Effective mRNA pulmonary delivery by dry powder formulation of PEGylated synthetic KL4 peptide

Effective mRNA pulmonary delivery by dry powder formulation of PEGylated synthetic KL4 peptide

Abstract

Pulmonary delivery of messenger RNA (mRNA) has considerable potential as therapy or vaccine for a range of lung diseases. Inhaled dry powder formulation of mRNA is particularly attractive as it has superior stability and dry powder inhaler is relatively easy to use. A safe and effective mRNA delivery vector as well as a suitable particle engineering method are required to produce a dry powder formulation that is respirable and mediates robust transfection in the lung. Here, we introduce a novel RNA delivery vector, PEG12KL4, in which the synthetic cationic KL4 peptide is attached to a monodisperse linear PEG of 12-mers. The PEG12KL4 formed nano-sized complexes with mRNA at 10:1 ratio (w/w) and mediated effective transfection on human lung epithelial cells. PEG12KL4/mRNA complexes were successfully formulated into dry powder by spray drying (SD) and spray freeze drying (SFD) techniques. Both SD and SFD powder exhibited satisfactory aerosol properties for inhalation. More importantly, the biological activity of the PEG12KL4 /mRNA complexes were successfully preserved after drying. Using luciferase mRNA, the intratracheal administration of the liquid or powder aerosol of PEG12KL4 /mRNA complexes at a dose of 5 μg mRNA resulted in luciferase expression in the deep lung region of mice 24 h post-transfection. The transfection efficiency was superior to naked mRNA or lipoplexes (Lipofectamine 2000), in which luciferase expression was weaker and restricted to the tracheal region only. There was no sign of inflammatory response or toxicity of the PEG12KL4 /mRNA complexes after single intratracheal administration. Overall, PEG12KL4 is an excellent mRNA transfection agent for pulmonary delivery. This is also the first study that successfully demonstrates the preparation of inhalable dry powder mRNA formulations with in vivo transfection efficiency, showing the great promise of PEG12KL4 peptide as a mRNA delivery vector candidate for clinical applications.

Keywords

Inhalation, mRNA transfection, PEGylation, Peptide, Spray drying, Spray freeze drying

Effect of Particle Formation Process on Characteristics and Aerosol Performance of Respirable Protein Powders.

Effect of Particle Formation Process on Characteristics and Aerosol Performance of Respirable Protein Powders

Brunaugh AD, Wu T, Kanapuram SR, Smyth HDC

Abstract

Pulmonary delivery of biopharmaceuticals may enable targeted local therapeutic effect and noninvasive systemic administration. Dry powder inhaler (DPI) delivery is an established patient-friendly approach for delivering large molecules to the lungs; however, the complexities of balancing protein stability with aerosol performance require that the design space of biopharmaceutical DPI formulations is rigorously explored. Utilizing four rationally selected formulations obtained using identical atomization conditions, an extensive study of the effect of the particle formation process (spray drying or spray freeze-drying) on powder properties, aerosol performance, and protein stability was performed. Multiple linear regression analysis was used to understand the relationship between powder properties, device dispersion mechanism, and aerosol performance. Spray drying and spray freeze-drying, despite the same spraying conditions, produced powders with vastly different physical characteristics, though similar aerosol performance. The resulting regression model points to the significance of particle size, density, and surface properties on the resulting aerosol performance, with these factors weighing differently according to the device dispersion mechanism utilized (shear-based or impaction-based). The physical properties of the produced spray dried and spray freeze-dried powders have differing implications for long-term stability, which will be explored extensively in a future study.

Keywords

Biopharmaceutical; dispersibility; particle engineering; pulmonary drug delivery; spray drying; spray freeze-drying

A spray freeze dried micropellet based formulation proof-of-concept for a yellow fever vaccine candidate

A spray freeze dried micropellet based formulation proof-of-concept for a yellow fever vaccine candidate

