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Stage-Specific Stability of Spray-Freeze-Dried Ovalbumin-Loaded Liposomes: Dissecting the Impact of Atomization, Freezing, and Drying of Cryoprotectant Performance

Xi Cheng, Mengyuan Li, Shen Yan, Shengyu Zhang, Xiao Dong Chen and Winston Duo Wu

Abstract

Objectives: Aqueous liposomal suspensions suffer from inherent physical and chemical instability, motivating the development of solid powder formulations. Spray-freeze-drying is a promising solidification technique; however, systematic investigations that isolate the impact of individual unit operations—atomization, freezing, and drying—on liposomal stability remain scarce. This study aimed to elucidate the stage-specific effects of each unit operation on the critical quality attributes of liposomal powders. Methods: Ovalbumin-loaded liposomes (O@L) prepared from DSPC, HSPC, or DPPC were processed using a self-developed microfluidic jet spray freezing tower coupled to a freeze dryer, with trehalose or mannitol as cryoprotectants. The effects of phospholipid composition, freezing temperature (−30 °C, −50 °C, and −70 °C), and freeze-drying cycle were assessed through the z-average, zeta potential, and encapsulation efficiency (EE) of the reconstituted liposomes, as well as the morphology, crystallinity, residual moisture, and hygroscopicity of the resulting powders. Results: Atomization induced protein leakage from the trehalose formulations, whereas mannitol better preserved EE. During freezing, trehalose provided superior cryoprotection, as mannitol partially crystallized and failed to uniformly coat the vesicles. After drying, however, vesicles frozen at −70 °C exhibited fusion attributable to non-uniform cryoprotectant distribution; a moderate freezing temperature (−50 °C) combined with a stepwise primary drying program proved optimal. Trehalose-based powders displayed higher residual moisture and irreversible crystallization above ~60% RH, whereas crystalline mannitol ensured low, reversible hygroscopicity. Conclusions: The optimal condition for a single unit operation does not guarantee the best overall outcome; rational spray-freeze-drying process design requires balancing stage-specific effects.

Keywords:

spray-freeze-drying; unit operation; liposomal powder; ovalbumin; cryoprotectant

Spray Freeze Drying for Solid Formulations of RNA Lipid Nanoparticles

K. Arte, Rachana Sapkota, Chanakya D. Patil, Yi-Jing Huang, E. Munson, Sajal M. Patel, Nicole M. Payton, Rushit N. Lodaya, Q. Zhou, L. Qu

Abstract

Spray-freeze drying (SFD) is a promising drying technique for stabilizing RNA lipid nanoparticles (RNA-LNPs) by converting them into solid-state formulations. This study examined the impact of the SFD process and the widely utilized stabilizing disaccharides, such as sucrose and trehalose, on the major properties (e.g. particle size, encapsulation efficiency) of RNA-LNPs post drying. Various process parameters, such as atomization, freezing, and drying temperature, were systematically evaluated, along with the impact of incorporating annealing post freezing step. Surface area measurements, solid-state particle size analysis, scanning electron microscopy, powder X-ray diffraction and solid-state NMR spectroscopy were employed to gain deeper insights into powder characteristics and matrix mobility. Our findings demonstrated that sucrose provided superior stabilization as compared to trehalose in our tested formulations and processes. Moreover, incorporation of the annealing process enhanced LNP stability. Results from the stability study revealed that the annealed 10% and 20% sucrose formulations maintained their stability, highlighting the potential of optimized SFD processing for producing solid formulations of RNA-LNPs. 

Three-fluid spray-drying vs. spray-freeze-drying of crosslinked alginate microspheres: Elucidating multiscale structure-property relationships for rational design.

Zi-Wei Nie, Chong-Zhi Qiu, Meng-Yuan Li, Shen Yan, Sheng-Yu Zhang, Xiao-Dong Chen, W. Wu

