One-Step Production of Protein-Loaded PLGA Microparticles via Spray Drying Using 3-Fluid Nozzle

Purpose

The aim of this study was to investigate the potential of using a spray-dryer equipped with a 3-fluid nozzle to microencapsulate protein drugs into polymeric microparticles.

Methods

Lysozyme and PLGA were used as a model protein and a model polymer, respectively. The effects of process and formulation variables, such as i) the type of organic solvent, ii) the feeding rate ratio of the outer PLGA-containing feed solution to inner lysozyme-containing feed solution, and iii) the mass ratio of PLGA to protein, on the properties (morphology, internal structure, protein surface enrichment and release profiles) of the spray dried microparticles were investigated to understand protein microencapsulation and particle formation mechanisms.

Results

The spherical, condensed microparticles were obtained with D₅₀ of 1.07–1.60 μm and Span in the range of 0.82–1.23. The lysozyme surface content decreased upon different organic solvents used as follows: acetonitrile?>?acetone?>?dichloromethane. Additionally, the lysozyme surface enrichment decreased slightly when increasing the feeding rate ratio of the outer feed solution to the inner feed solution from 4:1 to 10:1. Furthermore, it was observed that there was a correlation between the degree of burst release and the lysozyme surface enrichment, whereas the lysozyme loading content had no substantial impact on the release kinetics.

Conclusions

The present work demonstrates the potential of spray dryer equipped with a 3-fluid nozzle in microencapsulation of proteins into PLGA matrices with different characteristics by varying process and formulation parameters.     

Nanoparticles Based on a Hydrophilic Polyester with a Sheddable PEG Coating for Protein Delivery

Purpose

To investigate the effect of polyethylene glycol (PEG) in nanoparticles based on blends of hydroxylated aliphatic polyester, poly(D,L-lactic-co-glycolic-co-hydroxymethyl glycolic acid) (PLGHMGA) and PEG-PLGHMGA block copolymers on their degradation and release behavior.

Methods

Protein-loaded nanoparticles were prepared with blends of varying ratios of PEG-PLGHMGA (molecular weight of PEG 2,000 and 5,000 Da) and PLGHMGA, by a double emulsion method with or without using poly(vinyl alcohol) (PVA) as surfactant. Bovine serum albumin and lysozyme were used as model proteins.

Results

PEGylated particles prepared without PVA had a zeta potential ranging from ~ ?3 to ~?35 mV and size ranging from ~200 to ~600 nm that were significantly dependent on the content and type of PEG-block copolymer. The encapsulation efficiency of the two proteins however was very low (<30%) and the particles rapidly released their content in a few days. In contrast, all formulations prepared with PVA showed almost similar particle properties (size: ~250 nm, zeta potential: ~?1 mV), while loading efficiency for both model proteins was rather high (80–90%). Unexpectedly, independent of the type of formulation, the nanoparticles had nearly the same release and degradation characteristics. NMR analysis showed almost a complete removal of PEG in 5 days which explains these marginal differences.

Conclusions

Protein release and particle degradation are not substantially influenced by the content of PEG, likely because of the fast shedding of the PEG blocks. These PEG shedding particles are interesting system for intracellular delivery of drugs.     

Brushed Block Copolymer Micelles with pH-Sensitive Pendant Groups for Controlled Drug Delivery

Purpose

To investigate the effects of small aliphatic pendent groups conjugated through an acid-sensitive linker to the core of brushed block copolymer micelles on particle properties.

Methods

The brushed block copolymers were synthesized by conjugating five types of 2-alkanone (2-butanone, 2-hexanone, 2-octanone, 2-decanone, and 2-dodecanone) through an acid-labile hydrazone linker to poly(ethylene glycol)-poly(aspartate hydrazide) block copolymers.

Results

Only block copolymers with 2-hexanone and 2-octanone (PEG-HEX and PEG-OCT) formed micelles with a clinically relevant size (< 50 nm in diameter), low critical micelle concentration (CMC, < 20 μM), and drug entrapment yields (approximately 5 wt.%). Both micelles degraded in aqueous solutions in a pH-dependent manner, while the degradation was accelerated in an acidic condition (pH 5.0) in comparison to pH 7.4. Despite these similar properties, PEG-OCT micelles controlled the entrapment and pH-dependent release of a hydrophobic drug most efficiently, without altering particle size, shape, and stability. The molecular weight of PEG (12 kDa vs 5 kDa) induced no change in pH-controlled drug release rates of PEG-OCT micelles.

