Degradation of poly(lactic-co-glycolic acid) microspheres: effect of copolymer composition.

The in vitro degradation behaviour of a wide range of poly(D,L-lactic-co-glycolic acid) (PLGA) has been examined in terms of degree of degradation and morphological change during an incubation period of up to 53 d. Gel permeation chromatography and differential scanning calorimetry were employed to characterize their degradation profiles. It was found that amorphous PLGA exhibited a transient multiple crystallization behaviour of D- or L-lactic acid oligomers during degradation. This indicated that the hydrolytic scission of ester bonds tends to primarily target the linkage between glycolic acid and D- or L-lactic acid or glycolic acid. In addition, two distinctive glass transition temperatures appeared when these crystallization phenomena occurred, suggesting the transient presence of fast and slowly eroding polymer domains within microspheres during the degradation. This study supports the heterogeneous bulk degradation for PLGA microspheres which has been proposed recently for a large specimen.

Biodegradable polymeric microcellular foams by modified thermally induced phase separation method.

Thermally induced phase separation (TIPS) for the fabrication of porous foams based on various biodegradable polymers of poly(L-lactic acid) and its copolymers with D-lactic acid and/or glycolic acid is presented. Diverse foam morphologies were obtained by systematically changing several parameters involved in the TIPS process, such as polymer type and concentration, coarsening conditions, solvent/nonsolvent composition, and the presence of an additive. The produced foams had microcellular structures with average pore diameters ranging from 1 to 30 microns depending on the process parameters, which were characterized by scanning electron microscopy (SEM) and mercury intrusion porosimetry. Additionally, Pluronic F127 was used as an additive porogen to control the pore geometry and size.

Biodegradable polymeric micelles composed of doxorubicin conjugated PLGA-PEG block copolymer.

Biodegradable polymeric micelles containing doxorubicin in the core region were prepared from a di-block copolymer composed of doxorubicin-conjugated poly(DL-lactic-co-glycolic acid) (PLGA) and polyethyleneglycol (PEG). The di-block copolymer of PLGA-PEG was first synthesized and the primary amino group of doxorubicin was then conjugated to the terminal hydroxyl group of PLGA, which had been pre-activated using p-nitrophenyl chloroformate. The resulting polymeric micelles in aqueous solution were characterized by measurement of size, drug loading, and critical micelle concentration. The micelles containing chemically-conjugated doxorubicin exhibited a more sustained release profile than PEG-PLGA micelles containing physically-entrapped doxorubicin. The cytotoxic activity of the micelles against HepG2 cells was greater than free doxorubicin, suggesting that the micelles containing conjugated doxorubicin were more effectively taken up cellularly, by an endocytosis mechanism rather than by passive diffusion. Confocal microscopic observation and flow cytometry analysis supported the enhanced cellular uptake of the micelles.

Pegylation enhances protein stability during encapsulation in PLGA microspheres.

During encapsulation of proteins in biodegradable microspheres, a significant amount of the protein reportedly undergoes denaturation to form irreversible insoluble aggregates. Incomplete in vitro release of proteins from the microspheres is a common observation. An attempt was made to overcome this problem by pegylation of the protein to be encapsulated. Lysozyme, a model protein, was conjugated with methoxy polyethylene glycol (mPEG, MW 5000). The conjugate was characterized by SDS-PAGE, SE-HPLC, and MALDI-TOF mass spectroscopy. The pegylated lysozyme (Lys-mPEG) consisted of different isomers of mono-, di- and tri-pegylated with about 15% as native lysozyme. The specific activity of the protein was retained after pegylation (101.3+/-10.4%). The microsphere encapsulation process was simulated for pegylated and native lysozyme. Pegylated lysozyme exhibited much better stability than native lysozyme against exposure to organic solvent (dichloromethane), homogenization, and showed reduced adsorption onto the surface of blank PLGA microspheres. Release profiles of the two proteins from microspheres were very different. For native lysozyme, it was high initial release (about 50%) followed by a nearly no release (about 10% in 50 days). In contrast, Lys-mPEG conjugate showed a triphasic and near complete release over 83 days. This study shows that the pegylation of protein can provide substantial protection against the destabilization of protein during encapsulation.

A new gene delivery formulation of polyethylenimine/DNA complexes coated with PEG conjugated fusogenic peptide.

