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MATERIALS AND METHODS
Materials
• Polymers:
Poly(D,L-lactic-co-glycolic acid) (PLGA) (Resomer RG 502H and Resomer RG 504H: 50:50 lactic acid:glycolic acid, -COOH end groups; and Resomer RG 752H: 75:25 lactic acid:glycolic acid, -COOH end groups) and poly(D,L-lactic acid) (PLA) (Resomer R 202H: -COOH end groups) (Evonik, Darmstadt, Germany)
• Drugs and colored agents:
dexamethasone (Discovery Fine Chemicals, Dorset, UK); riboflavin (DSM Nutritional Products, Basel, Switzerland); Sudan-III-red (Merck, Darmstadt, Germany); methylene blue (Sigma Aldrich, Steinheim, Germany)
• Organic solvents and acids:
N-methyl-pyrrolidone (NMP), acetonitrile and tetrahydrofuran (Fisher Scientific, Ill-kirch, France); ethanol 96% (VWR, Fontenay-sous-Bois, France); formic acid (Riedel-de Haen, Seelze, Germany)
• Additives:
Carbopol® 980 Polymer (Lubrizol, Wickliffe, Ohio, USA); Poly(ethylene glycol) (PEG 400) (Acros organics, Geel, Belgium); stearic acid (Fisher Scientific, Illkirch, France); acetyltributyl citrate (ATBC) (Morflex, Greensboro, NC, USA); hydroxypropyl methyl cellulose (HPMC, Methocel E15 and K100; Colorcon, Dartford, UK); polyoxyethylene glycol sorbitan monooleate (Tween 80; Cooper, Melun, France)
Methods
In-situ forming PLGA implants for intraocular dexamethasone delivery
Preparation of the liquid formulations
Appropriate amounts of PLGA and dexamethasone were dissolved in NMP in glass vials under stirring at 500 rpm (Multipoint Stirrer, Thermo Scientific, Loughborough, UK) at room tem-perature for 60 min. Afterwards, the vials were kept without stirring for 1 h at room temperature in order to remove air bubbles. The formulations were stored at 2-8 °C, and allowed to reach room temperature prior to use.
In-situ formation of implants
Eppendorf vials were filled with 2.25 or 4.5 mL phosphate buffer pH 7.4 (USP 40) and kept at 37 °C overnight. One hundred µl of the liquid PLGA/dexamethasone/NMP formulations (pre-pared as described in section 2.2.) were injected into the vials using a syringe pump (2 mL/min; PHD 2000; Harvard Apparatus, Holliston, USA). Solvent exchange initiated polymer precipi-tation and in-situ implant formation. The Eppendorf vials were placed into a horizontal shaker (80 rpm, 37 °C; GFL 3033, Gesellschaft fuer Labortechnik, Burgwedel, Germany).
Characterization of in-situ formed implants
In vitro drug release: At determined time points, the phosphate buffer pH 7.4 was completely renewed. The amount of dexamethasone in the withdrawn bulk fluid was determined by HPLC-UV analysis, using a Thermo Fisher Scientific Ultimate 3000 Series HPLC, equipped with a LPG 3400 SD/RS pump, an auto sampler (WPS-3000 SL) and a UV-Vis detector (VWD-3400RS) (Thermo Fisher Scientific, Waltham, USA). Samples were centrifuged for 2.5 min at 10,000 rpm (Centrifuge Universal 320; Hettich, Tuttlingen, Germany), and filtered with a 0.45 µm PVDF syringe filter (Millex-HV, Merck Millipore, Tullagreen, Ireland). Fifty µL samples were injected into an A C18 RP column (Gemini 3 µm C18 110 Å, 100 mm x 4.6 mm; Phe-nomenex, Le Pecq, France). The mobile phase consisted of acetonitrile and water (33:67 v/v), the flow rate was 1.5 mL/min. Dexamethasone had a retention time of approximately 3.8 min, the detection wavelength was λ = 254 nm. The calibration curve was linear (R > 0.999) within the range of 0.06 to 0.00003 mg/mL. To determine the amount of dexamethasone potentially remaining in the implants after 35 d exposure to phosphate buffer pH 7.4, the remnants were freeze-dried for 3 d (Christ Epsilon 2–4 LSC; Martin Christ, Osterode, Germany) and the ly-ophilisates were dissolved in a mixture of acetonitrile and ethanol (2:1 v/v). The solutions were filtered using 0.45 µm PVDF filter syringes, and analyzed for their drug contents by HPLC-UV (as described above). In case of incomplete drug release at the end of the observation period, the “missing” amounts were experimentally recovered in the implant remnants. All experiments were conducted in triplicate. In addition, the pH of the release medium was measured at pre-determined time points using a pH meter (InoLab pH Level 1; WTW, Weilheim, Germany) (n = 3).
