Effect of additives on encapsulation efficiency, stability and bioactivity of entrapped lysozyme from biodegradable polymer particles

Low encapsulation efficiency, incomplete and erratic release profiles are the most common features of controlled released protein delivery systems employing biodegradable polymers. In the present study, lysozyme as a model protein was encapsulated in biodegradable microspheres using solvent evaporation method and the effect of amphiphilic stabilizer, a basic salt and a lyoprotectant on microparticle formulation was evaluated. Incorporation rat serum albumin (RSA) in the internal aqueous phase during emulsion increased the encapsulation efficiency of lysozyme and maintained the bioactivity. Use of NaHCO3 improved the encapsulation efficiency of lysozyme from 15-94%, but at the cost of reduced in vitro release characteristics. Incorporation of both RSA and NaHCO3 improved the bioactivity of lysozyme and decreased burst release of the protein from the polymer particle, but reduced the encapsulation efficiency from 90-70%. Addition of sucrose in the internal aqueous phase lowered the encapsulation efficiency which was restored by its addition in the external aqueous phase. Maintenance of internal aqueous phase pH close to the iso-electric point of the protein and osmotic balance between the internal aqueous phase and the external aqueous phase during solvent evaporation method helped in better encapsulation of the protein drug. In vitro release of the lysozyme correlated with the effect of different excipients on entrapment in polymer matrix. Entrapment efficiency as high as 76%, low burst effect and high bioactivity of the entrapped lysozyme was observed from the polymer particles. Use of RSA, sucrose and NaHCO3 helped in a co-operative way towards the formulation of particles entrapping bioactive lysozyme.     

Formulation and Characterization of Immunoreactive Tetanus Toxoid Biodegradable Polymer Particles

Poly lactide-co-glycolide and polylactide polymer particles entrapping immunoreactive tetanus toxoid (TT) were prepared with a view to developing a single shot controlled release vaccine formulation. Denaturation of TT by dichloromethane (DCM) during primary emulsification stage of particle formulation was minimized by incorporation of an optimal amount of rat serum albumin (RSA) in the internal aqueous phase. Incorporation of RSA as a stabilizer during the primary emulsification stage of polymer particle formulation protected the immunoreactivity of TT, enhanced its encapsulation efficiency and also led to uniform polymer particle formation. Use of sonication, both during primary and secondary emulsification processes, resulted in formation of nanoparticles whereas microparticles were formed when the secondary emulsion was carried out by homogenization. Immunoreactive TT particles made from different polymers incorporating stabilizers released antigen continuously for more than four months in vitro. Single injection of both type of particles encapsulating stabilized TT elicited anti-TT antibody titers in rats for more than five months, which was higher than that obtained with TT injected in saline. Anti-TT antibody titers in vivo were in accordance with the in vitro release characteristics of immunoreactive TT from the particles. Immune responses with hydrophobic polymer particles were better than those made using hydrophilic polymers. These results indicate the importance of protecting the immunoreactivity of TT during formation of polymer particles for sustained and improved antibody response.     

Influences of excipients on in vitro release and in vivo performance of tetanus toxoid loaded polymer particles.

