Molecular recognition by Kluyveromyces of amphotericin B-loaded, galactose-tagged, poly (lactic acid) microspheres

In an effort to develop a new way of drug delivery, especially for polyenic antifungal molecules, we have incorporated amphotericin B (AmB) into biodegradable galactosylated poly (L-lactic acid) (L-PLA) and poly (L-lactic-co-glycolic acid) (PLGA) microspheres. These drug carriers were prepared by solvent evaporation using an oil/water (o/w) emulsion. The ratio of galactosyl spacers with different chain lengths was 1.74-2.78%. The maximal quantity of AmB encapsulated reported to 100 mg of the galactosylated microspheres was 7.14 mg for L-PLA (encapsulation rate 45% of mole) and 6.42 mg for PLGA derivatives (encapsulation rate 81% of mole). In our yeast model, drug release depended on three factors: (i) presence of galactosylated antennae, (ii) length of galactosyl antenna and (iii) nature of the polymer. More of the AmB trapped in PLGA microspheres was released than from PLA microspheres. These novel functionalised microspheres could be required for the delivering of therapeutic agents according to their recognition to specific cells.     

Formation of Inhalable Rifampicin–Poly(l-lactide) Microparticles by Supercritical Anti-solvent Process

Formation of inhalable microparticles containing rifampicin and poly(l-lactide) (L-PLA) by using supercritical anti-solvent process (SAS) was investigated. The solutions of drug and polymer in methylene chloride were sprayed into supercritical carbon dioxide. The effect of polymer content and operating conditions, temperature, pressure, carbon dioxide molar fraction, and concentration of solution, on product characteristics were studied. The prepared microparticles were characterized with respect to their morphology, particle size and size distribution, drug content, drug loading efficiency, and drug release characteristic. Discrete, spherical microparticles were obtained at high polymer:drug ratios of 7:3, 8:2, and 9:1. The shape of L-PLA microparticles became more irregular and agglomerated with decreasing polymer content. Microparticles with polymer content higher than 60% exhibited volumetric mean diameter less than 5 μm, but percent drug loading efficiency was relatively low. Drug-loaded microparticles containing 70% and 80% L-PLA showed a sustainable drug release property without initial burst release. Operating temperature level influenced on mean size and size distribution of microparticles. The operating pressure and carbon dioxide molar fraction in the range investigated were unlikely to have an effect on microparticle formation. An increasing concentration of feed solution provided larger size microparticles. Rifampicin-loaded L-PLA microparticles could be produced by SAS in a size range suitable for dry powder inhaler formulation.     

CO2 and fluorinated solvent-based technologies for protein microparticle precipitation from aqueous solutions

Precipitation with a compressed or supercritical fluid antisolvent (PCA) has been used to produce microparticles of biologically active proteins, pharmaceuticals, and polymers. However, the application of PCA to a wider range of proteins is limited by the low mutual solubility of water (necessary to dissolve most proteins) and CO(2) (traditionally used as the compressed antisolvent). This investigation extends PCA to proteins in aqueous solutions by utilizing ethanol as a cosolvent to enhance the antisolvent properties of CO(2) toward aqueous systems. alpha-Chymotrypsin, a model protein, was precipitated from both compressed CO(2) and a liquid fluorinated antisolvent, a hydrofluoroether (HFE). The equilibrium phase behavior of the antisolvent/ethanol/water systems was examined to identify a one-phase region suitable for protein precipitation. Spherical protein microparticles with a primary particle size of approximately 0.2-0.6 microm were recovered using both the compressed CO(2) and fluorinated antisolvents. Although the proteins retained significant activity using both antisolvent systems, the HFE-precipitated chymotrypsin retained higher activity than the CO(2)-precipitated protein.     

