units in the PCL and the PEG segments. The aggregation behavior of the copolymers was thoroughly investigated by means of surface tension (critical micellar concentration), dynamic light scattering (size, size distribution and zeta potential) and transmission electron microscopy (morphology). In general, the greater the central PEG segment, the larger the micelles formed. The physical stability was intimately associated with the molecular weight and the composition. Then, the encapsulation of RIF in the different copolymer families was evaluated, and the physical stability of the drug-loaded aggregates characterized. The micellar size appears as the most crucial property, this phenomenon being primarily controlled by the molecular weight of the PEG central block. Having expressed this, sufficiently high CL/EO ratios (and long PCL segments) are also demanded to attain stable micellar systems with cores that are large enough to host the bulky RIF molecule. collapse abstract

Development and preclinical evaluation of acellular collagen scaffolding and autologous artificial connective tissue in the regeneration of oral mucosa wounds.

This work assessed wound healing response in rabbit oral lesions grafted with autologous artificial connective tissue or acellular collagen scaffolds. Autologous artificial oral connective tissue (AACT) was produced using rabbit fibroblasts and colla... expand abstractgen I scaffolds. Before implantation, AACT grafts were assayed to demonstrate the presence of fibroblasts and extracellular matrix components, as well as the expression of characteristic genes and secretion of chemokines, cytokines, and growth factors. AACT grafts were tested in the rabbits from which the fibroblasts were obtained, whereas acellular collagen type I scaffolds (CS) were evaluated in a separate group of rabbits. In both cases, contralateral wounds closed by secondary intention were used as controls. In a separate experiment, AACT-grafted wounds were directly compared with contralateral CS-grafted wounds in the same animals. Wound contraction and histological parameters were examined to evaluate closure differences between the treatments in the three animal experiments performed. Contraction of wounds grafted with AACT and CS was significantly lower than in their controls (p < 0.05). Additionally, AACT significantly lowered wound contraction when compared with CS (p < 0.05). Intriguingly, it was observed that AACT-grafted wounds initially displayed a significantly higher (p < 0.05)-albeit transient-inflammatory response than seen in CS-grafted wounds and secondary healed wounds. This suggests that an early inflammatory component may contribute to tissue regeneration. Altogether, the results suggest that AACT- and CS-grafted wounds favor regeneration of oral mucosa. collapse abstract

A highly concentrated and taste-improved aqueous formulation of efavirenz for a more appropriate pediatric management of the anti-HIV therapy.

Pediatric HIV is scarce in developed countries; 90% of pediatric HIV patients are in developing countries. In contrast, children represent 15% of the new infections in poor countries. Approximately 90% of the HIV-positive children do not have access ... expand abstractto antiretrovirals (ARVs). Without treatment, 50% of the patients die before the 2 years of age. Efavirenz (EFV, aqueous solubility approximately 4 microg/mL, 40-45% bioavailability), a non-nucleoside reverse transcriptase inhibitor (NNRTI), is a first-choice pediatric ARV. To assure therapeutic plasma concentrations, the low oral bioavailability demands the administration of relatively high EFV doses. Aqueous EFV irritates the oral mucosa, causing a Burning Mouth Syndrome (BMS). A triglyceride-based liquid formulation of EFV (30 mg/mL) is not commercially available worldwide, making the appropriate dose adjustment and the swallowing difficult. More importantly, clinical trials indicated that the oral bioavailability of this oily solution is lower than that of the solid one. Moreover, a relatively high inter-subject variability has been found. The present work reports the development and full characterization of a concentrated (20 mg/mL, 2%) and taste-masked aqueous formulation of EFV for a more appropriate management of the pediatric anti-HIV therapy. Formulations displayed high physicochemical stability over time under regular storage conditions. Release assays in vitro showed a burst effect (2 h) and zero-order kinetics later on (between 2 and 24 h), compatible with the oral administration route and release. Finally, taste tests performed by adult healthy volunteers indicated that the unique combination of flavors and sweeteners employed (i) reduced the intensity of the BMS and (ii) shortened its duration significantly. Overall results indicate that the cost-effective and scalable nanotechnological strategy proposed could enable the more convenient and compliant administration of lower EFV doses. Due to a better pharmacokinetic profile, this would result in similar plasma levels than higher doses administered in solid or triglyceride-soluble form. In this context, some reduction of the treatment cost can be envisioned. This could improve the access of less affluent pediatric patients to medication in poor countries. collapse abstract

Drug delivery systems and liver targeting for the improved pharmacotherapy of the hepatitis B virus (HBV) infection.

