Low Initial Burst Research Articles

A Novel Preparation Method for Octreotide Acetate–Loaded PLGA Microspheres with a High Drug‐Loading Capacity and a Low Initial Burst Release, and Its Studies on Relations between In Vitro and In Vivo Release

ABSTRACTIn this study, octreotide acetate (OTA)–loaded poly(lactide‐co‐glycolide) (PLGA) microspheres were prepared using an optimized double emulsion solvent evaporation method. The loading capacity (LC) was increased by raising the concentration of OTA in the inner aqueous phase (W1), and burst release was decreased (<10.0%) because the higher viscosity hindered the diffusion of OTA. The in vitro release profiles were closely correlated with in vivo release profiles calculated using the Wagner–Nelson method (r = 0.9876). To evaluate the drug release profiles rapidly, an accelerated release method was developed. In the accelerated release method, the acidic and alkaline buffers (the first 6 h, pH 4.0; the last 18 h, pH 9.6) were used as release media, respectively. Results indicated that the accelerated release profiles (24 h) were closely correlated with the in vivo release profiles (r = 0.9623). Therefore, through optimization of the experiment variables (buffer components and pH), an accelerated release method was developed to evaluate in vivo drug release from microspheres. © 2013 Wiley Periodicals, Inc. Adv Polym Technol 2013, 32, 21354; View this article online at wileyonlinelibrary.com. DOI 10.1002/adv.21354

The Anti-Melanoma Efficiency of the Intratumoral Injection of Cucurbitacin-Loaded Sustained-Release Carriers: A PLGA Particle System

A dilemma for current cancer therapy is surgical injury in addition to the toxicities and inefficiencies of systemic chemotherapy. Meanwhile, localized therapies have become a noticeable strategy. In this study, Cucurbitacin (Cuc)-loaded poly(lactic-co-glycolic acid) particles of different sizes (about 50 μm, 5 μm, and 270 nm, respectively) were prepared as the sustained-release system for intratumoral injection, and their physicochemical properties, in vitro cytotoxicity, particles-cells interactions, pharmacokinetics, and pharmacodynamics were systemically investigated for the first time. The results of cytotoxicity experiments and pharmacokinetic/pharmacodynamic studies indicated that the release patterns of the particles would strongly affect the biological efficiency not only at the cell level but also in two types of animal models. Cuc raw material and nanoparticles showed higher initial burst release in vitro, and higher drug concentration in tumor and plasma. Large particles (about 50 μm) showed lower initial burst and slow drug release, which would not supply enough amount of drug to inhibit the cancer cell growth during the whole treatment period. Particles with mean diameter of about 5 μm performed the best anti-melanoma efficiency both in vitro and in vivo. The release patterns, instead of the particles-cells interactions, would be the key factors to affect the biological effects of the particulate system for intratumoral injection.

Preparation and evaluation of double-walled microparticles prepared with a modified water-in-oil-in-oil-in-water (w1/o/o/w3) method

In this study, a modified water-in-oil-in-oil-in-water (w1/o/o/w3) method was developed to prepare double-walled microparticles containing ovalbumin (OVA). The microparticles were characterized with respect to their morphology, particle size, encapsulation efficiency, production yield, thermal properties and in vitro drug release. Microscopy observations clearly showed that microparticles have spherical shape and smooth surface. These microparticles were characterized to have double-walled structure, with a cavity in the centre. By using w1/o/o/w3 method, a significant decrease in mean particle size and a significant increase in encapsulation efficiency were obtained. The mean particle size and the encapsulation efficiency of double-walled microparticles were also affected by the changing amount of OVA and mass ratio of polymers. Microparticles prepared with two polymers exhibited a significantly lower initial burst release followed by sustained release compared to microparticles made from poly(d,l-lactide-co-glycolide) 50/50 only. It can be concluded that these microparticles can be a potential delivery system for therapeutic proteins.