Conclusion

Acid-labile small aliphatic pendant groups are useful to control the entrapment and release of a hydrophobic drug physically entrapped in the core of brushed block copolymer micelles.     

Surface Analysis of PEGylated Nano-Shields on Nanoparticles Installed by Hydrophobic Anchors

Purpose

This work describes a method for functionalisation of nanoparticle surfaces with hydrophilic “nano-shields” and the application of advanced surface characterisation to determine PEG amount and accumulation at the outmost 10 nm surface that is the predominant factor in determining protein and cellular interactions.

Methods

Poly(lactic-co-glycolic acid) (PLGA) nanoparticles were prepared with a hydrophilic PEGylated “nano-shield” inserted at different levels by hydrophobic anchoring using either a phospholipid-PEG conjugate or the copolymer PLGA-block-PEG by an emulsification/diffusion method. Surface and bulk analysis was performed including X-ray photoelectron spectroscopy (XPS), nuclear magnetic resonance spectroscopy (NMR) and zeta potential. Cellular uptake was investigated in RAW 264.7 macrophages by flow cytometry.

Results

Sub-micron nanoparticles were formed and the combination of (NMR) and XPS revealed increasing PEG levels at the particle surface at higher PLGA-b-PEG copolymer levels. Reduced cellular interaction with RAW 264.7 cells was demonstrated that correlated with greater surface presentation of PEG.

Conclusion

This work demonstrates a versatile procedure for decorating nanoparticle surfaces with hydrophilic “nano-shields”. XPS in combination with NMR enabled precise determination of PEG at the outmost surface to predict and optimize the biological performance of nanoparticle-based drug delivery.     

Preparation, Characterization, and Release of Amoxicillin from Electrospun Fibrous Wound Dressing Patches

Purpose

To produce electrospun polymeric fibrous wound dressing patches that can release the antibiotic drug amoxicillin in a controlled manner.

Methods

Poly(D,L-lactide-co-glycolide) acid (PLGA) fibrous dressings with entrapped amoxicillin were produced by electrospinning. The morphology and successful entrapment of amoxicillin in the PLGA fibrous dressings were validated by scanning electron microscopy (SEM) and Fourier Transform Infrared (FTIR) spectroscopy. The rate of drug release from the dressing patches was measured in various media for a period of 21 days using UV spectroscopy.

Results

PLGA fibres entrapping amoxicillin were collected for 300 s and then cut to form square patches with an average weight of 55 mg. Each dressing patch contained ~2 mg of amoxicillin. The mean fibre diameter was 2.2?±?0.4 μm. The drug release from the PLGA dressings was found to be different for each medium during the 21-day release period with the highest and lowest concentration of drug released observed when the dressings were immersed in simulated body fluid (SBF) and phosphate buffered saline (PBS), respectively.

Conclusions

The release profiles obtained in this study and the well-established biocompatibility of PLGA indicate that the fibre-based patches with entrapped amoxicillin fabricated in this work are very well suited for applications in wound healing and infection control.     

Gamma Irradiation of Active Self-Healing PLGA Microspheres for Efficient Aqueous Encapsulation of Vaccine Antigens

Purpose

To investigate the effect of γ-irradiation of poly(lactic-co-glycolic acid) (PLGA)/Al(OH)₃/0 or 5 wt% diethyl phthalate (DEP) microspheres for active self-healing encapsulation of vaccine antigens.

Methods

Microspheres were irradiated with ⁶⁰Co at 2.5 and 1.8 MRad and 0.37 and 0.20 MRad/h. Encapsulation of tetanus toxoid (TT) was achieved by mixing Al(OH)₃-PLGA microspheres with TT solution at 10–38°C. Electron paramagnetic resonance (EPR) spectroscopy was used to examine free radical formation. Glass transition temperature (T_g) and molecular weight of PLGA was measured by differential scanning calorimetry and gel permeation chromatography, respectively. Loading and release of TT were examined by modified Bradford, amino acid analysis, and ELISA assays.