A fusogenic peptide, KALA, was conjugated with poly(ethylene glycol) (PEG) for use as an endosome disruptive agent in the gene delivery formulation of polyethyleneimine (PEI). A maleimide terminated methoxy-PEG, a cysteine specific derivative, was reacted with KALA to produce a PEG-KALA conjugate. The conjugate was analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry, and its hemolytic activity was determined relative to KALA. Positively charged PEG-KALA conjugate was coated onto the surface of negatively charged DNA/PEI complexes to form net positively charged PEG-KALA/DNA/PEI complexes. They were 200-400 nm in diameter with increasing amount of PEG-KALA, whereas DNA/PEI complexes coated with KALA aggregated to a great extent. This was because PEG chains surrounding the surface of the complexes suppressed the inter-particle interaction that was mediated by cationic KALA. Transfection efficiency progressively increased as the amount of PEG-KALA to be coated was increased, suggesting that fusogenic activity of KALA contributes to enhancing the level of gene expression.

A new preparation method for protein loaded poly(D, L-lactic-co-glycolic acid) microspheres and protein release mechanism study.

A new method for encapsulating a model protein, lysozyme into hydrophilic uncapped poly(d,l-lactic-co-glycolic acid) (PLGA) microspheres was developed using an oil/water (O/W) single emulsion technique. Lysozyme powder, which was prepared from lyophilization after adjusting a lysozyme solution pH at 3, was molecularly dissolved in a co-solvent system composed of dimethylsulfoxide (DMSO) and methylene chloride. The resulting organic solution containing PLGA was directly emulsified into an aqueous phase, and the organic solvent phase was extracted and evaporated. Various lysozyme-loaded PLGA microspheres having different morphologies were obtained depending on the relative mixing ratio of the two co-solvents used. In vitro release experiments indicated that an initial lysozyme release rate from the microspheres was mainly controlled by ionic interaction between basic amino acid residues in lysozyme and free carboxylate groups in PLGA polymer chain ends, which was probed by incubating the microspheres in a series of media having different NaCl concentrations. However, the protein release leveled off after about 15 days' incubation. To determine the reason for the protein 'no-release' from biodegradable microspheres, a systematic analysis was carried out. By separately adding 0.5 M NaCl, 5 M guanidine HCl, or 5 mM sodium dodecyl sulfate into the release media during the non-release period, it was possible to selectively identify a specific protein non-release mechanism: ionic interaction, non-covalent aggregation, and/or surface adsorption, respectively. It was found that non-covalent aggregation and surface adsorption of lysozyme within the microspheres were the main cause of no further release, whereas ionic interaction between degrading polymer and protein played an insignificant role in the later stage of the release period. The greater amount of additional lysozyme release by sodium dodecyl sulfate than by guanidine hydrochloride suggested that protein surface adsorption was a more critical factor in protein release than aggregation.

In vitro and in vivo anti-tumor activities of nanoparticles based on doxorubicin-PLGA conjugates.

Doxorubicin was chemically conjugated to a terminal end group of poly(D,L-lactic-co-glycolic acid) [PLGA] by an ester linkage and the doxorubicin-PLGA conjugate was formulated into nanoparticles. A carboxylic acid end group of PLGA was conjugated to a primary hydroxyl group of doxorubicin. The primary amine group of doxorubicin was protected during the conjugation process and then deprotected. The nanoparticles containing the conjugate exhibited sustained doxorubicin release profiles over a 1-month period, whereas those containing unconjugated free doxorubicin showed a rapid doxorubicin release within 5 days. Doxorubicin release patterns could be controlled by conjugating doxorubicin to PLGA having different molecular weights. The conjugated doxorubicin nanoparticles showed increased uptake within a HepG2 cell line, which was quantitated by a flow cytometry and visualized by confocal microscopy. The nanoparticles exhibited slightly lower IC(50) value against the HepG2 cell line compared to that of free doxorubicin. In vivo anti-tumor activity assay also showed that a single injection of the nanoparticles had comparable activity to that of free doxorubicin administered by daily injection. The conjugation approach of doxorubicin to PLGA was potentially useful for the formulation of nanoparticles that requires targeting for cancer cells as well as sustained release at the site.

Conjugation of drug to poly(D,L-lactic-co-glycolic acid) for controlled release from biodegradable microspheres.

