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Fabrication of uniform enzyme-immobilized carbohydrate microparticles with high enzymatic activity and stability via spray drying and spray freeze drying

Shengyu Zhang, Hong Lei, Xingmin Gao, Xingxing Xiong, Winston Duo Wu, Zhangxiong Wu, Xiao Dong Chen

Abstract

Enzyme-immobilized particles with high enzymatic activities are fundamentally and practically important for many areas, such as pharmaceuticals, medicine, and biocatalysis. In this study, by selecting trypsin and trehalose as the representative enzyme and excipient, two techniques, spray drying (SD) and spray freeze drying (SFD), have been utilized to generate enzyme-immobilized particles and comparatively studied. In both methods, uniform enzyme-immobilized microparticles are successfully obtained by using a micro-fluidic aerosol nozzle (MFAN) as the monodisperse droplet generator. The particle morphology, size and inner structure are distinctly different between the SD- and SFD-derived trypsin/trehalose composite microparticles. The former shows crumpled morphology, smaller sizes and dense inner structure while the latter shows spherical and open porous morphology with larger particle sizes. The particle formation processes in both methods are discussed. The more surface-active and large-sized trypsin molecules tend to be accumulated at the air-liquid interface of drying droplets, leading to particle buckling in SD and the formation of thin surface trypsin-enriched layer in SFD. The trypsin enzymatic activity is highly related to the presence of trehalose and the processing method. For the pure trypsin microparticles, SFD leads to a better activity preservation than SD does due to the much higher temperature adopted in SD. The presence of trehalose can significantly protect the enzymatic activity of trypsin, reaching 97.7 ± 2.6% and 97.3 ± 1.6% activity preservation with the optimal trypsin/trehalose mass ratio of 1:1 for both the SD- and SFD-derived microparticles, respectively. The protection of the enzymatic activity originates from the hydrogen bonding formation between trypsin and trehalose and the formation of highly amorphous glass matrices, which decrease enzyme unfolding and aggregation. In terms of process operability, SD offers a rather simple and economic means to produce enzymatic microparticles of high activity with the appropriate dosage of trehalose.

Keywords

Particle processing, Drying technology, Uniform microparticles, Enzyme immobilization, Activity preservation

Spray freeze drying of small nucleic acids as inhaled powder for pulmonary delivery

Wanling Liang, Alan Y.L. Chan, Michael Y.T. Chow, Fiona F.K.Lo, Yingshan Qiu, Philip C.L. Kwok, Jenny K.W. Lam

Abstract

The therapeutic potential of small nucleic acids such as small interfering RNA (siRNA) to treat lung diseases has been successfully demonstrated in many in vivo studies. A major barrier to their clinical application is the lack of a safe and efficient inhaled formulation. In this study, spray freeze drying was employed to prepare dry powder of small nucleic acids. Mannitol and herring sperm DNA were used as bulking agent and model of small nucleic acid therapeutics, respectively. Formulations containing different solute concentration and DNA concentration were produced. The scanning electron microscope (SEM) images showed that the porosity of the particles increased as the solute concentration decreased. Powders prepared with solute concentration of 5% w/v were found to maintain a balance between porosity and robustness. Increasing concentration of DNA improved the aerosol performance of the formulation. The dry powder formulation containing 2% w/w DNA had a median diameter of 12.5 µm, and the aerosol performance study using next generation impactor (NGI) showed an emitted fraction (EF) and fine particle fraction (FPF) of 91% and 28% respectively. This formulation (5% w/v solute concentration and 2% w/w nucleic acid) was adopted subsequently to produce siRNA powder. The gel retardation and liquid chromatography assays showed that the siRNA remained intact after spray freeze drying even in the absence of delivery vector. The siRNA powder formulation exhibited a high EF of 92.4% and a modest FPF of around 20%. Further exploration of this technology to optimise inhaled siRNA powder formulation is warranted.

Keywords

Inhalation, Pulmonary delivery, Small interfering RNA, Spray freeze drying

System-level analysis of a novel air-cooled condenser using spray freezing of phase change materials

Ben Xu, Swanand Bhagwat, Hongxin Xu, Arif Rokoni, Matthew McCarthy, Ying Sun

Abstract

A comprehensive system-level analysis is performed for a novel air-cooled condenser based on spray freezing of phase change materials (PCMs). This novel air-cooled condenser uses PCMs to decouple the process of steam condensation and heat rejection to air in order to significantly improve air-side heat transfer and reduce steam condensation temperature as compared to conventional air-cooled condensers (ACCs). Melting of solid PCM particles in a two-phase PCM slurry flow anchors the steam condensation temperature close to the PCM melting point regardless of the change in ambient air temperature. Spray freezing of millimeter-sized liquid PCM droplets increases the air-side heat transfer coefficient by five times compared to the finned-tubed ACCs. A multiscale model, which directly captures the melting and settling of PCM particles at the microscopic level and accounts for phase change through energy source terms at the macroscopic level, has been developed to simulate the PCM slurry flow over heated tube bundles. Using this multiscale model, the effects of particle volume fraction, Reynolds number, and particle to steam tube diameter ratio on the averaged wall Nusselt number of the steam tubes are investigated. It is found that the averaged wall Nusselt number for a PCM slurry flow of 20% solid fraction achieves a 38% enhancement over the PCM single-phase flow of same Reynolds number. On the air side, the freezing/melting of PCM droplet/particle is approximated based on a 1-D transient heat conduction model and the air-side pressure drop across the PCM droplet array is determined using a 3-D k-ε turbulence model. The performance of this spray-freezing PCM ACC is compared against a baseline ACC of a 500 MWe power plant. It is found that, for comparable footprint area and ambient conditions, the spray-freezing PCM ACC reduces the initial temperature difference to as low as 16.8 °C and provides up to 10.8 MW net power production gain compared to the baseline ACC.

