Precision Particle Fabrication Research Articles

Long-acting injectable donepezil microspheres: Formulation development and evaluation

Novel formulations of donepezil (DNP)-loaded microspheres based on a bio-degradable polymer of poly(lactic-co-glycolic acid) (PLGA) with a one-month duration of effect were developed, aimed at reducing dosing frequency and adverse effects and improving patient adherence. The spherical and monodispersed DNP-loaded microspheres were precisely fabricated by the Inventage Lab Precision Particle Fabrication method (IVL-PPFM®) based on micro-electromechanical systems (MEMS) and microfluidic technology. The types of polymers and end-groups, the drug/polymer ratio (DPR), and the routes of administration for DNP were studied to ensure an effective concentration and desired duration. Laser-light particle size analysis and scanning electron microscopy were used to characterization. Also, non-clinical animal models of beagle dogs are used to optimize DNP formulations and evaluate their pharmacokinetic properties. The PK results showed that the DPR was a critical factor in determining the exposure level and duration of DNR release. Furthermore, the lactide ratio, which varied depending upon the type of polymer, determined the hydrophobic interaction and was also an important factor affecting the desired DNP release. Since DNP shows a large inter-species variation between dogs and humans, PK modeling and simulation of the reference drug (i.e., Aricept®) and DNP-loaded microspheres were used for formulation development to overcome and interpret these variations. In addition, the developed PK model was extrapolated to humans using the estimated PK parameter and published clinical pharmacology data for DNP. The predicted PK profile of the DNP-loaded microsphere in humans showed that the formulation with PLGA 7525A and the DPR of 1/9 could maintain drug concentration for a month and could control initial burst release. The data obtained from the study could be used as scientific evidence for decision-making in future formulation development.

Comparative Evaluation of Safety and Efficacy of a Novel Hyaluronic Acid-polynucleotide/Poly-L-lactic Acid Composite Dermal Filler

Poly-L-lactic acid (PLLA) is widely used in tissue engineering. The natural polymer hyaluronic acid (HA) shows excellent biocompatibility and affects cell signaling, proliferation, and differentiation. In addition, a polynucleotide (PN) induces cell growth of human skin fibroblasts and osteoblasts. In this study, we evaluated the properties, safety, and efficacy of a novel composite filler consisting of cross-linked HA with PN in combination with monodisperse PLLA microspheres manufactured using Inventage Lab Precision Particle Fabrication method. The composition of the filler and characteristics of the microspheres were examined via scanning electron microscopy, particle size analysis, gel permeation chromatography, and rheology and osmolality measurement. Additionally, safety and efficacy of HA-PN/PLLA composite filler were conducted in in vitro and in vivo. Analysis of PLLA microspheres revealed spherical surfaces and a narrower particle size distribution than that in PLLA filler. HA-PN/PLLA composite filler had higher viscosity and elasticity values and similar osmolality as compared to those of HA and PN fillers. The nontoxicity in in vitro and in vivo tests reflected that the composite filler may be safe for human use. In addition, the composite filler maintained a more stable volume than did HA filler for 24 weeks after administration in HWY/Slc hairless rats. Furthermore, the results support the effect of HA-PN/PLLA in restoring skin structure. Altogether, these data suggest that the novel composite filler might be a safe and effective option in terms of tissue integration, clinical management during delivery and high esthetic durability. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Uniform biodegradable microparticle systems for controlled release

Drug delivery methods can impact efficacy as much as the nature of the drug itself. Microparticles made of biodegradable polymers such as poly(D,l-lactide-co-glycolide) and poly(lactic acid) (PLA) have been studied extensively for controlled release of diverse drugs. By using a modified solvent extraction/evaporation method called precision particle fabrication (PPF), uniform microparticles such as single-wall microspheres, double-wall microspheres and liquid-core microcapsules have been fabricated with precise control of their geometric structures. By producing particles of uniform size, which has crucial impact on drug release behaviors, PPF-fabricated microparticles provide unique insights about drug release mechanism. Using small-molecule and macromolecule model drugs, our group demonstrated that physicochemical properties of the polymers and drugs and structural properties of the matrix can greatly impact drug distribution within microparticles, particle erosion and drug release rates. By careful selection of particle size and shell thickness, uniform microparticles can achieve “zero-order”, pulsatile or tandem release of drugs.

