Stabilization and Controlled Release of Bovine Serum Albumin Encapsulated in Poly(D,L-lactide) and Poly(ethylene glycol) Microsphere Blends

Purpose. The acidic microclimate in poly(D, L-lactide-co-glycolide) 50/50 microspheres has been previously demonstrated by our group as the primary instability source of encapsulated bovine serum albumin (BSA). The objectives of this study were to stabilize the encapsulated model protein, BSA, and to achieve continuous protein release by using a blend of: slowly degrading poly(D, L-lactide) (PLA), to reduce the production of acidic species during BSA release; and pore-forming poly(ethylene glycol) (PEG), to increase diffusion of BSA and polymer degradation products out of the polymer. Methods. Microspheres were formulated from blends of PLA (Mw 145,000) and PEG (Mw 10,000 or 35,000) by using an anhydrous oil-in-oil emulsion and solvent extraction (O/O) method. The polymer blend composition and phase miscibility were examined by FT-IR and DSC, respectively. Microsphere surface morphology, water uptake, and BSA release kinetics were also investigated. The stability of BSA encapsulated in microspheres was examined by losses in protein solubility, SDS-PAGE, IEF, CD, and fluorescence spectroscopy. Results. PEG was successfully incorporated in PLA microspheres and shown to possess partial miscibility with PLA. A protein loading level of 5% (w/w) was attained in PLA/PEG microspheres with a mean diameter of approximately 100 m. When PEG content was less than 20% in the blend, incomplete release of BSA was observed with the formation of insoluble, and primarily non-covalent aggregates. When 20%-30% PEG was incorporated in the blend formulation, in vitro continuous protein release over 29 days was exhibited. Unreleased BSA in these formulations was water-soluble and structurally intact. Conclusions. Stabilization and controlled relaease of BSA from PLA/PEG microspheres was achieved due to low acid and high water content in the blend formulation.     

5-Fluorouracil-loaded microspheres prepared by spray-drying poly(D,L-lactide) and poly(lactide-co-glycolide) polymers: Characterization and drug release

5-Fluorouracil (5-FU), a hydrosoluble anti-neoplastic drug, was encapsulated in microspheres of poly(D,L-lactide) (PLA) and poly(lactide-co-glycolide) (PLGA) polymers using the spray-drying technique, in order to obtain small size microspheres with a significant drug entrapment efficiency. Drug-loaded microspheres included between 47?±?11 and 67?±?12?µg 5-FU?mg^?1 microspheres and the percentage of entrapment efficiency was between 52?±?12 and 74?±?13. Microspheres were of small size (average diameter: 0.9?±?0.4–1.4?±?0.8?µm microspheres without drug; 1.1?±?0.5–1.7?±?0.9?µm 5-FU-loaded microspheres) and their surface was smooth and slightly porous, some hollows or deformations were observed in microspheres prepared from polymers with larger T_g. A fractionation process of the raw polymer during the formation of microspheres was observed as an increase of the average molecular weight and also of T_g of the polymer of the microspheres. The presence of 5-FU did not modify the T_g values of the microspheres. Significant interactions between the drug and each one of the polymers did not take place and total release of the included drug was observed in all cases. The time needed for the total drug release (28–129?h) was in the order PLA?>?PLGA 75/25?>?PLGA 50/50. A burst effect (17–20%) was observed during the first hour and then a period of constant release rate (3.52?±?0.82–1.46?±?0.26?µg 5-FU?h^?1 per milligram of microspheres) up to 8 or 13?h, depending on the polymer, was obtained.     