Nanostructured microspheres produced by supercritical fluid extraction of emulsions

The system poly(lactic-co-glycolic) acid/ piroxicam (PLGA/PX) was selected, as a model system, to evaluate the effectiveness of supercritical carbon dioxide (SC-CO(2)) extraction of the oily phase (ethyl acetate) from oil-in-water emulsions used in the production of polymer/drug microspheres for sustained drug release applications. The influence of process parameters like operating pressure and temperature, flow rate and contacting time between the emulsion and SC-CO(2) was studied with respect to the microsphere size, distribution and solvent residue. Different polymer concentrations in the oily phase were also tested in emulsions formulation to monitor their effects on droplets and microspheres size distribution at fixed mixing conditions. Spherical PLGA microspheres loaded with PX (10% w/w) with mean sizes ranging between 1 and 3 microm and very narrow size distributions were obtained due to the short supercritical processing time (30 min) that prevents the aggregation phenomena typically occurring during conventional solvent evaporation process. A solvent residue smaller than 40 ppm was also obtained at optimized operating conditions. DSC and SEM-EDX analyses confirmed that the produced microparticles are formed by a solid solution of PLGA and PX and that the drug is entrapped in an amorphous state into the polymeric matrix with an encapsulation efficiency in the range of 90-95%. Drug release rate studies showed very uniform drug concentration profiles, without any burst effect, confirming a good dispersion of the drug into the polymer particles.     

Chelation of calcium ions by poly(gamma-glutamic acid) from Bacillus subtilis(chungkookjang)

Many studies have clarified that poly(gamma-glutamic acid) (PGA) increases the solubility of Ca(2+), suggesting that PGA enhances calcium absorption in small intestine. However, there has been no report on the specific interaction between PGA and Ca(2+) in water. We studied the aqueous solution properties of PGA calcium salt (PGA-Ca complex). The chelating ability and binding strength of PGA for Ca(2+) were evaluated. PGA-Ca complex was soluble in water in contrast with the insolubility of poly(acrylic acid) (PAA) calcium salt and the chelating ability of PGA for Ca(2+) was almost the same than that of PAA. The globular conformation of PGA-Ca complex in water was estimated by SEC and viscosity measurements. The chelation of PGA for Ca(2+) was examined by 1H NMR. The present study showing the characteristics of PGA-Ca complex will provide useful information of the calcium absorption by PGA in vivo.     

Thermogelling aqueous solutions of alternating multiblock copolymers of poly(L-lactic acid) and poly(ethylene glycol)

We are reporting alternating multiblock copolymers of poly(L-lactic acid)/poly(ethylene glycol) aqueous solution (> 15 wt %) undergoing sol-gel-sol transition as the temperature increases from 20 to 60 degrees C. Micelles of the multiblock copolymers (in water) are about 20 nm in radius at low temperature. They are aggregated to a larger size as the temperature increases, which should play a critical role in the sol-to-gel transition. The transition temperature and gel window were affected by the molecular weight and composition of the multiblock copolymer. In particular, the aqueous solution of an alternating multiblock copolymer (Mn approximately 6700 daltons) prepared from poly(ethylene glycol) (Mn approximately 600 daltons) and poly(L-lactic acid) (Mn approximately 1300 daltons) showed a maximum modulus at body temperature (37 degrees C). The in situ gel forming ability of the polymer aqueous solution in vivo as well as in vitro indicates that it can be a promising injectable biomaterial.     

Long-term release of clodronate from biodegradable microspheres

This paper describes the formulation of a biodegradable microparticulate drug delivery system containing clodronate, a bisphosphonate intended for the treatment of bone diseases. Microspheres were prepared with several poly(D,L-lactide-co-glycolide) (PLGA) copolymers of various molecular weights and molar compositions and 1 poly(D,L-lactide) (PDLLA) homopolymer by a water-in-oil-in-water (w/o/w) double emulsion solvent evaporation procedure. Critical process parameters and formulation variables (ie, addition of stabilizing agents) were evaluated for their effect on drug encapsulation efficiency and clodronate release rate from microparticles Well-formed clodronate-loaded microspheres were obtained for all polymers by selecting suitable process parameters (inner water/oil volume ratio 1∶16, temperature-raising rate in the solvent evaporation step 1°C/min, 2% wt/vol NaCl in the external aqueous phase). Good yields were obtained in all batches of clodronate microspheres (above 60%); drug encapsulation efficiencies ranged between 49% and 75% depending on the polymer used. Clodronate release from all copolymer microspheres was completed in about 48 hours, while those from PDLLA microspheres required about 20 days. The change of microsphere composition by adding a surfactant such as Span 20 or a viscosing agent such as carboxymethylcellulose extended the long-term release up to 3 months. Clodronate was successfully entrapped in PLGA and PDLLA microspheres, and drug release could be modulated from 48 hours up to 3 months by suitable selection of polymer, composition, additives, and manufacturing conditions. Published: July 11, 2001.     

