Increasing Angiogenesis Factors in Hypoxic Diabetic Wound Conditions by siRNA Delivery: Additive Effect of LbL-Gold Nanocarriers and Desloratadine-Induced Lysosomal Escape.

Impaired wound healing in people with diabetes has multifactorial causes, with insufficient neovascularization being one of the most important. Hypoxia-inducible factor-1 (HIF-1) plays a central role in the hypoxia-induced response by activating angiogenesis factors. As its activity is under precise regulatory control of prolyl-hydroxylase domain 2 (PHD-2), downregulation of PHD-2 by small interfering RNA (siRNA) could stabilize HIF-1α and, therefore, upregulate the expression of pro-angiogenic factors as well. Intracellular delivery of siRNA can be achieved with nanocarriers that must fulfill several requirements, including high stability, low toxicity, and high transfection efficiency. Here, we designed and compared the performance of layer-by-layer self-assembled siRNA-loaded gold nanoparticles with two different outer layers-Chitosan (AuNP@CS) and Poly L-arginine (AuNP@PLA). Although both formulations have exactly the same core, we find that a PLA outer layer improves the endosomal escape of siRNA, and therefore, transfection efficiency, after endocytic uptake in NIH-3T3 cells. Furthermore, we found that endosomal escape of AuNP@PLA could be improved further when cells were additionally treated with desloratadine, thus outperforming commercial reagents such as Lipofectamine® and jetPRIME®. AuNP@PLA in combination with desloratadine was proven to induce PHD-2 silencing in fibroblasts, allowing upregulation of pro-angiogenic pathways. This finding in an in vitro context constitutes a first step towards improving diabetic wound healing with siRNA therapy.

Potential of Near-Infrared Chemical Imaging as Process Analytical Technology Tool for Continuous Freeze-Drying.

Near-infrared chemical imaging (NIR-CI) is an emerging tool for process monitoring because it combines the chemical selectivity of vibrational spectroscopy with spatial information. Whereas traditional near-infrared spectroscopy is an attractive technique for water content determination and solid-state investigation of lyophilized products, chemical imaging opens up possibilities for assessing the homogeneity of these critical quality attributes (CQAs) throughout the entire product. In this contribution, we aim to evaluate NIR-CI as a process analytical technology (PAT) tool for at-line inspection of continuously freeze-dried pharmaceutical unit doses based on spin freezing. The chemical images of freeze-dried mannitol samples were resolved via multivariate curve resolution, allowing us to visualize the distribution of mannitol solid forms throughout the entire cake. Second, a mannitol-sucrose formulation was lyophilized with variable drying times for inducing changes in water content. Analyzing the corresponding chemical images via principal component analysis, vial-to-vial variations as well as within-vial inhomogeneity in water content could be detected. Furthermore, a partial least-squares regression model was constructed for quantifying the water content in each pixel of the chemical images. It was hence concluded that NIR-CI is inherently a most promising PAT tool for continuously monitoring freeze-dried samples. Although some practicalities are still to be solved, this analytical technique could be applied in-line for CQA evaluation and for detecting the drying end point.

Successful batch and continuous lyophilization of mRNA LNP formulations depend on cryoprotectants and ionizable lipids.

The limited thermostability and need for ultracold storage conditions are the major drawbacks of the currently used nucleoside-modified lipid nanoparticle (LNP)-formulated messenger RNA (mRNA) vaccines, which hamper the distribution of these vaccines in low-resource regions. The LNP core contains, besides mRNA and lipids, a large fraction of water. Therefore, encapsulated mRNA, or at least a part of it, is subjected to hydrolysis mechanisms similar to unformulated mRNA in an aqueous solution. It is likely that the hydrolysis of mRNA and colloidal destabilization are critical factors that decrease the biological activity of mRNA LNPs upon storage under ambient conditions. Hence, lyophilization as a drying technique is a logical and appealing method to improve the thermostability of these vaccines. In this study, we demonstrate that mRNA LNP formulations comprising a reduction-sensitive ionizable lipid can be successfully lyophilized, in the presence of 20% w/v sucrose, both by conventional batch freeze-drying and by an innovative continuous spin lyophilization process. While the chemical structure of the ionizable lipid did not affect the colloidal stability of the LNP after lyophilization and redispersion in an aqueous medium, we found that the ability of LNPs to retain the mRNA payload stably encapsulated, and mediate in vivo and in vitro mRNA translation into protein, post lyophilization strongly depended on the ionizable lipid in the LNP formulation.

Spin Freezing and Its Impact on Pore Size, Tortuosity and Solid State.