The Stability of Recombinant Human Growth Hormone in Poly(lactic-co-glycolic acid) (PLGA) Microspheres

Purpose. The development of a sustained release formulation for recombinant human growth hormone (rhGH) as well as other proteins requires that the protein be stable at physiological conditions during its in vivo lifetime. Poly(lactic-co-glycolic acid) (PLGA) microspheres may provide an excellent sustained release formulation for proteins, if protein stability can be maintained. Methods. rhGH was encapsulated in PLGA microspheres using a double emulsion process. Protein released from the microspheres was assessed by several chromatrographic assays, circular dichroism, and a cell-based bioassay. The rates of aggregation, oxidation, diketopiperazine formation, and deamidation were then determined for rhGH released from PLGA microspheres and rhGH in solution (control) during incubation in isotonic buffer, pH 7.4 and 37°C. Results. rhGH PLGA formulations were produced with a low initial burst (<20%) and a continuous release of rhGH for 30 days. rhGH was released initially from PLGA microspheres in its native form as measured by several assays. In isotonic buffer, pH 7.4 and 37°C, the rates of rhGH oxidation, diketopiperazine formation, and deamidation in the PLGA microspheres were equivalent to the rhGH in solution, but aggregation (dimer formation) occured at a slightly faster rate for protein released from the PLGA microspheres. This difference in aggregation rate was likely due to the high protein concentration used in the encapsulation process. The rhGH released was biologically active throughout the incubation at these conditions which are equivalent to physiological ionic strength and pH. Conclusions. rhGH was successfully encapsulated and released in its fully bioactive form from PLGA microspheres over 30 days. The chemical degradation rates of rhGH were not affected by the PLGA microspheres, indicating that the internal environment of the microspheres was similar to the bulk solution. After administration, the microspheres should become fully hydrated in the subcutaneous space and should experience similar isotonic conditions and pH. Therefore, if a protein formulation provides stability in isotonic buffer, pH 7.4 and 37°C, it should allow for a safe and efficacious sustained release dosage form in PLGA microspheres.     

A Heterogeneously Structured Composite Based on Poly(lactic-co-glycolic acid) Microspheres and Poly(vinyl alcohol) Hydrogel Nanoparticles for Long-Term Protein Drug Delivery

Purpose. To prepare a heterogeneously structured composite based on poly (lactic-co-glycolic acid) (PLGA) microspheres and poly(vinyl alcohol) (PVA) hydrogel nanoparticles for long-term protein drug delivery. Methods. A heterogeneously structured composite in the form of PLGA microspheres containing PVA nanoparticles was prepared and named as PLGA-PVA composite microspheres. A model protein drug, bovine serum albumin (BSA), was encapsulated in the PVA nanoparticles first. The BSA-containing PVA nanoparticles was then loaded in the PLGA microspheres by using a phase separation method. The protein-containing PLGA-PVA composite microspheres were characterized with regard to morphology, size and size distribution, BSA loading efficiency, in vitroBSA release, and BSA stability. Results. The protein-containing PLGA-PVA composite microspheres possessed spherical shape and nonporous surface. The PLGA-PVA composite microspheres had normal or Gaussian size distribution. The particle size ranged from 71.5 m to 282.7 m. The average diameter of the composite microspheres was 180 m. The PLGA-PVA composite microspheres could release the protein (BSA) for two months. The protein stability study showed that BSA was protected during the composite microsphere preparation and stabilized inside the PLGA-PVA composite microspheres. Conclusions. The protein-containing PLGA-PVA composite may be suitable for long-term protein drug delivery.     

Preparation and Characterization of Poly (D,L-Lactide-Co-Glycolide) Microspheres for Controlled Release of Poly(L-Lysine) Complexed Plasmid DNA

Purpose. To produce and characterize controlled release formulations of plasmid DNA (pDNA) loaded in poly (D,L-lactide-co-glycolide) (PLGA) microspheres both in free form and as a complex with poly (L-lysine). Methods. Poly (L-lysine) (PLL) was used to form pDNA/PLL complexes with complexation ratio of 1:0.125 and 1:0.333 w/w to enhance the stability of pDNA during microsphere preparation and protect pDNA from nuclease attack. pDNA structure, particle size, zeta potential, drug loading, in vitro release properties, and protection from DNase I were studied. Results. The microspheres were found to be spherical with average particle size of 3.1-3.5 m. Drug loading of 0.6% was targeted. Incorporation efficiencies of 35.1% and 29.4-30.6% were obtained for pDNA and pDNA/PLL loaded microspheres respectively. Overall, pDNA release kinetics following the initial burst did not correlate with blank microsphere polymer degradation profile suggesting that pDNA release is convective diffusion controlled. The percentage of supercoiled pDNA in the pDNA and pDNA/PLL loaded microspheres was 16.6 % and 76.7-85.6% respectively. Unencapsulated pDNA and pDNA/PLL degraded completely within 30 minutes upon the addition of DNase I. Encapsulation of DNA/PLL in PLGA microspheres protected pDNA from enzymatic degradation. Conclusions. The results show that using a novel process, pDNA can be stabilized and encapsulated in PLGA microspheres to protect pDNA from enzymatic degradation.     