Evaluation of protective efficacy using a nonstructural protein NS1 in DNA vaccine–loaded microspheres against dengue 2 virus

Dengue virus results in dengue fever or severe dengue hemorrhagic fever/dengue shock syndrome in humans. The purpose of this work was to develop an effective antidengue virus delivery system, by designing poly (dl-lactic-co-glycolic) acid/polyethylene glycol (PLGA/PEG) microspheres using a double-emulsion solvent extraction method, for vaccination therapy based on locally and continuously sustained biological activity. Nonstructural protein 1 (NS1) in deoxyribonucleic acid (DNA) vaccine–loaded PLGA/PEG microspheres exhibited a high loading capacity (4.5% w/w), yield (85.2%), and entrapment efficiency (39%), the mean particle size 4.8 μm, and a controlled in vitro release profile with a low initial burst (18.5%), lag time (4 days), and continued released protein over 70 days. The distribution of protein on the microspheres surface, outer layer, and core were 3.0%, 28.5%, and 60.7%, respectively. A release rate was noticed to be 1.07 μg protein/mg microspheres/day of protein release, maintained for 42 days. The cumulative release amount at Days 1, 28, and 42 was 18.5, 53.7, and 62.66 μg protein/mg microspheres, respectively. The dengue virus challenge in mice test, in which mice received one dose of 20 μg NS1 protein content of microspheres, in comparison with NS1 protein in Al(OH)3 or PBS solution, was evaluated after intramuscular immunization of BALB/c mice. The study results show that the greatest survival was observed in the group of mice immunized with NS1 protein–loaded PLGA/PEG microspheres (100%). In vivo vaccination studies also demonstrated that NS1 protein–loaded PLGA/PEG microspheres had a protective ability; its steady-state immune protection in rat plasma changed from 4,443 ± 1,384 pg/mL to 10,697 ± 3,197 pg/mL, which was 2.5-fold higher than that observed for dengue virus in Al(OH)3 at 21 days. These findings strongly suggest that NS1 protein–loaded PLGA/PEG microspheres offer a new therapeutic strategy in optimizing the vaccine incorporation and delivery properties of these potential vaccine targeting carriers.

Development, Optimization & Evaluation of Porous Chitosan Scaffold Formulation of Gliclazide for the Treatment of Type-2 Diabetes Mellitus

In order to achieve high bioactivity, low systemic side effects and prevention of high critical hypoglycaemic conditions of antidiabetic drugs in the treatment of diabetes mellitus, sustained and controlled delivery system is crucial. In this study, a three-dimensional (3-D) porous scaffold formulation was developed with the ability to release antidiabetic drugs in a controlled fashion. The gliclazide (GLZ), was successfully incorporated into prefabricated 3-D porous matrix chitosan scaffolds using a freeze-drying method. Porosity, swelling, water absorption and drug entrapment efficiency was increased after the addition of PEG 4000. The release kinetics of GLZ from two different (2% and 3%) chitosan scaffold formulations investigated showed sustained and controlled delivery of GLZ. The GLZ release is strongly dependent upon the physical and chemical properties of the chitosan and PEG 4000. Scaffold with 3% chitosan discharge GLZ rapidly with a high initial burst release while scaffold with 2% chitosan can extend the release of GLZ to longer than 2 weeks with a low initial burst release. Compared to chitosan alone, the PEG 4000 incorporated on a 3-D scaffold had significantly reduced the initial burst release. In-vivo antidiabetic tests of chitosan (2%)-PEG (1.5%) scaffold formulation demonstrated its ability to reduce blood sugar level for a prolonged duration (7 days). From this study, we concluded that, versatile carrier system i.e. porous scaffold formulation is effective for the delivery of GLZ for diabetes mellitus and maintain the concentration levels over a prolonged period of time, when compared against the free drug.

Comparison of Microencapsulation by Emulsion-Solvent Extraction/Evaporation Technique Using Derivatives Cellulose and Acrylate-Methacrylate Copolymer as Carriers