Results

EPR spectroscopy results indicated absence of free radicals in PLGA microspheres after γ-irradiation. Antigen-sorbing capacity, encapsulation efficiency, and T_g of the polymer were also not adversely affected. When DEP-loaded microspheres were irradiated at 0.2 MRad/h, some PLGA pores healed during irradiation and PLGA healing during encapsulation was suppressed. The molecular weight of PLGA was slightly reduced when DEP-loaded microspheres were irradiated at the same dose rate. At the 0.37 MRad/h dose rate, these trends were not observed and the full immunoreactivity of TT was preserved during encapsulation and 1-month release. Gamma irradiation slightly increased TT initial burst release. The small increase in total irradiation dose from 1.8 to 2.5 MRad had insignificant effect on the polymer and microspheres properties analyzed.

Conclusions

Gamma irradiation is a plausible approach to provide a terminally sterilized, self-healing encapsulation PLGA excipient for vaccine delivery.     

Cyclosporine A Loaded Electrospun Poly(D,L-Lactic Acid)/Poly(Ethylene Glycol) Nanofibers: Drug Carriers Utilizable in Local Immunosuppression

Purpose

The present study aims to prepare poly(D,L-lactic acid) (PLA) nanofibers loaded by the immunosuppressant cyclosporine A (CsA, 10 wt%). Amphiphilic poly(ethylene glycol)s (PEG) additives were used to modify the hydrophobic drug release kinetics.

Methods

Four types of CsA-loaded PLA nanofibrous carriers varying in the presence and molecular weight (MW) of PEG (6, 20 and 35 kDa) were prepared by needleless electrospinning. The samples were extracted for 144 h in phosphate buffer saline or tissue culture medium. A newly developed and validated LC-MS/MS method was utilized to quantify the amount of released CsA from the carriers. In vitro cell experiments were used to evaluate biological activity.

Results

Nanofibers containing 15 wt% of PEG showed improved drug release characteristics; significantly higher release rates were achieved in initial part of experiment (24 h). The highest released doses of CsA were obtained from the nanofibers with PEG of the lowest MW (6 kDa). In vitro experiments on ConA-stimulated spleen cells revealed the biological activity of the released CsA for the whole study period of 144 h and nanofibers containing PEG with the lowest MW exhibited the highest impact (inhibition).

Conclusions

The addition of PEG of a particular MW enables to control CsA release from PLA nanofibrous carriers. The biological activity of CsA-loaded PLA nanofibers with PEG persists even after 144 h of previous extraction. Prepared materials are promising for local immunosuppression in various medical applications.

     

Polydopamine-Based Surface Modification for the Development of Peritumorally Activatable Nanoparticles

Purpose

To create poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), where a drug-encapsulating NP core is covered with polyethylene glycol (PEG) in a normal condition but exposes a cell-interactive TAT-modified surface in an environment rich in matrix metalloproteinases (MMPs).

Methods

PLGA NPs were modified with TAT peptide (PLGA-pDA-TAT NPs) or dual-modified with TAT peptide and a conjugate of PEG and MMP-substrate peptide (peritumorally activatable NPs, PANPs) via dopamine polymerization. Cellular uptake of fluorescently labeled NPs was observed with or without a pre-treatment of MMP-2 by confocal microscopy and flow cytometry. NPs loaded with paclitaxel (PTX) were tested against SKOV-3 ovarian cancer cells to evaluate the contribution of surface modification to cellular delivery of PTX.

Results

While the size and morphology did not significantly change due to the modification, NPs modified with dopamine polymerization were recognized by their dark color. TAT-containing NPs (PLGA-pDA-TAT NPs and PANPs) showed changes in surface charge, indicative of effective conjugation of TAT peptide on the surface. PLGA-pDA-TAT NPs and MMP-2-pre-treated PANPs showed relatively good cellular uptake compared to PLGA NPs, MMP-2-non-treated PANPs, and NPs with non-cleavable PEG. After 3 h treatment with cells, PTX loaded in cell-interactive NPs showed greater toxicity than non-interactive ones as the former could enter cells during the incubation period. However, due to the initial burst drug release, the difference was not as clear as microscopic observation.

Conclusions

PEGylated polymeric NPs that could expose cell-interactive surface in response to MMP-2 were successfully created by dual modification of PLGA NPs using dopamine polymerization.     

Optimization of Stability, Encapsulation, Release, and Cross-Priming of Tumor Antigen-Containing PLGA Nanoparticles

Purpose

In order to investigate Poly (lactic-co-glycolic acid) (PLGA) nanoparticles (NP) as potential vehicles for efficient tumor antigen (TA) delivery to dendritic cells (DC), this study aimed to optimize encapsulation/release kinetics before determining immunogenicity of antigen-containing NP.

