Ocular Biodistribution of Spironolactone after a Single Intravitreal Injection of a Biodegradable Sustained-Release Polymer in Rats.

Sustained-release formulations for ocular delivery are of increasing interest given their potential to significantly improve treatment efficacy and patient adherence. The objectives of this study were (i) to develop a sustained-release formulation of spironolactone (SPL) using a biodegradable and injectable polymer, hexyl-substituted poly-lactic acid (hexPLA) and (ii) to investigate the ocular biodistribution and tolerability of SPL and its metabolites in rats in vivo over 1 month following a single intravitreal injection (IVT inj). The concentrations of SPL and its two principal active metabolites, 7α-thiomethylspironolactone and canrenone (CAN), in the different ocular compartments were determined at different time points (3, 7, and 31 days after IVT inj) using a validated ultra-high-performance liquid chromatography-mass spectrometry method. Systemic exposure following a single IVT inj of 5% SPL-hexPLA formulation was evaluated by quantifying SPL and its metabolites in the plasma. Ocular tolerability of the formulation was evaluated using in vivo retinal imaging and histology. In vitro release studies revealed a sustained release of SPL from 5% SPL-hexPLA for up to 65 days. In vivo studies showed that SPL and its metabolites were detected in all ocular tissues at 3 and 7 days post-IVT inj. At 31 days post-IVT inj, SPL and CAN were mainly detected in the retina. These results also highlighted the clearance pathway of SPL and its metabolite involving the anterior and posterior routes in the first week (days 3 and 7), then mainly the posterior segment in the last week (day 31). This study showed that a single IVT inj of 5% SPL-hexPLA in rats enabled sustained delivery of therapeutic amounts of SPL for up to 1 month to the retina without systemic exposure. This formulation may be of interest for the local treatment of diseases involving overactivation of the mineralocorticoid receptor in the chorioretina such as chronic central serous chorioretinopathy.

Topical Administration of Spironolactone-Loaded Nanomicelles Prevents Glucocorticoid-Induced Delayed Corneal Wound Healing in Rabbits.

The objective was to investigate whether mineralocorticoid receptor antagonism using a novel topical micellar formulation of spironolactone could prevent glucocorticoid-induced delayed corneal wound healing in New Zealand white rabbits. Spironolactone micelles (0.1%, w/v) with a mean number weighted diameter of 20 nm were prepared using a pegylated copolymer (mPEG-dihexPLA) and showed a preliminary stability of at least 12 months at 5 °C. Preclinical studies in New Zealand white rabbits demonstrated that the 0.1% spironolactone micellar formulation was well-tolerated since no reaction was observed in the cornea following multiple daily instillation over 5 days. As expected, the preclinical studies also confirmed that dexamethasone significantly delayed epithelial wound healing as compared to untreated control (percentage re-epithelialization after day 4: 84.6 ± 13.9% versus 99.5 ± 1.0% for the control, p < 0.05). However, the addition of the 0.1% spironolactone micellar formulation significantly improved the extent of re-epithelialization, countering the dexamethasone induced delayed wound healing with a percentage re-epithelialization that was statistically equivalent to the control (96.9 ± 7.3% versus 99.5 ± 1.0%, p > 0.05). The biodistribution study provided insight into the ocular metabolism of spironolactone and hence the relative contributions of the parent molecule and its two principal metabolites, 7α-thiomethylspironolactone and canrenone, to the observed pharmacological effects. Comparison of the efficacies of spironolactone and potassium canrenoate (a water-soluble precursor of canrenone) in overcoming the dexamethasone-induced delayed wound healing confirmed that the former had greater efficacy. The results pointed to the greater potency of 7α-thiomethylspironolactone over canrenone as a mineralocorticoid receptor antagonist, which explained its superior ability in countering the glucocorticoid-induced overactivation that was responsible for the delayed wound healing. In conclusion, the preliminary results supported the above-mentioned hypothesis suggesting that coadministration of mineralocorticoid receptor antagonists to patients under glucocorticoid therapy might prevent the deleterious effects of glucocorticoids on complex corneal wound healing processes.