Pulsatile protein release from monodisperse liquid-core microcapsules of controllable shell thickness.

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

Controlled protein release from monodisperse biodegradable double-wall microspheres of controllable shell thickness

Biodegradable polymer microparticles are promising delivery depots for protein therapeutics due to their relatively simple fabrication and facile administration. Double-wall microspheres (DWMS) comprising a core and shell made of two distinct polymers may provide enhanced control of the drug release profiles. Using precision particle fabrication (PPF) technology, monodisperse DWMS were fabricated with model protein bovine serum albumin (BSA)-loaded poly(lactide-co-glycolide) (PLG) core and drug-free poly(d,l-lactic acid) (PDLL) shell of uniform thickness. Monolithic single-wall microspheres were also fabricated to mimic the BSA-loaded PLG core. Using ethyl acetate and dichloromethane as shell- and core-phase solvents, respectively, BSA was encapsulated selectively in the core region within DWMS with higher loading and encapsulation efficiency compared to using dichloromethane as core and shell solvents. BSA in vitro release rates were retarded by the presence of the drug-free PDLL shell. Moreover, increasing PDLL shell thickness resulted in decreasing BSA release rate. With a 14-μm thick PDLL shell, an extended period of constant-rate release was achieved.

Protein Encapsulation in and Release from Monodisperse Double-Wall Polymer Microspheres

Biodegradable polymer double-wall microspheres (DWMS) are promising vehicles for macromolecular therapeutics such as proteins and peptides. Using precision particle fabrication (PPF) technology, uniform DWMS with outer diameter approximately 55 μm were fabricated comprising poly(lactide-co-glycolide) cores encapsulating bovine serum albumin (BSA) and approximately 10 μm thick, drug-free, poly(lactic acid) (PLA) shells of varying PLA molecular weight. Also, monolithic single-wall microspheres (SWMS) were fabricated to mimic the BSA-loaded core. The use of relatively fast-extracting ethyl acetate and slowly extracting dichloromethane as shell- and core-phase solvents, respectively, was found to produce DWMS with well-defined core-shell structure, high BSA encapsulation efficiency, and the desired localization of protein in the particle core. Initial protein distribution, particle erosion, and in vitro protein release from DWMS and SWMS were examined. The presence of a BSA-free shell in DWMS decreased the protein release rate and extended the duration of release from approximately 50 days to 70-80 days, demonstrating the capacity of such DWMS to provide enhanced control of protein delivery rates.

Combined modality doxorubicin-based chemotherapy and chitosan-mediated p53 gene therapy using double-walled microspheres for treatment of human hepatocellular carcinoma

The therapeutic efficiency of combined chemotherapy and gene therapy on human hepatocellular carcinoma HepG2 cells was investigated using double-walled microspheres that consisted of a poly(d,l-lactic-co-glycolic acid) (PLGA) core surrounded by a poly(l-lactic acid) (PLLA) shell layer and fabricated via the precision particle fabrication (PPF) technique. Here, double-walled microspheres were used to deliver doxorubicin (Dox) and/or chitosan-DNA nanoparticles containing the gene encoding the p53 tumor suppressor protein (chi-p53), loaded in the core and shell phases, respectively. Preliminary studies on chi-DNA nanoparticles were performed to optimize gene transfer to HepG2 cells. The transfection efficiency of chi-DNA nanoparticles was optimal at an N/P ratio of 7. In comparison to the 25-kDa branched polyethylenimine (PEI), chitosan showed no inherent toxicity towards the cells. Next, the therapeutic efficiencies of Dox and/or chi-p53 in microsphere formulations were compared to free drug(s) and evaluated in terms of growth inhibition, and cellular expression of tumor suppressor p53 and apoptotic caspase 3 proteins. Overall, the combined Dox and chi-p53 treatment exhibited enhanced cytotoxicity as compared to either Dox or chi-p53 treatments alone. Moreover, the antiproliferative effect was more substantial when cells were treated with microspheres than those treated with free drugs. High p53 expression was maintained during a five-day period, and was largely due to the controlled and sustained release of the microspheres. Moreover, increased activation of caspase 3 was observed, and was likely to have been facilitated by high levels of p53 expression. Overall, double-walled microspheres present a promising dual anticancer delivery system for combined chemotherapy and gene therapy.