Protein instability during microencapsulation has been one of the major hurdles of biodegradable polymer particles-based vaccine delivery systems. In the present work, effect of serum albumin, sucrose and sodium bicarbonate on surface morphology, entrapment efficiency, in vitro release and in vivo performance tetanus toxoid (TT) loaded PLA particles were investigated. Use of serum albumin as well as high concentration of protein antigen ( approximately 60mg/ml) helped in protecting the immunoreactivity of the antigen during primary emulsification step of particle formulation. Incorporation of sucrose in the internal aqueous phase led to the reduction in encapsulation efficiency of TT from 43.8+/-4.3% to 27.3+/-3.6% in PLA particles and resulted with formation of particles having irregular surface characteristics. Addition of sodium bicarbonate along with sucrose during primary emulsion led to slight improvement in encapsulation efficiency of TT (34.3+/-3.2%) but affected the in vivo performance in terms of serum anti-TT antibody titers from single point immunization. Restoration of osmotic balance by adding equivalent amount of sucrose in external aqueous phase helped in preventing multiple emulsion instability and subsequently improved the encapsulation efficiency of TT to 63.1+/-4.2%. Maximum entrapment efficiency of TT up to 69.2+/-5.1% was achieved when serum albumin, sucrose and sodium bicarbonate were used in internal aqueous phase and sucrose was used in the external aqueous phase. Polymer particles entrapping tetanus toxoid along with optimal stabilizers showed burst release of immunoreactive antigen (>40% in early period) and elicited high and sustained anti-TT antibody titers from single point intramuscular immunization. Anti-TT antibody titers were further enhanced upon immunization of admixture of PLA particles and alum. Choice and use of stabilizers during particle formulation thus need careful considerations not only to protect the immunoreactivity of the antigen, but also to produce stable, uniform particles for optimal in vivo performances.     

Effect of poly(ethylene glycol) content and formulation parameters on particulate properties and intraperitoneal delivery of insulin from PLGA nanoparticles prepared using the double-emulsion evaporation procedure

Abstract

Context: Size, encapsulation efficiency and stability affect the sustained release from nanoparticles containing protein-type drugs.

Objectives: Insulin was used to evaluate effects of formulation parameters on minimizing diameter, maximizing encapsulation efficiency and preserving blood glucose control following intraperitoneal (IP) administration.

Methods: Homogenization or sonication was used to incorporate insulin into poly(D,L-lactic-co-glycolic acid) (PLGA) nanoparticles with increasing poly(ethylene glycol) (PEG) content. Effects of polymer type, insulin/polymer loading ratio and stabilizer in the internal aqueous phase on physicochemical characteristics of NP, in vitro release and stability of encapsulated insulin were investigated. Entrapment efficiency and release were assessed by radioimmunoassay and bicinconnic acid protein assay, and stability was evaluated using SDS-PAGE. Bioactivity of insulin was assessed in streptozotocin-induced, insulin-deficient Type I diabetic mice.

Results: Increasing polymeric PEG increased encapsulation efficiency, while the absence of internal stabilizer improved encapsulation and minimized burst release kinetics. Homogenization was shown to be superior to sonication, with NP fabricated from 10% PEG–PLGA having higher insulin encapsulation, lower burst release and better stability. Insulin-loaded NP maintained normoglycaemia for 24?h in diabetic mice following a single bolus, with no evidence of hypoglycemia.

Conclusions: Insulin-loaded NP prepared from 10% PEG–PLGA possessed therapeutically useful encapsulation and release kinetics when delivered by the IP route.     

Design and evaluation of a novel nanoparticulate-based formulation encapsulating a HIP complex of lysozyme

Formulation development of protein therapeutics using polymeric nanoparticles has found very little success in recent years. Major formulation challenges include rapid denaturation, susceptibility to lose bioactivity in presence of organic solvents and poor encapsulation in polymeric matrix. In the present study, we have prepared hydrophobic ion pairing (HIP) complex of lysozyme, a model protein, using dextran sulfate (DS) as a complexing polymer. We have optimized the process of formation and dissociation of HIP complex between lysozyme and DS. The effect of HIP complexation on enzymatic activity of lysozyme was also studied. Nanoparticles were prepared and characterized using spontaneous emulsion solvent diffusion method. Furthermore, we have also investigated release of lysozyme from nanoparticles along with its enzymatic activity. Results of this study indicate that nanoparticles can sustain the release of lysozyme without compromising its enzymatic activity. HIP complexation using a polymer may also be employed to formulate sustained release dosage forms of other macromolecules with enhanced encapsulation efficiency.     

Why Does PEG 400 Co-Encapsulation Improve NGF Stability and Release from PLGA Biodegradable Microspheres?