Cyclosporine A-Loaded Nanocarriers for Topical Treatment of Murine Experimental Autoimmune Uveoretinitis.

In the present study, tissue distribution and the therapeutic effect of topically applied cyclosporine A (CsA)-loaded methoxy-poly(ethylene-glycol)-hexyl substituted poly(lactic acid) (mPEGhexPLA) nanocarriers (ApidSOL) on experimental autoimmune uveitis (EAU) were investigated. The CsA-loaded mPEGhexPLA nanocarrier was tolerated well locally and showed no signs of immediate toxicity after repeated topical application in mice with EAU. Upon unilateral CsA treatment, CsA accumulated predominantly in the corneal and sclera-choroidal tissue of the treated eye and in lymph nodes (LN). This regimen reduced EAU severity in treated eyes compared to PBS-treated controls. This improvement was accompanied by reduced T-cell count, T-cell proliferation, and IL-2 secretion of cells from ipsilateral LN. In conclusion, topical treatment with CsA-loaded mPEGhexPLA nanocarriers significantly improves the outcome of EAU.

Polymeric micelle nanocarriers for the cutaneous delivery of tacrolimus: a targeted approach for the treatment of psoriasis.

Tacrolimus (TAC) suffers from poor cutaneous bioavailability when administered topically using conventional vehicles with the consequence that although it is indicated for the treatment of atopic dermatitis, it has poor efficacy against psoriasis. The aim of this work was to formulate TAC loaded polymeric micelles using the biodegradable and biocompatible methoxy-poly(ethylene glycol)-dihexyl substituted polylactide (MPEG-dihexPLA) diblock copolymer and to investigate their potential for targeted delivery of TAC into the epidermis and upper dermis. Micelle formulations were characterized with respect to drug content, stability, and size. An optimal 0.1% micelle formulation was developed and shown to be stable over a period of 7 months at 4 °C; micelle diameters ranged from 10 to 50 nm. Delivery experiments using human skin and involving quantification by UHPLC-MS/MS demonstrated that this formulation resulted in significantly greater TAC deposition in skin than that with Protopic (0.1% w/w; TAC ointment), (1.50 ± 0.59 and 0.47 ± 0.20 µg/cm(2), respectively). The cutaneous biodistribution profile of TAC in the upper 400 µm of tissue (at a resolution of 20 µm) demonstrated that the increase in cutaneous drug levels was due to improved TAC deposition in the stratum corneum, viable epidermis, and upper dermis. Given that there was no increase in the amount of TAC in deeper skin layers or any transdermal permeation, the results suggested that it would be possible to increase TAC levels selectively in the target tissue without increasing systemic absorption and the risk of side effects in vivo. Micelle distribution and molecular penetration pathways were subsequently visualized with confocal laser scanning microscopy (CLSM) using a fluorescently labeled copolymer and fluorescent dyes. The CLSM study indicated that the copolymer was unable to cross the stratum corneum and that release of the micelle "payload" was dependent on the molecular properties of the "cargo" as evidenced by the different behaviors of DiO and fluorescein. A preferential deposition of micelles into the hair follicle was also confirmed by CLSM. Overall, the results indicate that MPEG-dihexPLA micelles are highly efficient nanocarriers for the selective cutaneous delivery of tacrolimus, superior to the marketed formulation (Protopic). Furthermore, they may also have significant potential for targeted delivery to the hair follicle.

Recent advances in mucosal immunization using virus-like particles.

