Comparative study of DNA encapsulation into PLGA microparticles using modified double emulsion methods and spray drying techniques

Recently, several research groups have shown the potential of microencapsulated DNA as adjuvant for DNA immunization and in tissue engineering approaches. Among techniques generally used for microencapsulation of hydrophilic drug substances into hydrophobic polymers, modified WOW double emulsion method and spray drying of water-in-oil dispersions take a prominent position. The key parameters for optimized microspheres are particle size, encapsulation efficiency, continuous DNA release and stabilization of DNA against enzymatic and mechanical degradation. This study investigates the possibility to encapsulate DNA avoiding shear forces which readily degrade DNA during this microencapsulation. DNA microparticles were prepared with polyethylenimine (PEI) as a complexation agent for DNA. Polycations are capable of stabilizing DNA against enzymatic, as well as mechanical degradation. Further, complexation was hypothesized to facilitate the encapsulation by reducing the size of the macromolecule. This study additionally evaluated the possibility of encapsulating lyophilized DNA and lyophilized DNA/PEI complexes. For this purpose, the spray drying and double emulsion techniques were compared. The size of the microparticles was characterized by laser diffractometry and the particles were visualized by scanning electron microscopy (SEM). DNA encapsulation efficiencies were investigated photometrically after complete hydrolysis of the particles. Finally, the DNA release characteristics from the particles were studied. Particles with a size of <10?µm which represent the threshold for phagocytic uptake could be prepared with these techniques. The encapsulation efficiency ranged from 100–35% for low theoretical DNA loadings. DNA complexation with PEI 25?kDa prior to the encapsulation process reduced the initial burst release of DNA for all techniques used. Spray-dried particles without PEI exhibited high burst releases, whereas double emulsion techniques showed continuous release rates.     

Mechanisms of burst release from pH-responsive polymeric microparticles

Objectives Microencapsulation of drugs into preformed polymers is commonly achieved through solvent evaporation techniques or spray drying. We compared these encapsulation methods in terms of controlled drug release properties of prepared microparticles and investigated the underlying mechanisms responsible for the ‘burst release’ effect. Methods Using two different pH‐responsive polymers with a dissolution threshold of pH 6 (Eudragit L100 and AQOAT AS‐MG), hydrocortisone, a model hydrophobic drug, was incorporated into microparticles below and above its solubility within the polymer matrix. Key findings Although, spray drying was an attractive approach due to rapid particle production and relatively low solvent waste, the oil‐in‐oil microencapsulation method was superior in terms of controlled drug release properties from the microparticles. Slow solvent evaporation during the oil‐in‐oil emulsification process allowed adequate time for drug and polymer redistribution in the microparticles and reduced uncontrolled drug burst release. Electron microscopy showed that this slower manufacturing procedure generated nonporous particles whereas thermal analysis and X‐ray diffractometry showed that drug loading above the solubility limit of the drug in the polymer generated excess crystalline drug on the surface of the particles. Raman spectral mapping illustrated that drug was homogeneously distributed as a solid solution in the particles when loaded below saturation in the polymer with consequently minimal burst release. Conclusions Both the manufacturing method (which influenced particle porosity and density) and drug:polymer compatibility and loading (which affected drug form and distribution) were responsible for burst release seen from our particles     

Comparison of post-emulsification freeze drying or spray drying processes for the microencapsulation of plasmid DNA

In this work, methods used to microencapsulate plasmid DNA in a biodegradable polymer were compared for their effects on the physicochemical characteristics of DNA-loaded microparticles and on the release and integrity of encapsulated DNA. Microparticles were formulated by either w/o/w emulsification and freeze-drying (EFD) or by w/o/w emulsification and spray-drying (ESD). The influence of both manufacturing processes on particle morphology, charge, release characteristics and biological activity of encapsulated DNA was evaluated. Particles produced by emulsification/spray-drying exhibited more diversity in shape and size than those produced by emulsification/freeze-drying. These particles also exhibited higher plasmid DNA encapsulation efficiency than particles produced by emulsification/freeze-drying. The fractional DNA release rates were similar over the first 25 days for both formulations, release rate declining more rapidly at later times for the ESD product. Mammalian cell transfection assays confirmed the biological activity of encapsulated DNA extracted from both types of particles, with significantly higher transfection levels being observed for ESD particles. Application of a double emulsion (w/o/w) before spray drying resulted in higher encapsulation levels (> 90%) relative to previous literature values, which used single (w/o) emulsions before spray drying. The emulsification/spray-drying technique described here appears to be a rapid and efficient method for the preparation of PLGA microparticles loaded with plasmid DNA.     