Determination of the drug solubility
The solubility of dexamethasone (as received) in phosphate buffer pH 7.4 at 37 °C was deter-mined in agitated glass flasks. An excess amount of dexamethasone powder (approximately 30 mg) was exposed to 80 mL bulk fluid, kept at 37 °C under horizontal shaking (80 rpm; GFL 3033). Samples were withdrawn, filtered (0.45 µm PVDF syringe filter), diluted and analyzed for their drug content by HPLC-UV (as described above, using an injection volume of 20 µL) until equilibrium was reached. Each experiment was conducted in triplicate.
Often neglected: PLGA/PLA swelling orchestrates drug release – HME implants
Implant preparation
Appropriate amounts of polymer (PLGA or PLA) and drug (dexamethasone) were mixed for 5 min at 98 rpm in a Turbula Shaker-Mixer (T2A, Willy A. Bachofen, Basel, Switzerland), fol-lowed by 5 min manual blending in a mortar with a pestle. The mixtures were filled into 5 mL syringes (Injekt Luer Lock Solo, B Braun, Melsungen, Germany), equipped with a shortened (1.5 cm) 16G needle. Figure 2.1 shows schematically the experimental set-up used to prepare the implants by hot melt extrusion. Briefly, a syringe was fixed in a holder. The water was kept at 95 °C. After 5 min, the content of the syringe was molten, and a texture analyzer (TAXT plus, Stable Micro Systems, Surrey, UK), equipped with a 50 kg load cell, was used to drive the syringe plunger downwards at a speed of 0.6 mm/min. The obtained extrudates were man-ually cut into cylinders (5 mm length), using a heated blade.
Figure 2.1: Schematic presentation of the experimental set-up used to prepare dexamethasone implants by hot melt extrusion.
Implant characterization
The diameter of the implants was measured with a SMZ-U microscope (Nikon, Tokyo, Japan), equipped with an AxioCam ICc1 camera and the Axiovision Zeiss Software (Carl Zeiss, Jena Germany).
The practical drug loading was determined as follows: Samples were dissolved in 5 mL of a 1:4 (v/v) ethanol:acetonitrile mixture. The drug content of the solutions was analyzed using a Thermo Fisher Scientific Ultimate 3000 Series HPLC, equipped with a LPG 3400 SD/RS pump, an auto sampler (WPS-3000 SL) and a UV-Vis detector (VWD-3400RS) (Thermo Fisher Sci-entific, Waltham, USA). Samples were centrifuged for 2.5 min at 10,000 rpm (Centrifuge Uni-versal 320; Hettich, Tuttlingen, Germany) and filtered with a 0.45 µm PVDF syringe filter (Millex-HV, Merck Millipore, Tullagreen, Ireland). Ten µL samples were injected into an A C18 RP column (Gemini 3 µm C18 110 Å, 100 mm x 4.6 mm; Phenomenex, Le Pecq, France). The mobile phase consisted of a 33:67 (v/v) acetonitrile:water mixture, the flow rate was 1.5 mL/min. Dexamethasone had a retention time of approximately 3.8 min, the detection wave-length was λ = 254 nm. The calibration curve was linear (R > 0.999) within the range of 0.06 to 0.00003 mg/mL. All experiments were conducted in triplicate. Mean values +/- standard deviations are reported.
In vitro drug release: Implants were placed into Eppendorf vials (1 implant per vial), filled with 4 mL phosphate buffer pH 7.4 (USP 40, 37 °C) and horizontally shaken at 80 rpm and 37 °C (GFL 3033, Gesellschaft fuer Labortechnik, Burgwedel, Germany). At pre-determined time points, the release medium was completely replaced. The amount of dexamethasone in the with-drawn bulk fluid was determined by HPLC-UV analysis, as described above (injection volume: 100 µL). If implant remnants remained at the end of the observation period, the amount of potentially “not released” dexamethasone was determined as follows: The remnants were freeze-dried for 3 d (Christ Epsilon 2–4 LSC; Martin Christ, Osterode, Germany) and the ly-ophilisates were dissolved in a 4:1 (v/v) acetonitrile:ethanol mixture. The solutions were filtered (0.45 µm PVDF filter syringes) and analyzed for their drug contents by HPLC-UV (as described above). Note that these experiments showed that in case of incomplete drug release at the end of the observation period, the amounts that had not been released, were experimentally recov-ered in the implant remnants (100 % mass balance). All experiments were conducted in tripli-cate. Mean values +/- standard deviations are reported. In addition, the pH of the release me-dium was measured at pre-determined time points using a pH meter (InoLab pH Level 1; WTW, Weilheim, Germany) (n = 3, mean values +/- standard deviations are reported).
Polymer degradation: Implants were treated as for drug release studies. At pre-determined time points, specimen were withdrawn, freeze-dried for 3 d (Christ Epsilon 2–4 LSC) and the lyoph-ilisates were dissolved in tetrahydrofuran (at a concentration of 1.5 mg/mL). The average pol-ymer molecular weight (Mw) of the PLGA and PLA in the samples was determined by Gel Permeation Chromatography (GPC, Separation Modules e2695 and e2695D, 2419 RI Detector, Empower GPC software; Waters, Guyancourt, France), using a PLGel 5 µm MIXED-D col-umn, 7.5 x 300 mm (Agilent Technologies, Interchim, Montluçon, France). The injection vol-ume was 50 µL. Tetrahydrofuran was the mobile phase (flow rate: 1 mL/min). Polystyrene standards with molecular weights between 1,090 and 70,950 Da (Polymer Labaratories, Varian, Les Ulis, France) were used to prepare the calibration curve. All experiments were conducted in triplicate. Mean values +/- standard deviations are reported.