Mucosal immunization offers the promises of eliciting a systemic and mucosal immune response, as well as enhanced patient compliance. Mucosal vaccination using defined antigens such as proteins and peptides requires delivery systems that combine good safety profiles with strong immunogenicity, which may be provided by virus-like particles (VLP). VLP are assembled from viral structural proteins and thus are devoid of any genetic material. They excel by mimicking natural pathogens, therefore providing antigen-protecting particulate nature, inherent immune-cell stimulatory mechanisms, and tissue-specific targeting depending on their parental virus. Nevertheless, despite of promising preclinical results, VLP remain rarely investigated in clinical studies. This review is intended to give an overview of obstacles and promises of VLP-based mucosal immunization as well as to identify strategies to further improve VLP while maintaining a good safety and tolerability profile.

Paclitaxel-eluting biodegradable synthetic vascular prostheses: a step towards reduction of neointima formation?

BACKGROUND: Clinical small-caliber vascular prostheses are unsatisfactory. Reasons for failure are early thrombosis and late intimal hyperplasia. We thus prepared biodegradable small-caliber vascular prostheses using electrospun polycaprolactone (PCL) with slow-releasing paclitaxel (PTX), an antiproliferative drug. METHODS AND RESULTS: PCL solutions containing PTX were used to prepare nonwoven nanofibre-based 2-mm ID prostheses. Mechanical morphological properties and drug loading, distribution, and release were studied in vitro. Infrarenal abdominal aortic replacement was carried out with nondrug-loaded and drug-loaded prostheses in 18 rats and followed for 6 months. Patency, stenosis, tissue reaction, and drug effect on endothelialization, vascular remodeling, and neointima formation were studied in vivo. In vitro prostheses showed controlled morphology mimicking extracellular matrix with mechanical properties similar to those of native vessels. PTX-loaded grafts with suitable mechanical properties and controlled drug-release were obtained by factorial design. In vivo, both groups showed 100% patency, no stenosis, and no aneurysmal dilatation. Endothelial coverage and cell ingrowth were significantly reduced at 3 weeks and delayed at 12 and 24 weeks in PTX grafts, but as envisioned, neointima formation was significantly reduced in these grafts at 12 weeks and delayed at 6 months. CONCLUSIONS: Biodegradable, electrospun, nanofibre, polycaprolactone prostheses are promising because in vitro they maintain their mechanical properties (regardless of PTX loading), and in vivo show good patency, reendothelialize, and remodel with autologous cells. PTX loading delays endothelialization and cellular ingrowth. Conversely, it reduces neointima formation until the end point of our study and thus may be an interesting option for small caliber vascular grafts.

Degradation and healing characteristics of small-diameter poly(epsilon-caprolactone) vascular grafts in the rat systemic arterial circulation.

BACKGROUND: Long-term patency of conventional synthetic grafts is unsatisfactory below a 6-mm internal diameter. Poly(epsilon-caprolactone) (PCL) is a promising biodegradable polymer with a longer degradation time. We aimed to evaluate in vivo healing and degradation characteristics of small-diameter vascular grafts made of PCL nanofibers compared with expanded polytetrafluoroethylene (ePTFE) grafts. METHODS AND RESULTS: We prepared 2-mm-internal diameter grafts by electrospinning using PCL (M(n)=80, 000 g/mol). Either PCL (n=15) or ePTFE (n=15) grafts were implanted into 30 rats. Rats were followed up for 24 weeks. At the conclusion of the follow-up period, patency and structural integrity were evaluated by digital subtraction angiography. The abdominal aorta, including the graft, was harvested and investigated under light microscopy. Endothelial coverage, neointima formation, and transmural cellular ingrowth were measured by computed histomorphometry. All animals survived until the end of follow-up, and all grafts were patent in both groups. Digital subtraction angiography revealed no stenosis in the PCL group but stenotic lesions in 1 graft at 18 weeks (40%) and in another graft at 24 weeks (50%) in the ePTFE group. None of the grafts showed aneurysmal dilatation. Endothelial coverage was significantly better in the PCL group. Neointimal formation was comparable between the 2 groups. Macrophage and fibroblast ingrowth with extracellular matrix formation and neoangiogenesis were better in the PCL group. After 12 weeks, foci of chondroid metaplasia located in the neointima of PCL grafts were observed in all samples. CONCLUSIONS: Small-diameter PCL grafts represent a promising alternative for the future because of their better healing characteristics compared with ePTFE grafts. Faster endothelialization and extracellular matrix formation, accompanied by degradation of graft fibers, seem to be the major advantages. Further evaluation of degradation and graft healing characteristics may potentially lead to the clinical use of such grafts for revascularization procedures.