Clénet D, Hourquet V, Woinet B, Ponceblanc H, Vangelisti M

Abstract

The stability of live-attenuated viruses is very challenging due to thermal sensitivity; therefore, solid form is usually required (often freeze-dried products). Micropellet technology is a lyophilization technology that has the potential to provide greater flexibility in the presentation of a given vaccine particularly in multi-dose format or in combination of different vaccines. As a novel vaccine alternative process, this spray freeze-dried (SFD) micropellet technology was evaluated using as a model a yellow fever virus produced in Vero cells (vYF). Screening of excipients was performed in order to optimize physico-chemical properties of the micropellets. Sugar/polymer-based formulations induced high glass transition temperature (Tg), adequate breaking force and attrition resistance of the SFD micropellets. These mechanical parameters and their stability are of considerable importance for the storage, the transport but also the filling process of the SFD micropellets. By adding excipients required to best preserve virus infectivity, an optimal sugar/polymer-based formulation was selected to build micropellets containing vYF. Monodisperse and dried micropellets with a diameter of about 530 µm were obtained, exhibiting similar potency to conventional freeze-dried product in terms of vYF infectious titer when both solid forms were kept under refrigerated conditions (2-8°C). Comparable kinetics of degradation were observed for vYF formulated in micropellets or as conventional freeze-dried product during an accelerated stability study using incubations at 25°C and 37°C over several weeks. The results from this investigation demonstrate the ability to formulate live-attenuated viruses in micropellets. Pharmaceutical applications of this novel vaccine solid form are discussed.

Keywords

Micropellets; formulation screening; freeze-drying; live-attenuated vaccine stability

Microwave heating synthesis and luminescence of NaY(WO4)2:(Ho3+, Yb3+) phosphors

Microwave heating synthesis and luminescence of NaY(WO4)2:(Ho3+, Yb3+) phosphors

Yang Yang, Hao Feng, Xiuguo Zhang

Abstract

NaY(WO4)(2):(Ho3+, Yb3+) phosphors were synthesized by microwave heating in an EDTA-mediated process. The samples were characterized by XRD, FT-IR, SEM, and fluorescence spectrophotometer. The XRD and FT-IR results show that the samples have the tetragonal phase. The SEM results show that obtained samples have octahedral morphology. ‘Oriented attachment’ and ‘Ostwald ripening’ play key roles in the formation of octahedrons. The UC emission spectra of NaY(WO4)(2):(Ho3+, Yb3+) phosphors under 980 nm excitation show two intense bands corresponding to F-5(4) + S-5(2) -> I-5(8) and F-5(5) -> I-5(8) transitions of Ho3+ ions. Based on the energy level diagrams of Yb3+ and Ho3+, as well as the results of power dependence of UC emission intensities, possible excitation path ways for different bands are deduced. The green and red UC emissions of NaY(WO4)(2):(Ho3+, Yb3+) phosphors originate from the two-photon process. Also, the samples have good thermal stability.

Preparation and characterization of nano amitriptyline hydrochloride particles by spray freeze drying

Preparation and characterization of nano amitriptyline hydrochloride particles by spray freeze drying

Hu Y, Ma C, Sun M, Guo C, Shen J, Wang J, Nie F, Gao B.

Abstract

Aim: To investigate the enhancement of bioavailability by the usage of drug nanoparticles for increasing the efficacy of antidepressant therapeutic value. Materials & methods: Nano-amitriptyline HCI (AMT·HCl) particles were successfully prepared via a simple spray freeze drying (SFD) method.

Results: The as-prepared nanoparticles are amorphous instead of crystalline. The mean size of AMT·HCl nanoparticles is 90 nm. In in vitro evaluation, AMT·HCl nanoparticles have greatly improved the dissolution compared with pure bulk materials, which have potential for enhancing human bioavailability and diminishing toxic effect. A nanoparticle formation mechanism was also proposed.

Conclusion: These findings promote the development of antidepressant therapeutic evaluation based on the usage of AMT·HCl nanoparticles by SFD method and indicate that SFD is an alternative for a range of nanoparticle preparation in industrial pharmacy.

Keywords

evaluation; amitriptyline hydrochloride; antidepressants; bioavailability; nanocrystals; spray freeze drying

Atmospheric spray freeze drying of sugar solution with phage D29

Atmospheric spray freeze drying of sugar solution with phage D29

Alvin Ly, Nicholas B. Carrigy, Hui Wang, Melissa Harrison, Dominic Sauvageau, Andrew R Martin, Reinhard Vehring, Warren H Finlay