Methods

Various techniques were used to liberate TA from cell lines. Single (gp100) and multiple (B16-tumor lysate containing gp100) antigens were encapsulated within differing molecular weight PLGA co-polymers. Differences in morphology, encapsulation/release and biologic potency were studied. Findings were adopted to encapsulate fresh tumor lysate from patients with advanced tumors and compare stimulation of tumor infiltrating lymphocytes (TIL) against that achieved by soluble lysate.

Results

Four cycles of freeze-thaw + 15 s sonication resulted in antigen-rich lysates without the need for toxic detergents or protease inhibitors. The 80KDa polymer resulted in maximal release of payload and favorable production of immunostimulatory IL-2 and IFN-γ. NP-mediated antigen delivery led to increased IFN-γ and decreased immunoinhibitory IL-10 synthesis when compared to soluble lysate.

Conclusions

Four cycles of freeze-thaw followed by 15 s sonication is the ideal technique to obtain complex TA for encapsulation. The 80KDa polymer has the most promising combination of release kinetics and biologic potency. Encapsulated antigens are immunogenic and evoke favorable TIL-mediated anti-tumor responses.     

Computer Modeling Assisted Design of Monodisperse PLGA Microspheres with Controlled Porosity Affords Zero Order Release of an Encapsulated Macromolecule for 3 Months

Purpose

The aim of this study was the development of poly(D,L-lactide-co-glycolide) (PLGA) microspheres with controlled porosity, to obtain microspheres that afford continuous release of a macromolecular model compound (blue dextran).

Methods

PLGA microspheres with a size of around 40 μm and narrow size distribution (span value of 0.3) were prepared with a double emulsion membrane emulsification method. Gene expression programming (GEP) analysis was applied to design and formulate a batch of microspheres with controlled porosity that shows continuous release of blue dextran.

Results

Low porous microspheres with a high loading efficiency were formed at high polymer concentrations (30% w/w in the oil phase) and were characterized with a burst release <10% and a three-phasic release profile of blue dextran. Increasing porosity (10% w/w polymer concentrations), a sustained release of blue dextran was obtained albeit with up to 40% of burst release. The desired formulation, calculated by GEP, resulted in microspheres with 72% loading efficiency and intermediate porosity. Blue dextran was indeed released continuously in almost a zero order manner over a period of 3 months after an initial small burst release of 9%.

Conclusions

By fine-tuning the porosity, the release profile of PLGA microspheres for macromolecules can be predicted and changed from a three-phasic to a continuous release.     

PLGA/Liposome Hybrid Nanoparticles for Short-Chain Ceramide Delivery

Purpose

Rapid premature release of lipophilic drugs from liposomal lipid bilayer to plasma proteins and biological membranes is a challenge for targeted drug delivery. The purpose of this study is to reduce premature release of lipophilic short-chain ceramides by encapsulating ceramides into liposomal aqueous interior with the aid of poly (lactic-coglycolicacid) (PLGA).

Methods

BODIPY FL labeled ceramide (FL-ceramide) and BODIPY-TR labeled ceramide (TR-ceramide) were encapsulated into carboxy-terminated PLGA nanoparticles. The negatively charged PLGA nanoparticles were then encapsulated into cationic liposomes to obtain PLGA/liposome hybrids. As a control, FL-ceramide and/or TR ceramide co-loaded liposomes without PLGA were prepared. The release of ceramides from PLGA/liposome hybrids and liposomes in rat plasma, cultured MDA-MB-231 cells, and rat blood circulation was compared using fluorescence resonance energy transfer (FRET) between FL-ceramide (donor) and TR-ceramide (acceptor).

Results

FRET analysis showed that FL-ceramide and TR-ceramide in liposomal lipid bilayer were rapidly released during incubation with rat plasma. In contrast, the FL-ceramide and TR-ceramide in PLGA/liposome hybrids showed extended release. FRET images of cells revealed that ceramides in liposomal bilayer were rapidly transferred to cell membranes. In contrast, ceramides in PLGA/liposome hybrids were internalized into cells with nanoparticles simultaneously. Upon intravenous administration to rats, ceramides encapsulated in liposomal bilayer were completely released in 2 min. In contrast, ceramides encapsulated in the PLGA core were retained in PLGA/liposome hybrids for 4 h.

Conclusions

The PLGA/liposome hybrid nanoparticles reduced in vitro and in vivo premature release of ceramides and offer a viable platform for targeted delivery of lipophilic drugs.     