Implant morphology: Implants were treated as for drug release studies. At pre-determined time points, specimen were withdrawn and freeze-dried as described above. Pictures were taken with an optical image analysis system (Nikon SMZ-U), equipped with a Zeiss camera (AxioCam ICc1).
Determination of drug solubility
The solubility of dexamethasone (as received) in phosphate buffer pH 7.4 at 37 °C was deter-mined in agitated glass flasks. An excess amount of dexamethasone powder (approximately 30 mg) was exposed to 80 mL bulk fluid, and kept at 37 °C under horizontal shaking (80 rpm; GFL 3033). At pre-determined time points, samples were withdrawn, immediately filtered (0.45 µm PVDF syringe filter), diluted and analyzed for their drug content by HPLC-UV analysis (as described above, using an injection volume of 20 µL). Measurements were performed until equilibrium was reached. Each experiment was conducted in triplicate. Mean values +/- stand-ard deviations are reported.
Table of contents :
INTRODUCTION GENERALE
Contexte de la recherche
Objectifs de la recherche
Présentation de ce travail
GENERAL INTRODUCTION
Research Context
Research Objectives
Presentation of the work
CHAPTER 1: INTRODUCTION
1.1 The eye
1.1.1 Anatomy of the eye
1.1.2 Dynamic and static ocular barriers
1.1.3 Blood-ocular barriers
1.1.4 Ocular clearance
1.2 Diseases of the eye
1.2.1 Diabetic retinopathy
1.2.2 Age-related macular degeneration
1.2.3 Uveitis
1.3 Therapy of the eye
1.3.1 Laser treatment
1.3.2 Anti-VEGF factors
1.3.3 Corticosteroid
1.4 Ocular drug delivery
1.4.1 Topical
1.4.2 Periocular
1.4.3 Suprachoroidal
1.4.4 Systemic/oral
1.4.5 Intravitreal
1.5 Ocular implants for sustained drug release
1.5.1 Non-biodegradable implants
1.5.2 Biodegradable implants
1.6 Poly(D,L-lactic-co-glycolic)acid
1.6.1 Physico-chemical properties
1.6.2 Biodegradation and biocompatibility
1.6.3 Sterilization
1.7 Techniques prolonging drug release using PLGA
1.7.1 Microparticles
1.7.2 In-situ forming implants
1.7.3 Pre-formed implants
References
CHAPTER 2: MATERIALS AND METHODS
2.1 Materials
2.2 Methods
2.2.1 In-situ forming PLGA implants for intraocular drug delivery
2.2.1.1 Preparation of the liquid formulations
2.2.1.2 In-situ formation of implants
2.2.1.3 Characterization of in-situ formed implants
2.2.1.4 Determination of the drug solubility
2.2.2 Often neglected: PLGA/PLA swelling orchestrates drug release – HME implants
2.2.2.1 Implant formation
2.2.2.2 Implant characterization
2.2.2.3 Determination of drug solubility
2.2.2 Coloring of PLGA implants to better understand drug release mechanisms……
2.2.3.1 Implant preparation
2.2.3.2 Implant characterization
2.2.3.3 Determination of the solubility of riboflavin
2.2.4 In-situ forming PLGA implants: How additives affect swelling and drug release.
2.2.4.1 Preparation of the liquid formulation
2.2.4.2 In-situ implant formation
2.2.4.3 Characterization of in-situ formed implants
CHAPTER 3: RESULTS AND DISCUSSION
Part 1: In-situ forming PLGA implants for intraocular drug delivery
3.1.1 Importance of the volume of the release medium
3.1.2 Impact of the drug loading
3.1.3 Impact of the PLGA molecular weight
3.1.4 Impact oft he polymer concentration
3.1.5 Conclusion
Part 2: Often neglected: PLGA/PLA swelling orchestrates drug release – HME implants
3.2.1 Morphology
3.2.2 PLGA (RG 502H)-based implants
3.2.3 PLGA (RG 752H)-based implants
3.2.4 PLA (R 202H)-based implants
3.2.5 The orchestrating role of PLGA/PLA swelling for drug release
3.2.6 Conclusion
Part 3: Coloring of PLGA implants to better understand drug release mecha-nisms
3.3.1 Pre-formed (HME) implants
3.3.2 In-situ forming implants
3.3.3 Conclusion
Part 4: In-situ forming PLGA implants: How additives affect swelling and drug release
3.4.1 Carbopol
3.4.2 PEG 400
3.4.3 HPMC
3.4.4 Stearic acid
3.4.5 ATBC
3.4.6 Conclusion
References
GENERAL CONCLUSION AND FURTURE PERSPECTIVES
General Conclusion
Future perspectives…