Purpose. The aim of this work was to understand the mechanism by which co-encapsulated PEG 400 improved the stability of NGF and allowed a continuous release from PLGA 37.5/25 microspheres. Methods. Microparticles were prepared according to the double emulsion method. PEG 400 was added with NGF in the internal aqueous phase (PEG/PLGA ratio 1/1 and 1.8/1). Its effect was investigated through interfacial tension studies. Protein stability was assessed by ELISA. Results. A novel application of PEG in protein stabilization during encapsulation was evidenced by adsorption kinetics studies. PEG 400 limited the penetration of NGF in the interfacial film of the primary emulsion. Consequently, it stabilized the NGF by reducing the contact with the organic phase. In addition, it avoided the NGF release profile to level off by limiting the irreversible NGF anchorage in the polymer layers. On the other hand, the amount of active NGF released in the early stages was increased. During microparticle preparation, NaCl could be added in the external aqueous phase to modify the structure of microparticles. This allowed to reduce the initial release rate without affecting the protein stability always encountered in the absence of PEG. Conclusions. PEG 400 appeared of major interest to achieve a continuous delivery of NGF over seven weeks from biodegradable microparticles prepared by the double emulsion technique.     

Formulation and characterization of immunoreactive tetanus toxoid biodegradable polymer particles

Poly lactide-co-glycolide and polylactide polymer particles entrapping immunoreactive tetanus toxoid (TT) were prepared with a view to developing a single shot controlled release vaccine formulation. Denaturation of TT by dichloromethane (DCM) during primary emulsification stage of particle formulation was minimized by incorporation of an optimal amount of rat serum albumin (RSA) in the internal aqueous phase. Incorporation of RSA as a stabilizer during the primary emulsification stage of polymer particle formulation protected the immunoreactivity of TT, enhanced its encapsulation efficiency and also led to uniform polymer particle formation. Use of sonication, both during primary and secondary emulsification processes, resulted in formation of nanoparticles whereas microparticles were formed when the secondary emulsion was carried out by homogenization. Immunoreactive TT particles made from different polymers incorporating stabilizers released antigen continuously for more than four months in vitro. Single injection of both type of particles encapsulating stabilized TT elicited anti-TT antibody titers in rats for more than five months, which was higher than that obtained with TT injected in saline. Anti-TT antibody titers in vivo were in accordance with the in vitro release characteristics of immunoreactive TT from the particles. Immune responses with hydrophobic polymer particles were better than those made using hydrophilic polymers. These results indicate the importance of protecting the immunoreactivity of TT during formation of polymer particles for sustained and improved antibody response.     

Evaluation of different buffers on plasmid DNA encapsulation into PLGA microparticles

Double emulsion solvent evaporation is a widely used method to prepare poly(dl-lactide-co-glycolide) (PLGA) microparticles encapsulating plasmid DNA. There are inherent problems associated with preparing plasmid DNA in this form, in particular the DNA is liable to degrade during manufacture and the resulting powder has low encapsulation efficiencies. This study compares the use of two buffers, 0.1 M NaHCO₃ and 0.07 M Na₂HPO₄ and the effect these have on the encapsulation efficiency and other critical parameters associated with these encapsulated DNA materials. Both buffers preserved the conformation of the original plasmid DNA during the homogenization process, but those made with 0.07 M Na₂HPO₄ had higher encapsulation efficiencies, as well as smaller diameters, compared with those made with 0.1 M NaHCO₃ (encapsulation efficiencies of 40.72–45.65%, and mean volume diameters of 2.96–4.45 μm). Buffers with a range of pH from 5 to 12 were investigated, and it was demonstrated that pH 9 was the point at which the highest amount of supercoiled DNA was balanced with the highest encapsulation efficiency. To simulate in vitro release, it was shown that microparticles made with 0.07 M Na₂HPO₄ had lower DNA release rates than those made with 0.1 M NaHCO₃. These results demonstrate that the use of different buffers can aid in retaining the conformation of plasmid DNA, and can also modulate the amount of DNA encapsulated and the release profiles of microparticles.     