Spin freeze-drying, as a part of a continuous freeze-drying technology, is associated with a much higher drying rate and a higher level of process control in comparison with batch freeze-drying. However, the impact of the spin freezing rate on the dried product layer characteristics is not well understood at present. This research focuses on the relation between spin-freezing and pore size, pore shape, dried product mass transfer resistance and solid state of the dried product layer. This was thoroughly investigated via high-resolution X-ray micro-computed tomography (µCT), scanning electron microscopy (SEM), thermal imaging and solid state X-ray diffraction (XRD). It was concluded that slow spin-freezing rates resulted in the formation of highly tortuous structures with a high dried-product mass-transfer resistance, while fast spin-freezing rates resulted in lamellar structures with a low tortuosity and low dried-product mass-transfer resistance.

A NIR-Based Study of Desorption Kinetics during Continuous Spin Freeze-Drying.

The pharmaceutical industry is progressing toward the development of more continuous manufacturing techniques. At the same time, the industry is striving toward more process understanding and improved process control, which requires the implementation of process analytical technology tools (PAT). For the purpose of drying biopharmaceuticals, a continuous spin freeze-drying technology for unit doses was developed, which is based on creating thin layers of product by spinning the solution during the freezing step. Drying is performed under vacuum using infrared heaters to provide energy for the sublimation process. This approach reduces drying times by more than 90% compared to conventional batch freeze-drying. In this work, a new methodology is presented using near-infrared (NIR) spectroscopy to study the desorption kinetics during the secondary drying step of the continuous spin freeze-drying process. An inline PLS-based NIR calibration model to predict the residual moisture content of a standard formulation (i.e., 10% sucrose) was constructed and validated. This model was then used to evaluate the effect of different process parameters on the desorption rate. Product temperature, which was controlled by a PID feedback mechanism of the IR heaters, had the highest positive impact on the drying rate during secondary drying. Using a higher cooling rate during spin freezing was found to significantly increase the desorption rate as well. A higher filling volume had a smaller negative effect on the drying rate while the chamber pressure during drying was found to have no significant effect in the range between 10 and 30 Pa.

Development and Application of a Mechanistic Cooling and Freezing Model of the Spin Freezing Step within the Framework of Continuous Freeze-Drying.

During the spin freezing step of a recently developed continuous spin freeze-drying technology, glass vials are rapidly spun along their longitudinal axis. The aqueous drug formulation subsequently spreads over the inner vial wall, while a cold gas flow is used for cooling and freezing the product. In this work, a mechanistic model was developed describing the energy transfer during each phase of spin freezing in order to predict the vial and product temperature change over time. The uncertainty in the model input parameters was included via uncertainty analysis, while global sensitivity analysis was used to assign the uncertainty in the model output to the different sources of uncertainty in the model input. The model was verified, and the prediction interval corresponded to the vial temperature profiles obtained from experimental data, within the limits of the uncertainty interval. The uncertainty in the model prediction was mainly explained (>96% of uncertainty) by the uncertainty in the heat transfer coefficient, the gas temperature measurement, and the equilibrium temperature. The developed model was also applied in order to set and control a desired vial temperature profile during spin freezing. Applying this model in-line to a continuous freeze-drying process may alleviate some of the disadvantages related to batch freeze-drying, where control over the freezing step is generally poor.

Macrophage reprogramming into a pro-healing phenotype by siRNA delivered with LBL assembled nanocomplexes for wound healing applications.

Excessive inflammatory responses in wounds are characterized by the presence of high levels of pro-inflammatory M1 macrophages rather than pro-healing M2 macrophages, which leads to delayed wound healing. Macrophage reprogramming from the M1 to M2 phenotype through knockdown of interferon regulatory factor 5 (irf5) has emerged as a possible therapeutic strategy. While downregulation of irf5 could be achieved by siRNA, it very much depends on successful intracellular delivery by suitable siRNA carriers. Here, we report on highly stable selenium-based layer-by-layer (LBL) nanocomplexes (NCs) for siRNA delivery with polyethyleneimine (PEI-LBL-NCs) as the final polymer layer. PEI-LBL-NCs showed good protection of siRNA with only 40% siRNA release in a buffer of pH = 8.5 after 72 h or in simulated wound fluid after 4 h. PEI-LBL-NCs also proved to be able to transfect RAW 264.7 cells with irf5-siRNA, resulting in successful reprogramming to the M2 phenotype as evidenced by a 3.4 and 2.6 times decrease in NOS-2 and TNF-α mRNA expression levels, respectively. Moreover, irf5-siRNA transfected cells exhibited a 2.5 times increase of the healing mediator Arg-1 and a 64% increase in expression of the M2 cell surface marker CD206+. Incubation of fibroblast cells with conditioned medium isolated from irf5-siRNA transfected RAW 264.7 cells resulted in accelerated wound healing in an in vitro scratch assay. These results show that irf5-siRNA loaded PEI-LBL-NCs are a promising therapeutic approach to tune macrophage polarization for improved wound healing.