Ketotifen-loaded microspheres prepared by spray-drying poly(D,L-lactide) and poly(D,L-lactide-co-glycolide) polymers: characterization and in vivo evaluation

Ketotifen (KT) was encapsulated into poly(D,L-lactide) (PLA) and poly(D,L-lactide-co-glycolide) (PLGA 50/50) by spray-drying to investigate the use of biodegradable drug-loaded microspheres as delivery systems in the intraperitoneal cavity. Ketotifen stability was evaluated by HPLC, and degradation was not observed. Drug entrapment efficiency was 74 +/- 7% (82 +/- 8 microg KT/mg for PLA) and 81 +/- 6% (90 +/- 7 microg KT/mg for PLGA 50/50). PLA microspheres released ketotifen (57% of encapsulated KT) in 350 h at two release rates (221 microg/h, 15 min to 2 h; 1.13 microg/h, 5-350 h). A quicker release of ketotifen took place from PLGA 50/50 microspheres (67.4% of encapsulated KT) in 50 h (322 microg/h, 15 min to 2 h; 16.18 microg/h, 5-50 h). After intraperitoneal administration (10 mg KT/kg b.w.), microsphere aggregations were detected in adipose tissue. Ketotifen concentration was determined in plasma by HPLC. The drug released from PLA and PLGA 50/50 microspheres was detected at 384 and 336 h, respectively. Noncompartmental analysis was performed to determine pharmacokinetic parameters. The inclusion of ketotifen in PLGA and PLA microspheres resulted in significant changes in the plasma disposition of the drug. Overall, these ketotifen-loaded microspheres yielded an intraperitoneal drug release that may be suitable for use as delivery systems in the treatment of inflammatory response in portal hypertension.     

Peptide Acylation by Poly(α-Hydroxy Esters)

Purpose. Poly(lactic acid) (PLA) and poly(lactic-co-glycolic acid) (PLGA) microspheres were investigated concerning the possible acylation of incorporated peptides. Methods. Atrial natriuretic peptide (ANP) and salmon calcitonin (sCT) were encapsulated into PLA and PLGA microspheres. Peptide integrity was monitored by HPLC-MS analysis during microsphere degradation for four weeks. sCT fragmentation with endoproteinase Glu-C was used for identifying modified amino acids. Peptide stability in lactic acid solutions was investigated to elucidate possible mechanisms for preventing peptide acylation. Results. Both peptides were acylated by lactic and glycolic acid units inside degrading microspheres in a time-dependent manner. After 21 days, 60% ANP and 7% sCT inside PLA microspheres were acylated. Fragmentation of sCT with endoproteinase Glu-C revealed that besides the N-terminal amine group, lysine, tyrosine or serine are further possible targets to acylation. Stability studies of the peptides in lactic acid solutions suggest that oligomers are the major acylation source and that lower oligomer concentration and higher pH substantially decreased the reaction velocity. Conclusions. The use of PLA and PLGA for drug delivery needs substantially more circumspection. As, according to FDA standards, the potential hazards of peptide acylation products need to be assessed, our findings may have significant implications for products already on the market. Techniques to minimize the acylation reaction are suggested.     