A Polysorbate-Based Non-Ionic Surfactant Can Modulate Loading and Release of β-Lactoglobulin Entrapped in Multiphase Poly(DL-Lactide-co-glycolide) Microspheres

Purpose. The goal of the present paper was to investigate the role of a surfactant, Tween 20, in the modulation of the entrapment and release of -lactoglobulin (BLG) from poly (DL-lactide-co-glycolide) microspheres. Methods. Poly(DL-lactide-co-glycolide) microspheres containing BLG were prepared by a water-in-oil-in-water emulsion solvent procedure. Tween 20 was used as a surfactant in the internal aqueous phase of the primary emulsion. BLG entrapment efficiency and burst release were determined. Displacement of BLG from microsphere surface was followed by confocal microscopy observations and zeta potential measurements, whereas morphological changes were observed by freeze-fracture electron microscopy. Results. Tween 20 was shown to increase 2.8 fold the encapsulation efficiency of BLG without any modification of the stability of the first emulsion and the viscosity of the internal aqueous phase. In fact, Tween 20 was shown to be responsible for removing the BLG molecules that were adsorbed on the particle surface or very close to the surface as shown by confocal microscopy and zeta potential measurements. Tween 20 reduced the number of aqueous channels between the internal aqueous droplets as well as those communications with the external medium. Thus, the more dense structure of BLG microspheres could explain the decrease of the burst release. Conclusions. These results constitute a step forward in the improvement of existing technology in controlling protein encapsulation and delivery from microspheres prepared by the multiple emulsion solvent evaporation method.     

Effects of formulation parameters on encapsulation efficiency and release behavior of thienorphine loaded PLGA microspheres

To develop a long-acting injectable thienorphine biodegradable poly (d, l-lactide-co-glycolide) (PLGA) microsphere for the therapy of opioid addiction, the effects of formulation parameters on encapsulation efficiency and release behavior were studied. The thienorphine loaded PLGA microspheres were prepared by o/w solvent evaporation method and characterized by HPLC, SEM, laser particle size analysis, residual solvent content and sterility testing. The microspheres were sterilized by gamma irradiation (2.5 kGy). The results indicated that the morphology of the thienorphine PLGA microspheres presented a spherical shape with smooth surface, the particle size was distributed from 30.19?±?1.17 to 59.15?±?0.67μm and the drug encapsulation efficiency was influenced by drug/polymer ratio, homogeneous rotation speed, PVA concentration in the water phase and the polymer concentration in the oil phase. These changes were also reflected in drug release. The plasma drug concentration vs. time profiles were relatively smooth for about 25 days after injection of the thienorphine loaded PLGA microspheres to beagle dogs. In vitro and in vivo correlation was established.     

Investigation on process parameters involved in preparation of poly-DL-lactide-poly(ethylene glycol) microspheres containing Leptospira Interrogans antigens

Block copolymer, poly-DL-lactide-poly(ethylene glycol) (PELA) with 11.5% of poly(ethylene glycol) (PEG) content was prepared by bulk ring-opening polymerization using stannous chloride as initiator. PELA microspheres with entrapped Leptospira Interrogans antigens, outer membrane protein (OMP) were elaborated by solvent extraction method based on the formation of multiple w/o/w emulsion, and the resulting microspheres were characterized with respect to particle size, OMP entrapment and morphology characteristics. The purpose of the present study is to perform the optimization of preparative parameters for OMP-loaded PELA micropsheres to control particle size and improve the OMP encapsulation efficiency. Of all the parameters investigated, the polymer concentration of organic phase and the external aqueous phase volume play major roles on particle size, while the organic phase volume, internal aqueous phase volume and the addition of surfactant into the internal aqueous phase display considerable effects on OMP loading efficiency. A small volume of internal aqueous phase and intermediate volumes of organic phase and external aqueous phase were favorable to achieve micropsheres with a size of 1-2 microns and high antigen encapsulation efficiency (70-80%). In vitro OMP release profiles from PELA microspheres consist of a small burst release followed by a gradual release phase. The OMP release rate shows some relations with the porous and water-swollen inner structure of the microspheres matrix. The presence of surfactant in microspheres accelerates OMP release, but the OMP entrapment within microspheres shows limited effects on the release profile.     