In spite of the progress made in vaccine and antiviral therapy development, hepatitis B virus (HBV) infection is still the most common cause of liver cirrhosis and hepatocellular carcinoma, with more than 400 million people chronically infected world... expand abstractwide. Antiviral therapy with nucleos(t)ide analogues and/or immunomodulating peptides is the only option to control and prevent the progression of the disease in chronic hepatitis B (CHB)-infected patients. So far, the current antiviral monotherapy remains unsatisfactory because of the low efficacy and the development of drug resistance mutants. Moreover, viral rebound is frequently observed following therapy cessation, since covalent closed circular DNA (cccDNA) is not removed from hepatocytes by antiviral therapy. First, this review describes the current pharmacotherapy for the management of CHB and the new drug candidates being investigated. Then, the challenges in the development of drug delivery systems for the targeting of antiviral drugs to the liver parenchyma are discussed. Finally, perspectives in the design of a more efficient pharmacotherapy to eradicate the virus from the host are addressed. collapse abstract

Repair of bone defect using bone marrow cells and demineralized bone matrix supplemented with polymeric materials.

We present a novel, reverse thermo-responsive (RTR) polymeric osteogenic composite comprising demineralized bone matrix (DBM) and unmanipulated bone marrow cells (BMC) for repair of bone defects. The polymers investigated were low viscosity aqueous s... expand abstractolutions at ambient temperature, which gel once they heat up and reach body temperature. Our goal to supplement DBM-BMC composite with RTR polymers displaying superior rheological properties, was to improve graft integrity and stability, during tissue regeneration. The osteogenic composite when implanted under kidney capsule of mice, proved to be biocompatible and biodegradable, with no residual polymer detected in the newly formed osteohematopoietic site. Implantation of the osteogenic composite into a large area of missing area of parietal bone of the skull of rats, resulted in an extensive remodeling of DBM particles, fully reconstituted hematopoietic microenvironment and well integrated normal flat bone within thirty days. The quality and shape of the newly created bone were comparable to the original bone and neither local or systemic inflammatory reactions nor fibrosis at the junction of the new and old calvarium could be documented. Furthermore, combined laser capture microdissection (LCM) technique and PCR analysis of male BMC in female rats confirmed the presence of male derived cells captured from the repaired/ regenerated flat bone defect. The use of active self sufficient osteogenic DBM-BMC composite supported by a viscous polymeric scaffold for purposive local hard tissue formation, may have a significant potential in enhancement of bone regeneration and repair following trauma, degenerative or inflamatory lesion, iatrogenic interventions and cosmetic indications. collapse abstract

Novel long-term anticonvulsant treatment with gabapentin without causing memory impairment in mice.

We previously reported that administration of a single dose of gabapentin (GBP) immediately after training improves memory of mice in an inhibitory avoidance task (IA), whereas GBP administered repeatedly for 7 days impairs memory. This is in accorda... expand abstractnce with the observation that long-term clinical treatment with GBP may be associated with adverse cognitive side effects. In the present work we used a GBP-loaded poly(epsilon-caprolactone) implant, allowing controlled release of the drug and maintenance of constant plasma levels over 1 week. When GBP-loaded implants were inserted subcutaneously into mice, immediately after training in the IA task, memory consolidation was enhanced. Moreover, GBP released from implants had an anticonvulsant action against pentylenetetrazole-induced seizures. These results suggest that maintenance of stable GBP plasma levels could protect against seizures without causing memory impairment. Hence, the adverse cognitive effects might be avoided by stabilizing plasma levels of the drug. collapse abstract

Efavirenz-loaded polymeric micelles for pediatric anti-HIV pharmacotherapy with significantly higher oral bioavailaibility.

