Lipid nanoparticles for local delivery of mRNA to the respiratory tract: Effect of PEG-lipid content and administration route.

Design of inhalable mRNA therapeutics is promising because local administration in the respiratory tract is minimally invasive and induces a local response. However, several challenges related to administration via inhalation and respiratory tract barriers have so far prevented the progress of inhaled mRNA therapeutics. Here, we investigated factors of importance for lipid nanoparticle (LNP)-mediated delivery of mRNA to the respiratory tract. We hypothesized that: (i) the PEG-lipid content is important for providing colloidal stability during aerosolization and for mucosal delivery, (ii) the PEG-lipid contentinfluences the expression of mRNA-encoded protein in the lungs, and (iii) the route of administration (nasal versus pulmonary) affects mRNA delivery in the lungs. In this study, we aimed to optimize the PEG-lipid content for mucosal delivery and to investigatethe effect of administration route on the kinetics of protein expression. Our results show that increasing the PEG-lipid content improves the colloidal stability during the aerosolization process, but has a negative impact on the transfection efficiencyin vitro. The kinetics of protein expressionin vivois dependent on the route of administration, and we found that pulmonaryadministration of mRNA-LNPs to mice results inmore durable protein expression than nasaladministration. These results demonstrate that the design of the delivery system and the route of administration are importantfor achieving high mRNA transfection efficiency in the respiratory tract.

Engineering aspects of lipid-based delivery systems: In vivo gene delivery, safety criteria, and translation strategies.

Defects in the genome cause genetic diseases and can be treated with gene therapy. Due to the limitations encountered in gene delivery, lipid-based supramolecular colloidal materials have emerged as promising gene carrier systems. In their non-functionalized form, lipid nanoparticles often demonstrate lower transgene expression efficiency, leading to suboptimal therapeutic outcomes, specifically through reduced percentages of cells expressing the transgene. Due to chemically active substituents, the engineering of delivery systems for genetic drugs with specific chemical ligands steps forward as an innovative strategy to tackle the drawbacks and enhance their therapeutic efficacy. Despite intense investigations into functionalization strategies, the clinical outcome of such therapies still needs to be improved. Here, we highlight and comprehensively review engineering aspects for functionalizing lipid-based delivery systems and their therapeutic efficacy for developing novel genetic cargoes to provide a full snapshot of the translation from the bench to the clinics. We outline existing challenges in the delivery and internalization processes and narrate recent advances in the functionalization of lipid-based delivery systems for nucleic acids to enhance their therapeutic efficacy and safety. Moreover, we address clinical trials using these vectors to expand their clinical use and principal safety concerns.

Line-field confocal optical coherence tomography for in vivo visualization of morphological characteristics of squamous cell carcinoma in a murine model.

OBJECTIVES: Non-invasive imaging with line-field confocal optical coherence tomography (LC-OCT) can support the diagnosis of squamous cell carcinoma (SCC) through visualization of morphological characteristics specific to skin cancer. We aimed to visualize prominent morphological characteristics of SCC using LC-OCT in a well-established murine SCC model. MATERIALS AND METHODS: Nine hairless mice were exposed to ultraviolet radiation three times weekly for 9 months to induce SCC development. Visible SCC tumors (n = 9) were imaged with LC-OCT and the presence of 10 well-described morphological characteristics of SCC were evaluated in the scans by two physicians with adjudication by a third. RESULTS: Overall, murine morphological characteristics resembled corresponding features previously reported in human SCCs. Interrupted dermal-epidermal junction occurred in 100% of tumors. In epidermis, the most frequently observed characteristics were severe epidermal dysplasia (100%) and tumor budding (89%). Common dermal characteristics included broad strands (100%) and collagen alterations (78%). CONCLUSION: LC-OCT imaging can be used to non-invasively visualize morphological characteristics specific to SCC in an in vivo preclinical model.

Reconstituted dry powder formulations of ZnO-adjuvanted ovalbumin induce equivalent antigen specific antibodies but lower T cell responses than ovalbumin adjuvanted with Alhydrogel® or cationic adjuvant formulation 01 (CAF®01).

Most licensed human vaccines are based on liquid dosage forms but have poor storage stability and require continuous and expensive cold-chain storage. In contrast, the use of solid vaccine dosage forms produced by for example spray drying, extends shelf life and eliminates the need for a cold chain. Zinc oxide (ZnO)-based nanoparticles display immunomodulatory properties, but their adjuvant effect as a dry powder formulation is unknown. Here, we show that reconstituted dry powder formulations of ZnO particles containing the model antigen ovalbumin (OVA) induce antigen-specific CD8+ T-cell and humoral responses. By systematically varying the ratio between ZnO and mannitol during spray drying, we manufactured dry powder formulations of OVA-containing ZnO particles that displayed: (i) a spherical or wrinkled surface morphology, (ii) an aerodynamic diameter and particle size distribution optimal for deep lung deposition, and (iii) aerosolization properties suitable for lung delivery. Reconstituted dry powder formulations of ZnO particles were well-tolerated by Calu-3 lung epithelial cells. Furthermore, almost equivalent OVA-specific serum antibody responses were stimulated by reconstituted ZnO particles, OVA adjuvanted with Alhydrogel®, and OVA adjuvanted with the cationic adjuvant formulation 01 (CAF®01). However, reconstituted dry powder ZnO particles and OVA adjuvanted with Alhydrogel® induced significantly lower OVA-specific CD8+CD44+ T-cell responses in the spleen than OVA adjuvanted with CAF®01. Similarly, reconstituted dry powder ZnO particles activated significantly lower percentages of follicular helper T cells and germinal center B cells in the draining lymph nodes than OVA adjuvanted with CAF®01. Overall, our results show that reconstituted dry powder formulations of ZnO nanoparticles can induce antigen-specific antibodies and can be used in vaccines to enhance antigen-specific humoral immune responses against subunit protein antigens.