Effects of Antibiotic Physicochemical Properties on Their Release Kinetics from Biodegradable Polymer Microparticles

Purpose

This study investigated the effects of the physicochemical properties of antibiotics on the morphology, loading efficiency, size, release kinetics, and antibiotic efficacy of loaded poly(DL-lactic-co-glycolic acid) (PLGA) microparticles (MPs) at different loading percentages.

Methods

Cefazolin, ciprofloxacin, clindamycin, colistin, doxycycline, and vancomycin were loaded at 10 and 20 wt% into PLGA MPs using a water-in-oil-in water double emulsion fabrication protocol. Microparticle morphology, size, loading efficiency, release kinetics, and antibiotic efficacy were assessed.

Results

The results from this study demonstrate that the chemical nature of loaded antibiotics, especially charge and molecular weight, influence the incorporation into and release of antibiotics from PLGA MPs. Drugs with molecular weights less than 600 Da displayed biphasic release while those with molecular weights greater than 1,000 Da displayed triphasic release kinetics. Large molecular weight drugs also had a longer delay before release than smaller molecular weight drugs. The negatively charged antibiotic cefazolin had lower loading efficiency than positively charged antibiotics. Microparticle size appeared to be mainly controlled by fabrication parameters, and partition and solubility coefficients did not appear to have an obvious effect on loading efficiency or release. Released antibiotics maintained their efficacy against susceptible strains over the duration of release. Duration of release varied between 17 and 49 days based on the type of antibiotic loaded.

Conclusions

The data from this study indicate that the chemical nature of antibiotics affects properties of antibiotic-loaded PLGA MPs and allows for general prediction of loading and release kinetics.     

BSA-PLGA-Based Core-Shell Nanoparticles as Carrier System for Water-Soluble Drugs

Purpose

Preparation, optimization and in vitro evaluation of core-shell nanoparticles comprising of a hydrophilic core of BSA surrounded by a hydrophobic shell of PLGA for loading water-soluble drugs.

Methods

A double emulsion method was optimized for preparation of BSA-PLGA based core-shell nanoparticles. Proof of concept for core-shell type structure was established by visual techniques like confocal microscopy and TEM. Characterization was done for particle size, encapsulation efficiency, drug loading and in vitro drug release. Cellular uptake was assessed using confocal microscopy, bio-TEM and HPLC assay, and cytotoxic activity was tested by MTT assay in MG-63 osteosarcoma cells.

Results

The optimized core-shell nanoparticles showed a particle size of 243 nm (PDI-0.13) and encapsulation efficiency of 40.5% with a drug loading of 8.5% w/w. In vitro drug release studies showed a sustained release for 12 h. Cellular uptake studies indicated a rapid and efficient uptake within 2 h. TEM studies indicated that the core-shell nanoparticles were localized in cytoplasm region of the cells. Gemcitabine loaded core-shell nanoparticles showed enhanced cytotoxicity against MG-63 cells as compared to marketed formulation of gemcitabine (GEMCITE®).

Conclusion

These results indicate that core-shell nanoparticles can be a good carrier system for delivering hydrophilic drugs like gemcitabine successfully to the cells with enhanced efficacy.

Polymeric Plerixafor: Effect of PEGylation on CXCR4 Antagonism,Cancer Cell Invasion,and DNA Transfection

Purpose

To determine the effect of PEG modification on pharmacologic and gene delivery properties of polymeric CXCR4 antagonist based on Plerixafor.

Methods

Polymeric Plerixafor (PAMD) was synthesized from Plerixafor (AMD3100) and grafted with different amounts of PEG (2 kDa). CXCR4 antagonism of the synthesized polymers was determined using receptor redistribution assay. Inhibition of cancer cell invasion by the polyplexes of the synthesized polymers was assessed using Boyden-chamber method. Transfection activity of DNA polyplexes formed with the synthesized polymers was evaluated in U2OS osteosarcoma and B16F10 melanoma cells.

Results

Our results demonstrate that modification of PAMD with PEG decreased toxicity of the polymers, while preserving their CXCR4 antagonism. Polyplexes prepared with PEG-PAMD inhibited invasion of cancer cells to an extent similar to the commercial CXCR4 antagonist Plerixafor. Negative effect of PEG on transfection activity of PEG-PAMD polyplexes could be overcome by using polyplexes formulated with a mixture of PAMD and PEG-PAMD.