Novel Polymeric Micelles Based on the Amphiphilic Diblock Copolymer Poly(N-vinyl-2-pyrrolidone)-block-poly(D,L-lactide)

Purpose. The purpose of this work was to synthesize a new amphiphilic diblock copolymer of poly(N-vinyl-2-pyrrolidone and poly(D,L-lactide) (PVP-b-PDLLA) capable of self-assembling into polymeric micelles with multiple binding sites and high entrapment efficiency. Methods. The copolymer was synthesized by ring-opening polymerization of D,L-lactide initiated by potassium PVP hydroxylate. It was characterized by gel permeation chromatography, ¹H- and ¹³C-NMR spectroscopy. The ability of the copolymer to self-assemble was demonstrated by dynamic and static light scattering, spectrofluorimetry and ¹H-NMR. The hydrophobic model drug indomethacin was incorporated into the polymeric micelles by a dialysis procedure. Results. A series of amphiphilic diblock copolymers based on PVP-b-PDLLA were successfully synthesized. The critical association concentrations in water were low, always below 15 mg/L. Micellar size was generally bimodal with a predominant population between 40 and 100 nm. PVP-b-PDLLA micelles were successfully loaded with the poorly water-soluble drug indomethacin and demonstrated an entrapment efficiency higher than that observed with control poly(ethylene glycol)-b-PDLLA micelles. It was hypothesized that specific interactions with the hydrophilic outer shell could contribute to the increase in drug loading. Conclusion. PVP-b-PDLLA micelles appear to exhibit multiple binding sites and thus represent a promising strategy for the delivery of a variety of drugs.     

The Stabilization and Encapsulation of Human Growth Hormone into Biodegradable Microspheres

Purpose. To produce and evaluate sustained-acting formulations of recombinant human growth hormone (rhGH) made by a novel microencapsulation process. Methods. The protein was stabilized by forming an insoluble complex with zinc and encapsulated into microspheres of poly (D,L-lactide co-glycolide) (PLGA) which differed in polymer molecular weight (8–3 1kD), polymer end group, and zinc content. The encapsulation procedure was cryogenic, non-aqueous, and did not utilize surfactants or emulsification. The rhGH extracted from each of these microsphere formulations was analyzed by size-exclusion, ion-exchange and reversed-phase chromatography, SDS-polyacrylamide gel electrophoresis, peptide mapping, and cell proliferation of a cell line expressing the hGH receptor. In addition, the in vivorelease profile was determined after subcutaneous administration of the microspheres to rats and juvenile rhesus monkeys. Results. Protein and bioactivity analyses of the rhGH extracted from three different microsphere formulations showed that the encapsulated protein was unaltered relative to the protein before encapsulation. In vivo, microsphere administration to rats or monkeys induced elevated levels of serum rhGH for up to one month, more than 20-fold longer than was induced by the same amount of protein injected subcutaneously as a solution. The rate of protein release differed between the three microsphere formulations and was determined by the molecular weight and hydrophobicity of the PLGA. The serum rhGH profile, after three sequential monthly doses of the one formulation examined, was reproducible and showed no dose accumulation. Conclusions. Using a novel process, rhGH can be stabilized and encapsulated in a solid state into PLGA microspheres and released with unaltered properties at different rates.     

Preparation and Characterization of a Composite PLGA and Poly(Acryloyl Hydroxyethyl Starch) Microsphere System for Protein Delivery

Purpose. To prepare and characterize a novel composite microsphere system based on poly(D,L-lactide-co-glycolide) (PLGA) and poly(acryloyl hydroxyethyl starch) (acHES) hydrogel for controlled protein delivery. Methods. Model proteins, bovine serum albumin, and horseradish peroxidase were encapsulated in the acHES hydrogel, and then the protein-containing acHES hydrogel particles were fabricated in the PLGA matrix by a solvent extraction or evaporation method. The protein-loaded PLGA-acHES composite microspheres were characterized for protein loading efficiency, particle size, and in vitro protein release. Protein stability was examined by size-exclusion chromatography, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and monitoring the enzymatic activity. Results. Scanning electron microscopy showed discrete PLGA microspheres containing many acHES particles. The composite microspheres were spherical and smooth in size range of 39-93 m. The drug loading efficiency ranged from 51 to 101%. The composite microspheres showed more favorable in vitro release than conventional PLGA microspheres. The composite microspheres showed 20% less initial with a gradual sustained release compared to high burst (60%) followed by a very slow release with the conventional PLGA microspheres. The composite microspheres also stabilized encapsulated proteins from the loss of activity during the microsphere preparation and release. Proteins extracted from the composite microspheres showed good stability without protein degradation products and structural integrity changes in the size-exclusion chromatography and SDS-PAGE analyses. Horseradish peroxidase extracted from microspheres retained more than 81% enzymatic activity. Conclusion. The PLGA-acHES composite microsphere system could be useful for the controlled delivery of protein drugs.     