Children constitute the most challenging population in anti-HIV/AIDS pharmacotherapy. Efavirenz (EFV; aqueous solubility 4 microg/ml, bioavailability 40-45%) is a first-line agent in the pediatric therapeutic cocktail. The liquid formulation of EFV i... expand abstracts not available worldwide, preventing appropriate dose adjustment and more convenient administration. The bioavailability of liquid EFV is lower than that of the solid formulation. Improving the bioavailability of the drug would reduce the cost of treatment and enable less affluent patients to access this drug. Aim: To encapsulate EFV in polymeric micelles to improve the aqueous solubility and the the oral bioavailability of the drug. Methods: EFV was incorporated into the core of linear and branched poly(ethylene oxide)-poly(propylene oxide) block copolymer micelles. The size and size distribution of the drug-loaded aggregates were characterized by dynamic light scattering and the morphology by transmission electron microscopy. The bioavailability of the EFV-loaded micellar system (20 mg/ml) was assessed in male Wistar rats (40 mg/kg) and compared to that of a suspension prepared with the content of EFV capsules in 1.5% carboxymethylcellulose PBS solution (pH 5.0), and an EFV solution in a medium-chain triglyceride (Miglyol((R)) 812). Results: This work demonstrates that the encapsulation of EFV, which is poorly water soluble, into polymeric micelles of different poly(ethylene oxide)-poly(propylene oxide) block copolymers significantly improves the oral bioavailability of the drug, and reduces the interindividual variability. Conclusion: This strategy appears a very promising one towards the development of a liquid aqueous EFV formulation for the improved pediatric HIV pharmacotherapy. collapse abstract

Poloxamine-based nanomaterials for drug delivery.

Poloxamines (Tetronic) are X-shaped amphiphilic block copolymers formed by four arms of poly(ethylene oxide)-poly(propylene oxide) (PEO-PPO) blocks bonded to a central ethylenediamine moiety. Such a structure confers multi-responsive behaviour, namel... expand abstracty temperature and pH-sensitiveness. At relatively low concentrations but above the critical micellar concentration (CMC), poloxamines generate polymeric micelles. Due to the presence of a hydrophobic core, these nanocarriers are useful in the solubilization and stabilization of poorly water-soluble drugs. Moreover, chemical modification of the micellar core is feasible. These remarkable and unique features, compared to the well-known linear poloxamers, have motivated an increasing interest in the study and application of the branched derivatives in different emerging disciplines. The present review concisely overviews the most important developments comprising the application of poloxamines in drug delivery, mainly as micellar carriers capable of enhancing drug solubility and stability, and also as surface modifiers in the technology of stealth polymeric nanoparticles. Their potential for the administration of drugs by different routes and the improvement of the drug bioavailability and therapeutic effect are discussed. collapse abstract

New old challenges in tuberculosis: potentially effective nanotechnologies in drug delivery.

Tuberculosis (TB) is the second most deadly infectious disease. Despite potentially curative pharmacotherapies being available for over 50 years, the length of the treatment and the pill burden can hamper patient lifestyle. Thus, low compliance and a... expand abstractdherence to administration schedules remain the main reasons for therapeutic failure and contribute to the development of multi-drug-resistant (MDR) strains. Pediatric patients constitute a high risk population. Most of the first-line drugs are not commercially available in pediatric form. The design of novel antibiotics attempts to overcome drug resistance, to shorten the treatment course and to reduce drug interactions with antiretroviral therapies. On the other hand, the existing anti-TB drugs are still effective. Overcoming technological drawbacks of these therapeutic agents as well as improving the effectiveness of the drug by targeting the infection reservoirs remains the central aims of Pharmaceutical Technology. In this framework, nanotechnologies appear as one of the most promising approaches for the development of more effective and compliant medicines. The present review thoroughly overviews the state-of-the-art in the development of nano-based drug delivery systems for encapsulation and release of anti-TB drugs and discusses the challenges that are faced in the development of a more effective, compliant and also affordable TB pharmacotherapy. collapse abstract

Nanotechnology solutions for infectious diseases in developing nations. Preface.

Episcleral implants for topotecan delivery to the posterior segment of the eye.

Purpose. Intravenous or periocular topotecan has been proposed as new treatment modality for patients with advanced intraocular retinoblastoma, but systemic topotecan lactone exposure induced by both approaches may cause toxicity. The purpose of this... expand abstract study was to develop a topotecan-loaded ocular delivery system to minimize systemic exposure and achieve selective transscleral penetration. Methods. Biocompatible polymer implants containing low (0.3 mg) or high (2.3 mg) topotecan load were manufactured and characterized in vitro. Adrenaline (500 mug) was coloaded to induce local vasoconstriction in vivo in 2 of 4 animal groups. Implants were inserted into the episclera of rabbits, and topotecan (lactone and total) concentrations in ocular tissues and plasma were determined over a period of 48 hours. Results. In vitro, implants released 30% to 50% of the loaded drug within 48 hours and 45% to 70% by day 10. In vivo, topotecan lactone was highly accumulated in locally exposed ocular tissues (ranging from 10(5) to 10(6) ng/g in sclera and choroid and 10(2) to10(3) ng/g in retina) over 48 hours with all the formulations studied. Low vitreous topotecan lactone levels (approximately 5 ng/mL) were found in animals receiving concomitant local vasoconstriction and high load implants. Topotecan lactone concentrations in plasma and in contralateral eyes were minimal or undetectable as a marker of tissue selectivity of the proposed strategy. Conclusions. These studies may contribute to improving the efficacy and safety of chemotherapy treatments for retinoblastoma and may support the role of the local vasculature and tissues promoting drug clearance and local accumulation during transscleral drug delivery. collapse abstract