Evaluation of the tablet core factors influencing the release kinetics and the loadability of push-pull osmotic systems.

Push-pull osmotic systems have been developed to deliver poorly soluble drugs in a modified-release fashion. The aim of this study was to investigate the influence of the tablet core factors on the drug release kinetics and loadability. The release kinetics was efficiently modulated by varying either the proportion of osmotic agent or the drug layer polymer grade as an alternative to change the membrane characteristics. High osmotic agent proportions and viscous-grade polymers were recommended to formulate high drug loads up to 20% without losing both the release completeness and the zero-order drug release kinetics.

Impact of covalently Nile Red and covalently Rhodamine labeled fluorescent polymer micelles for the improved imaging of the respective drug delivery system.

Novel fluorescently labeled poly(ethylene glycol)-poly(hydroxyoctanoic acid) (MPEG-PHOA) block-copolymers were synthesized for the improved visualization of the deriving polymeric micelle drug delivery system. Albeit commonly used, one has to be aware that by simple incorporation of Nile Red (hydrophobic) or Rhodamine B (hydrophilic) as fluorescent compounds in nanocarriers (e.g., nanoparticles, liposomes or micelles) for imaging applications, these fluorescent probes can diffuse out of the carrier system and lead to artefacts due to the concomitant fluorescence loss or areal distribution. In order to inhibit such an uncontrolled diffusion, the Nile Red derivative 2-((9-(diethylamino)-5-oxo-5H-benzo[a]phenoxazin-2-yl)oxy)acetic acid was synthesized and covalently attached to the MPEG-PHOA block-copolymer via a mild Mitsunobu reaction to yield the desired MPEG-PHOA-Nile Red polymer for micelle preparations. Rhodamine B was coupled via its native carboxylic acid group with the copolymer MPEG-PHOA under mild conditions using DMAP, EDC, and NHS. For the proof of concept, aqueous solutions of composite micelles made of 0.5% (w/w fluorescence dye) MPEG-PHOA-dye and MPEG-PHOA copolymers were prepared ("spiking" of the non-labeled base MPEG-PHOA micelles) and characterized by transmission electron microscopy (TEM), dialysis and fluorescence spectrometry. The fluorescence intensity of the Nile Red in the solutions was followed up at physiological temperatures and pH values (37 °C, pH = 7.4 PBS buffer 0.01 M) over a period of 8 weeks. The labeled and non-labeled micelle formulations were tested in vitro in cells (Rhodamine-micelle formulations), then in vivo in a case study of an ophthalmic application (Nile Red micelle formulations). Both in vitro and in vivo experiments revealed a significant improvement of fluorescence stability of the MPEG-PHOA-dye formulations, facilitating the investigations on tracing the micelles and their stability. The results clearly demonstrate the value of the novel Nile Red and Rhodamine derivatives, whose simple synthesis and covalent attachment may easily be transferred to other nanosized polymeric drug delivery systems, e.g., MPEGylated or non-MPEGylated PLA/PLGA nanoparticles and be envisioned for novel theranostic systems.