Keywords

Phase change material; Dry cooling; Slurry flow and heat transfer; Power plant cooling

Spray-freeze-drying in the manufacture of pharmaceuticals

Abstract

Over the last decade, the development of new drug delivery methods and devices for dry powder inhalation1, needle-free intradermal powder injection2 or sustained parenteral drug delivery3 has led to an increasing demand for powder formulations incorporating an active pharmaceutical ingredient (API)4,5.

In contrast to the production and handling of powders for oral dosage forms, methods to prepare stable biopharmaceutical powders are limited due to the sensitivity of peptides and proteins to powder processing conditions4,6. Furthermore, bulk properties such as size distribution or density of the final particles are different depending on the application4,5,7. Particles for dry powder inhalation, for example, should have particles of less than 5 µm in diameter and a narrow size distribution5, whereas particles for needle-free ballistic injection must be 30-60µm with a density greater than 0.7 g/ml7 for a successful intradermal delivery. One of the most commonly used methods of drying protein formulations is freeze-drying8-11 but, because it does not involve droplet formulation, the final dry cake can only be reduced to particles by subsequent mechanical milling or grinding4. Some reported disadvantages associated with this method of powder manufacturing include the following:

Production of particles with diameters above 1 mm
Broad particle size distributions
Changes of solid state and degradation of the peptide or protein due to heat generation during inter-particle collision12,13

Protein Spray-Freeze Drying. Effect of Atomization Conditions on Particle Size and Stability

Henry R CostantinoLaleh FirouzabadianKen HogelandChichih WuChris BeganskiKaren G CarrasquilloMelissa CórdovaKai GriebenowStephen E ZaleMark A Tracy

Abstract

Purpose. To investigate the effect of atomization conditions on particle size and stability of spray-freeze dried protein.

Methods. Atomization variables were explored for excipient-free (no zinc added) and zinc-complexed bovine serum albumin (BSA). Particle size was measured by laser diffraction light scattering following sonication in organic solvent containing poly(lactide-co-glycolide) (PLG). Powder surface area was determined from the N2 vapor sorption isotherm. Size-exclusion chromatography (SEC) was used to assess decrease in percent protein monomer. Fourier-transform infrared (FTIR) spectroscopy was employed to estimate protein secondary structure. PLG microspheres were made using a non-aqueous, cryogenic process and release of spray-freeze dried BSA was assessed in vitro.

Results. The most significant atomization parameter affecting particle size was the mass flow ratio (mass of atomization N2 relative to that for liquid feed). Particle size was inversely related to specific surface area and the amount of protein aggregates formed. Zinc-complexation reduced the specific surface area and stabilized the protein against aggregation. FTIR data indicated perturbations in secondary structure upon spray-freeze drying for both excipient-free and zinc-complexed protein.

Conclusions. Upon sonication, spray-freeze dried protein powders exhibited friability, or susceptibility towards disintegration. For excipient-free protein, conditions where the mass flow ratio was > ∼0.3 yielded sub-micron powders with relatively large specific surface areas. Reduced particle size was also linked to a decrease in the percentage of protein monomer upon drying. This effect was ameliorated by zinc-complexation, via a mechanism involving reduction in specific surface area of the powder rather than stabilization of secondary structure. Reduction of protein particle size was beneficial in reducing the initial release (burst) of the protein encapsulated in PLG microspheres.

Keywords

particle size, PLG microspheres, protein delivery, spray-freeze drying, stability

Spray-freeze-drying of nanosuspensions: the manufacture of insulin particles for needle-free ballistic powder delivery