Drug delivery systems in HIV pharmacotherapy: what has been done and the challenges standing ahead.

Worldwide, over 40 million people are infected with the Human Immunodeficiency Virus (HIV). The High Activity Antiretroviral Therapy (HAART) combines at least three antiretroviral (ARV) drugs and, for over a decade, has been used to extend the lifesp... expand abstractan of the HIV-infected patients. Chronic intake of HAART is mandatory to control HIV infection. The frequent administration of several drugs in relatively high doses is a main cause of patient incompliance and a hurdle toward the fulfillment of the pharmacotherapy. High adherence to HAART does not lead to complete HIV virus elimination from the host. Intracellular and anatomical viral reservoirs are responsible for the perpetuation of the infection. Active transport mechanisms involving proteins of the ATP-binding cassette superfamily prevent the penetration of ARV drugs into the brain and may account for the limited bioavailability after oral administration. A new research that addresses from simple organoleptic or technological problems to more complex issues involving the targeting of specific tissues and organs has emerged. With the aim to reduce dosing frequency, to improve the compliance of the existing pharmacotherapy and to target viral reservoirs, the design of drug delivery systems (DDS) is becoming complementary to new drug discovery. Based on the common molecular features that characterize the different families of ARV drugs, the present review describes state-of-the-art ARV DDS and thoroughly discusses the challenges in the development of medicines with enhanced biopharmaceutical properties. In addition, a number of specific issues such as pediatric HAART, preventive pharmacotherapy and specific HIV-associated ethical issues are addressed in an integrative manner. Finally, the impact of such novel drug development on the Pharmaceutical Technology field is discussed. collapse abstract

Indinavir-loaded pH-sensitive microparticles for taste masking: toward extemporaneous pediatric anti-HIVAIDS liquid formulations with improved patient compliance.

The aim of this work was to develop indinavir pediatric anti-HIV/AIDS formulations enabling convenient dose adjustment, ease of oral administration, and improved organoleptic properties by means of the generation of drug-loaded microparticles made of... expand abstract a polymer that is insoluble under intake conditions and dissolves fast in the stomach in order to completely release the active agent. Indinavir-loaded microparticles made of a pH-dependent polymeric excipient soluble at pH < 5, Eudragit E100, were prepared using a double emulsion solvent diffusion technique and the in vitro release profiles characterized. Finally, taste masking properties were evaluated in blind randomized sensory experiments by ten healthy human volunteers. The use of a w/o/o emulsion system resulted in indinavir loads around 90%. Thermal analysis of the microparticles by differential scanning calorimetry revealed that indinavir appeared mainly dispersed at the molecular level. Concentrations of residual organic solvents as determined by gas chromatography were below the upper limits specified by the European Pharmacopeia for pharmaceutical oral formulations. Then, the behavior of drug-containing microparticles in aqueous media at different pH values was assessed. While they selectively dissolved in gastric-like medium, in tap water (intake conditions), the matrix remained almost unchanged and efficiently prevented drug dissolution. Finally, sensoring taste tests performed by volunteers indicated that systems with indinavir loads approximately 15% displayed acceptable taste. This work explored the production of indinavir-containing microparticles based on a common pharmaceutical excipient as a means for the improvement of medicines of drugs involved in the treatment of HIV/AIDS. For systems containing about 15% drug, taste studies confirmed the acceptability of the formulation. In pediatric regimes, this composition would require an acceptable amount of formulation (0.7-1.5 g). collapse abstract

Self-associative behavior and drug-solubilizing ability of poloxamine (tetronic) block copolymers.