Microencapsulation is a useful method to prolong a drug release from dosage forms and to reduce its adverse effect (1) among various available methods. The microencapsulation of hydrophilic active ingredients requires the use of a polar dispersing phase such as a mineral oil. Acetone/paraffin systems are conventionally used. The current study aimed to investigate two different microencapsulation techniques comparatively, water in oil in oil (w/o/o) and oil in oil (o/o), for theophylline (TH) loaded ethylcellulose (EC), cellulose acetate butyrate (CAB), Eudragit RS and RL microspheres with regard to loading efficiency, release and degradation kinetics. Microspheres were prepared by the emulsification method by solvent diffusion/evaporation technique and different polymers which were incorporated into microspheres to control the release rate of drug. Theophylline (TH) was chosen as a model drug. The emulsion technique was investigated for to prepare theophylline microparticles. EC and CAB and acrylatemethacrylate copolymer corresponding to the above ratios were selected as microparticles wall materials. The effects of type polymers on the physical characteristics and dissolution of the microparticles were also studied. However, the TH loading efficiency (for w/o/o emulsion about 90.64% and o/o emulsion about 73.90/5 to 95.90%) and the TH release kinetics were influenced by the microencapsulation technique. The results demonstrated that the o/o microspheres (containing of CAB) was most appropriate, providing a high encapsulation efficiency (95.90%) and low initial burst release (6.45%). The microspheres prepared with CAB polymer showed faster dissolution rate than other polymers with 0.75: 1 drug to polymer ratio. The double emulsion technique with EC as wall material gave the high dissolution efficiency (80.48%) of microcapsules. Eudragit RS microspheres showed higher yield (90%). The release of TH from CAB and Eudragit RL walled microcapsules was slow whilst the release from those of EC and Eudragit RS were faster. The type of polymer and the drug to polymer ratio were found to be the key factors affecting the release profile which could lead to microspheres with desired release behavior.

Multi-arm histidine copolymer for controlled release of insulin from poly(lactide-co-glycolide) microsphere

For long-term, sustained protein delivery, a new, star-shaped block copolymer composed of methoxy poly(ethylene glycol) (mPEG), branched oligoethylenimine (bOEI), and poly(l-histidine) (pHis) was synthesized via the multi-initiation and ring-opening polymerization (ROP) of His N-carboxy anhydride (NCA) on bOEI with a PEG conjugation. The resulting mPEG-bOEI-pHis (POH) had strong buffering capacity within the neutral-to-acidic pH range and was complexed with insulin (Ins) via an electrostatic attraction plus hydrophobic interactions, resulting in the formation of a dual-interaction complex (DIC, weight ratio 2) of approximately 30–60 nm in size. This DIC tolerated high salt concentrations without destabilization, supporting the existence of hydrophobic interactions, and protected Ins from the organic solvent/water interface. The DIC in poly(lactide-co-glycolide) microspheres (PLGA MS) as a long-term Ins delivery formulation was evenly distributed via a double-emulsion method. The DIC-loaded PLGA MS offered a higher Ins loading and a lower initial burst than Ins-loaded PLGA MS. This formulation possessed near zero-order release kinetics (for at least one month). In streptozotocin (STZ)-induced diabetic rats, a DIC-loaded PLGA MS formulation was able to maintain blood-glucose levels at 200–350 mg/dL for the first two weeks and even lower levels (100–200 mg/dL) for the next two weeks. Thus, a new POH polymer and its complex with a drug protein could have potential biological application as a long-term, sustained protein delivery system.

Long-acting formulation of a new muscarinic receptor antagonist for the treatment of overactive bladder

A new muscarinic receptor antagonist, 5-hydroxymethyl tolterodine (5-HMT), was successfully encapsulated into PLGA microspheres. With an increase of PLGA concentration from 15% to 40%, encapsulation efficiency of 5-HMT increased from 55.39% to 76.32%, and the particle size of microsphere increased from 34.33 to 70.48 µm. Increasing the homogenisation speed from 850 to 2300 rpm, the particle size was reduced about 65%.The in vitro and in vivo studies in beagle dogs show that the release profile of 5-HMT-loaded microspheres (5-HMT MS) prepared with 503H is characterised by a low initial burst followed by slow release that lasted for 2 weeks. A Cmax of 1.617 ± 0.392 ng/mL was found on the sixth day. When evaluated for inhibition of the carbachol-induced contraction of rat urinary bladder, 5-HMT MS showed a much longer and more potent effect than tolterodine tablets. The mean urination time of the rats in the 5-HMT MS group was significantly decreased (p < 0.05 or p < 0.01) to less than 2 weeks.