Formulation and in vitro/in vivo evaluation of terbutaline sulphate incorporated in PLGA (25/75) and L-PLA microspheres

Terbutaline sulphate (TBS) is widely used in the treatment of bronchial asthma, chronic bronchitis and emphysema. Because of its short biological half life and dosing schedule, a long acting TBS formulation is required to improve patient compliance. The objective of this study was to develop a TBS containing biodegradable microsphere formulation. Poly(D, L-lactide-co-glyco-lide) (PLGA) and poly(L-lactic acid) (L-PLA) were chosen as matrix materials. A solvent evaporation method was used for preparation of microspheres. Surface morphology, particle size distribution and encapsulation efficiency were investigated. In vitro release studies were performed in pH 7.4 phosphate buffer. In vivo distribution of microspheres were studied in the Swiss albino male mice. All microspheres were spherical in shape and had a porous surface with mean diameters of 9–21 μm. The encapsulation efficiency was influenced by the polymer type, but not the molecular weight. About 90% of the initial amount was trapped in PLGA microspheres, and the remainder was on the surface. In the case of L-PLA, 50% of the total drug was associated with the surface of microspheres. The in vitro release pattern was biphasic characterized by an initial burst phase followed by a slower phase. The L-PLA microspheres released ～92% of the initial payload in 72 h. On the other hand, TBS release was increased with an increase in the molecular weight of PLGA. Biodistribution of L-PLA microspheres was characterized by an initially high uptake (35%) by the lungs. All these results suggest that L-PLA and PLGA microspheres have the potential to be used for passive lung targeting.     

Determinants of Release Rate of Tetanus Vaccine from Polyester Microspheres

Controlled-release formulations based on poly(lactic) (PLA) and poly(lactic/glycolic) acid (PLGA) microspheres containing tetanus vaccine were designed. The polymers forming the microspheres were L-PLA of different molecular weights and DL-PLGA, 50:50. These microspheres were prepared by two solvent elimination procedures, both using a double emulsion, and were characterized for size, morphology, and toxoid release kinetics. The influence of formulation variables such as polymer type, vaccine composition, and vaccine/polymer ratio was also investigated. Both techniques yielded microspheres with similar size, morphology, and release properties. Microsphere size was dependent on the type of polymer and the presence of the surfactant L--phosphatidylcholine, which led to a reduction in microsphere size. On the other hand, the release kinetics of encapsulated protein were affected by the polymer properties (ratio lactic/glycolic acid and molecular weight) as well as by the vaccine composition, vaccine loading, and microsphere size. Moreover, for some formulations, a decrease in microsphere size occurred simultaneously, with an increase in porosity leading to an augmentation of release rate. The changes in the PLA molecular weight during in vitro release studies indicated that release profiles of tetanus toxoid from these microspheres were only marginally influenced by polymer degradation. A significant fraction of protein (between 15 and 35%) was initially released by diffusion through water-filled channels. In contrast, the decrease in the PLGA molecular weight over the first 10 days of incubation suggested that erosion of the polymer matrix substantially affects protein release from these microspheres. Among all formulations developed, two differing in microsphere size, polymer hydrophobicity, and release profile were selected for in vivo administration to mice. Administration of both formulations resulted in tetanus neutralizing antibody levels that were higher than those obtained after administration of the fluid toxoid.     