4D Micro-Computed X-ray Tomography as a Tool to Determine Critical Process and Product Information of Spin Freeze-Dried Unit Doses.

Maintaining chemical and physical stability of the product during freeze-drying is important but challenging. In addition, freeze-drying is typically associated with long process times. Therefore, mechanistic models have been developed to maximize drying efficiency without altering the chemical or physical stability of the product. Dried product mass transfer resistance ( R p ) is a critical input for these mechanistic models. Currently available techniques to determine R p only provide an estimation of the mean R p and do not allow measuring and determining essential local (i.e., intra-vial) R p differences. In this study, we present an analytical method, based on four-dimensional micro-computed tomography (4D- µ CT), which enables the possibility to determine intra-vial R p differences. Subsequently, these obtained R p values are used in a mechanistic model to predict the drying time distribution of a spin-frozen vial. Finally, this predicted primary drying time distribution is experimentally verified via thermal imaging during drying. It was further found during this study that 4D- µ CT uniquely allows measuring and determining other essential freeze-drying process parameters such as the moving direction(s) of the sublimation front and frozen product layer thickness, which allows gaining accurate process knowledge. To conclude, the study reveals that the variation in the end of primary drying time of a single vial could be predicted accurately using 4D- µ CT as similar results were found during the verification using thermal imaging.

In-Situ X-ray Imaging Of Sublimating Spin-Frozen Solutions.

Spin-freeze-drying is a promising technique to enable long-term storage of pharmaceutical unit doses of aqueous drug solutions. To investigate the sublimation of the ice during the primary phase of freeze-drying, X-ray imaging can yield crucial temporally resolved information on the local dynamics. In this paper, we describe a methodology to investigate the sublimation front during single unit-dose freeze-drying using 4D in-situ X-ray imaging. Three spin-frozen samples of different solutions were imaged using this methodology and the process characteristics were analysed and reduced to two-dimensional feature maps.

Lyophilization and nebulization of pulmonary surfactant-coated nanogels for siRNA inhalation therapy.

RNA interference (RNAi) enables highly specific silencing of potential target genes for treatment of pulmonary pathologies. The intracellular RNAi pathway can be activated by cytosolic delivery of small interfering RNA (siRNA), inducing sequence-specific gene knockdown on the post-transcriptional level. Although siRNA drugs hold many advantages over currently applied therapies, their clinical translation is hampered by inefficient delivery across cellular membranes. We previously developed hybrid nanoparticles consisting of an siRNA-loaded nanosized hydrogel core (nanogel) coated with Curosurf®, a clinically used pulmonary surfactant (PS). The latter enhances both particle stability as well as intracellular siRNA delivery, which was shown to be governed by the PS-associated surfactant protein B (SP-B). Despite having a proven in vitro and in vivo siRNA delivery potential when prepared ex novo, clinical translation of this liquid nanoparticle suspension requires the identification of a long-term preservation strategy that maintains nanoparticle stability and potency. In addition, to achieve optimal pulmonary deposition of the nanocomposite, its compatibility with state-of-the-art pulmonary administration techniques should be evaluated. Here, we demonstrate that PS-coated nanogels can be lyophilized, reconstituted and subsequently nebulized via a vibrating mesh nebulizer. The particles retain their physicochemical integrity and their ability to deliver siRNA in a human lung epithelial cell line. The latter result suggests that the functional integrity of SP-B in the PS coat towards siRNA delivery might be preserved as well. Of note, successful lyophilization was achieved without the need for stabilizing lyo- or cryoprotectants. Our results demonstrate that PS-coated siRNA-loaded nanogels can be lyophilized, which offers the prospect of long-term storage. In addition, the formulation was demonstrated to be suitable for local administration with a state-of-the-art nebulizer for human use upon reconstitution. Hence, the data presented in this study represent an important step towards clinical application of such nanocomposites for treatment of pulmonary disease.

Dual chamber cartridges in a continuous pharmaceutical freeze-drying concept: Determination of the optimal dynamic infrared heater temperature during primary drying.