Effect of formulation and processing factors on the characteristics of biodegradable microcapsules of zidovudine

Abstract

Biodegradable microcapsules of zidovudine (AZT) were prepared using poly-(lactide/glycolide) by the solvent evaporation technique. The objective of this project was to focus on the effect of several formulation and processing factors on the efficiency of encapsulation, surface morphology, and drug release profiles. When the drug was incorporated as powder or as aqueous suspension containing a high amount of insoluble particles, to the organic phase the surface of the microcapsules was appeared to be wrinkled. The efficiency of encapsulation decreased when AZT powder was dispersed directly into the organic solvent instead of adding as an aqueous solution. When the relative volume of the aqueous phase containing 1% PVA was changed from 25 up to 125% of the volume of the organic phase, the efficiency of encapsulation, surface morphology, and release profiles did not change significantly. The efficiency of encapsulation decreased from 9 to 3·8% when the drug loading was increased from 10 to 50% of the weight of the polymer.     

Role of a novel multifunctional excipient poly(ethylene glycol)-block-oligo(vinyl sulfadimethoxine) in controlled release of lysozyme from PLGA microspheres

This study investigated the effect of ion-pairing of anionic polyelectrolytes: our novel poly(ethylene glycol)-block-oligo(vinyl sulfadimethoxine) (PEG-OVSDM) and poly(ethylene glycol)-block-poly(l-aspartic acid) (PEG-PAA) with cationic lysozyme on retention of protein stability during emulsification. Soluble lysozyme recovery after exposure to the deleterious interface was 42-88% (when ion-paired with PEG-OVSDM, PEG-OVSDM concentration dependent) compared to only 30% for free lysozyme. PEG-OVSDM provided a higher stabilization of lysozyme than PEG-PAA (36-60%). Lysozyme when recovered in the aqueous phase and analyzed by chromatography, enzymatic assay, fluorescence, and mass spectrometry showed no significant physicochemical change when compared with a lysozyme standard. Lysozyme was incorporated into poly(lactide-co-glycolide) (PLGA) microspheres via the typical double emulsion method. Incorporation of lysozyme complexes led to a higher encapsulation efficiency and loading amount, and a lower incidence of insoluble lysozyme aggregates compared to the control microspheres containing lysozyme only. More significantly, ion-pairing was able to dramatically reduce the initial lysozyme release to 18% compared with 50% from control microspheres and provided an overall better control of protein release. PEG-PAA was less effective than PEG-OVSDM in controlling the release probably due to weaker interactions between this polyelectrolyte and lysozyme. Manipulation of such polyelectrolyte-protein complexation may play a role in protein-controlled delivery.     

In vitro modified release of acyclovir from ethyl cellulose microspheres

The aim of this study was to demonstrate a sustained-release microparticulate dosage form for acyclovir via an in vitro study. Ethyl cellulose was selected as a model encapsulation material. All of the microspheres were prepared by an oil-in-water solvent evaporation technique. A 2³ full factorial experiment was applied to study the effects of the viscosity of polymer, polymer/drug ratio, and polymer concentration on the drug encapsulation efficiency and the dissolution characteristics. The encapsulation efficiency of acyclovir in microspheres was in the range of 20.0-56.6%. Increase in the viscosity of ethyl cellulose and the ratio of CH₂Cl₂/ethyl cellulose increased drug encapsulation efficiency. The drug continuously released from microspheres for at least 12 h, and the release rate depended on the pH of the release medium. The sustained release characteristic was more prominent in the simulated intestine fluid than in the simulated gastric fluid. A faster release of drug was observed when a high viscosity polymer was used. The decomposition of acyclovir significantly decreased when encapsulated by ethyl cellulose, especially when stored at 37 and 50 °C.     