Sustained release of melatonin from poly (lactic‐co‐glycolic acid) (PLGA) microspheres to induce osteogenesis of human mesenchymal stem cells in vitro

Melatonin promotes bone formation and prevents bone degradation via receptor-dependent or receptor-independent actions. The aim of this study is to encapsulate melatonin into poly (lactic-co-glycolic acid) (PLGA) microspheres (PLGA-MEL-MS) and create a melatonin sustained release system, then to evaluate its effect on the osteogenesis of human mesenchymal stem cells (hMSCs) in vitro. PLGA-MEL-MS were prepared by single emulsion solvent evaporation technique. Scanning electron microscopy demonstrated the incorporation of melatonin did not disturb the conventional generation of PLGA microspheres in size and morphology. In vitro drug release assay showed that PLGA-MEL-MS exhibited a biphasic drug release pattern: a low initial burst release effect with approximately 40% drug release at the first 3 days and a relatively retarded and continuous release with about 85% drug release over the 25 days. Cell proliferation assay demonstrated that PLGA-MEL-MS had no apparent effect on proliferation of human MSCs. In an osteogenesis assay, PLGA-MEL-MS obviously enhanced alkaline phosphatase (ALP) mRNA expression and increased ALP activity compared to that in the control group. Meanwhile, several markers of osteoblast differentiation were also significantly upregulated, including runx2, osteopontin, and osteocalcin. Furthermore, quantificational alizarin red-based assay demonstrated that PLGA-MEL-MS significantly enhanced calcium deposit of hMSCs compared to the controls. Therefore, this simple melatonin sustained release system can control released melatonin to generate a microenvironment with a relatively stable concentration of melatonin for a period of time to support osteogenic differentiation of hMSCs in vitro. This suggests that this system may be used as bone growth stimulator in bone healing in vivo.

Cerasomal doxorubicin with long-term storage stability and controllable sustained release

Liposomal nanohybrid cerasomes display a remarkable ability to maintain their size and retain encapsulated doxorubicin (DOX) over a period of 90days under storage conditions in solution compared with liposomes and liposils. Cerasomes retained 92.1±2.9% of the drug payload after 90days storage, much more than liposomes (35.2±2.5%) and liposils (53.2±5.5%). Under physiologically relevant conditions cerasomes exhibit a low initial burst in the first 5h and subsequent sustained release of DOX over the next 150h. Moreover, the magnitude of the initial burst and the rate of sustained release of DOX from cerasomes can be modulated by incorporating dipalmitoylphosphatidylglycerol (DPPG) in the cerasome structure and altering the ratios of the cerasome-forming lipid and phospholipids. Consequently, a wide range of release profiles can be achieved by altering the vesicle composition. Finally, human ovarian cancer cells are effectively killed by DOX released from cerasomes. Together these results suggest that cerasomes may be a promising drug delivery system for the long-term storage and controllable sustained release of the anticancer drug DOX.

PEG/RGD-modified magnetic polymeric liposomes for controlled drug release and tumor cell targeting

Polymeric liposomes (PEG/RGD-MPLs), composed of amphiphilic polymer octadecyl-quaternized modified poly (γ-glutamic acid) (OQPGA), PEGylated OQPGA, RGD peptide grafted OQPGA and magnetic nanoparticles, was prepared successfully. These PEG/RGD-MPLs could be used as a multifunctional platform for targeted drug delivery. The results showed that PEG/RGD-MPLs were multilamellar spheres with nano-size (50–70nm) and positive surface charge (28–42mV). Compared with magnetic conventional liposomes (MCLs), PEG/RGD-MPLs exhibited sufficient size and zeta potential stability, low initial burst release and less magnetic nanoparticles leakage. The cell uptake results suggested that the PEG/RGD-MPLs (with RGD and magnetic particles) exhibited more drug cellular uptake than non RGD and non magnetism carriers in MCF-7cells. MTT assay revealed that PEG/RGD-MPLs showed lower in vitro cytotoxicity to GES-1cells at ≤100μg/mL. These data indicated that the multifunctional PEG/RGD-MPLs may be an alternative formulation for drug delivery system.