The applicability of DCCs in a continuous freeze-drying concept based on spin freezing and infrared heating was evaluated. Maximum applicable filling volume was evaluated. Secondly the mechanistic model for the determination of the optimal dynamic infrared heater temperature during primary drying of regular vials during continuous freeze-drying was adapted and validated for DCCs. Finally, since spin frozen DCCs may be more prone to choked flow due to the small neck opening and the large product surface area, it was evaluated if the choked flow constraints in the model could be increased to improve the efficiency of the drying process. The experiments revealed that the maximum allowable filling volume for spin freezing at the current experimental setup was 0.8 ml which is 80% of the maximum filling volume. Applying the mechanistic model for the determination of the optimal dynamic infrared heater temperature during primary drying of the studied DCCs and experimentally verifying this determined infrared heater temperature trajectory resulted in an elegant freeze-dried product without visual signs of collapse. The experimentally determined primary drying time agreed with the one calculated based on the mechanistic model. Choked flow did not occur during the continuous freeze-drying of DCCs containing 3% sucrose or 3% mannitol.

Dry amorphisation of mangiferin, a poorly water-soluble compound, using mesoporous silica.

Mangiferin, a poorly water soluble compound, was processed via a dry amorphisation technique (ball milling) in combination with mesoporous silica to enhance the solubility of mangiferin. The amorphous samples were prepared by mixing 1:1 (w/w) Syloid® XDP 3050 silica-mangiferin mixtures using a planetary mono mill at different milling speeds and milling times according to a 32 full factorial experimental design. The prepared samples were characterized for dissolution profile, particle size distribution using laser diffraction particle size analyzer, thermal characteristics using DSC, crystalline characteristics using XRD and molecular interactions using FTIR and ss-NMR. The samples were tested for stability at stress conditions (40 °C/75%RH) for up to 6 months in open and closed containers. To improve stability of the samples, mixtures of 1:1:2 mangiferin-polymer (Soluplus or HPMC)-silica samples were also prepared and analyzed. Amorphisation of mangiferin is possible using dry amorphisation by ball milling with mesoporous silica in a short amount of time. The amorphisation rate of the samples improved with the energy input of the milling process. The samples prepared with high energy input resulted in amorphous samples and showed a better stability at the stress conditions for up to 3 months. Solubility of these samples increased from 0.32 to 0.50 mg/ml and the particle size decreased from 35.5 µm to around 7 µm. The spectral analysis suggest presence of interactions between the silica material and the compound. The amorphous stability was improved with addition of polymer, even though the solubility of the samples was lower.

The relevance of shear, sedimentation and diffusion during spin freezing, as potential first step of a continuous freeze-drying process for unit doses.

Recently, a continuous freeze-drying process for the production of unit doses was presented and evaluated. In this concept, the freezing step is modified compared to traditional batch freeze-drying, as glass vials filled with a liquid formulation, are rotated around their longitudinal axis while cooled and frozen with a cold, sterile and inert gas (i.e. spin freezing). Finally, a thin frozen product layer spread over the entire vial wall is achieved. The aim of this paper is twofold: firstly, the relation between the rotation velocity and the relative difference between top and bottom of the frozen product layer thickness was determined for different vial types. Secondly, the impact of shear and centrifugal forces generated during spinning was examined, to find out whether they might cause pharmaceutical instability and sedimentation, respectively. Mechanistic and experimental evaluation showed that shear has no effect on proteins. Calculations showed that the sedimentation and diffusion velocity is too low to cause inhomogeneity in the product layer. In addition, Global Sensitivity Analysis (GSA) and Uncertainty Analysis (UA) were performed in order to account for the uncertainty of the used mechanistic model.

Modelling the primary drying step for the determination of the optimal dynamic heating pad temperature in a continuous pharmaceutical freeze-drying process for unit doses.

In the pharmaceutical industry, traditional freeze-drying of unit doses is a batch-wise process associated with many disadvantages. To overcome these disadvantages and to guarantee a uniform product quality and high process efficiency, a continuous freeze-drying process is developed and evaluated. The main differences between the proposed continuous freeze-drying process and traditional freeze-drying can be found firstly in the freezing step during which the vials are rotated around their longitudinal axis (spin freezing), and secondly in the drying step during which the energy for sublimation and desorption is provided through the vial wall by conduction via an electrical heating pad. To obtain a more efficient drying process, the energy transfer has to be optimised without exceeding the product and process limits (e.g. cake collapse, choked flow). Therefore, a mechanistic model describing primary drying during continuous lyophilisation of unit doses based on conduction via heating pads was developed allowing the prediction of the optimal dynamic power input and temperature output of the electric heating pads. The model was verified by experimentally testing the optimal dynamic primary drying conditions calculated for a model formulation. The primary drying endpoint of the model formulation was determined via in-line NIR spectroscopy. This endpoint was then compared with the predicted model based endpoint. The mean ratio between the experimental and model based predicted drying time for six verification runs was 1.05±0.07, indicating a good accordance between the model and the experimental data.