Preparation and characterization of poly(D,L-lactide-co-glycolide) microspheres for controlled release of human growth hormone

The purpose of this research was to assess the physicochemical properties of a controlled release formulation of recombinant human growth hormone (rHGH) encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) composite microspheres. rHGH was loaded in poly(acryloyl hydroxyethyl) starch (acHES) microparticles, and then the protein-containing microparticles were encapsulated in the PLGA matrix by a solvent extraction/evaporation method. rHGH-loaded PLGA microspheres were also prepared using mannitol without the starch hydrogel microparticle microspheres for comparison. The detection of secondary structure changes in protein was investigated by using a Fourier Transfer Infrared (FTIR) technique. The composite microspheres were spherical in shape (44.6±2.47 μm), and the PLGA-mannitol microspheres were 39.7±2.50 μm. Drug-loading efficiency varied from 93.2% to 104%. The composite microspheres showed higher overall drug release than the PLGA/mannitol microspheres. FTIR analyses indicated good stability and structural integrity of HGH localized in the microspheres. The PLGA-acHES composite microsphere system could be useful for the controlled delivery of protein drugs.     

Layered open pore poly(L-lactic acid) nanomorphology

This paper reports on specific open and interconnected CO(2) foams of poly(L-lactic acid). The effect of varying gas concentration and hence physical changes induced by CO(2) has been investigated and thus used to generate specific structures. The developed morphologies have a skin core structure with larger pores in the core and open and interconnected smaller pores in the skin.     

Preparation of budesonide and budesonide-PLA microparticles using supercritical fluid precipitation technology

The objective of this study was to prepare and characterize microparticles of budesonide alone and budesonide and polylactic acid (PLA) using supercritical fluid (SCF) technology. A precipitation with a compressed antisolvent (PCA) technique employing supercritical CO₂ and a nozzle with 100-μm internal diameter was used to prepare microparticles of budesonide and budesonide-PLA. The effect of various operating variables (temperature and pressure of CO₂ and flow rates of drug-polymer solution and/or CO₂) and formulation variables (0.25%, 0.5%, and 1% budesonide in methylene chloride) on the morphology and size distribution of the microparticles was determined using scanning electron microscopy. In addition, budesonide-PLA particles were characterized for their surface charge and drug-polymer interactions using a zeta meter and differential scanning calorimetry (DSC), respectively. Furthermore, in vitro budesonide release from budesonide-PLA microparticles was determined at 37°C. Using the PCA process, budesonide and budesonide-PLA microparticles with mean diameters of 1 to 2 μm were prepared. An increase in budesonide concentration (0.25%–1% wt/vol) resulted in budesonide microparticles that were fairly spherical and less aggiomerated. In addition, the size of the microparticles increased with an increase in the drug-polymer solution flow rate (1.4–4.7 mL/min) or with a decrease in the CO₂ flow rate (50–10 mL/min). Budesonide-PLA microparticles had a drug loading of 7.94%, equivalent to ∼80% encapsulation efficiency. Budesonide-PLA microparticles had a zeta potential of— 37±4 mV, and DSC studies indicated that SCF processing of budesonide-PLA microparticles resulted in the loss of budesonide crystallinity. Finally, in vitro drug release studies at 37°C indicated 50% budesonide release from the budesonide-PLA microparticles at the end of 28 days. Thus, the PCA process was successful in producing budesonide and budesonide-PLA microparticles. In addition, budesonide-PLA microparticles sustained budesonide release for 4 weeks.     

Drying Using Supercritical Fluid Technology as a Potential Method for Preparation of Chitosan Aerogel Microparticles