Abstract

Crosslinked alginate microspheres (CLAMs) are promising drug carriers, but conventional two-fluid atomization is limited by premix clogging and formulation complexity. To address these limitations, this study employed a three-fluid atomization strategy to fabricate CLAMs via spray-drying and spray-freeze-drying, enabling elucidation of multiscale structure-property relationships. With a three-fluid nozzle, sodium alginate and CaCl2·2H2O solutions were fed separately, enabling in situ crosslinking upon atomization. A series of CLAMs loaded with diclofenac sodium (DS) were fabricated via subsequent hot-air drying (SD-DS@CLAMs) or freezing followed by vacuum freeze drying (SFD-DS@CLAMs). SD-DS@CLAMs appeared as dense particles with a wrinkled surface, whereas SFD-DS@CLAMs possessed a porous, spherical morphology. In both systems, DS was incorporated in an amorphous state via physical entrapment. Moisture sorption was governed by crosslinking degree, drug hydrophobicity, and drying-dependent microstructure, with porous SFD particles enhancing capacity and reducing hysteresis. Notably, the crosslinking degree of the microspheres exhibited a non-monotonic trend driven by the competition between component segregation and reaction window. Crucially, rapid gel network formation upon hydration decoupled release kinetics from particle morphology, resulting in consistent Fickian diffusion profiles. While spray-drying offers high efficiency, spray-freeze-drying shows great potential for protecting heat-sensitive components and creating porous structures. Based on the current formulation design, these two methods provide complementary fabrication strategies that can be flexibly selected based on specific needs without altering release behavior, thereby expanding the design and application of CLAMs delivery systems.

Single-Step Solvent Evaporation Via Spray-Drying for the Production of Ph-Sensitive Polymeric Nanospheres for Dermatological Applications

A. Cardoso, B. Machado, S. S. Guterres, H. R. Marcelino

Abstract

Polymeric nanospheres are promising drug delivery systems capable of controlling the release of active molecules. However, their inherent colloidal instability necessitates the use of drying techniques to enhance physicochemical stability and microbial resistance. However, conventional multi-step production processes hinder industrial-scale-up due to increased complexity and longer processing times. This work aimed to develop a single-step spray-drying process to obtain Eudragit® L100 nanospheres in powder form, thereby eliminating the need for traditional solvent evaporation, and to investigate the effects of incorporating these nanospheres into hydroxyethyl cellulose gels for dermatological applications. Eudragit® L100 was selected for its controlled release at approximately pH 6.00, facilitating delivery to specific areas, particularly on barrier-disrupted skin. Nanospheres were produced via nanoprecipitation followed by direct spray-drying to reduce production time. The nanosuspension and redispersed dried formulations were characterized by particle size, polydispersity index, morphology, and zeta potential. Before drying, the nanospheres had an average size (Z-average) of 114 nm and a polydispersity index (PdI) of 0.19 and successfully preserved their properties after a single drying step (Z-average = 140 nm and PdI = 0.2). Zeta potential was altered (p < 0.05) but remained high (-23 to -17 mV), indicating electrostatic stabilization. Gel formulations exhibited dose-dependent pseudoplastic behavior, with consistency decreasing proportionally to nanosphere concentration. Finally, moderate to strong correlations were found between rheology and textural analysis. This single-step solvent removal methodology represents a significant manufacturing advancement, offering reduced processing time and costs. The pH-responsive nanospheres showed excellent redispersibility and gel compatibility, establishing their potential as carriers for dermatological systems.

Development and Evaluation of a Novel Inhalable Liposomal Powder Co-Encapsulating ASSNAC and Pirfenidone via Spray Freeze-Drying for Targeted Pulmonary Fibrosis Therapy

Qin-Xiu Zhang, Shou-Wei Sun, Miao-Miao Lu, Run-Xin Qin, Jun-Xuan Ren, Lian-Jie Yao, Dian-Long Jia, Jin-Kyu Chang, Xiao-Hong Chu, Rui Wang, Fang Liu, Jun Li