The incidence of the structural features on the self-assembly of different poloxamines (the conventional sequential Tetronic 304, 901, 904, 908, 1107, 1301, and 1307; a reverse-sequential counterpart Tetronic 150R1; and a chemically modified derivati... expand abstractve, N-methylated Tetronic 1107) was thoroughly studied in 10 mM HCl by means of pi-A isotherm, surface tension, and pyrene fluorescence measurements. The size and size distribution of the aggregates were investigated by dynamic and static light scattering, and the morphology was probed by transmission electron microscopy. The abilities of the different derivatives to solubilize the drug simvastatin were also evaluated. Poloxamines with both higher PO/EO ratio and molecular weight (T1301 and T150R1) led to micelles with larger and more hydrophobic cores, particularly adequate for hosting hydrophobic molecules and protecting the labile lactone form of simvastatin from hydrolysis. On the other hand, the hydroxy acid form of simvastatin interacted with the central ethylenediamine group under alkaline pH (T304) or when a permanent positive charge due to methylation was present. Micelles of long poloxamine molecules containing large PPO blocks (with 23-29 units, namely, T1301, T1307, and T150R1), particularly the one that also has long PEO blocks, were the most physically stable toward dilution. collapse abstract

Lactoyl-poloxaminecollagen matrix for cell-containing tissue engineering modules.

Collagen-containing crosslinked, remodelable poloxamine derivatives were produced by introducing very short oligo(lactic acid) segments through the reaction of poloxamine with L-lactide and the later addition of unsaturated bonds by the reaction of m... expand abstractodified poloxamine with methacryloyl chloride. Degradation studies on discs indicated a faster weight loss in comparison to the stability of lactoyl-free samples. Cell-containing modules (both HepG2 cells and two different umbilical vein smooth muscle cell (UVSMC) cell-types) were produced. Live/Dead assay showed high survival levels for both HepG2 and UVSMC cell types after crosslinking. While nondegradable modules did not change shape over time, lactoyl-poloxamine matrices showed a gradual shrinkage and size decrease and an increase in the roughness of the surface. These findings were consistent with the expected degradability of the lactoyl derivative. A UVSMC cell line (CRL-2481) embedded in a LA-poloxamine/collagen matrix showed the characteristic elongated shape at day 9. UVSMC primary cells behaved in a manner similar to that seen in collagen gels: these cells formed isolated clusters through the matrix that gradually lost viability. On tissue culture polystyrene the same cells aggregated and did not reach confluence. Modules with embedded CRL-2481 UVSMC led to a better initial adhesion of endothelial cells and a higher extent of surface coverage than seen with the UVSMC-free system. With embedded primary UVSMC, some EC attachment and formation of gap junctions was seen. The pattern was not well organized. With further improvement (and characterization), the lactoyl poloxamine derivative is potentially useful as a scaffold for modular tissue engineering, when tissue remodeling is an important consideration. collapse abstract

In vitroin vivo characterization of melt-molded gabapentin-loaded poly(epsilon-caprolactone) implants for sustained release in animal studies.

Gabapentin (GBP) is a water soluble low molecular weight drug with anticonvulsivant and antinociceptive activity. In animal models, systemic administration regimes resembling chronic exposure to this drug (50mg/kg, twice a day during one week), induc... expand abstracte memory impairment. Aiming to gain further insight on the mechanisms involved in this process, a monolithic implant that releases constant plasma levels during one-week was designed. GBP-loaded poly(epsilon-caprolactone) matrices were produced by means of a simple and reproducible melt-molding/compression procedure. In vitro release studies firstly comprised uncoated implants that displayed release profiles according to a pseudo-first order model. In order to further regulate the release, two-sided coated implants where drug-free layers would perform as membranes controlling the delivery rate were prepared. A more moderated burst effect and a relatively linear (zero-order) release between days 1 and 7 were apparent. Implants were investigated in vivo and the plasma levels monitored during 10 days. Findings indicated that after a more pronounced release during day 1 and the achievement of the levels in blood comparable to a twice-a-day intraperitoneal management, relatively constant levels were attained until day 7. Overall results support the usefulness of this manufacturing method for the production of implants to attain more prolonged GBP release profiles in memory animal studies. collapse abstract

Triclosan-loaded poloxamine micelles for enhanced topical antibacterial activity against biofilm.