Poly(d,l-lactic-co-glycolic acid) (PLGA) was chemically conjugated to a model drug, N-(9-fluorenylmethoxycarbonyl-N-tert-butoxycarbonyl-l-tryptophan (Fmoc-Trp(Boc)) via an ester linkage. Various coupling reaction conditions were tested to optimize the conjugation process between a hydroxyl terminal group of PLGA and a carboxylic acid group of Fmoc-Trp(Boc). Two different lactic/glycolic acid compositions of PLGA (50/50 and 75/25) were used for the conjugation. The Fmoc-Trp(Boc)-PLGA conjugates were formulated into microspheres by a solvent evaporation technique for controlled release of Fmoc-Trp(Boc) over an one month period. A linear constant release of Fmoc-Trp(Boc) and its water-soluble PLGA oligomer conjugates was observed over an extended period without any initial burst effect, while unconjugated Fmoc-Trp(Boc) encapsulated within microspheres exhibited a rapid release profile. In addition, Fmoc-Trp(Boc) release rate solely depended on the PLGA composition that affected polymer degradation rate. The release rate of Fmoc-Trp(Boc) conjugated with fast degrading 50/50 PLGA was more rapid than that conjugated with relatively slow degrading 75/25 PLGA. This study demonstrates that PLGA-drug conjugation approach is a new and novel strategy to control the drug release rate from PLGA microspheres by utilizing the chemical degradation rate of PLGA backbone.

DNA transfection using linear poly(ethylenimine) prepared by controlled acid hydrolysis of poly(2-ethyl-2-oxazoline).

A series of linear poly(ethylenimine) (L-PEI) containing varying amounts of cationic charge density in its backbone was produced by controlled hydrolysis of poly(2-ethyl-2-oxazoline) (PEtOz) for using as a nonviral DNA transfection agent. The effects of cationic charge density and molecular weight of the L-PEI on the cytotoxicity and transfection efficiency were studied. The efficiency of transfection was monitored by using a luciferase reporter gene system. Gel retardation assay and dynamic light scattering (DLS) showed that the condensation capacity of L-PEI was suitable for transfection. Highly compacted L-PEI/DNA complex ( approximately 150 nm) was obtained with a surface charge value of around +28.4 mV. Cell cytotoxicity was affected to a great extent by the hydrolysis percent of L-PEI as well as by the molecular weight. Transfection efficiency of luciferase plasmid DNA against NIH 3T3 fibroblast was largely dependent upon the hydrolysis percent (charge density) in the polymer backbone and the molecular weight of the L-PEI, but independent of the total amount of cationic charges used for DNA condensation. L-PEI with a hydrolysis percent of 88.0% exhibited comparable transfection efficiency to that of commonly used branched PEI.

Protein release microparticles based on the blend of poly(D,L-lactic-co-glycolic acid) and oligo-ethylene glycol grafted poly(L-lactide).

Bovine serum albumin (BSA), a model protein drug, was encapsulated with a microparticle based on the blend of poly(D,L-lactic-co-glycolic acid) (PLGA) and poly(L-lactide)-g-oligo(ethylene glycol) (PLLA-g-oligoEG). Effects of PLLA-g-oligoEG in the blend on degradation, characteristic properties, and release behavior of the microparticle were studied. Drug loading efficiency increased with increase in the graft frequency of oligoEG in the graft copolymer in the blend. The release of BSA was found to be more efficient for microparticles based on the blend than on the PLGA, which is due to the faster protein diffusion through the swollen phase of the hydrogel-like structure. The microparticles based on the blend showed a slower degradation and a lower pH shift compared to that of PLGA.

In vitro release profiles of eristostatin from biodegradable polymeric microspheres: protein aggregation problem.

Eristostatin, a low molecular weight polypeptide (MW 5725), was encapsulated within two biodegradable poly(lactic acid-glycolic acid) microspheres. In vitro release profiles from the microspheres exhibited fast initial release for up to several days, followed by very slow or no release. The later stage of the slow release was due to protein aggregation within the microspheres. A simple, noninvasive method to detect the protein aggregation within the microspheres has been developed: extraction of radiolabeled protein from the microspheres by a DC electric field into a polyacrylamide gel and subsequent exposure to a gamma-ray sensitive film. Direct application of the degrading microspheres onto the sample loading zone of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) could successfully extract the monomeric and oligomeric proteins into the autoradiogram of a gel slab, while unextractable protein aggregates within the microspheres could be directly visualized in their loading position.

Estimation of temperature-dependent pore size in poly(N-isopropylacrylamide) hydrogel beads.