Conclusion

Modification of PAMD with PEG is a viable strategy to preserve the desirable CXCR4 antagonism and ability to inhibit cancer cell invasion of PAMD, while improving safety and colloidal stability of the PAMD polyplexes.     

Microspheres Prepared with PLGA Blends for Delivery of Dexamethasone for Implantable Medical Devices

Purpose

To develop and characterize microspheres using poly (lactic-co-glycolic acid) (PLGA) blends (PLGA5050 (25 KD) and PLGA6535 (70 KD)) for dexamethasone delivery to prevent foreign body response to implantable biosensors.

Methods

A single emulsion based oil/water solvent evaporation/extraction method was used to prepare microspheres.

Results

All the microspheres prepared exhibited the typical triphasic release profile, but with different initial burst release, lag phase and zero order release rates. The burst release was reduced when the two PLGA were mixed at a molecular level, whereas increase in burst release was observed when phase separation occurred. Microspheres prepared using PLGA blends had significantly shorter lag phase. The activation energy (Ea) of dexamethasone release from microspheres was similar to the Ea value of PLGA degradation. The release kinetics were significantly enhanced under accelerated conditions (45 and 53°C) without altering the release mechanism of the post-burst phase. A rank order correlation between accelerated and “real-time” release kinetics was observed.

Conclusions

Polymer blends of PLGA can produce microspheres with reduced lag time. The accelerated release testing conditions investigated can discriminate the formulations and predict “real-time” release. Such accelerated release testing can be used as a rapid screening method to facilitate formulation development.     

Thermosensitive mPEG-b-PA-g-PNIPAM Comb Block Copolymer Micelles: Effect of Hydrophilic Chain Length and Camptothecin Release Behavior

Purpose

Block copolymer micelles are extensively used as drug controlled release carriers, showing promising application prospects. The comb or brush copolymers are especially of great interest, whose densely-grafted side chains may be important for tuning the physicochemical properties and conformation in selective solvents, even in vitro drug release. The purpose of this work was to synthesize novel block copolymer combs via atom transfer radical polymerization, to evaluate its physicochemical features in solution, to improve drug release behavior and to enhance the bioavailablity, and to decrease cytotoxicity.

Methods

The physicochemical properties of the copolymer micelles were examined by modulating the composition and the molecular weights of the building blocks. A dialysis method was used to load hydrophobic camptothecin (CPT), and the CPT release and stability were detected by UV–vis spectroscopy and high-performance liquid chromatography, and the cytotoxicity was evaluated by MTT assays.

Results

The copolymers could self-assemble into well-defined spherical core-shell micelle aggregates in aqueous solution, and showed thermo-induced micellization behavior, and the critical micelle concentration was 2.96–27.64 mg L^?1. The micelles were narrow-size-distribution, with hydrodynamic diameters about 128–193 nm, depending on the chain length of methoxy polyethylene glycol (mPEG) blocks and poly(N-isopropylacrylamide) (PNIPAM) graft chains or/and compositional ratios of mPEG to PNIPAM. The copolymer micelles could stably and effectively load CPT but avoid toxicity and side-effects, and exhibited thermo-dependent controlled and targeted drug release behavior.

Conclusions

The copolymer micelles were safe, stable and effective, and could potentially be employed as CPT controlled release carriers.     

Spray-Dried Chitosan Microparticles for Cellular Delivery of an Antigenic Protein: Physico-chemical Properties and Cellular Uptake by Dendritic Cells and Macrophages

Purpose

Spray-dried chitosan microparticles for cellular delivery of antigen to dendritic cells (DC) and macrophages (M?) were investigated.

Methods

Chitosan microparticles were prepared by spray drying. For comparison, poly(lactic-co-glycolic acid) (PLGA) and poly(α-butyl cyanoacrylate) (BCA) micro-/nanoparticles were generated. Bovine serum albumin (BSA) was used as a model antigen. The particles were characterized in terms of size, morphology, surface charge, surface composition, protein content, entrapment efficiency, in vitro release, and protein integrity. Additionally, they were subject to cell viability and cellular uptake study with DC and M?.