Abstract

Background: Pulmonary fibrosis (PF) is a progressive lung disease with limited therapies. Oral Pirfenidone (PFD), an approved anti-fibrotic, shows poor lung bioavailability and significant toxicity. S-Allylmercapto-N-acetylcysteine (ASSNAC) has been demonstrated to possess significant anti-inflammatory and antioxidant properties. Methods: In this study, dry powder inhaler formulations (DPIs) co-encapsulating ASSNAC and PFD in liposomes are reported. The formulation was prepared via spray freeze-drying (SFD) using L-leucine as both a cryoprotectant and a surface morphology modifier. The interfacial enrichment of L-leucine during atomization and freezing created a hydrophobic, corrugated surface that lowered particle surface energy, prevented liquid/solid bridge formation, and reduced hygroscopicity. Results: The resulting liposomal powders exhibited optimal aerosol performance: fine particle fraction (FPF) of 61.08% ± 2.45% and mass median aerodynamic diameter (MMAD) of 2.22 ± 0.11 µm. Cellular studies demonstrated that dual-loaded liposomes were efficiently taken up by Beas-2B and HFL-1 cells, with no cytotoxicity (cell viability > 90%) and negligible hemolysis. In the TGF-β1-induced in vitro fibrosis model, the combined treatment exerted prominent therapeutic effects. This regimen preserved normal epithelial morphology, accelerated wound repair, suppressed fibroblast invasiveness, and lowered the expression levels of Collagen I, Collagen III, and α-SMA. In bleomycin-induced pulmonary fibrosis rats, inhaled ASSNAC + PFD liposomal powder markedly relieved lung tissue injury and collagen accumulation. It restored redox balance and lowered pulmonary TGF-β1, hydroxyproline and collagen III levels. The formulation blocked TGF-β1 signaling and fibrotic gene expression. Conclusions: Pulmonary administration avoids oral pirfenidone-induced liver and stomach toxicity. Collectively, the ASSNAC + PFD co-encapsulated liposomal dry powder produced by SFD represents a safe and efficacious therapeutic strategy for PF.

Development of Erlotinib Nanoliposome-Loaded Dry Powder Inhaler for Enhanced Pulmonary Delivery

Mayur R. Nikam, D. Sonawane, A. Pawar

Abstract

Objectives: To develop erlotinib nanoliposome-loaded dry powder inhaler formulations for targeted pulmonary delivery in non-small cell lung cancer, addressing the limitations of poor aqueous solubility, systemic toxicity, and suboptimal tumor localization associated with conventional erlotinib therapy. Methods: Six DPI formulations (IF1–IF6) were prepared by co-spray drying of pre-optimized erlotinib nanoliposomes with varying concentrations of lactose monohydrate, mannitol, and L-leucine as carrier excipients. Formulations were systematically evaluated for physicochemical properties, powder flow, aerodynamic performance using Andersen Cascade Impactor, in vitro drug release in simulated lung fluid, solid-state characterization, and accelerated stability under ICH Q1A(R2) conditions. The optimized batch was selected based on superior aerodynamic performance and overall physicochemical attributes. Results: Particle size D50 ranged from 4.82±0.18 μm (IF1) to 2.98±0.22 μm (IF6), with entrapment efficiency of 84.6–88.1% post spray drying. Formulation IF5 was identified as optimal, demonstrating MMAD of 3.18±0.21 μm, FPF of 56.4±1.9%, emitted dose of 88.2±1.4%, and cumulative drug release of 93.4±2.7% over 12 hours in simulated lung fluid. Drug release followed Hixson-Crowell kinetics (R²=0.9986) with anomalous non-Fickian diffusion confirmed by Korsmeyer-Peppas exponent n=0.7153. Accelerated stability studies confirmed no statistically significant changes in critical quality attributes over six months (p>0.05). Conclusion: The developed nanoliposomal DPI system offers a clinically significant advancement in site-specific pulmonary erlotinib delivery, with potential to improve therapeutic outcomes and reduce systemic adverse effects in NSCLC patients, warranting further in vivo pharmacokinetic and efficacy validation.

Improving the Quality of Lyophilized Animal Vaccines: A Review of Optimization Strategies for Freeze-Drying Processes

Fang-Jing Huang, Jia-Jie Jiao, Ze-Xi Wang, Hong-Ying Jie, Jia-Hao He, Xiao-Xin Zuo, Yan-Hong Zhao, Yu Lu, Jian-Hong Gu, Fang Lyu

Abstract

Simple Summary Lyophilization is a well-established technique for the long-term preservation of live vaccines, offering advantages such as superior protection and ease of transportation. However, the quality of lyophilized animal vaccines remains inconsistent, and their widespread application is limited due to high economic costs, the complexity of formulation design, and incompatibility between lyophilization protocols and vaccine solutions. We elaborate on the fundamental principles and procedural steps of the freeze-drying process. Synthesizing the research findings and experience of our team and the broader field, we enumerate the critical factors that determine the quality of lyophilized animal vaccines. This paper aims to elucidate the preparation principles, current status, application value, and future perspectives of lyophilized animal vaccines, with the aspiration of promoting the high-quality application of freeze-drying technology in the field of animal vaccine manufacturing.