Formulation of sustained-release microspheres of granulocyte macrophage colony stimulating factor by freezing-induced phase separation with dextran and encapsulation with blended polymers

This study aimed to assess the potential merits of formulating sustained-release microspheres of recombinant human granulocyte macrophage colony stimulating factor (rhGM-CSF) via freezing-induced phase separation (FIPS) of the protein with dextran followed by encapsulation with binary mixture of poly(lactic-co-glycolic acid) (PLGA) 2A (MW∼12K) and 3A (MW∼47K) or of PLGA2A and polylactic acid (PLA; MW∼83K). The formulated dextran particles and microspheres were characterized in vitro for loading, aggregation, bioactivity and release behavior of the protein where appropriate. rhGM-CSF retained about 60% of bioactivity with no significant aggregation after each formulation step. Encapsulation of protein-loaded dextran particles attained only 80% with the PLGA2A and PLGA3A blend, but 100% with the PLGA2A and PLA mixture. The former formulation exhibited a triphasic in-vitro release profile typical of PLGA microspheres while the latter revealed a much lower initial burst followed by a steady and complete release of rhGM-CSF with preserved bioactivity over a 15-day period.

PLGA microspheres containing bee venom proteins for preventive immunotherapy

Bee venom (BV) allergy is potentially dangerous for allergic individuals because a single bee sting may induce an anaphylactic reaction, eventually leading to death. Currently, venom immunotherapy (VIT) is the only treatment with long-lasting effect for this kind of allergy and its efficiency has been recognized worldwide. This therapy consists of subcutaneous injections of gradually increasing doses of the allergen. This causes patient lack of compliance due to a long time of treatment with a total of 30–80 injections administered over years. In this article we deal with the characterization of different MS-PLGA formulations containing BV proteins for VIT. The PLGA microspheres containing BV represent a strategy to replace the multiple injections, because they can control the solute release. Physical and biochemical methods were used to analyze and characterize their preparation. Microspheres with encapsulation efficiencies of 49–75% were obtained with a BV triphasic release profile. Among them, the MS-PLGA 34 kDa-COOH showed to be best for VIT because they presented a low initial burst (20%) and a slow BV release during lag phase. Furthermore, few conformational changes were observed in the released BV. Above all, the BV remained immunologically recognizable, which means that they could continuously stimulate the immune system. Those microspheres containing BV could replace sequential injections of traditional VIT with the remarkable advantage of reduced number of injections.

Intracellular salicylic acid is involved in signal cascade regulating low ammonium-induced taxoid biosynthesis in suspension cultures of Taxus chinensis

It was previously reported that low initial ammonium (2 mM) in medium had significant stimulating effects on the biosynthesis of taxuyunnanine C (Tc) by Taxus chinensis cells. However, the secondary metabolism induction mechanism of the low initial ammonium is yet unknown in plant cells. To provide an insight into the defense signals response to the low initial ammonium, oxidative burst and intracellular salicylic acid (SA) were detected, and their influences on the expression of important genes in taxoid biosynthetic pathway were examined in the cell cultures of T. chinensis. Induced H(2)O(2) production, elevated phenylalanine ammonia-lyase (PAL) activity, and enhanced SA biosynthesis were observed. Interestingly, inhibition of SA biosynthesis by paclobutrazol and (BOC-aminooxy) acetic acid significantly depressed the Tc stimulation and up-regulation of Tc biosynthetic genes of geranylgeranyl diphosphate synthase and taxadiene synthase. The role of intracellular SA in regulating Tc biosynthesis was further confirmed by applying exogenous SA in normal ammonium (20 mM) medium. The results indicated that SA acted as a signal in low initial ammonium-induced Tc biosynthesis. A signal transduction cascade from defense signal response to activated transcription of taxoid biosynthetic genes and enhanced Tc production is proposed.

Biodegradable insulin-loaded PLGA microspheres fabricated by three different emulsification techniques: Investigation for cartilage tissue engineering

Growth, differentiation and migration factors facilitate the engineering of tissues but need to be administered with defined gradients over a prolonged period of time. In this study insulin as a growth factor for cartilage tissue engineering and a biodegradable PLGA delivery device were used. The aim was to investigate comparatively three different microencapsulation techniques, solid-in-oil-in-water (s/o/w), water-in-oil-in-water (w/o/w) and oil-in-oil-in-water (o/o/w), for the fabrication of insulin-loaded PLGA microspheres with regard to protein loading efficiency, release and degradation kinetics, biological activity of the released protein and phagocytosis of the microspheres. Insulin-loaded PLGA microspheres prepared by all three emulsification techniques had smooth and spherical surfaces with a negative zeta potential. The preparation technique did not affect particle degradation nor induce phagocytosis by human leukocytes. The delivery of structurally intact and biologically active insulin from the microspheres was shown using circular dichroism spectroscopy and a MCF7 cell-based proliferation assay. However, the insulin loading efficiency (w/o/w about 80%, s/o/w 60%, and o/o/w 25%) and the insulin release kinetics were influenced by the microencapsulation technique. The results demonstrate that the w/o/w microspheres are most appropriate, providing a high encapsulation efficiency and low initial burst release, and thus these were finally used for cartilage tissue engineering. Insulin released from w/o/w PLGA microspheres stimulated the formation of cartilage considerably in chondrocyte high density pellet cultures, as determined by increased secretion of proteoglycans and collagen type II. Our results should encourage further studies applying protein-loaded PLGA microspheres in combination with cell transplants or cell-free in situ tissue engineering implants to regenerate cartilage.