Abstract

Therapeutic bacteriophages offer a potential alternative approach in the treatment of drug resistant bacteria. In the present study, we examine the ability of atmospheric spray freeze-drying (ASFD) to process bacteriophage D29 into a solid dry formulation. Bacteriophage D29 is of particular interest due to its ability to infect Mycobacterium tuberculosis. A sugar solution containing bacteriophage D29 was sprayed and instantly frozen in a cold chamber. Cold drying gas was then passed through the chamber at a high flow rate and atmospheric pressure. Convective transport combined with the low temperature of the drying gas results in sublimation of ice, yielding a free-flowing, porous powder. The bacteriophages were atmospheric spray freeze-dried in solutions with varying concentrations of trehalose and mannitol. A solution of trehalose and mannitol at a mass ratio of 7:3 and a total mass concentration of 100 mg/mL led to powder with 4.9 ± 0.1% moisture content and an acceptable titer reduction of ∼0.6 logs. In comparison, a pure trehalose solution and a 1:1 ratio of trehalose and mannitol both had titer reductions of >1.5 logs. Spectroscopic analysis showed that trehalose in the powder was amorphous while mannitol completely crystallized during the drying process, both of which are desirable for preserving phage viability and storage in powders. The results highlight the potential for using ASFD as an alternative process in preserving biopharmaceutical products.

Inert particles as process aid in spray-freeze drying

Inert particles as process aid in spray-freeze drying

Fan Zhang, Xiaoyu Ma, Xiusheng Wu, Qing Xu, Wei Tian, Zhanyong Li

Abstract

Spray-freeze drying (SFD) is a novel and advanced drying technology in the production of high-value foods and pharmaceuticals. However, long drying time is still a disadvantage for the SFD applications. This constraint could be alleviated using inert particles. This article provides the experimental results on SFD of whole milk in an integrated spray-freeze and vacuum freeze drying equipment. The effects were quantified in terms of the SFD drying time and the properties of the obtained milk powder in relation to the size of stainless steel balls used as inert particles. It is shown that the drying time could be reduced to some extent due to inert particles embedded and the milk powder of reduced size has high porous microstructure.

Development of Inhalable Dry Gene Powders for Pulmonary Drug Delivery by Spray-Freeze-Drying

Development of Inhalable Dry Gene Powders for Pulmonary Drug Delivery by Spray-Freeze-Drying

Edina Vranic, Merima Sirbubalo, Amina Tucak, Jasmina Hadžiabdić, Ognjenka Rahić, Alisa Elezovic

Abstract

There is considerable potential for pulmonary gene therapy as a treatment for a number of conditions for which current treatment is inadequate. Delivering genes directly to the lungs by dry powder inhalers (DPIs) have attracted much attention due to better stability of genes. Formulating genes as powders for aerosol delivery is a challenge as it requires not only flowability and dispersibility of the powders but also maintaining gene stability and biological activity during manufacturing and delivery. In this review, we aim to provide an overview about the potentials of spray-freeze-drying (SFD) for the development of inhalable dry gene powders for pulmonary drug delivery. We will discuss the main steps involved within the production process (i.e., spraying, freezing and drying) and introduce different SFD methods which can successfully be used for the production of porous particles whose physical and aerosol characteristics are considered to be ideal for use in pulmonary drug delivery.

Establishment of evaluation method for gene-silencing by serial pulmonary administration of siRNA and pDNA powders: Naked siRNA inhalation powder suppresses luciferase gene expression in lung

Establishment of evaluation method for gene-silencing by serial pulmonary administration of siRNA and pDNA powders: Naked siRNA inhalation powder suppresses luciferase gene expression in lung

Takaaki Ito, Tomoyuki Okuda, Ryo Takayama, Hirokazu Okamoto

Abstract

In order to evaluate the in vivo effect of inhaled formulations, it is a gold standard to create a lung metastasis model by intravenously injecting cancer cells into an animal. Because the cancer grows from the blood vessel side, there is a possibility of underestimating the effect of an inhaled formulation administered to the lung epithelium side. In addition, the metastasis model has disadvantages in terms of preparation time and expense. The present study aimed to establish a new method to evaluate the effect of an inhaled small interfering RNA (siRNA) formulation that is more correct, more rapid, and less expensive. We investigated whether siRNA can suppress gene expression of plasmid DNA (pDNA) by serial pulmonary administration of siRNA and pDNA powders prepared by spray-freeze-drying. We revealed that formulations of dry siRNA powder significantly suppressed gene expression of pDNA powder compared with a control group with no siRNA. Naked siRNA inhalation powder with no vector showed the suppression of gene expression equivalent to that of an siRNA-polyethyleneimine complex without damaging tissues. These results show that the present method is suitable for evaluating the gene-silencing effect of inhaled siRNA powders.