From Liquid Feeds to Functional Powders: Process Engineering, Quality, Valorisation and Emerging Innovations in Spray Drying of Fruits and Vegetables

E. K. Anshida Beegum, S. Shameena, P. R. Geetha Lekshmi, Athulya S. Kumar, S. S. Sumi

Abstract

Spray drying is an effective and scalable technique for transforming highly perishable fruit and vegetable juices, extracts and processing by-products into stable powders with improved handling, storage, convenience and application potential. This review synthesises recent advances in spray drying of fruit and vegetable products, moving beyond a description of the drying process towards an integrated appraisal of process engineering, formulation strategies, powder quality, storage stability, by-product valorisation and emerging technologies. The literature indicates that drying temperature, feed flow rate, airflow rate, atomisation conditions and carrier type and concentration collectively determine powder recovery and key quality attributes, including moisture content, water activity, hygroscopicity, colour, solubility, bulk density, flowability and reconstitution behaviour. Maltodextrin and gum arabic remain widely employed carrier agents because they improve powder formation, reduce stickiness and moisture uptake and support storage stability, although their effectiveness varies with feed composition and processing conditions. Water activity below 0.30 is generally associated with improved microbiological and physicochemical stability of fruit and vegetable powders. Conventional spray drying nevertheless remains constrained by thermal degradation of heat-sensitive compounds, wall deposition, stickiness, caking and losses of valuable bioactive components. Emerging approaches, including vacuum, ultrasound-assisted, dehumidified air, electrostatic, nano and pulse combustion spray drying, provide opportunities to improve heat and mass transfer, drying efficiency, powder recovery and bioactive retention. Spray drying of peels, pomace, seeds and other processing residues further expands opportunities for waste reduction and value-added functional ingredients. Future priorities include integrated optimisation of process and formulation variables, predictive and data-driven process control, sustainable carrier systems, energy-efficient drying, long-term storage evaluation and pilot- and industrial-scale validation to facilitate the commercial development of stable, functional and economically viable fruit and vegetable powders.

Drygels – A promising porous solid state carriers as a Novel Pharmaceutical Dosage Form

S. R, Dhanusiya V, Yogashruthi S

Abstract

Traditional gel-based dosage forms, such as hydrogels and organogels, have been utilized extensively in topical and semi-solid pharmaceutical formulations. Nevertheless, constraints include inadequate compatibility with hydrophobic active pharmaceutical ingredients (APIs), vulnerability to microbial contamination, restricted shelf life, and insufficient mechanical stability have prompted the advancement of alternate delivery strategies. Drygels, which include aerogels, xerogels, and cryogels, represent a promising category of porous solid-state carriers derived from gel precursors via controlled drying or freeze-processing methods.These characteristics facilitate the effective loading of hydrophobic pharmaceuticals in an amorphous form, markedly enhancing water solubility and oral bioavailability. Prominent production techniques encompass supercritical CO₂ drying, freeze-drying, and ambient-pressure evaporative drying, each yielding unique structural attributes. Drygels exhibit extensive applicability in oral, transdermal, pulmonary, nasal, and implantable drug delivery systems, as well as biosensing. Drygels constitute a revolutionary platform in pharmaceutical formulation, providing improved performance and versatility. This review rigorously analyzes their composition, manufacturing techniques, gelation mechanisms and stability properties.

Metamorphosis in Drying Techniques for Biopharmaceuticals.

Aditya Vikas Mane, Karina Arora, Nitya Srivastava, Prince Kumar, Gurvinder Singh, A. Yadav, Rajesh Kumar

Abstract

Biopharmaceutical products such as proteins, peptides, and nucleic acid-based therapeutics exhibit limited physicochemical stability in aqueous formulations. The process of developing and producing these bioproducts is often difficult and requires careful attention, particularly when it comes to drying for long-term storage and distribution of the final product. Conventional drying techniques like freeze-drying and spray drying have been widely used to enhance product stability, but are limited to their time and high-energy consumption. Over the years, significant technological advances in drying processes have emerged to address these limitations and improve process efficiency. This review discusses the principles and applications of both conventional and emerging drying techniques used in biopharmaceutical manufacturing, with particular emphasis on advanced approaches such as spin freeze drying, spray freeze drying, microwave-assisted drying, active freeze drying, and continuous freeze drying of suspended vials. The advantages, limitations, and potential of these technologies in improving biopharmaceutical formulations, their stability, processing efficiency, and industrial scalability are critically examined.

Keywords:

Spray freeze-drying