Self-assembled mPEG-hexPLA polymeric nanocarriers for the targeted cutaneous delivery of imiquimod.

mPEG-hexPLA micelles have shown their ability to improve delivery and cutaneous bioavailability of a wide range of poorly water soluble and lipophilic molecules. Although poorly water soluble, imiquimod (IMQ) is only moderately lipophilic and it was decided to investigate whether mPEG-hexPLA polymeric micelles could be used as a drug delivery system for this "less than ideal" candidate for encapsulation. Nanosized IMQ micelles (dn = 27 nm) were formulated and characterized. Moreover, the innovative use of size exclusion chromatography allowed the exact drug localization inside the formulation to be determined; it appeared that the use of acetic acid to solubilize IMQ led to a higher IMQ content outside the micelle than inside. IMQ micelles (0.05%) were formulated in a gel using carboxymethyl cellulose (CMC). In vitro application of this formulation to porcine and human skin led to promising delivery results. IMQ deposition in human skin was 1.4 ±â€¯0.4 µg/cm2 while transdermal permeation was only 79 ±â€¯19 ng/cm2: the formulation displayed >17-fold selectivity for cutaneous deposition over transdermal permeation. The optimized 0.05% gel significantly outperformed Aldara® cream (containing 5% IMQ) formulation in terms of delivery efficiency to human skin (2.85 ±â€¯0.74% vs 0.04 ±â€¯0.01%). Despite IMQ being only partially incorporated in the micelles, the biodistribution profile showed that the optimized 0.05% gel delivered as much as 518.2 ±â€¯173.3 ng/cm2 (1.04 ±â€¯0.35% of the applied dose) to the viable epidermis and 236.4 ±â€¯88.2 ng/cm2 (0.47 ±â€¯0.18% of the applied dose) to the upper dermis where the target antigen presenting cells reside. In contrast, for Aldara® cream, the delivery efficiencies in those layers were less than 0.02%. The optimal 0.05% gel thus allowed therapeutically relevant drug levels to be achieved in target tissues despite a 100-fold dose reduction.

Effect of implantation site on outcome of tissue-engineered vascular grafts.

OBJECTIVE: Vascular prostheses for small caliber bypass grafts in cardiac and vascular diseases or for access surgery are still missing. Poly (Ɛ-caprolactone) (PCL) has been previously investigated by our group and showed good biocompatibility and mechanical properties in vitro and rapid endothelialisation, cellular infiltration and vascularisation in vivo yielding optimal patency in the abdominal aortic position. The aim of the present study is to evaluate our PCL graft in the carotid position and to compare its outcome to the grafts implanted in the abdominal aortic position. METHODS: PCL grafts (1 mm ID/10 mm long) were implanted into the left common carotid artery in 20 Sprague-Dawley rats and compared to our previously published series of abdominal aortic implants. The animals were followed up to 3, 6, 12 and 24 weeks. At each time point, in vivo compliance, angiography and histological examination with morphology were performed. RESULTS: PCL grafts showed good mechanical properties and ease of handling. The average graft compliance was 14.5 ±â€¯1.7%/ mmHg compared to 7.8 ±â€¯0.9% for the abdominal position and 45.1 ±â€¯3.2%/ mmHg for the native carotid artery. The overall patency for the carotid position was 65% as compared to 100% in the abdominal position. Complete endothelialisation was achieved at 3 weeks and cell invasion was more rapid than in the aortic position. In contrast, intimal hyperplasia (IH) and vascular density were less pronounced than in the aortic position. CONCLUSION: Our PCL grafts in the carotid position were well endothelialised with early cellular infiltration, higher compliance, lower IH and calcification compared to the similar grafts implanted in the aortic position. However, there was a higher occlusion rate compared to our abdominal aorta series. Anatomical position, compliance mismatch, flow conditions may answer the difference in patency seen.

Poly-Lactic Acid-Based Biopolymer Formulations Are Safe for Sustained Intratympanic Dexamethasone Delivery.