Formulation and process parameters affecting protein encapsulation into PLGA microspheres during ethyl acetate-based microencapsulation process

The objective of this study was to investigate formulation and process parameters affecting protein encapsulation into PLGA microspheres during an ethyl acetate-based double emulsion microencapsulation process. Lysozyme was used as a model protein throughout this study. An aqueous lysozyme solution was emulsified in ethyl acetate containing 0.6 approximately 1.2 g PLGA75 : 25. The primary emulsion was then transferred quickly to an aqueous phase to make a water-in-oil-in-water emulsion. Ethyl acetate quenching was performed on the double emulsion stirred for 5, 15, 30 or 45 min. The resultant microspheres were further hardened, collected and dried overnight under vacuum. The bicinchoninic acid assay was carried out to determine the quantity of lysozyme present in the aqueous continuous phase and inside the microspheres. While the primary emulsion was stirred without quenching, lysozyme in the inner water phase continued diffusing across the ethyl acetate phase into the aqueous continuous phase. Emulsion droplets were also broken into smaller ones with ongoing stirring; this event also contributed to lysozyme leaking out of the inner water phase. The amount of lysozyme leaching to the aqueous continuous phase ranged from 4.79 +/- 2.1 to 51.9 +/- 5.3% under the experimental condition. Ethyl acetate quenching stopped the primary emulsion droplets from being fragmented into smaller ones and caused PLA75 : 25 precipitation to form microspheres. As a result, the rate of ethyl acetate removal influenced lysozyme encapsulation efficiency, as well as microsphere size. Depending on the timing of ethyl acetate quenching, lysozyme encapsulation efficiencies were found to be 9.89 +/- 4.53 approximately 75.82 +/- 6.55%. Optimization of the onset of ethyl acetate quenching and formulations could permit attainment of a desirable protein encapsulation efficiency.     

Encapsulation of BSA using a modified W/O/O emulsion solvent removal method

A systematic investigation of protein encapsulation in polylactic-co-glycolic-acid (PLGA) was carried out using the formation of a w/o/o emulsion followed by solvent removal. Various factors were studied, including composition of the suspension medium and the relative amounts of aqueous phase containing protein to polymer solution. High yields of microsphere fabrication were achieved by using silicon oil containing methylene chloride as a suspension medium instead of pure silicon oil, with minimal loss of polymer and protein drug (<2%). The amount of aqueous phase influenced the process and successful encapsulation was obtained if the volume ratios of aqueous phase to polymer solution were less than 5% (v/v) at a wide range of polymer concentration (2–15%?g?ml^?1). Protein encapsulation by this w/o/o emulsion and solvent removal method has a high yield of microsphere fabrication and protein encapsulation (98%). In addition, it provides an easy way to control the release rate of protein encapsulated in microspheres by modulating their porosity in fabrication process.     

Preparation of polymeric particles in CO2 medium using non-toxic solvents: Formulation and comparisons with a phase separation method

The aim of this work was to elaborate formulation strategies to encapsulate a protein into biodegradable polymeric particles for sustained release purpose. In this paper, two encapsulation methods will be presented, one dealing with a phase separation phenomenon while the other involving an emulsification/extraction process in CO₂ medium. In those methods, only non-volatile injectable solvents such as glycofurol or isosorbide dimethyl ether were used to dissolve the polymer. Moreover, experimental designs were built up to help us to go further in the understanding of the processes and to better predict output responses in design space. Spherical particles were successfully generated with a satisfactory encapsulation yield. Further characterization steps such as in vitro, in vivo releases will be carried out to validate the interest of our encapsulation methods in the development of drug delivery systems.     