Our research group is interested in the study of different technological approaches to treat hospital biofilm as a means to constrain nosocomial-acquired infections. The present work investigated the effect of the incorporation of the antibacterial a... expand abstractgent triclosan (TS) into polymeric micelles of poloxamine T1107 (MW=15 kDa, 70 wt% PEO). The aggregation phenomenon was primarily investigated by means of Critical Micellar Concentration in a broad range of pH. Then, the effect of the polymer concentration on the micellar size was evaluated by Dynamic Light Scattering. Solubility levels increased up to 4 orders of magnitude. The drug inclusion affected the micellization, resulting in size increase and micellar fusion. This phenomenon was only apparent in TS-saturated systems. TS-loaded aggregates proved to be active in vitro against a broad spectrum of bacteria but more importantly, also against two representative clinical pathogens: methicilin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Enterococcus faecalis (VREF). While the former was sensitive to even very low TS levels attainable in poloxamine-free aqueous media, the later was inhibited only when exposed to higher drug levels affordable exclusively using an inclusion system. These findings indicated the release of the drug from the reservoir. Finally, the activity of a TS-containing 5% poloxamine combination of pH 7.4 was assessed on biofilms of Staphylococcus epidermidis. Results showed a significant decrease (p<0.001) in the number of Colony-Formation Units when the biofilm was exposed to the TS/poloxamine as compared to the limited activity of the polymer-free TS control. collapse abstract

Poly(ethylene oxide)-poly(propylene oxide) block copolymer micelles as drug delivery agents: improved hydrosolubility, stability and bioavailability of drugs.

The low solubility in biological fluids displayed by about 50% of the drugs still remains the main limitation in oral, parenteral, and transdermal administration. Among the existing strategies to overcome these drawbacks, inclusion of hydrophobic dru... expand abstractgs into polymeric micelles is one of the most attractive alternatives. Amphiphilic poly(ethylene oxide)-poly(propylene oxide) block copolymers are thermoresponsive materials that display unique aggregation properties in aqueous medium. Due to their ability to form stable micellar systems in water, these materials are broadly studied as hydrosolubilizers for poorly water-soluble drugs. The present review provides a concise description of the most important applications of PEO-PPO-based copolymers in the Pharmaceutical Technology field as means for attaining improved solubility, stability, release, and bioavailability of drugs. collapse abstract

Collagenpoloxamine hydrogels: cytocompatibility of embedded HepG2 cells and surface-attached endothelial cells.

The effects of cross-linked poloxamine hydrogels on the cellular function of embedded HepG2 cells and surface-attached endothelial cells were assessed. HepG2 cells embedded within collagen/poloxamine-methacrylate gel survived photo-cross-linking (MTT... expand abstract viability, 78%). There was a gradual increase in cell number during the first week. The cumulative secretion of alpha1-antitrypsin by HepG2 cells showed an almost linear profile. However, lower levels for the collagen/poloxamine-methacrylate matrix were observed when compared with collagen. Endothelial cells attached poorly to poloxamine gels without collagen (alamarBlue reduction ranged from 36 to 63%) and did not spread well. The addition of collagen led to spread cells and alamarBlue reduction levels of 75-93% (24 h after seeding). On day 5, some detachment was noted through analysis of vascular endothelial cadherin staining. Finally, the collagen-containing matrix was used to prepare cylindrical modules containing HepG2 cells to show the utility of this material in modular tissue constructs. A fluorescent cytoplasmic tracer, Vybrant CFDA SE, showed that embedded cells remained viable for more than 2 months, confirming the good cytocompatibility of collagen/poloxamine-methacrylate in the form of modules. The suitability of these modules for preparing uniform, scaleable, and vascularized constructs remains to be demonstrated. collapse abstract

Smart hydrogels for in situ generated implants.

The objective of this study was to explore the use of reverse thermo-responsive (RTG) polymers for generating implants at their site of performance, following minimally invasive surgical procedures. Aiming at combining syringability and enhanced mech... expand abstractanical properties, a new family of injectable RTG-displaying polymers that exhibit improved mechanical properties was created, following two different strategies: (1) to synthesize high-molecular-weight polymers by covalenty joining poly(ethylene glycol) and poly(propylene glycol) chains using phosgene as the coupling molecule and (2) to cross-link poly(ethylene oxide) (PEO)-poly(propylene oxide) (PPO)-PEO triblocks after end-capping them with triethoxysilane or methacrylate reactive groups. While the methacrylates cross-linked rapidly, the triethoxysilane groups enabled the system to cross-link gradually over time. The chain-extended PEO/PPO copolymers had molecular weights in the 39 000-54 000 interval and exhibited improved mechanical properties. Reverse thermo-responsive systems displaying gradually increasing mechanical properties were generated by cross-linking triethoxysilane-capped (EO)(99)-(PO)(67)-(EO)(99) (F127) triblocks. Over time, the ethoxysilane groups hydrolyzed and created silanol moieties that subsequently condensated. With the aim of further improving their mechanical behavior, F127 triblocks were reacted with methacryloyl chloride and the resulting dimethacrylate was subsequently cross-linked in an aqueous solution at 37 degrees C. The effect of the concentration of the F127 dimethacrylate on the mechanical properties and the porous structure of the cross-linked matrixes produced was assessed. Rheometric studies revealed that the cross-linked hydrogels attained remarkable mechanical properties and allowed the engineering of robust macroscopic constructs, such as large tubular structures. The microporosity of the matrixes produced was studied by scanning electron microscopy. Monolayered conduits as well as structures comprising two and three layers were engineered in vitro, and their compliance and burst strength were determined. collapse abstract