The pore sizes of various temperature-sensitive hydrogel beads have been estimated at different temperatures by a gel filtration method using several probe molecules of known molecular weight. The hydrogel beads were prepared by inverse suspension copolymerizations of a series of N-isopropylacrylamide (NIPAAm) and acrylamide (AAm) monomers with a small amount of cross-linker. These hydrogel beads exhibit LCST (lower critical solution temperature) behavior in aqueous solution, that is, expanding and swelling when cooled below the LCST and shrinking and collapsing when heated above the LCST. The pore sizes of these temperature-sensitive hydrogel beads are affected significantly by both the temperature and the gel composition, but not much by cross-linker concentration.

Conjugation of trypsin by temperature-sensitive polymers containing a carbohydrate moiety: thermal modulation of enzyme activity.

Novel temperature-sensitive polymers containing glucose units in their backbone were synthesized and covalently conjugated to trypsin. A series of copolymers based on N-isopropylacrylamide (NIPAAm) and glucosyoxylethyl methacrylate (GEMA) were prepared by using 4,4'-azobis(4-cyanovaleric acid) as an initiator, which resulted in one terminal carboxylic acid group per polymer chain. The polymers were conjugated to primary amine groups of trypsin with water-soluble carbodiimide as a coupling agent, which led to a star-shaped conformation. The polymer-enzyme conjugation was confirmed and characterized by size exclusion and reversed-phase chromatography. Almost of all amine groups in trypsin available for the conjugation were consumed and, consequently, a very dense layer of copolymers was actually coated around the enzyme surface. The conjugated enzymes exhibited reversible precipitation/resolubilization behaviors over a wide range of temperatures, depending on the content of GEMA in the copolymer. They also demonstrated no detectable self-digestion (autolysis) process, but the unconjugated enzyme showed very severe autolysis that led to a rapid inactivation in aqueous solution. When bovine serum albumin was used as a substrate, the protein substrate was not attacked by the conjugated enzyme, but completely digested by the unconjugated enzyme. This result was presumably caused by a steric repulsion process of the attached polymer chains around the enzyme toward the protein substrate. However, the enzyme retained sufficient activity against a low molecular weight substrate. Interestingly, the conjugated enzymes demonstrated very peculiar enzyme activity-temperature profiles, with two apparent optimal temperatures, indicating that a temperature-controlled collapse and flocculation of the copolymers around the enzyme surface modulated the mass transfer rates of substrate to the active site of the enzyme. The conjugated enzymes also exhibited improved thermal stability with increasing the amount of carbohydrate units in the polymer chain.

Protein-fatty acid complex for enhanced loading and stability within biodegradable nanoparticles.

Lysozyme was hydrophobically modified with a fatty acid, sodium oleate, via an ion-pairing mechanism. Ionic binding between an anionic carboxylic group of sodium oleate and basic amino groups in lysozyme was primarily utilized to form lysozyme-oleate complex. The complex formation was pH dependent. The lysozyme-oleate complex dissolved in an organic solvent exhibited much higher conformational stability at elevated temperature compared with free lysozyme in the same solvent. The complex was formulated into biodegradable nanoparticles by a spontaneous emulsion and solvent diffusion method. The resultant formulation showed near 100% encapsulation efficiency of lysozyme within nanoparticles with < 100 nm in diameter with a narrow size distribution. Lysozyme could be loaded into the nanoparticles up to 18.6% (w/w) with concomitantly increased particle sizes. This study demonstrates a new formulation method of biodegradable nanoparticles with highly efficient encapsulation of proteins, which are potentially useful for oral protein delivery including mucosal vaccination.

Hydrophobic ion pair formation between leuprolide and sodium oleate for sustained release from biodegradable polymeric microspheres.

Leuprolide acetate, an analogue of luteinizing hormone-releasing hormone (LH-RH), was hydrophobically ion paired with a long chain fatty acid, sodium oleate, in an aqueous solution. Solution behaviors of the complex formed between leuprolide and sodium oleate were investigated in terms of aqueous solubility, turbidity, particle size, and zeta potential as a function of molar ratio between the two species. It was found that with increasing the stoichiometric molar amounts of sodium oleate to leuprolide approached up to 2.5-3, the solution became gradually turbid with increasing particle sizes, indicating leuprolide precipitation as a result of hydrophobic ion pairing. On the other hand, beyond that critical molar ratio range, the solution turned into clear with much reduced particle size, indicative of micelle formation. The hydrophobically modified leuprolide-oleate complex was lyophilized and directly encapsulated within biodegradable poly(D, L-lactic-co-glycolic acid) (PLGA) microspheres via a single oil-in-water (O/W) emulsion method. Microsphere morphology, leuprolide release behavior, and polymer mass erosion profiles were examined in comparison to the PLGA microspheres prepared with free leuprolide.