Reduction in Burst Release from Poly(D,L-Lactide-Co-Glycolide) Microparticles by Solvent Treatment

A high initial burst release of an antisense oligonucleotides drug from poly(lactide-co-glycolide) (PLGA) microparticles prepared by the multiple emulsion (w/o/w) method was significantly reduced, when the microparticles were treated with a polymer non-solvent (ethanol)/water mixture. The ethanol/water mixture acted like a plasticizer and significantly decreased the glass transition temperature (Tg) of the PLGA. Thus, PLGA microparticles in the wet state became soft, and the surface pores were fused together during treatment. As a result, microparticles with reduced surface pores that released a low initial burst were obtained. The reduction in the initial burst was more significant for the smaller microparticles ( < 50 μm) and decreased with increasing microparticle size. A less significant reduction in the initial burst was observed when the microparticles were treated with a polymer solvent (e.g., acetone)/water mixture. Keywords: Microparticles, Initial burst, Poly(lactide-co-glycolide), Glass transition temperature, Solvent treatment, PLGA, probe sonication, Shaker, UV-spectrophotometry, PC UV-Vis scanning spectropho-tometer, Scanning Electron Microscopy, SEM, Differential Scanning Calorimetry, DSC, Scanning electron micrographs, polymer matrix, ethyl acetate, methylene chloride, 2-pyrrolidone

PEGylated TNF-related apoptosis-inducing ligand (TRAIL)-loaded sustained release PLGA microspheres for enhanced stability and antitumor activity

The purpose of this work was to develop an effective PEGylated TNF-related apoptosis-inducing ligand (PEG-TRAIL) delivery system for antitumor therapy based on local injection to tumor sites that has a sustained effect without protein aggregation or an initial release burst. The authors designed poly (lactic-co-glycolic) acid (PLGA) microspheres that deliver PEG-TRAIL locally and continuously at tumor sites with sustained biological activity and compared its performance with that of TRAIL microspheres. TRAIL or PEG-TRAIL was microencapsulated into PLGA microspheres using a double-emulsion solvent extraction method. Prepared TRAIL and PEG-TRAIL microspheres showed entirely spherical, smooth surfaces. However, PEG-TRAIL microspheres exhibited a 2.07-fold higher encapsulation efficiency than TRAIL microspheres, and exhibited a tri-phasic in vitro release profile with a lower initial burst (15.8%) than TRAIL microspheres (42.7%). Furthermore, released PEG-TRAIL showed a continued ability to induce apoptosis over 14 days. In vivo pharmacokinetic studies also demonstrated that PEG-TRAIL microspheres had a sustained release profile (18 days), and that the steady-state concentration of PEG-TRAIL in rat plasma was reached at day 3 and maintained until day 15; its steady-state concentration in rat plasma changed from 1444.3 ± 338.4 to 2697.7 ± 419.7 pg/ml. However, TRAIL microspheres were released out within 2 days after administration. Finally, in vivo antitumor tests revealed that tumor growths were significantly more inhibited by a single dose of PEG-TRAIL microspheres than TRAIL microspheres when delivered at 300 μg of TRAIL/mouse. Tumors taken from mouse treated with PEG-TRAIL microspheres showed 78.3% tumor suppression at 24 days, and this was 3.02-fold higher than that observed for TRAIL microspheres (25.9% tumor inhibition). Furthermore, these improved pharmaceutical characteristics of PEG-TRAIL microspheres resulted in superior therapeutic effects without detectable side effects. These findings strongly suggest that PEG-TRAIL microspheres offer a new therapeutic strategy for the treatment of cancers.