Heiko Schiffter, Jamie Condliffe, Sebastian Vonhoff

Abstract

The feasibility of preparing microparticles with high insulin loading suitable for needle-free ballistic drug delivery by spray-freeze-drying (SFD) was examined in this study. The aim was to manufacture dense, robust particles with a diameter of around 50 µm, a narrow size distribution and a high content of insulin. Atomization using ultrasound atomizers showed improved handling of small liquid quantities as well as narrower droplet size distributions over conventional two-fluid nozzle atomization. Insulin nanoparticles were produced by SFD from solutions with a low solid content (<10 mg ml−1) and subsequent ultra-turrax homogenization. To prepare particles for needle-free ballistic injection, the insulin nanoparticles were suspended in matrix formulations with a high excipient content (>300 mg ml−1) consisting of trehalose, mannitol, dextran (10 kDa) and dextran (150 kDa) (abbreviated to TMDD) in order to maximize particle robustness and density after SFD. With the increase in insulin content, the viscosity of the nanosuspensions increased. Liquid atomization was possible up to a maximum of 250 mg of nano-insulin suspended in a 1.0 g matrix. However, if a narrow size distribution with a good correlation between theoretical and measurable insulin content was desired, no more than 150 mg nano-insulin could be suspended per gram of matrix formulation. Particles were examined by laser light diffraction, scanning electron microscopy and tap density testing. Insulin stability was assessed using size exclusion chromatography (SEC), reverse phase chromatography and Fourier transform infrared (FTIR) spectroscopy. Densification of the particles could be achieved during primary drying if the product temperature (Tprod) exceeded the glass transition temperature of the freeze concentrate (Tg′) of −29.4°C for TMDD (3∶3∶3∶1) formulations. Particles showed a collapsed and wrinkled morphology owing to viscous flow of the freeze concentrate. With increasing insulin loading, the d (v, 0.5) of the SFD powders increased and particle size distributions got wider. Insulin showed a good stability during the particle formation process with a maximum decrease in insulin monomer of only 0.123 per cent after SFD. In accordance with the SEC data, FTIR analysis showed only a small increase in the intermolecular β-sheet of 0.4 per cent after SFD. The good physical stability of the polydisperse particles made them suitable for ballistic injection into tissue-mimicking agar hydrogels, showing a mean penetration depth of 251.3 ± 114.7 µm.

Keywords

spray-freeze-drying, needle-free injection, insulin delivery, nanosuspensions, protein formulation

A spray-freezing approach to reduced graphene oxide/MoS2 hybrids for superior energy storage

Tao Cheng, Jin Xu, Ziqi Tan, Jianglin Ye, Zhuchen Tao, Zhenzhen Du, Ying Wu, Shuilin Wu, Hengxing Ji, Yan Yu. Yanwu Zhu

Abstract

A three-dimensional (3D) architectural hybrid, composed of reduced graphene oxide (RGO) and ultrathin MoS2 layers, is fabricated by a facile spray-freezing method. The spray-freezing to liquid nitrogen rapidly freezes the precursor droplets which avoids phase separation and restacking of MoS2 and RGO platelets, and the following drying/annealing results in the porous 3D structure. The as-prepared 3D architectural RGO/MoS2 hybrid has a high surface area of 128 m2 g−1, a porous structure and a good electrical conductivity. In LIBs, the capacity of RGO/MoS2 anode (with an optimized MoS2 content of 55 wt%) remains 1197 mAh g−1 after 400 cycles of measurement at a current density of 1 A g−1 and it remains 892 mAh g−1 over 400 cycles at a current density of 2 A g−1. A capacity of 723 mAh g−1 is obtained at a current of 10 A g−1. As for the anode (with an optimized MoS2 content of 74 wt%) in SIBs, a high initial discharge capacity of 1315 mAh g−1, a superior rate capacity of 470 mAh g−1 at 1 A g−1 and an excellent cycling stability (518 mAh g−1 after 200 cycles at 0.5 A g−1) are demonstrated.

Keywords

Reduced graphene oxide, Molybdenum disulfide, Lithium ion batteries, Sodium ion  batteries, Electrochemistry

Development of Inhalable Dry Powder Formulations Loaded with Nanoparticles Maintaining Their Original Physical Properties and Functions

Okuda T

Abstract

Functional nanoparticles, such as liposomes and polymeric micelles, are attractive drug delivery systems for solubilization, stabilization, sustained release, prolonged tissue retention, and tissue targeting of various encapsulated drugs. For their clinical application in therapy for pulmonary diseases, the development of dry powder inhalation (DPI) formulations is considered practical due to such advantages as: (1) it is noninvasive and can be directly delivered into the lungs; (2) there are few biocomponents in the lungs that interact with nanoparticles; and (3) it shows high storage stability in the solid state against aggregation or precipitation of nanoparticles in water. However, in order to produce effective nanoparticle-loaded dry powders for inhalation, it is essential to pursue an innovative and comprehensive formulation strategy in relation to composition and powderization which can achieve (1) the particle design of dry powders with physical properties suitable for pulmonary delivery through inhalation, and (2) the effective reconstitution of nanoparticles that will maintain their original physical properties and functions after dissolution of the powders. Spray-freeze drying (SFD) is a relatively new powderization technique combining atomization and lyophilization, which can easily produce highly porous dry powders from an aqueous sample solution. Previously, we advanced the optimization of components and process conditions for the production of SFD powders suitable to DPI application. This review describes our recent results in the development of novel DPI formulations effectively loaded with various nanoparticles (electrostatic nanocomplexes for gene therapy, liposomes, and self-assembled lipid nanoparticles), based on SFD.

Keywords

aerosol delivery; dry powder inhaler; powder-particle design; reconstituted nanoparticle; spray-freeze drying