Supercritical fluid technology offers several advantages in preparation of microparticles. These include uniformity in particle size, morphology, and drug distribution without degradation of the product. One of the recent advantages is preparation of porous aerogel carrier with proper aerodynamic properties. In this study, we aimed to prepare chitosan aerogel microparticles using supercritical fluid (SCF) technology and compare that with microparticles produced by freeze drying (FD). Loading the prepared carriers with a model drug (salbutamol) was also performed. Comparisons of the particle properties and physicochemical characterizations were undertaken by evaluating particle size, density, specific surface area, and porosity. In vitro drug release studies were also investigated. The effect of many variables, such as molecular weight of chitosan oligomers, concentrations of chitosan, and concentrations of tripolyphosphate on the release, were also investigated. Chitosan aerogels were efficiently produced by SCF technology with an average particle size of 10 μm with a tapped density values around 0.12 g/mL, specific surface area (73–103) m²/g, and porosity (0.20–0.29) cc/g. Whereas, microparticles produced by FD method were characterized as cryogels with larger particle size (64 microns) with clear cracking at the surface. Sustained release profile was achieved for all prepared microparticles of salbutamol produced by the aforementioned methods as compared with pure drug. The results also demonstrates that chitosan molecular weight, polymer concentration, and tripolyphosphate concentration affected the release profile of salbutamol from the prepared microparticles. In conclusion, SCF technology was able to produce chitosan aerogel microparticles loaded with salbutamol that could be suitable for pulmonary drug delivery system.KEY WORDS: aerodynamic, aerogels, chitosan, salbutamol, supercritical fluid technology     

Condensed DNA particles formed in a PCR with plasmid templates: An electron microscopy study

Suspensions of microparticles produced in a polymerase chain reaction on plasmid templates using different primers have been studied by electron microscopy. In all of the samples, microspheres of two types have been detected: of a spherical or ellipsoid form (smooth, without thorns) and of an ellipsoid form with big thorns and/or outgrowths; the diameter of the microspheres varies from 1 to 3 μm. Along with microspheres, so-called three-dimensional network structures of various shapes formed by three-dimensional nanoparticles of a particular topology have been observed. In some samples, discs a few microns in diameter and several dozen nanometers in thickness have been detected. It has been shown that the quantity of netshaped structures and discs sharply increases during asymmetric PCR. In addition, DNA nanofilaments (nanowires), electron-dense point particles (nanodots), electron-translucent nanoparticles in the form of rags, and large electron-dense spherical nanoparticles have been found. Suspensions of microparticles after their quick (5 min) heating at 94°C have been examined. The partial melting of smooth microspheres (without thorns) has been established: they lose a part of the DNA so that the details of their structure (ultrastructure) can be discerned. Simultaneously, clusters of nanowires appear after the heating. The molecular mechanisms of the formation of nanoand microparticles are discussed.     

Structural study of poly(L-lactic acid) spherulites

Spherulites of poly(L-lactic acid) (PLLA) and of its blends with atactic poly(3-hydroxybutyrate) (a-PHB, from 10 to 75 wt %) were investigated by microfocus X-ray diffraction using synchrotron radiation. Radial scans in 5 microm steps with 3 microm beam diameter were performed. In PLLA, tens of identical diffraction images were collected. The unit cell a-axis was radially oriented, and the other axes lacked any specific orientation. In contrast, all PLLA/a-PHB blends showed a periodic change of diffraction pattern with increasing distance from the spherulite center. In all cases, the a-axis lay along the radius, while the b- and c-axes rotated about a with a defined periodicity. The unit cell twisting frequency increased with a-PHB content and closely matched the band spacing observed by polarized optical microscopy, which changed from 250 to 60 microm when the amount of a-PHB increased from 10 to 75 wt %. Concomitantly, a gradual broadening of all X-ray reflections was observed.     

Enhanced growth of animal and human endothelial cells on biodegradable polymers

Biodegradable polymers such as poly(L-lactic acid) (PLLA), poly(glycolic acid) (PGA) and PGA coated with PLLA are being employed for cell transplantation and for in vivo regeneration of vascular tissue. Ingrowth and organization of fibrovascular tissue inside polymer scaffolds lead to the occlusion of the regenerated blood vessel. In order to provide regulatory mechanisms to control the development of an inner capsule, endothelialization of these materials is necessary. To achieve this, we employed a novel ammonia plasma technique to surface modified PLLA substrates. Human endothelial cell (HUVEC) and rabbit microvascular endothelial cell (RbMVEC) growth was studied on modified PLLA and control PLLA. Our studies show that modified PLLA and fibronectin (Fn)-coated modified PLLA exhibited statistically significant improvement in HUVEC and RbMVEC growth (P<0.001) when compared to PLLA and Fn-coated PLLA. Therefore, ammonia plasma treatment gives us the unique capability of modifying prosthetic biomaterials of various constructs with the eventual transplantation of mammalian cells to be used in tissue engineering or as biological implants.     