Reservoir-type microcapsules prepared by the solvent exchange method: effect of formulation parameters on microencapsulation of lysozyme

A new microencapsulation technique based on the solvent exchange method was implemented using an ultrasonic atomizer system to encapsulate a protein drug in mild conditions. The reservoir-type microcapsules encapsulating lysozyme as a model protein were prepared by inducing collisions between the aqueous droplets containing lysozyme and the droplets of organic solvent with dissolved poly(lactic acid-co-glycolic acid) (PLGA). The main focus of the study was to examine formulation variables on the size and the encapsulation efficiency of the formed microcapsules. The formulation variables examined were concentrations of mannose in the aqueous cores, NaCl in the aqueous collection medium, and PLGA in organic solvent. The mean diameter of the microcapsules ranged from 40 microm to 100 microm. Smaller microcapsules showed lower encapsulation efficiencies. The resulting microcapsules released native lysozyme in a sustained manner, and the release rate was dependent on the formulation conditions, such as the concentration and molecular weight of the polymer used. The solvent exchange method does not induce lysozyme aggregation and loss of its biological activity. The solvent exchange method, implemented by the ultrasonic atomizer system, provides an effective tool to prepare reservoir-type microcapsules for delivering proteins.     

Response Surface Methodology to Obtain β-Estradiol Biodegradable Microspheres for Long-Term Therapy of Osteoporosis

The purpose of this work was to evaluate the main and interaction effects of formulation factors on the drug encapsulation efficiency of β-estradiol biodegradable microspheres by applying response surface methodology. A secondary purpose was to obtain an optimized formula for long-term therapy of osteoporosis. A three factor, three level Box-Behnken experimental design was used to get 15 experimental runs. The independent variables were drug/polymer ratio (X1), dispersing agent concentration (X2), and deaggregating agent concentration (X3). The dependent variables were percentage encapsulation efficiency (Y1), cumulative percent drug released (Y2), and percentage yield of the microspheres (Y3). The formulations were prepared by emulsion solvent evaporation technique using ethyl acetate as organic solvent. The optimized formulation was maximized for encapsulation efficiency and further characterized for the particle size distribution, scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier transform infrared (FT-IR). The mathematical relationship obtained between X1, X2, X3, and Y1 was:

Y1 = ?129.85 + 29.35X₁ + 129.99X₂ + 64.82X₃ ? 3.2X₁X₂ ? 0.29X₁X₃ ? 35.83X₂X₃ ? 2.05X₁² ? 13.23X₂² ? 5.92X₃² (R2 = 0.99)

The equation showed that X1, X2, and X3 affect Y1 positively but interaction between any two of these factors affects Y1 negatively. The most significant interaction was between X2 and X3. The finding indicated that controlled releases β-estradiol biodegradable microspheres with high encapsulation efficiency and low pulsatile release can be prepared and the quantitative response surface methodology applied helped in understanding the effects and the interaction effects between the three factors applied.     

Protein-loaded poly(epsilon-caprolactone) microparticles. I. Optimization of the preparation by (water-in-oil)-in water emulsion solvent evaporation.

The aim of this work was to optimize protein entrapment in pure poly(epsilon-caprolactone) (PCL) microparticles (MP) using the (water-in-oil)-in water solvent evaporation technique and bovine serum albumin (BSA) as drug model. Therefore, the preparative variables such as polymer solvent, protein/polymer ratio, polymer molecular weight, internal aqueous/organic phases ratio, organic/external aqueous phase ratio, and nature of the emulsifier were evaluated on microparticle characteristics such as BSA entrapment, entrapment efficiency, size and morphology. The in vitro release profiles of BSA from such MP in two different media with or without sodium dodecyl sulphate (SDS) were investigated. In optimum conditions, smooth and spherical pure PCL MP with high encapsulation efficiency (50.29 +/- 5.01%) were prepared. The release profiles of BSA in the release media were significantly different and faster in the presence of SDS. Moreover, they exhibited a relatively low burst effect after 24h (<30%) followed by a continuous release over 28 days. Due to PCL's numerous desirable characteristics, such MP could be an exciting alternative for the controlled release of proteinaceous compounds.