Water-soluble betamethasone-loaded poly(lactide-co-glycolide) hollow microparticles as a sustained release dosage form

In this study, betamethasone disodium phosphate-loaded microparticles were fabricated for sustained release using poly(lactide-co-glycolide) (PLGA) by spray drying and emulsion solvent evaporation/extraction techniques. Encapsulation efficiencies ranged from 59-80% using a water-in-oil-in-oil (W/O/O) double emulsion technique and more than 90% for a spray-drying method were obtained. This was a significant improvement compared to fabrication by a water-in-oil-in-water (W/O/W) double emulsion process, which had an encapsulation efficiency of less than 15%. Multiple-phase and biphasic release profiles were observed for microparticles of PLGA 50/50 and PLGA of higher lactide contents, respectively. The PLGA 50/50 hollow microparticles fabricated using the W/O/O double emulsion technique provided a sustained release of betamethasone disodium phosphate over 3 weeks.     

Water-soluble betamethasone-loaded poly(lactide-co-glycolide) hollow microparticles as a sustained release dosage form

In this study, betamethasone disodium phosphate-loaded microparticles were fabricated for sustained release using poly(lactide-co-glycolide) (PLGA) by spray drying and emulsion solvent evaporation/extraction techniques. Encapsulation efficiencies ranged from 59–80% using a water-in-oil-in-oil (W/O/O) double emulsion technique and more than 90% for a spray-drying method were obtained. This was a significant improvement compared to fabrication by a water-in-oil-in-water (W/O/W) double emulsion process, which had an encapsulation efficiency of less than 15%. Multiple-phase and biphasic release profiles were observed for microparticles of PLGA 50/50 and PLGA of higher lactide contents, respectively. The PLGA 50/50 hollow microparticles fabricated using the W/O/O double emulsion technique provided a sustained release of betamethasone disodium phosphate over 3 weeks.     

Spray-drying preparation of microparticles containing cationic PLGA nanospheres as gene carriers for avoiding aggregation of nanospheres

Preparation of nano-sized particles using lyophilization, which is a standard drying technique for high-molecular-weight compounds such as bioactive peptides, proteins, plasmid DNA and siRNA, often results in particle aggregation. In this study, spray-drying was applied for preparation of cationic PLGA nanospheres as gene delivery vectors in order to minimize aggregation and loss of gene transfection efficiency. PLGA nanoparticle emulsions were prepared by dropping an acetone/methanol mixture (2/1) containing PLGA and a cationic material, such as PEI, DOTMA, DC-Chol or CTAB, into distilled water with constant stirring. The PLGA nanosphere emulsion was dried with mannitol by spray-drying, and mannitol microparticles containing PLGA nanospheres were obtained. Mean particle diameter of spray dried PLGA particles was 100-250 nm, which was similar to that of the nano-emulsion before drying, whereas the lyophilized PLGA particles showed increased particle diameter due to particle aggregation. PEI, DOTMA and DC-Chol were useful for maintaining nanoparticle size and conferring positive charge to nanospheres. Transfection of pDNA (pCMV-Luc) using these spray-dried cationic PLGA nanospheres yielded high luciferase activity in COS-7 cells, particularly with PLGA/PEI nanospheres. The present spray-drying technique is able to provide cationic PLGA nanospheres, and may improve redispersal and handling properties.     

Colon-specific delivery of 5-aminosalicylic acid from chitosan-Ca-alginate microparticles

Chitosan-Ca-alginate microparticles for colon-specific delivery and controlled release of 5-aminosalicylic acid after peroral administration were prepared using spray drying method followed by ionotropic gelation/polyelectrolyte complexation. Physicochemical characterization pointed to the negatively charged particles with spherical morphology having a mean diameter less than 9 microm. Chitosan was localized dominantly in the particle wall, while for alginate, a homogeneous distribution throughout the particles was observed. (1)H NMR, FTIR, X-ray and DSC studies indicated molecularly dispersed drug within the particles with preserved stability during microencapsulation and in simulated in vivo drug release conditions. In vitro drug release studies carried out in simulated in vivo conditions in respect to pH, enzymatic and salt content confirmed the potential of the particles to release the drug in a controlled manner. The diffusional exponents according to the general exponential release equation indicated anomalous (non-Fickian) transport in 5-ASA release controlled by a polymer relaxation, erosion and degradation. Biodistribution studies of [(131)I]-5-ASA loaded chitosan-Ca-alginate microparticles, carried out within 2 days after peroral administration to Wistar male rats in which TNBS colitis was induced, confirmed the dominant localization of 5-ASA in the colon with low systemic bioavailability.     