HYPOTHESIS AND BACKGROUND: The clinical treatment of sudden sensorineural hearing loss currently relies on the administration of steroids, either systemically or via intratympanic injections. Intratympanic injections bypass the hemato-cochlear barrier, reducing its systemic side effects. The efficacy of the injections is limited through rapid drug clearance via the Eustachian tube, and through nonoptimal properties of slow-release drug carriers. A new slow-release drug delivery vehicle based on hexyl-substituted-poly-lactic-acid (HexPLA), with the highest possible safety profile and complete bio-degradability, has been evaluated for safety and efficacy in a standardized guinea pig model of intratympanic injection. METHODS: A total of 83 animals received through retrobullar injection either empty Nile-red-colored HexPLA vehicle, 5%-dexamethasone-HexPLA, 5%-dexamethasone suspension, or a sham operation. Long-term residence time of vehicle, biocompatibility, click- and pure-tone hearing thresholds, and dexamethasone levels in the perilymph were prospectively assessed. RESULTS: At 1 week after injection, HexPLA vehicle was morphologically present in the middle ear and perilymph levels in the 5%-dexamethasone-HexPLA were on average 2 to 3 µg/ml and one order of magnitude higher compared with those of the 5%-dexamethasone suspension group. No significant postoperative morphological or functional changes were observed up to 3 months postdelivery. CONCLUSIONS: HexPLA is safe, fully biocompatible, and efficient for sustained high-dose, intratympanic delivery of dexamethasone at least for 1 week and therefore of high interest for the treatment of sudden sensorineural hearing loss and other acute inner ear diseases. Due to the favorable chemical properties, a wide range of other drugs can be loaded into the vehicle further increasing its potential value for otological applications.

A novel thermoresponsive hydrogel based on chitosan.

Injectable thermosetting chitosan hydrogels are attractive systems for drug delivery and tissue engineering that combine biodegradability, biocompatibility and the ability to form in situ gel-like implants. Thermally-induced gelation relies advantageously on biopolymer secondary interactions, avoiding potentially toxic polymerization reactions that may occur with in situ polymerizing formulations. In view of a biomedical use, such formulations have to be sterilizable and storable on extended periods without losing their thermosetting properties. These two key features have been studied in the present paper. Chitosans from two different sources were added with several phosphate-free polyols or polyoses as gelling agents. Despite a reduction in chitosan molecular weight following autoclaving, the hydrogels prepared with autoclaved chitosan showed the desired thermosetting properties. Hence, chitosan steam sterilization combined with aseptic preparation of the hydrogel allows a sterile formulation to be obtained. Whereas thermosetting hydrogels were shown to be unstable when refrigerated, freezing was shown to be conceivable as a storage method. When trehalose or mannitol was used as stabilizing agent, the formulation reconstituted from a lyophilizate displayed thermosetting properties and was still injectable, paving the way to the development of a clinically utilizable, novel chitosan thermosetting hydrogel.

Nanoparticles for drug delivery: the need for precision in reporting particle size parameters.

Polymeric drug-loaded nanoparticles have been extensively studied in the field of drug delivery. Biodistribution depends on the physicochemical properties of particles, especially size. The global message from the literature is that small particles have an enhanced ability to reach their target. The present review highlights the difficulties in validating the data from biodistribution studies without accurate particle size determination.

Self-assembling polymeric nanocarriers to target inflammatory lesions in ulcerative colitis.

We have developed a self-assembling polymeric nanocarrier to deliver the potent immunosuppressive drug Cyclosporine A (CsA) to inflammatory lesions in ulcerative colitis (UC) patients. Our nanocarrier has a high drug loading capacity and efficiently targets its CsA payload to the diseased tissue after local administration. Tissue drug levels were several orders of magnitude higher in animals suffering from a trinitrobenzene-sulfonic acid (TNBS) - induced colitis, compared to healthy control animals; no drug was detectable in the plasma, underlining the localized delivery strategy. An efficient reduction in inflammation score was obtained with a CsA dose of 1mg/mL. Therapeutic efficacy was comparable to 5-aminosalicylic acid (5-ASA), the positive control treatment in the TNBS-induced colitis model. Repetitive treatment of healthy animals with CsA nanocarriers for seven days was well tolerated with no alterations in colon histology.