Ligand-Specific Targeting of Microspheres to Phagocytes by Surface Modification with Poly(L-Lysine)-Grafted Poly(Ethylene Glycol) Conjugate

Purpose. The purpose of this study was to demonstrate specific receptor-mediated targeting of phagocytes by functional surface coatings of microparticles, shielding from nonspecific phagocytosis and allowing ligand-specific interactions via molecular recognition. Methods. Coatings of the comb polymer poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) were investigated for potential to inhibit 1) nonspecific spreading of human blood-derived macrophages (MOs) and dendritic cells (DCs) on glass and 2) nonspecific phagocytosis of PLL-g-PEG-coated, carboxylated polystyrene (PS) or biodegradable poly(D,L-lactide-co-glycolide) (PLGA) microspheres. Coating was performed by adsorption of positively charged PLL-g-PEG on negatively charged microparticles or plasma-cleaned glass through electrostatic interaction. The feasibility of ligand-specific interactions was tested with a model ligand, RGD, conjugated to PEG chains of PLL-g-PEG to form PLL-g-PEG-RGD and compared with inactive ligand conjugate, PLL-g-PEG-RDG. Results. Coatings with PLL-g-PEG largely impaired the adherence and spreading of MOs and DCs on glass. The repellent character of PLL-g-PEG coatings drastically reduced phagocytosis of coated PS and PLGA microparticles to 10% in presence of serum. With both MOs and DCs, we observed ligand-specific interactions with PLL-g-PEG-RGD coatings on glass and PS and PLGA microspheres. Ligand specificity was abolished when using inactive ligand conjugate PLL-g-PEG-RDG, whereas repellency of coating was maintained. Conclusions. Coatings of PLL-g-PEG-ligand conjugates provide a novel technology for ligand specific targeting of microspheres to MOs and DCs while reducing nonspecific phagocytosis.     

Stabilization of 10-Hydroxycamptothecin in Poly(lactide-co-glycolide) Microsphere Delivery Vehicles

Purpose. The purpose of this study was to investigate the potential of poly(lactide-co-glycolide) (PLGA) microspheres to stabilize and deliver the analogue of camptothecin, 10-hydroxycamptothecin (10-HCPT). Methods. 10-HCPT was encapsulated in PLGA 50:50 microspheres by using an oil-in-water emulsion-solvent evaporation method. The influence of encapsulation conditions (i.e., polymer molecular weight (M_w), polymer concentration, and carrier solvent composition) on the release of 10-HCPT from microspheres at 37°C under perfect sink conditions was examined. Analysis of the drug stability in the microspheres was performed by two methods:i) by extraction of 10-HCPT from microspheres and ii). by sampling release media before lactone— carboxylate conversion could take place. Results. Microspheres made, of low M_w polymer (inherent viscosity 0.15 dl/g) exhibited more continuous drug release than those prepared from polymers of higher M_w (i.v. = 0.58 and 1.07 dl/g). In addition, a high polymer concentration and the presence of cosolvent in the carrier solution to dissolve 10-HCPT were both necessary in the microsphere preparation in order to eliminate a large initial burst of the released 10-HCPT. An optimal microsphere formulation released 10-HCPT slowly and continuously for over two months with a relatively small initial burst of the released drug. Both analytical methods used to assess the stability of 10-HCPT revealed that the unreleased camptothecin analogue in the microspheres remained in its active lactone form (>95%) over the entire 2-month duration of study. Conclusions. PLGA carriers such as those described here may be clinically useful to stabilize and deliver camptothecins for the treatment of cancer.     

The Acidic Microclimate in Poly(lactide-co-glycolide) Microspheres Stabilizes Camptothecins