蛋白质/多肽药物聚乳酸/乳酸-羟基乙酸共聚物微球研究进展

缓释微粒给药系统是蛋白质/多肽药物传输系统的一个重要研究方向，聚乳酸和乳酸-羟基乙酸共聚物是制备缓释微球最常用的载体材料。蛋白质/多肽药物聚乳酸/乳酸-羟基乙酸共聚物微球常用的制备方法包括溶剂萃取/挥发法(复乳法)、相分离法和喷雾干燥法。本文总结了微球制备中面临的难点如蛋白质/多肽药物稳定性、包封率、药物突释和药物吸附等问题，并综述了保持药物结构稳定性和生物活性、提高包封率、改善药物释放曲线等微球制备方法和进展。     

喷雾冷冻干燥技术制备盐酸伊立替康脂质体冻干微粒及其理化性质考察

目的:考察喷雾冷冻干燥(SFD)技术制备脂质体冻干微粒的可行性。方法:以盐酸伊立替康为模型药物,采用硫酸铵梯度法制备盐酸伊立替康脂质体,SFD技术制备脂质体冻干微粒;以喷嘴高度、物料流速、雾滴/液氮质量比为因素,应用Box-Behnk-enDesign(BBD)试验考察三者之间的配比对微粒包封率的影响以优化SFD工艺,并对所制备的脂质体及冻干微粒的理化性质进行了考察。结果:SFD优化工艺为物料流速5.5mL·min-1,喷嘴高度18.5cm,雾滴/液氮质量比3.7%,由此制备的脂质体冻干微粒的外观和再分散性好,平均粒径、粒度分布、主药含量及包封率等理化性质与原脂质体基本保持一致,且放置6个月后与原脂质体溶液比较稳定性更好。结论:SFD技术制备脂质体冻干微粒具有可行性,并提高了脂质体的稳定性。     

Preparation of Polymeric Submicron Particle-Containing Microparticles Using a 4-Fluid Nozzle Spray Drier

Purpose We studied a novel method for preparing polymeric submicron particle-containing microparticles using a 4-fluid nozzle spray drier. Method Ethylcellulose (EC) and poly(lactic-co-glycolic acid) (PLGA), either alone or in combination with polyethylenimine (PEI), were used as polymers to produce submicron particles, and mannitol (MAN) was used as a water-soluble carrier for the microparticles. The polymer and MAN solutions were supplied through different liquid passages of a 4-fluid nozzle and then dried to obtain MAN microparticles containing EC or PLGA submicron particles. The polymer/MAN ratio was controlled by changing the concentration of the polymer and MAN solutions. EC or PLGA microparticles were observed via scanning electron microscopy, and the size of microparticles was determined by image analysis. The particle size distribution of EC or PLGA submicron particles was measured with a super dynamic light scattering spectrophotometer. Results The method generated submicron-sized (<1 μm) particles of EC and PLGA. The mean diameters of EC and PLGA particles at a polymer/MAN ratio of 1:10 were 631 and 490 nm, respectively. The mean diameter of PLGA particles decreased as the PLGA/MAN ratio was reduced, reaching ∼200 nm at a PLGA/MAN ratio of 1:100. The mean diameter of PLGA/PEI particles at PLGA/PEI/MAN ratios of 1:0.5:10 and 1:0.5:100 were 525 and 223 nm, respectively, and their zeta potentials were +50.8 and +58.2 mV, respectively. The size of EC submicron particles could be controlled by varying the spray conditions. Conclusions This study demonstrated that it is possible to prepare polymeric submicron particles dispersed in MAN microparticles in a single process using the 4-fluid nozzle spray drying method. Cationic PLGA particles with a diameter of ∼200 nm could be prepared by adding PEI, suggesting the possibility of its use as a carrier for delivering DNA into cells. The precipitation of EC may occur by the mutual dispersion and mixing of solvents after collision of EC and MAN mists by antisolvent effect, thereby producing MAN microparticles containing EC submicron particles.     