Synergies of VEGF inhibition and photodynamic therapy in the treatment of age-related macular degeneration.

PURPOSE: Photodynamic therapy (PDT) and the administration of compounds acting against vascular endothelial growth factor (anti-VEGF) are approved for the treatment of choroidal neovascularization (CNV) secondary to age-related macular degeneration (AMD). Experimental evidence that the combined use of both treatment options may improve therapeutic outcome is presented. METHODS: Fertilized chick eggs were incubated until day 12 of embryo development (EDD12) and were treated by PDT using two different photosensitizing agents (liposomal formulation of BPD-MA; m-THPP encapsulated in polymeric nanoparticles) and were visualized using an epifluorescence microscope. Vascular occlusion of the treated zones of the chorioallantoic membrane (CAM) was assessed by fluorescence angiography 24 and 48 hours after treatment. Alternatively, PDT-treated areas were exposed to a soluble VEGF receptor antagonist (sFlt-1) 6 hours after treatment and were analyzed. RESULTS: Vascular occlusion in the PDT-treated areas was observed with both photosensitizers 24 hours after treatment. Reperfusion of preexisting blood vessels and first signs of revascularization were visible 48 hours after PDT. Topical administration of sFlt-1 to the treated areas augmented occlusion and limited subsequent angiogenesis in a dose-dependent manner. CONCLUSIONS: The combined use of PDT and of agents targeting angiogenic cytokines may synergistically improve therapeutic outcome after combined treatment in patients with CNV secondary to AMD.

Polymeric photosensitizer prodrugs for photodynamic therapy.

A targeting strategy based on the selective enzyme-mediated activation of polymeric photosensitizer prodrugs (PPP) within pathological tissue has led to the development of agents with the dual ability to detect and treat cancer. Herein, a detailed study of a simple model system for these prodrugs is described. We prepared "first-generation" PPP by directly tethering the photosensitizer (PS) pheophorbide a to poly-(L)-lysine via epsilon amide links and observed that by increasing the number of PS on a polymer chain, energy transfer between PS units improved leading to better quenching efficiency. Fragmentation of the PPP backbone by trypsin digestion gave rise to a pronounced fluorescence increase and to more efficient generation of reactive oxygen species upon light irradiation. In vitro tests using the T-24 bladder carcinoma cell line and ex vivo experiments using mouse intestines illustrated the remarkable and selective ability of these PPP to fluoresce and induce phototoxicity upon enzymatic activation. This work elucidated the basic physicochemical parameters, such as water solubility and quenching/activation behavior, required for the future elaboration of more adaptable "second-generation" PPP, in which the PS is tethered to a proteolytically stable polymer backbone via enzyme-specific peptide linkers. This polymer architecture offers great flexibility to tailor make the PPP to target any pathological tissue known to over-express a specific enzyme.

Poly(hexyl-substituted lactides): novel injectable hydrophobic drug delivery systems.