Microencapsulation of dissociable human growth hormone aggregates within poly(D,L-lactic-co-glycolic acid) microparticles for sustained release.

For the sustained release formulation of recombinant human growth hormone (rhGH), dissociable rhGH aggregates were microencapsulated within poly(D,L-lactic-co-glycolic acid) (PLGA) microparticles. rhGH aggregates were first produced by adding a small volume of aqueous rhGH solution into a partially water miscible organic solvent phase (ethyl acetate, EtAc) containing PLGA. These rhGH aggregates were then microencapsulated within PLGA polymer phase by extracting EtAc into an aqueous phase pre-saturated with EtAc. Release profiles of rhGH from these microparticles were greatly affected by changing the volume of incubation medium. The released rhGH species consisted of mostly monomeric form having a correct conformation. This study reveals that sustained rhGH release could be achieved by microencapsulating reversibly dissociable protein aggregates within biodegradable polymers.

Effect of formulation and processing variables on the characteristics of microspheres for water-soluble drugs prepared by w/o/o double emulsion solvent diffusion method.

80% except for acetaminophen, due to its lower solubility in water and higher solubility in corn oil. The release profile of the drug was pH dependent. In acidic medium, the release rate was much slower, however, the drug was released quickly at pH 7.4. Tacrine showed unexpected release profiles, probably due to ionic interaction with polymer matrix and the shell structure and the highest release rate was obtained at pH 2.0. The prepared microspheres had a sponge-like inner structure with or without central hollow core and the surface was dense with no apparent pores.

Thermal cycling effects on the bioreactor performances of immobilized beta-galactosidase in temperature-sensitive hydrogel beads.

The enzyme beta-galactosidase was immobilized in thermally reversible hydrogel beads that exhibit a reversible expansion and collapse of the gel volume in response to temperature. The kinetic performances of immobilized enzyme bead reactors were studied during thermal cycling operation. A periodic cycling of temperature for the packed-bed reactor induced a cyclic swelling and deswelling of the hydrogel beads. A temperature cycling operation around the phase transition temperature of the gel matrix enhanced the overall enzymatic conversion of the substrate, compared to its upper and lower isothermal operations. The effects on the overall conversion of various thermal cycling operational conditions, such as cycling range and heating and cooling rates, were investigated.

Effect of temperature cycling on the activity and productivity of immobilized beta-galactosidase in a thermally reversible hydrogel bead reactor.

The enzyme beta-galactosidase has been immobilized within thermally reversible hydrogel beads that exhibit LCST (lower critical solution temperature) behavior. The hydrogel beads containing the immobilized enzymes swell and expand below the LCST and deswell and shrink above the LCST. This behavior is reversible. The enzyme was physically entrapped in a crosslinked hydrogel of a copolymer of N-isopropylacrylamide (NIPAAm) and acrylamide (AAm), and formed as beads in an inverse suspension polymerization. The beads were placed in a packed bed column reactor which was operated in a continuous, single pass mode, either isothermally at 30 or 35 degrees C, or with temperature cycling between 30 and 35 degrees C. The thermal cycling significantly enhanced overall reactor enzyme activity relative to isothermal operation at either the higher or lower temperature. It is postulated that mass transfer rates within the hydrogel beads are greatly enhanced by the movement of water in and out of the beads during the expansion or collapse of the polymer chain network as temperature is cycled.

Protein release from poly(lactic-co-glycolic acid) microspheres: protein stability problems.

The enzyme, carbonic anhydrase, has been incorporated within poly(lactic-co-glycolic acid) microspheres using a double emulsion and solvent evaporation technique. The protein stability problems during the microsphere formulation procedure and during the release period were examined in relation to the protein release kinetics over a 2 month period. Different protein release profiles could be obtained depending on the polymers used. The protein release kinetics exhibited an initial fast release followed by a slow release, resulting in an incomplete protein release although the microspheres degraded significantly. The very slow release kinetics were attributed to the protein aggregation and non-specific adsorption within the microspheres. It was found that the protein was significantly denatured and aggregated during the double emulsion formulation step. Several excipients such as albumin, poly(ethylene oxide), Pluronic F-127, and gelatin, which were loaded along with the protein within microspheres, demonstrated better protein release kinetics partly due to an increase in the protein stability. The released protein from these fast degrading microspheres, however, was severely hydrolyzed and lost its catalytic activity, caused by the accumulation of degradation products in the medium.