Methylation of poloxamine for enhanced cell adhesion.

Aiming at producing a synthetic collagen-mimetic material that is stiffer than collagen but that like collagen allows both cell encapsulation and cell growth on the surface, a positively charged poloxamine hydrogel was prepared by methylating the ter... expand abstracttiary amine groups of a four-arm poly(ethylene oxide)-poly(propylene oxide) block copolymer derivative (Tetronic 1107). This derivative was subsequently reacted with methacryloyl isocyanate, rendering positively charged materials that are further cross-linkable by a photointiated free radical polymerization. Different hydrogels containing methylated poloxamine methacrylate concentrations between 6% and 18% were produced and characterized by means of water uptake and viscoelastic properties. A sharp increase in water content was observed in distilled water during the first week; some of the gels showed water uptakes as high as 2 times the initial wet weight. In PBS, this effect was less prominent due to the decrease in the osmotic gradient. Also, a gradual increase of both the storage modulus (G') and the loss modulus (G") resulted from increasing the polymer concentration: for example, G' values ranged between 70 and 23000 Pa for 6% and 18% methylated poloxamine methacrylate hydrogels (at 1 Hz, 100 Pa of oscillatory stress). HepG2 cells embedded in different compositions and exposed to UV light displayed good viability levels after the cross-linking, unlike a previously reported attempt at creating a synthetic collagen-mimetic material. A well-spread endothelial cell morphology was apparent on methylated poloxamine films after preincubation in serum-containing medium, while on unmodified poloxamine methacrylate hydrogels cells attached poorly. However, EC did not attach well to the same material when fabricated not as films but as cylindrical modules as needed for the modular construct for which this material was intended. Thus, for this apparently more challenging geometry, it was necessary to combine collagen with the methylated poloxamine to have good attachment of EC on the surface of modules as well as films. While the challenge of creating a synthetic alternative to collagen as a stiffer cell-compatible substrate remains, methylated poloxamine displays many of the attributes that make it a useful material for tissue engineering. collapse abstract

Surface study of collagenpoloxamine hydrogels by a 'deep freezing' ToF-SIMS approach.

In order to determine the presence of collagen molecules at the surface of a collagen-modified poloxamine hydrogel (a semi-interpenetrating network), the surface composition was studied using Time-of-Flight Secondary Ion Mass Spectra (ToF-SIMS). Coll... expand abstractagen was added to the poloxamine hydrogel (poloxamine is a commercially available four-arm poly(ethylene oxide)/poly(propylene oxide) block copolymer, PEO/PPO) to promote the attachment of endothelial or liver cells. X-ray photoelectron spectroscopy (XPS) of dry samples showed a sharp increase in the N content from 0.6% in a pure poloxamine hydrogel to 8.8% in the collagen-containing material. Afterwards, the surface was studied by a 'deep freezing' ToF-SIMS approach under progressive heating from -120 to -60 degrees C. The positive spectrum of collagen/poloxamine at -65 degrees C displayed distinct signals corresponding to different amino acid fragments such as CH4N+ (30 m/z, Gly), C3HN2+ (43 m/z, Arg), C2H6N+ (44 m/z, Ala) and C4H5N2+(81m/z, His) and others corresponding to the PEO and PPO blocks of poloxamine. In addition, the negative spectrum showed peaks at 26 m/z (CN-), 32 m/z (S-) and 42 m/z (CNO-) characteristic of fragments of the collagen molecule. Imaging experiments indicated the homogeneous distribution of the collagen on the surface. These results supported the use of ToF-SIMS for the surface characterization of hydrated hydrogels and confirmed the collagen presence as the means whereby cells attach to the modified poloxamine matrix. collapse abstract

Semi-synthetic collagenpoloxamine matrices for tissue engineering.