Preparation of peptide-loaded polymer microparticles using supercritical carbon dioxide

In this study, peptide-loaded microparticles were prepared using an aerosol solvent extraction system (ASES) by employing supercritical carbon dioxide as an antisolvent. The effects of the molecular weight of poly(Llactide) (PLLA), poly(ethylene glycol) (PEG), the block length of methoxy poly(ethylene glycol)-b-poly(L-lactide) (mPEG-PLLA), the blending of PLLA and PEG, and the drug-to-polymer feed ratio on the formation of leuprolide acetate (LA)-loaded microparticles and their release characteristics were investigated. Scanning electron microscope observations showed that the LA-loaded polymer particles had a spherical morphology with a smooth surface. The entrapment efficiency of LA in the ASES-processed microparticles was found to be extremely high (about 99%), whereas the initial release rate of the LA-loaded microparticles was very low for PLLA. The release rate of LA was observed to increase as the PEG block length of mPEG-PLLA and/or the drug content in the microparticles increased. When PLLA was blended with PEG, the release rate of LA from the PLLA/PEG microparticles was significantly faster compared with the corresponding mPEG-PLLA copolymer.     

L-pyroglutamate spontaneously formed from L-glutamate inhibits growth of the hyperthermophilic archaeon Sulfolobus solfataricus

Identification of physiological and environmental factors that limit efficient growth of hyperthermophiles is important for practical application of these organisms to the production of useful enzymes or metabolites. During fed-batch cultivation of Sulfolobus solfataricus in medium containing L-glutamate, we observed formation of L-pyroglutamic acid (PGA). PGA formed spontaneously from L-glutamate under culture conditions (78 degrees C and pH 3.0), and the PGA formation rate was much higher at an acidic or alkaline pH than at neutral pH. It was also found that PGA is a potent inhibitor of S. solfataricus growth. The cell growth rate was reduced by one-half by the presence of 5.1 mM PGA, and no growth was observed in the presence of 15.5 mM PGA. On the other hand, the inhibitory effect of PGA on cell growth was alleviated by addition of L-glutamate or L-aspartate to the medium. PGA was also produced from the L-glutamate in yeast extract; the PGA content increased to 8.5% (wt/wt) after 80 h of incubation of a yeast extract solution at 78 degrees C and pH 3.0. In medium supplemented with yeast extract, cell growth was optimal in the presence of 3.0 g of yeast extract per liter, and higher yeast extract concentrations resulted in reduced cell yields. The extents of cell growth inhibition at yeast extract concentrations above the optimal concentration were correlated with the PGA concentration in the culture broth. Although other structural analogues of L-glutamate, such as L-methionine sulfoxide, glutaric acid, succinic acid, and L-glutamic acid gamma-methyl ester, also inhibited the growth of S. solfataricus, the greatest cell growth inhibition was observed with PGA. We also observed that unlike other glutamate analogues, N-acetyl-L-glutamate enhanced the growth of S. solfataricus. This compound was stable under cell culture conditions, and replacement of L-glutamate with N-acetyl-L-glutamate in the medium resulted in increased cell density.     

Continuous supercritical emulsions extraction: a new technology for biopolymer microparticles production

Supercritical emulsion extraction (SEE) was recently proposed for the production of biopolymer microparticles starting from oil‐in‐water emulsions. This technology can improve the product quality because of the fast and selective extraction of the dispersed oily phase by using supercritical carbon dioxide (SC‐CO₂). However, until now, SEE was proposed in batch configuration, sharing with the traditional processes an intrinsically discontinuous operation and problems of batches reproducibility and process yield. In this study, by using a countercurrent packed column, the SEE process was proposed in a continuous operating mode (SEE‐CM) for the production of poly‐lactic‐co‐glycolic acid (PLGA) microparticles. The new process design takes advantage of the large contact area between the SC‐CO₂ and emulsion allowing the production of PLGA microparticles with controlled and narrow size distributions in only few minutes. SEE‐CM operating parameters such as pressure, temperature, and flow rate ratios were analyzed and the process efficiency in terms of recovered material and its size distribution compared with SEE (batch mode operation) and conventional evaporation technology. PLGA microparticles showed a mean particle size between 1–3 µm (depending on the droplet sizes) with a SD that was always smaller than that associated with particles produced by discontinuous processes. Single and double emulsions were successfully treated and the microparticles physico‐chemical properties showed no morphological and structural differences between the SEE‐CM‐produced microparticles and the ones obtained by conventional evaporation technology. Biotechnol. Bioeng. 2011; 108:676–686. © 2010 Wiley Periodicals, Inc.     