Prospects of pharmaceuticals and biopharmaceuticals loaded microparticles prepared by double emulsion technique for controlled delivery

Several methods and techniques are potentially useful for the preparation of microparticles in the field of controlled drug delivery. The type and the size of the microparticles, the entrapment, release characteristics and stability of drug in microparticles in the formulations are dependent on the method used. One of the most common methods of preparing microparticles is the single emulsion technique. Poorly soluble, lipophilic drugs are successfully retained within the microparticles prepared by this method. However, the encapsulation of highly water soluble compounds including protein and peptides presents formidable challenges to the researchers. The successful encapsulation of such compounds requires high drug loading in the microparticles, prevention of protein and peptide degradation by the encapsulation method involved and predictable release, both rate and extent, of the drug compound from the microparticles. The above mentioned problems can be overcome by using the double emulsion technique, alternatively called as multiple emulsion technique. Aiming to achieve this various techniques have been examined to prepare stable formulations utilizing w/o/w, s/o/w, w/o/o, and s/o/o type double emulsion methods. This article reviews the current state of the art in double emulsion based technologies for the preparation of microparticles including the investigation of various classes of substances that are pharmaceutically and biopharmaceutically active.     

Comparison of process parameters for microencapsulation of plasmid DNA in poly(D,L-lactic-co-glycolic) acid microspheres

Poly(D,L-lactic-co-glycolic) acid (PLGA) microspheres containing plasmid DNA encoding the firefly luciferase gene were prepared using the water-in-oil-in-water (w/o/w) double emulsion and solvent evaporation method. In this study, we investigated the effects of three process parameters on DNA microencapsulation: (1) emulsification method used to generate the primary emulsion, (2) water/oil ratio during formation of the first emulsion, and (3) surfactant concentration used in the preparation of the second emulsion. The resulting formulations were also analyzed for microsphere size, encapsulation efficiency, and kinetics of DNA release. We found that although each process alteration resulted in encapsulation of biologically active, structurally intact DNA, the surfactant and water/oil ratio significantly affected the size, release kinetics and encapsulation efficiency of plasmid DNA.     

Comparative study of poly (lactic-co-glycolic acid)-poly ethyleneimine-plasmid DNA microparticles prepared using double emulsion methods

Controlled release of plasmid DNA (pDNA) from biodegradable poly lactic-co-glycolic acid (PLGA) microparticles has the potential to enhance transgene expression. However, barriers to this approach include limited encapsulation efficiency, pDNA damage during fabrication and confinement of the microparticles inside phagolysosomal compartments. Combining PLGA with poly ethyleneimine (PEI) can improve protection of pDNA during fabrication, increase encapsulation efficiencies and impart the PLGA microparticles with the capacity to escape the phagolysosomal compartments. This study compares three promising formulation methods for preparing PLGA PEI pDNA microparticles and evaluates for buffering capacity, cellular uptake, transfection efficiency and toxicity. In the first method, PLGA PEI pDNA microparticles are prepared by entrapping pDNA in blended PLGA/PEI using the double emulsion water-in-oil-in-water solvent evaporation technique (PA). In a second approach, PEI-pDNA polyplexes are prepared and then entrapped in PLGA microparticles using a double emulsion solvent evaporation method (PB). Microparticles prepared using formulation methods PA and PB are then compared against PLGA microparticles with PEI conjugated to the surface using carbodiimide chemistry (PC); 0.5% PVA is identified as the optimum concentration of surfactant for generating the strongest transfection efficiencies. N:P ratios of 5 and 10 are selected for preparation of each group. Gel electrophoresis demonstrates that all PLGA microparticle formulations have strong pDNA binding capacity. An MTT assay shows that in vitro cytotoxicity of PLGA PEI microparticles is significantly lower than PEI alone. PLGA PEI pDNA microparticles mediate higher cellular uptake efficiency and consequently higher transgene expression than unmodified PLGA microparticles in COS7 and HEK293 cells. Preparing PEI-pDNA polyplexes prior to entrapment in PLGA microparticles (PB) results in the highest pDNA loading. This is 2.5-fold higher than pDNA loading in unmodified PLGA microparticles. PLGA PEI pDNA microparticles prepared using method PB generates the strongest transfection efficiencies, which are 500-fold higher than unmodified PLGA pDNA microparticles in HEK293 cells and 1800-fold higher in COS-7 cells. The highest transfection efficiencies generated from microparticles prepared using method PB is achieved using an N:P ratio of 5.     