Monodisperse double-walled microspheres loaded with chitosan-p53 nanoparticles and doxorubicin for combined gene therapy and chemotherapy

We have designed and evaluated a dual anticancer delivery system to provide combined gene therapy and chemotherapy. Double-walled microspheres consisting of a poly(d,l-lactic-co-glycolic acid) (PLGA) core surrounded by a poly(lactic acid) (PLA) shell were fabricated via the precision particle fabrication (PPF) technique. We make use of the advantages of double-walled microspheres to deliver chitosan–DNA nanoparticles containing the gene encoding the p53 tumor suppressor protein (chi-p53) and/or doxorubicin (Dox), loaded in the shell and core phases, respectively. Different molecular weights of PLA were used to form the shell layer for each formulation. The microspheres were monodisperse with a mean diameter of 65 to 75μm and uniform shell thickness of 8 to 17μm. Blank and Dox-loaded microspheres typically exhibited a smooth surface with relatively few small pores, while chi-microspheres containing p53 nanoparticles, with and without Dox, presented rough and porous surfaces. The encapsulation efficiency of Dox was significantly higher when it was encapsulated alone compared to co-encapsulation with chi-p53 nanoparticles. The encapsulation efficiency of chi-p53 nanoparticles, on the other hand, was not affected by the presence of Dox. As desired, chi-p53 nanoparticles were released first, followed by simultaneous release of chi-p53 nanoparticles and Dox at a near zero-order rate. Thus, we have demonstrated that the PPF method is capable of producing double-walled microspheres and encapsulating dual agents for combined modality treatment, such as gene therapy and chemotherapy.

PPF microsphere depot sustains NSAID blood levels with infusion‐like kinetics without ‘burst’

Management of pain is of paramount importance for a myriad of indications in human and animal health. Unfortunately, the administration of pain therapeutics is often complicated by insufficient control over pharmacokinetic profiles as a result of frequent oral dosing. Attempts to sustain and tightly control the concentration of these drugs in the blood via controlled release injectable formulations have typically resulted in drug 'burst', followed by marginal control over the ensuing pharmacokinetics. Here, precision particle fabrication (PPF) technology was used to produce uniform microspheres encapsulating the nonsteroidal anti-inflammatory drug meloxicam. After subcutaneous injection, plasma concentrations of meloxicam were held constant in canines for more than 2 weeks without initial drug burst. Pharmacokinetic profiles were accurately modeled using equations typically applied to steady-state infusion. PPF microsphere depots of pain therapeutics or other compounds may ultimately improve safety and sustain the efficacy of medications where such controlled uniform exposure would be therapeutically beneficial.

Production and characterization of polymer microspheres containing trace explosives using precision particle fabrication technology

Well characterized test materials are essential for validating the performance of current trace explosive detection systems. These test materials must replicate trace explosive contamination in the form of small particles with characteristic diameters in the micrometer range. In this work, Precision Particle Fabrication was used to fabricate monodisperse polymer microspheres that contain high explosives. Three high explosives were successfully incorporated into the microspheres. Ion mobility spectrometry confirmed that the encapsulation efficiency was typically greater than 50%, with some suspected loss to the aqueous phase during production. This study demonstrates that, with this technique, polymer microspheres containing explosives can be produced with sufficient encapsulation, along with tightly controlled particle size distributions at high production rates. These microspheres have proven to be a valuable test material for trace explosive detectors because of their highly precise size, shape and explosive composition.