Purpose. The camptothecin (CPT) analogue, 10-hydroxycamptothecin (10-HCPT) has been shown previously to remain in its acid-stable (and active) lactone form when encapsulated in poly(lactide-co-glycolide) (PLGA) microspheres (1). The purpose of this study was to determine the principal mechanism(s) of 10-HCPT stabilization. Methods. CPTs were encapsulated in PLGA 50:50 microspheres by standard solvent evaporation techniques. Microspheres were eroded in pH 7.4 buffer at 37°C. The ratio of encapsulated lactone to carboxylate was determined by HPLC as a function of time, initial form of drug encapsulated, fraction of co-encapsulated Mg(OH)₂, CPT lipophilicity, and drug loading. Two techniques were developed to assess the microclimate pH, including: i) measurement of H⁺ content of the dissolved microspheres in an 80:20 acetonitrile/H₂O mixture and ii) confocal microscopy of an encapsulated pH-sensitive dye, fluorescein. Results. The encapsulated carboxylate converted rapidly to the lactone after exposure to the release media, indicating the lactone is favored at equilibrium in the microspheres. Upon co-encapsulation of Mg(OH)₂, the trend was reversed, i.e., the lactone rapidly converted to the carboxylate form. Measurement of -log(hydronium ion activity) (pa*_H) of dissolved microspheres with pH-electrode and pH mapping with fluorescein revealed the presence of an acidic microclimate. From the measurements of H+ and water contents of particles hydrated for 3 days, a microclimate pH was estimated to be in the neighborhood of 1.8. The co-encapsulation of Mg(OH)₂ could both increase the pa*_H reading and neutralize pH in various regions of the microsphere interior. Varying the drug lipophilicity and loading revealed that the precipitation of the lactone could also stabilize CPT. Conclusions. PLGA microspheres prepared by the standard solvent evaporation techniques develop an acidic microclimate that stabilizes the lactone form of CPTs. This microclimate may be neutralized by co-encapsulating a base such as Mg(OH)₂, as suggested by previous work with poly(ortho esters) (2).     

Controlled Delivery Systems for Proteins Based on Poly(Lactic/Glycolic Acid) Microspheres

This paper describes an investigation of the use of poly(lactic/glycolic acid) polymers for long-term delivery of high molecular weight, water-soluble proteins. Poly(lactic/glycolic acid) (PLGA) microspheres, containing (fluorescein isothiocyanate)-labeled bovine serum albumin and (fluorescein isothiocyanate)-labeled horseradish peroxidase, were prepared by a modified solvent evaporation method using a double emulsion. The microspheres were spherical with diameters of 55–95 µm and encapsulated more than 90% of the protein. The preparation method was gentle and maintained enzyme activity and protein solubility. Stability studies showed that the encapsulation of an enzyme inside PLGA microspheres can protect them from activity loss. When not placed inside PLGA microspheres, (fluorescein isothiocyanate)-labeled horseradish peroxidase lost 80% of its activity in solution at 37°C in a few days, whereas inside the PLGA microspheres it retained more than 55% of its activity after 21 days of incubation at 37°C. In vitro release studies revealed that different release profiles (i.e., near-constant or biphasic) and release rates can be achieved by simply modifying factors in the preparation procedure such as mixing rate and volume of inner water and organic phases. Degradation studies by scanning electron microscopy and gel-permeation chromatography suggested that the mechanism responsible for protein release is mainly through matrix erosion.     

Surface Modification of Poly(lactide-co-glycolide) Nanospheres by Biodegradable Poly(lactide)-Poly(ethylene glycol) Copolymers

The modification of surface properties of biodegradable poly(lactide-co-glycolide) (PLGA) and model polystyrene nanospheres by poly(lactide)-poly(ethlene glycol) (PLA:PEG) copolymers has been assessed using a range of in vitro characterization methods followed by in vivo studies of the nanospheres biodistribution after intravenous injection into rats. Coating polymers with PLA:PEG ratio of 2:5 and 3:4 (PEG chains of 5000 and 2000 Da, respectively) were studied. The results reveal the formation of a PLA: PEG coating layer on the particle surface resulting in an increase in the surface hydrophilicity and decrease in the surface charge of the nanospheres. The effects of addition of electrolyte and changes in pH on stability of the nanosphere dispersions confirm that uncoated particles are electrostatically stabilized, while in the presence of the copolymers, steric repulsions are responsible for the stability. The PLA:PEG coating also prevented albumin adsorption onto the colloid surface. The evidence that this effect was observed for the PLA:PEG 3:4 coated nanospheres may indicate that a poly(ethylene glycol) chain of 2000 Da can provide an effective repulsive barrier to albumin adsorption. The in vivo results reveal that coating of PLGA nanospheres with PLA:PEG copolymers can alter the biodistribution in comparison to uncoated PLGA nanospheres. Coating of the model polystyrene nanospheres with PLA:PEG copolymers resulted in an initial high circulation level, but after 3 hours the organ deposition data showed values similar to uncoated polystyrene spheres. The difference in the biological behaviour of coated PLGA and polystyrene nanospheres may suggest a different stability of the adsorbed layers on these two systems. A similar biodistribution pattern of PLA:PEG 3:4 to PEG 2:5 coated particles may indicate that poly(ethylene glycol) chains in the range of 2000 to 5000 can produce a comparable effect on in vivo behaviour.     