A novel Method for the selective recovery and purification of γ‐polyglutamic acid from Bacillus licheniformis fermentation broth

Microbially produced gamma‐polyglutamic acid (γ‐PGA) is a commercially important biopolymer with many applications in biopharmaceutical, food, cosmetic and waste‐water treatment industries. Owing to its increasing demand in various industries, production of γ‐PGA is well documented in the literature, however very few methods have been reported for its recovery. In this paper, we report a novel method for the selective recovery and purification of γ‐PGA from cell‐free fermentation broth of Bacillus licheniformis. The cell‐free fermentation broth was treated with divalent copper ions, resulting in the precipitation of γ‐PGA, which was collected as a pellet by centrifugation. The pellet was resolubilized and dialyzed against de‐ionized water to obtain the purified γ‐PGA biopolymer. The efficiency and selectivity of γ‐PGA recovery was compared with ethanol precipitation method. We found that 85% of the original γ‐PGA content in the broth was recovered by copper sulfate‐induced precipitation, compared to 82% recovery by ethanol precipitation method. Since ethanol is a commonly used solvent for protein precipitation, the purity of γ‐PGA precipitate was analyzed by measuring proteins that co‐precipitated with γ‐PGA. Of the total proteins present in the broth, 48% proteins were found to be co‐precipitated with γ‐PGA by ethanol precipitation, whereas in copper sulfate‐induced precipitation, only 3% of proteins were detected in the final purified γ‐PGA, suggesting that copper sulfate‐induced precipitation offers better selectivity than ethanol precipitation method. Total metal content analysis of the purified γ‐PGA revealed the undetectable amount of copper ions, whereas other metal ions detected were in low concentration range. The purified γ‐PGA was characterized using infrared spectroscopy. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Preparation of biodegradable microspheres and matrix devices containing naltrexone

In this study, the use of biodegradable polymers for microencapsulation of naltrexone using solvent evaporation technique is investigated. The use of naltrexone microspheres for the preparation of matrix devices is also studied. For this purpose, poly(L-lactide) (PLA) microspheres containing naltrexone prepared by solvent evaporation technique were compressed at temperatures above the Tg of the polymer. The effect of different process parameters, such as drug/polymer ratio and stirring rate during preparation of microspheres, on the morphology, size distribution, and in vitro drug release of microspheres was studied. As expected, stirring rate influenced particle size distribution of microspheres and hence drug release profiles. By increasing the stirring speed from 400 to 1200 rpm, the mean diameter of microspheres decreased from 251 μm to 104 μm. The drug release rate from smaller microspheres was faster than from larger microspheres. However, drug release from microspheres with low drug content (20% wt/wt) was not affected by the particle size of microspheres. Increasing the drug content of microspheres from 20% to 50% wt/wt led to significantly faster drug release from microspheres. It was also shown that drug release from matrix devices prepared by compression of naltrexone microspheres is much slower than that of microspheres. No burst release was observed with matrix devices. Applying higher compression force, when compressing microspheres to produce tablets, resulted in lower drug release from matrix devices. The results suggest that by regulating different variables, desired release profiles of naltrexone can be achieved using a PLA microparticulate system or matrix devices.     

Hydrazide-functionalized poly(2-hydroxyethyl methacrylate) microspheres for immobilization of horseradish peroxidase

Nonporous cross-linked poly(2-hydroxyethyl methacrylate-co-ethylene dimethacrylate) (poly(HEMA-co-EDMA)) microspheres were prepared by dispersion polymerization of HEMA and EDMA. The polymerization was performed in toluene/2-methylpropan-1-ol in the presence of cellulose acetate butyrate as a steric stabilizer and dibenzoyl peroxide initiator. The particle size may be increased by decreasing the toluene/2-methylpropan-1-ol ratio and by increasing polymerization temperature. Adipohydrazide was attached to the microspheres activated with 2,4,6-trichloro-1,3,5-triazine. After periodate oxidation of its carbohydrate moieties, horseradish peroxidase was coupled to the hydrazide-functionalized poly(HEMA-co-EDMA) microparticles up to 7.3 microgram of enzyme/g of carrier without a significant loss of its activity. Immobilized peroxidase was found to be stable, retaining more than 97% of its initial activity when stored for 23 days after the preparation.