Poly(hexyl-substituted lactides) (PHLA) as new hydrophobic polyesters with controlled molecular weights and narrow distributions were synthesized by ring-opening polymerization (ROP) using tin(II) 2-ethylhexanoate (Sn(Oct)(2)) and benzyl alcohol as catalyst and initiator. Glass transition temperatures (T(g)) and zero shear viscosities (eta(0)) at 25 degrees C could be modulated from T(g)= -42 degrees C to -10 degrees C and 40 to 4850 Pa s, respectively, by varying the polymer molecular weight and the number of hexyl groups along the polymer chain. Degradation studies were performed in terms of both mass and molecular weight loss in the course of time. The degradation mechanism is shown to be of the "bulk erosion" type, and comparable to standard poly(D,L-lactide) (PLA). Despite the increased steric hindrance in the poly(monohexyl-substituted lactide) (PmHLA) due to the hexyl side groups, its degradation rate at pH 7.4 and 37 degrees C was found to be slightly higher than observed for the analogue standard PLA. This could be attributed to the flexible rubbery state of the hexyl-substituted polymer (T(g) approximately -15 degrees C) at the physiological temperature, which is favoring the degradation in comparison to the rigid and glassy standard PLA (T(g) approximately 40 degrees C). In contrast, degradation studies performed at 60 degrees C, where both polymers are above their glass transition temperature, confirmed that the degradation rate is lower for the sterically more hindered PmHLA. The degradation products were analyzed by ESI-MS. Hydrolysis lead first to the corresponding oligo-ester fragments and finally to the nontoxic 2-hydroxyoctanoic acid and lactic acid. Tetracycline was tested as a model drug for release studies. This drug was found to be released faster and in higher amounts in its active form from the PHLA matrix than from standard PLA. The results presented in this work demonstrate the potential of these hydrophobic polylactide-based semisolid materials as an alternative to conventional PLA/PLGA for injectable drug delivery systems.

Fluorescent biodegradable PLGA particles with narrow size distributions: preparation by means of selective centrifugation.

Size is the most studied parameter in the field of nanoparticle characterization but few studies have been performed on biodegradable particles with well-defined sizes. The aim of this work was to prepare fluorescent biodegradable polymeric particles having well-defined sizes and well-characterized surface properties. Poly(D,L-lactide-co-glycolide acid) particles were prepared by the emulsion evaporation process. Filtration and centrifugation were used to produce particle fractions in the narrow size range from polydispersed batches, and the efficiency of separation was compared. Selective centrifugation allowed for the preparation of five classes of particles having narrow size distribution (0.1, 0.3, 0.6, 1 and 2 microm). Particles were characterized in terms of size distribution, surface morphology, charge, residual surfactant and hydrophilicity. The results showed similar surface properties for all the batches. 3,3'-Dioctadecyloxacarbo-cyanine perchlorate has been successfully incorporated as a fluorescent dye and its ability to remain associated with the particles during cell culture experiments has been proven. Such particles may be used as an adequate tool for studying cellular uptake.

Tolerance of high and low amounts of PLGA microspheres loaded with mineralocorticoid receptor antagonist in retinal target site.

Mineralocorticoid receptor (MR) contributes to retinal/choroidal homeostasis. Excess MR activation has been shown to be involved in pathogenesis of central serous chorioretinopathy (CSCR). Systemic administration of MR antagonist (MRA) reduces subretinal fluid and choroidal vasodilation, and improves the visual acuity in CSCR patients. To achieve long term beneficial effects in the eye while avoiding systemic side-effects, we propose the use of biodegradable spironolactone-loaded poly-lactic-co-glycolic acid (PLGA) microspheres (MSs). In this work we have evaluated the ocular tolerance of MSs containing spironolactone in rat' eyes. As previous step, we have also studied the tolerance of the commercial solution of canrenoate salt, active metabolite of spironolactone. PLGA MSs allowed in vitro sustained release of spironolactone for 30days. Rat eyes injected with high intravitreous concentration of PLGA MSs (10mg/mL) unloaded and loaded with spironolactone maintained intact retinal lamination at 1month. However enhanced glial fibrillary acidic protein immunostaining and activated microglia/macrophages witness retinal stress were observed. ERG also showed impaired photoreceptor function. Intravitreous PLGA MSs concentration of 2mg/mL unloaded and loaded with spironolactone resulted well tolerated. We observed reduced microglial/macrophage activation in rat retina compared to high concentration of MSs with normal retinal function according to ERG. Spironolactone released from low concentration of MSs was active in the rat retina. Low concentration of spironolactone-loaded PLGA MSs could be a safe therapeutic choice for chorioretinal disorders in which illicit MR activation could be pathogenic.