Collagen-containing poloxamine hydrogels were produced with the aim of overcoming the low stiffness displayed by collagen gels that are not otherwise chemically crosslinked. Matrices were obtained by functionalization of a four-arm PEO-PPO block copo... expand abstractlymer (poloxamine, Tetronic) with methcrylate groups and subsequent free radical polymerization of water solutions of the modified polymer in the presence of collagen. The resulting matrices had a sharp increase in stiffness, when compared to pure collagen gels. For example, whereas collagen had a storage modulus (G') around 70 Pa and a loss modulus (G'') of 10 Pa, a crosslinked collagen/poloxamine system containing 8.3% crosslinked poloxamine had G' and G'' values of 7400 and 1000 Pa, respectively. HepG2 cells were seeded within the gels before the crosslinking and the viability levels estimated by AlamarBlue assay were between 65% and 91% for systems containing 0.04-0.09 wt% photoinitiator. HepG2 and endothelial cells also adhered to and spread on the surface of the collagen-containing specimens, suggesting their potential utility in tissue engineering. collapse abstract

PEO-PPO-PEO-based poly(ether ester urethane)s as degradable reverse thermo-responsive multiblock copolymers.

Aiming at developing biodegradable thermo-responsive polymers that display enhanced rheological properties, a family of PEO-PPO-PEO based poly(ether ester urethane)s, was developed. The materials were produced following a two-step synthetic pathway. ... expand abstractThe PEO-PPO-PEO triblocks were first end-capped with LA or CL oligo(ester)s whereby pentablocks were produced. Then, the different precursors were chain extended using hexamethylene diisocyanate to create the respective polymers. The length and type of the ester block influenced the behavior of the molecules in water, especially their viscosity versus temperature response. The gelation temperature increased from 23 degrees C for a 20wt% F127 solution to 26 and 31 degrees C for pentablocks with 4.4 and 7.5 lactoyl units, respectively. Materials containing longer LA units failed to show any reverse thermo-responsiveness. The presence of the oligo(ester) blocks also reduced the viscosity of the gel at 37 degrees C. While F127 displayed a viscosity of around 28,000Pas, pentablocks containing 4.4 and 7.5 LA units showed values of 15,400 and 12,600Pas. Also, the viscosity at 37 degrees C as well as the gelation temperature decreased as the molecular weight of the oligo(ester)s increased. Finally, the degradation process of the gels was studied by monitoring their viscosity at body temperature and determining the molecular weight of the polymers, over time. Polymers were tailored so to combine high initial viscosity values with diverse degradation rates, as a function of the length and type of the oligo(ester) present along the polymeric backbone. collapse abstract

Poloxamine hydrogels with a quaternary ammonium modification to improve cell attachment.

Four-arm poly(ethylene oxide) and poly(propylene oxide) (PEO-PPO) block copolymer (poloxamine, Tetronic 1107) hydrogels were modified with positively charged groups with the aim of overcoming the low cell adhesion properties of these PEO-rich systems... expand abstract. Different matrices containing poloxamine-methacrylate (6-12%) and a quaternary ammonium methacrylate ([2-(methacryloyloxy)ethyl]-trimethylammonium chloride [MAETAC], 0-0.48 M) were produced by a photo-initiated free radical copolymerization. A sharp increase in water content for MAETAC-containing gels was seen during the first 2 days of incubation in distilled water; some of the gels showed water uptakes as high as 12 times the initial wet weight. In phosphate-buffered saline (PBS), this effect was less pronounced because of the decrease in the osmotic gradient. In addition, a gradual increase of both the storage and the loss modulus of the gels resulted from increasing the MAETAC content [e.g., G' values increased from 13,500 Pa for 12% poloxamine-methacrylate gels without MAETAC to 151,000 Pa for 0.48M MAETAC contents (at 1 Hz, 100 Pa of oscillatory stress)]. Although on pure poloxamine-methacrylate gels HepG2 and HUVEC cells attached poorly, on MAETAC-containing specimens a well-spread morphology and confluent monolayers were obtained, at least after preincubation in serum containing medium. Although not having all the cell compatibility of collagen, these fully synthetic quaternary ammonium-modified PEO-rich gels may have some utility in tissue-engineering applications where stiff gels and cell attachment after gelation are desired. collapse abstract