Microencapsulate Aspergillus niger peptidases from agroindustrial waste wheat bran: spray process evaluation and stability

The aim of this work was to obtain microencapsulated stable Aspergillus niger peptidases by post fermentation spray drying. The enzymatic extract was evaluated before and after spray drying microencapsulation to verify the effects of five different process parameters on the extract enzymatic activity, i.e. air flow, extract feed rate, drying temperature, homogenising time and weight ratio of extract to encapsulation material. The optimal conditions were determined by desirability functions and experimentally confirmed. Additionally, the stability of the microparticles was assessed during 60 days at 4?°C, 25?°C and 40?°C. The results revealed that the microparticles stored at 4?°C retained approximately 100% of their proteolytic activity at nine days of storage. Considering the industrial adaptation of the bioprocess and the prospect of commercial application of the proteases, the evaluation of different parameters for drying enzymes is required as a valuable alternative to obtain biotechnological products with high added value.     

Using water-soluble chitosan for flavour microencapsulation in food industry

The aim of this work is to investigate the possibility of producing flavour microparticles, by means of water-soluble chitosan, considering all the advantages of this natural polymer (non-toxic, biocompatibility, biodegradability, anticholesterolemic), with or without a crosslinking agent (tripolyphosphate (TPP)). The microparticles were prepared by spray drying and characterized by their particle size, surface morphology and zeta potential. Structural analysis of the surface of the particles was performed by scanning electron microscopy (SEM). Significant differences were found in the surface structure of the crosslinked and non-crosslinked particles. Chemical characterization of the microparticles was performed by Fourier transform infrared spectroscopy (FTIR), the results being significant and proving the success of flavour microencapsulation. This work shows that it is possible to encapsulate peach flavours using water-soluble chitosan, through a spray-drying process.     

Encapsulation and stabilization of nerve growth factor into poly(lactic-co-glycolic) acid microspheres.

The development of a stable sustained-release formulation of recombinant human nerve growth factor (rhNGF) for the treatment of neuronal diseases is described. The protein was encapsulated into poly(lactic-co-glycolic) acid (PLGA) microspheres using a spray freeze drying technique. Liquid nitrogen and cold ethanol were used to spray-freeze-dry solid rhNGF that had been suspended in a solution of PLGA dissolved in ethyl acetate. When excipients such as sugar (trehalose), surfactant (pluronic F68), and poly(ethylene glycol) (PEG) were added to the PLGA formulation to protect rhNGF from degradation during spray freeze drying, the protein degraded via aggregation during in vitro release. The formation of an insoluble rhNGF-zinc complex prior to encapsulation into PLGA microspheres stabilized the protein during both microencapsulation and release. In this study, we have demonstrated that the addition of zinc acetate in a 1:12 rhNGF-to-zinc acetate molar ratio in a solid rhNGF formulation (4 mM sodium bicarbonate at pH 7.4) improves stability of rhNGF during release at 37 degrees C (physiological temperature). The stabilization may be due to rhNGF complexation with zinc to form stable aggregates. The PLGA formulation consisting of 10% rhNGF encapsulated in 12 kDa PLGA (50:50 lactide/glycolide) provided a continuous release of 14 days. The low initial burst (approximately 1%) and controlled-release rate were achieved by the addition of 3 or 6% solid zinc carbonate to the polymer phase during microencapsulation.     

Comparison of a novel spray congealing procedure with emulsion-based methods for the micro-encapsulation of water-soluble drugs in low melting point triglycerides

The particle size characteristics and encapsulation efficiency of microparticles prepared using triglyceride materials and loaded with two model water-soluble drugs were evaluated. Two emulsification procedures based on o/w and w/o/w methodologies were compared to a novel spray congealing procedure. After extensive modification of both emulsification methods, encapsulation efficiencies of 13.04% tetracycline HCl and 11.27% lidocaine HCl were achievable in a Witepsol®-based microparticle. This compares to much improved encapsulation efficiencies close to 100% for the spray congealing method, which was shown to produce spherical particles of ～58 μm. Drug release studies from a Witepsol® formulation loaded with lidocaine HCl showed a temperature-dependent release mechanism, which displayed diffusion-controlled kinetics at temperatures ～25°C, but exhibited almost immediate release when triggered using temperatures close to that of skin. Therefore, such a system may find application in topical semi-solid formulations, where a temperature-induced burst release is preferred.     