重组降血压肽缓释微球的制备与体外释放

目的采用复乳溶剂蒸发法制备重组降血压肽(rAHP)缓释微球。方法以聚乳酸(PLA)为缓释材料,利用正交设计优化微球制备的最佳工艺条件,并考察了微球的体外释药特性。结果微球制备的最优工艺为:油相中PLA的浓度为7.5%、初乳搅拌速度为900 r/min、内水相与油相体积比为1∶10,外水相聚乙烯醇124浓度为5%;按此工艺制备的微球粒径跨度小、分布均匀,包封率为81.35%,载药量在10.92%,微球得率在80.26%,微球的平均粒径分布范围在75～80μm之间;载药微球在磷酸盐缓冲液中0.5 h内的累积释药量为17.5%,第15天累积释药率达到98.6%。结论该微球制备工艺成熟,包封率高,符合我国药典对缓释制剂的指导原则要求。     

5-Fluorouracil-loaded microspheres prepared by spray-drying poly(D,L-lactide) and poly(lactide-co-glycolide) polymers: characterization and drug release

5-Fluorouracil (5-FU), a hydrosoluble anti-neoplastic drug, was encapsulated in microspheres of poly(D,L-lactide) (PLA) and poly(lactide-co-glycolide) (PLGA) polymers using the spray-drying technique, in order to obtain small size microspheres with a significant drug entrapment efficiency. Drug-loaded microspheres included between 47 +/- 11 and 67 +/- 12 microg 5-FU mg(-1) microspheres and the percentage of entrapment efficiency was between 52 +/- 12 and 74 +/- 13. Microspheres were of small size (average diameter: 0.9 +/- 0.4-1.4 +/- 0.8 microm microspheres without drug; 1.1 +/- 0.5-1.7 +/- 0.9 microm 5-FU-loaded microspheres) and their surface was smooth and slightly porous, some hollows or deformations were observed in microspheres prepared from polymers with larger Tg. A fractionation process of the raw polymer during the formation of microspheres was observed as an increase of the average molecular weight and also of Tg of the polymer of the microspheres. The presence of 5-FU did not modify the Tg values of the microspheres. Significant interactions between the drug and each one of the polymers did not take place and total release of the included drug was observed in all cases. The time needed for the total drug release (28-129 h) was in the order PLA > PLGA 75/25 > PLGA 50/50. A burst effect (17-20%) was observed during the first hour and then a period of constant release rate (3.52 +/- 0.82-1.46 +/- 0.26 microg 5-FU h(-1) per milligram of microspheres) up to 8 or 13 h, depending on the polymer, was obtained.     

Use of hydrodynamic flow focusing for the generation of biodegradable camptothecin-loaded polymer microspheres

The present study was conducted to investigate the use of hydrodynamic flow focusing for the generation of biodegradable polymer microspheres encapsulating the anticancer drug camptothecin. Poly(D,L-lactide-co-glycolide) (PLGA) and poly(L-lactide) (PLA) were used as the matrix materials. Camptothecin was dissolved in the disperse phase and microspheres with a mean size between 2 and 3 microm generated using hydrodynamic flow focusing. When up to 1 wt.% of the drug was added to PLA, the drug encapsulation efficiency was 64%. For PLGA, the drug encapsulation efficiency was between 39 and 46%. Drug release from PLA particles was rapid and complete within 6 h, while drug release from PLGA particles showed no burst effect and followed a first order release profile. The encapsulated camptothecin stayed in its active lactone form, as shown by HPLC, and was able to exert cell toxic effects as shown by a cell viability assay. Hydrodynamic flow focusing is a promising tool for the preparation of drug-releasing biodegradable microspheres typically made by solvent evaporation and/or solvent extraction, as indicated by the successful encapsulation of the anticancer drug camptothecin.