Results

Size of chitosan, PLGA, and BCA micro-/nanoparticles ranged between 3.11–7.18, 0.94–6.26, and 0.30–6.34 μm, respectively. Particle morphology and in vitro protein release varied, depending on polymer type, particle composition and preparation process parameters. Chitosan microparticles were cationic, while PLGA microparticles were neutral. BCA micro-/nanoparticles were either anionic or cationic, according to polymerization pH. Protein content and entrapment efficiency of chitosan and PLGA microparticles were relatively consistent. Only integrity and conformational structure of protein encapsulated in chitosan microparticles were completely retained. Chitosan and PLGA microparticles were non-toxic to DC and M?, but the former were internalized more efficiently.

Conclusions

Spray-dried chitosan microparticles delivered the antigen efficiently to DC and M?.     

Nucleic Acid Aptamers as Stabilizers of Proteins: The Stability of Tetanus Toxoid

Purpose

Exposure of tetanus toxoid to moisture leads to its aggregation and reduction of potency. The aim of this work was to use SELEX (systematic evolution of ligands by exponential enrichment) protocol and select aptamers which recognize tetanus toxoid (M_r ~150 kDa) with high affinity.

Methods

Colyophilized preparations of tetanus toxoid and specific aptamers were encapsulated in PLGA microspheres and sustained release of the antigen was observed up to 55 days using different techniques.

Results

The total protein released was between 40–55% (24–45% residual antigenicity) in the presence of the aptamers as compared to 25% (11% residual antigenicity) for the antigen alone. We show that instead of inhibiting absorption of moisture, the aptamers blocked the protein unfolding upon absorption of moisture, inhibiting the initiation of aggregation. When exposed to accelerated storage conditions, some of the RNA sequences were able to inhibit moisture-induced aggregation in vitro and retain antigenicity of tetanus toxoid.

Conclusions

Nucleic acid aptamers represent a novel class of protein stabilizers which stabilize the protein by interacting directly with it. This mechanism is unlike that of small molecules which alter the medium properties and hence depend on the stress condition a protein is exposed to.     

Development and In Vitro-In Vivo Evaluation of Polymeric Implants for Continuous Systemic Delivery of Curcumin

Purpose

The introduction of curcumin into clinics is hindered by its low water solubility and poor bioavailability. To overcome these limitations, we developed curcumin implants using poly (??-caprolactone) as the polymeric matrix.

Methods

Implants were prepared by melt-extrusion method; in vitro drug release was optimized for effects of polymer composition, drug load, surface area and water-soluble additives. Implants were also tested under in vivo conditions for cumulative curcumin release, and liver concentration was correlated with its efficacy to modulate selected xenobiotic-metabolizing enzymes (CYP1A1 and GSTM).

Results

Drug release from implants followed biphasic release pattern with Higuchi kinetics and was proportional to the surface area of implants. Drug release increased proportionately from 2 to 10% (w/w) drug load, and incorporation of 10% (w/w) of water-soluble additives (F-68, PEG 8000 and cyclodextrin) did not significantly alter the drug release. In vivo drug release was found to be ??1.8 times higher than in vitro release. Curcumin was detected at 60?±?20 ng/g in the liver after four days of implantation and was almost constant (8?C15 ng/g) for up to 35 days. This time-dependent drop in curcumin level was found to be due to induction of CYP1A1 and GSTM (??) enzymes which led to increased metabolism of curcumin.

Conclusion

Our data showed that these implants were able to release curcumin for long duration and to modulate liver phase I and phase II enzymes, demonstrating curcumin??s biological efficacy delivered via this delivery system.     

Pulsatile Protein Release from Monodisperse Liquid-Core Microcapsules of Controllable Shell Thickness

Purpose

Pulsatile delivery of proteins, in which release occurs over a short time after a period of little or no release, is desirable for many applications. This paper investigates the effect of biodegradable polymer shell thickness on pulsatile protein release from biodegradable polymer microcapsules.

Methods

Using precision particle fabrication (PPF) technology, monodisperse microcapsules were fabricated encapsulating bovine serum albumin (BSA) in a liquid core surrounded by a drug-free poly(lactide-co-glycolide) (PLG) shell of uniform, controlled thickness from 14 to 19 μm.

Results

When using high molecular weight PLG (Mw 88 kDa), microparticles exhibited the desired core-shell structure with high BSA loading and encapsulation efficiency (55–65%). These particles exhibited very slow release of BSA for several weeks followed by rapid release of 80–90% of the encapsulated BSA within 7 days. Importantly, with increasing shell thickness the starting time of the pulsatile release could be controlled from 25 to 35 days.

Conclusions

Biodegradable polymer microcapsules with precisely controlled shell thickness provide pulsatile release with enhanced control of release profiles.