Comparison of a novel spray congealing procedure with emulsion-based methods for the micro-encapsulation of water-soluble drugs in low melting point triglycerides

The particle size characteristics and encapsulation efficiency of microparticles prepared using triglyceride materials and loaded with two model water-soluble drugs were evaluated. Two emulsification procedures based on o/w and w/o/w methodologies were compared to a novel spray congealing procedure. After extensive modification of both emulsification methods, encapsulation efficiencies of 13.04% tetracycline HCl and 11.27% lidocaine HCl were achievable in a Witepsol-based microparticle. This compares to much improved encapsulation efficiencies close to 100% for the spray congealing method, which was shown to produce spherical particles of approximately 58 microm. Drug release studies from a Witepsol formulation loaded with lidocaine HCl showed a temperature-dependent release mechanism, which displayed diffusion-controlled kinetics at temperatures approximately 25 degrees C, but exhibited almost immediate release when triggered using temperatures close to that of skin. Therefore, such a system may find application in topical semi-solid formulations, where a temperature-induced burst release is preferred.     

Stability and release characteristics of poly(D,L-lactide-co-glycolide) encapsulated CaPi-DNA coprecipitation

The aims of this work were to determine the stability of DNA-calcium-phosphate coprecipitation (CaPi-DNA) against various conditions during double emulsification microencapsulation and assess the release and physicochemical characteristics of poly(D,L-lactide-co-glycolide) (PLGA) microparticles loading CaPi-DNA. CaPi-DNA prepared at pH 6.5 showed a good stability with over 60% CaPi-DNA remained after emulsification, but no more than 40% at pH 8.0. Polyvinyl alcohol (PVA, 1-5%) could make over 80% CaPi-DNA (pH 7.0) preserved after homogenization. The dichloromethane (DCM), mixture of DCM and ethyl acetate, ether and n-hexane (1:1) exhibited neglectable influence on CaPi-DNA under homogenization. PLGA had influenced on CaPi-DNA without any additional stabilizer, in particular, PLGA (75:25, 4%, w/v) demonstrated a profound damage with only about 10% of the original CaPi-DNA remained. PLGA microparticles loading CaPi-DNA were spherical in shape with size range of 2.0-5.0microm, and entrapment efficiency 30-50%. CaPi-DNA was found to increase the stability of pDNA in PLGA microparticles without losing its structure integrity. The release of CaPi-DNA from microparticles showed a low burst release (<7.5%) within 24h and following sustained release process. The amount of cumulated CaPi-DNA release over 30 days was: 17.6% for PLGA (lactide:glycolide=50:50), 27.3% for PLGA (65:35) and 44.8% for PLGA (75:25) microparticles, respectively. The encapsulation of CaPi-DNA in microparticles could significantly protect CaPi-DNA from degradation of nuclease with average over 80% of total DNA recovery. These results suggested that the encapsulation of CaPi-DNA in PLGA microparticles could improve stability of pDNA.     

Vancomycin biodegradable poly(lactide-co-glycolide) microparticles for bone implantation. Influence of the formulation parameters on the size, morphology, drug loading and in vitro release

The present study investigates vancomycin microencapsulation in biodegradable PLAGA microparticles. To optimize encapsulation efficiency by the double emulsion (w/o/w) solvent evaporation/extraction process, two parameters were studied: surfactant (Span 80) rate and external aqueous phase saturation. In vitro dissolution studies, laser granulometry and scanning electron microscopy were performed to characterize the microparticles. The best results were obtained by stabilizing the first emulsion with 0.5% Span 80 and saturating the external phase with sodium chloride. Such parameters allowed a 95% drug encapsulation efficiency. This process yielded round microparticles with a mean diameter of approximately 170 microm and presenting a smooth surface without any pores. Moreover, this formulation induces a sustained drug release at a constant rate over a period of 10 days. Such materials could be associated with biphasic calcium phosphate granules to form an antibiotic-loaded injectable bone substitute offering a long-term activity in situ.