Microsphere-Based Seamless Scaffolds Containing Macroscopic Gradients of Encapsulated Factors for Tissue Engineering

Spatial and temporal control of bioactive signals in three-dimensional (3D) tissue engineering scaffolds is greatly desired. Coupled together, these attributes may mimic and maintain complex signal patterns, such as those observed during axonal regeneration or neovascularization. Seamless polymer constructs may provide a route to achieve spatial control of signal distribution. In this study, a novel microparticle-based scaffold fabrication technique is introduced as a method to create 3D scaffolds with spatial control over model dyes using uniform poly(D,L-lactide-co-glycolide) microspheres. Uniform microspheres were produced using the Precision Particle Fabrication technique. Scaffolds were assembled by flowing microsphere suspensions into a cylindrical glass mold, and then microspheres were physically attached to form a continuous scaffold using ethanol treatment. An ethanol soak of 1 h was found to be optimum for improved mechanical characteristics. Morphological and physical characterization of the scaffolds revealed that microsphere matrices were porous (41.1 +/- 2.1%) and well connected, and their compressive stiffness ranged from 142 to 306 kPa. Culturing chondrocytes on the scaffolds revealed the compatibility of these substrates with cell attachment and viability. In addition, bilayered, multilayered, and gradient scaffolds were fabricated, exhibiting excellent spatial control and resolution. Such novel scaffolds can serve as sustained delivery devices of heterogeneous signals in a continuous and seamless manner, and may be particularly useful in future interfacial tissue engineering investigations.

Monodisperse Gelatin Microspheres as a Drug Delivery Vehicle: Release Profile and Effect of Crosslinking Density

Uniform gelatin microspheres (GMS) of a wet size of 100 microm in diameter were fabricated by the electric field assisted precision particle fabrication (E-PPF) method and crosslinked with different glutaraldehyde (GA) concentrations to study the effect of the crosslinking density on drug release. The drug release profiles of the crosslinked GMS were studied along with the intraparticle drug distribution and the particle degradation characteristics. Due to the concentration gradient of GA along the diffusion path into the GMS, the crosslinking density is higher on the GMS surface, making it less susceptible to degradation. As a result, the GMS with higher GA concentrations (0.375-0.875%) exhibited a highly resistant surface toward enzymatic degradation. On the other hand, the amount of drug complexation at the surface decreases as the GA concentration increases, which can be attributed to the lowered basicity of gelatin caused by the increased crosslinking density. These factors collectively affect the drug release kinetics and give rise to similar release profiles for GMS above a GA concentration of 0.375%.

NanoCipro encapsulation in monodisperse large porous PLGA microparticles

Pulmonary drug delivery of controlled release formulations may provide an effective adjunct approach to orally delivered antibiotics for clearing persistent lung infections. Dry powder formulations for this indication should possess characteristics including; effective deposition to infected lung compartments, persistence at the infection site, and steady release of antibiotic. Large porous particles (∼ 10–15μm) have demonstrated effective lung deposition and enhanced lung residence as a result of their large diameter and reduced clearance by macrophages in comparison to small microparticles (∼ 1–5μm). In this report, Precision Particle Fabrication technology was used to create monodisperse large porous particles of poly( d, l-lactic-co-glycolic acid) (PLGA) utilizing oils as extractable porogens. After extraction, the resulting large porous PLGA particles exhibited a low density and a web-like or hollow interior depending on porogen concentration and type, respectively. Ciprofloxacin nanoparticles (nanoCipro) created by homogenization in dichloromethane, possessed a polymorph with a decreased melting temperature. Encapsulating nanoCipro in large porous PLGA particles resulted in a steady release of ciprofloxacin that was extended for larger particle diameters and for the solid particle morphology in comparison to large porous particles. The encapsulation efficiency of nanoCipro was quite low and factors impacting the entrapment of nanoparticles during particle formation were elucidated. A dry powder formulation with the potential to control particle deposition and sustain release to the lung was developed and insight to improve nanoparticle encapsulation is discussed.