Reduction in burst release after coating poly(D,L-lactide-co-glycolide) (PLGA) microparticles with a drug-free PLGA layer

The high initial burst release of a highly water-soluble drug from poly (D,L-lactide-co-glycolide) (PLGA) microparticles prepared by the multiple emulsion (w/o/w) solvent extraction/evaporation method was reduced by coating with an additional polymeric PLGA layer. Coating with high encapsulation efficiency was performed by dispersing the core microparticles in peanut oil and subsequently in an organic polymer solution, followed by emulsification in the aqueous solution. Hardening of an additional polymeric layer occurred by oil/solvent extraction. Peanut oil was used to cover the surface of core microparticles and, therefore, reduced or prevented the rapid erosion of core microparticles surface. A low initial burst was obtained, accompanied by high encapsulation efficiency and continuous sustained release over several weeks. Reduction in burst release after coating was independent of the amount of oil. Either freshly prepared (wet) or dried (dry) core microparticles were used. A significant initial burst was reduced when ethyl acetate was used as a solvent instead of methylene chloride for polymer coating. Multiparticle encapsulation within the polymeric layer increased as the size of the core microparticles decreased (< 50 µm), resulting in lowest the initial burst. The initial burst could be controlled well by the coating level, which could be varied by varying the amount of polymer solution, used for coating.

Preparation and in vitro characterisation of Ehrlichia ruminantium plasmid DNA and proteins encapsulated into and DNA adsorbed onto biodegradable microparticles

Four E. ruminantium 1H12 open reading frames and their proteins known to protect sheep against heartwater needle challenge were encapsulated into, or adsorbed onto poly( d, l-lactide-co-glycolide) microparticles. Microspheres with smooth surface and smaller than 5 μm diameters were produced, with high adsorption and encapsulation efficiencies. Gel electrophoresis showed that neither encapsulation nor adsorption affected the stability of the DNA or proteins. Cationic microparticles released ∼40% of plasmid DNA on day 1 while PLGA 50:50-COOH microparticles co-encapsulating plasmid DNA and polyvinyl alcohol only started to release from days 12–28. Recombinant proteins were released from PLGA 85:15 and homopolymer R 203 S microparticles in a biphasic manner with a high initial burst release (∼45–80%). In contrast, PLGA 50:50 microparticles had low (15–65%) initial burst release followed by (25–80%) release by days (days 28–42). A cocktail of these microparticles could therefore be used as single-dose auto-booster vaccine.

Evaluation of succinylated pullulan for long-term protein delivery in poly(lactide-co-glycolide) microspheres

Two types of pullulan with different succinylations were synthesized for an evaluation as additives for long-term protein delivery in poly(lactide-co-glycolide) microspheres (PLGA MS). Negatively charged succinylated pullulan (SP) forms an ionic complex with cationic protein (lysozyme; Lys) via an ionic interaction. SP2 (2 succinyl groups per 10 glucose units in pullulan) constructed a better nano-size complex with lysozyme (Lys) in distilled water (DW) than SP1 (1 succinyl group per 10 glucose units in pullulan). To assess the long-term delivery capability, PLGA MS complexes with different Lys:SP2 ratios (1:1 (LMS 1), 1:2 (LMS 2) and 1:3 (LMS 3), as wt% of Lys:SP2) were prepared with water in oil in water (W/O/W). The complex loaded PLGA MS showed a higher loading efficiency and a lower insoluble Lys content than PLGA without SP (LMS 0), indicating that SP helps stabilize Lys at the organic/water (O/W) interface. In evaluating the release pattern of Lys, LMS 1, 2, and 3 all demonstrated a low initial burst and complete release behavior (reaching almost 100% after 27 days), whereas the total amount of Lys released from LMS 0 did not reach 80% during the same time period. During a 14-day release test, the stability of Lys was confirmed by RP-HPLC. In the case of LMS 0, an unexpected peak (retention time 9.2 min) was created, which was not observed in LMS 1, 2, and 3. This suggests that SP suppresses the denaturation of Lys in PLGA MS. These results show that SP, as an additive in PLGA MS, has potential for the long-term delivery of therapeutic proteins. Open image in new window