Macromolecule Release from Monodisperse PLG Microspheres: Control of Release Rates and Investigation of Release Mechanism

Novel macromolecular therapeutics such as peptides, proteins, and DNA are advancing rapidly toward the clinic. Because of typically low oral bioavailability, macromolecule delivery requires invasive methods such as frequently repeated injections. Parenteral depots including biodegradable polymer microspheres offer the possibility of reduced dosing frequency but are limited by the inability to adequately control delivery rates. To control release and investigate release mechanisms, we have encapsulated model macromolecules in monodisperse poly(D,L-lactide-co-glycolide) (PLG) microspheres using a double-emulsion method in combination with the precision particle fabrication technique. We encapsulated fluorescein-dextran (F-Dex) and sulforhodamine B-labeled bovine serum albumin (R-BSA) into PLG microspheres of three different sizes: 31, 44, and 80 microm and 34, 47, and 85 microm diameter for F-Dex and R-BSA, respectively. The in vitro release profiles of both compounds showed negligible initial burst. During degradation and release, the microspheres hollowed and swelled at critical time points dependant upon microsphere size. The rate of these events increased with microsphere size resulting in the largest microspheres exhibiting the fastest overall release rate. Monodisperse microspheres may represent a new delivery system for therapeutic proteins and DNA and provide enhanced control of delivery rates using simple injectable depot formulations.

Uniform Biodegradable Hydrogel Microspheres Fabricated by a Surfactant‐Free Electric‐Field‐Assisted Method

Uniform biodegradable hydrogel microspheres (HMS) with precisely controlled size have been fabricated using an electric-field-assisted precision particle fabrication technique. Particle agglomeration was prevented by charging the hydrogel drops and allowing Coulomb repulsion to separate them. As a result, surfactant-free and non-toxic particle fabrication was possible and the resulting microspheres were most suitable for biomedical and food-related applications. Due to the size uniformity, the present HMS may serve as a convenient yet most accurate vehicle for controlled delivery of therapeutic agents and other active ingredients.

Monodisperse Liquid-filled Biodegradable Microcapsules

Encapsulation of liquids into biodegradable polymer microcapsules has been a challenging task due to production limitations stemming from solution viscosity, phase stabilization, molecular localization, and scalable production. We report an extension of Precision Particle Fabrication (PPF) technology for the production of monodisperse liquid-filled microcapsules containing an oil or aqueous core and contrast these results to double-walled microspheres. PPF technology utilizes a coaxial nozzle to produce a liquid core jet surrounded by a polymer annular jet, which is further encompassed by a non-solvent carrier stream, typically 0.5% wt/vol polyvinyl alcohol in water. Jet diameters are controlled by the volumetric flow rate of each phase. The compound jet is then disrupted into uniform core/shell droplets via a controllable acoustic wave and shell material is hardened by solvent extraction. Monodisperse polymeric microcapsules demonstrated a narrow size distribution and the formation of a continuous shell leading to efficient encapsulation of various liquid cores. The intermingling of core and shell phases and the localization of different molecular probes (fluorescent dyes and fluorescently labeled proteins) to the core or shell phase provided additional evidence of phase separation and molecular partitioning, respectively. We also demonstrate the pulsatile release of bovine serum albumin encapsulated in an aqueous core. PPF technology provided exceptional control of the overall size and shell thickness of microcapsules filled with various types of oil or water. This technique may enable advanced delivery profiles of pharmaceuticals or nutraceuticals.

Three-month, zero-order piroxicam release from monodispersed double-walled microspheres of controlled shell thickness.

Double-walled microspheres represent an increasingly important class of drug delivery devices that provide enhanced control of drug delivery schedules. Clearly, the overall particle size and shell thickness are important parameters in modulating the drug release rates. Precision particle fabrication technology has been used to fabricate double-walled microspheres of predefined uniform diameters of 40-60 microm exhibiting a poly(D,L-lactide-co-glycolide) (PLG) core and poly(L-lactide) (PL) shell of controllable thickness from approximately 2 to 10 microm. The release of a model small-molecule drug, piroxicam, from uniform microspheres of pure PLG and PL is compared to the release from double-walled microspheres exhibiting different PL shell thicknesses. The presence of the PL shell enveloping a PLG core essentially eliminated the initial "burst" of piroxicam that was observed when the drug was released from pure PLG microspheres. In addition, increasing the PL shell thickness shifted the release profile from a biphasic shape for pure PLG microspheres to zero-order piroxicam release over 3 months for the thickest (approximately 10 microm) PL shell.