Malaria Biomimetic for Tumor Targeted Drug Delivery.

Malaria infected erythrocytes utilize the parasite protein VAR2CSA to bind to a unique presentation of chondroitin sulfate (CS) for their placenta specific tropism. Interestingly, many cancers express a similar form of CS, thereby termed oncofetal CS (ofCS). The distinctive tropism of malaria infected erythrocytes and the identification of oncofetal CS, therefore, represent potentially potent tools for cancer targeting. Here we describe an intriguing drug delivery platform that effectively mimics infected erythrocytes and their specificity for ofCS. We used a lipid catcher-tag conjugation system for the functionalization of erythrocyte membrane-coated drug carriers with recombinant VAR2CSA (rVAR2). We show that these malaria mimicking erythrocyte nanoparticles (MMENPs) loaded with docetaxel (DTX) specifically target and kill melanoma cells in vitro. We further demonstrate effective targeting and therapeutic efficacy in a xenografted melanoma model. These data thus provide a proof of concept for the use of a malaria biomimetic for tumor targeted drug delivery. Given the broad presentation of ofCS found across various types of malignancies, this biomimetic may therefore show potential as a broadly targeted cancer therapy against multiple tumor indications.

Lipid Nanoparticle-Enabled Intracellular Delivery of Prime Editors.

Prime editing is an advanced gene editing platform with potential to correct almost any disease-causing mutation. As genome editors have evolved, their size and complexity have increased, hindering delivery technologies with low-carrying capacity and endosomal escape. We formulated an array of lipid nanoparticles (LNPs) containing prime editors (PEs). We were able to encapsulate PEs in LNPs and confirmed the presence of PE mRNA and two different guide RNAs using HPLC. In addition, we developed a novel reporter cell line for rapid identification of LNPs suited for prime editing. A 54% prime editing rate was observed with enhanced LNPs (eLNPs) containing the cholesterol analog ß-sitosterol at optimal ratios of RNA cargoes. eLNPs displayed a polyhedral morphology and a more fluid membrane state that led to improved endosomal escape, eventually causing onset of editing within 9 h and reaching maximum efficiency after 24 h. Hence, PEs delivered using LNPs can propel a new wave of therapies for many additional targets potentially enabling a range of new applications.

Comparative study of lipid nanoparticle-based mRNA vaccine bioprocess with machine learning and combinatorial artificial neural network-design of experiment approach.

To develop a combinatorial artificial-neural-network design-of-experiment (ANN-DOE) model, the effect of ionizable lipid, an ionizable lipid-to-cholesterol ratio, N/P ratio, flow rate ratio (FRR), and total flow rate (TFR) on the outcome responses of mRNA-LNP vaccine were evaluated using a definitive screening design (DSD) and machine learning (ML) algorithms. Particle size (PS), PDI, zeta potential (ZP), and encapsulation efficiency (EE) of mRNA-LNP were optimized within a defined constraint (PS 40-100 nm, PDI ≤ 0.30, ZP≥(±)0.30 mV, EE ≥ 70 %), fed to ML algorithms (XGBoost, bootstrap forest, support vector machines, k-nearest neighbors, generalized regression-Lasso, ANN) and prediction was compared to ANN-DOE model. Increased FRR decreased the PS and increased ZP, while increased TFR increased PDI and ZP. Similarly, DOTAP and DOTMA produced higher ZP and EE. Particularly, a cationic ionizable lipid with an N/P ratio ≥ 6 provided a higher EE. ANN showed better predictive ability (R2 = 0.7269-0.9946), while XGBoost demonstrated better RASE (0.2833-2.9817). The ANN-DOE model outperformed both optimized ML models by R2 = 1.21 % and RASE = 43.51 % (PS prediction), R2 = 0.23 % and RASE = 3.47 % (PDI prediction), R2 = 5.73 % and RASE = 27.95 % (ZP prediction), and R2 = 0.87 % and RASE = 36.95 % (EE prediction), respectively, which demonstrated that ANN-DOE model was superior in predicting the bioprocess compared to independent models.

Method of manufacturing CAF®09 liposomes affects immune responses induced by adjuvanted subunit proteins.

The ability to induce antigen-specific CD4+ and CD8+T-cell responses is one of the fundamental requirements when developing new efficacious vaccines against challenging infectious diseases and cancer. However, no adjuvants are currently approved for human subunit vaccines that induce T-cell immunity. Here, we incorporated a Toll-like receptor 4 agonist, i.e., the ionizable lipidoid L5N12, in the liposomal cationic adjuvant formulation 09 (CAF®09), and found that modified CAF®09 liposomes possess preserved adjuvant function as compared to unmodified CAF®09. CAF®09 consists of the cationic lipid dimethyldioctadecylammonium (DDA), monomycoloyl glycerol analogue 1 (MMG-1), and polyinosinic:polycytidylic acid [poly(I:C)]. By using the microfluidic mixing technology for liposome preparation, we gradually replaced DDA with L5N12, while keeping the molar ratios of MMG-1 and poly(I:C) constant. We found that this type of modification resulted in colloidally stable liposomes, which were significantly smaller and displayed reduced surface charge as compared to unmodified CAF®09, prepared by using the conventional thin film method. We showed that incorporation of L5N12 decreases the membrane rigidity of CAF®09 liposomes. Furthermore, vaccination with antigen adjuvanted with L5N12-modified CAF®09 or antigen adjuvanted with unmodified CAF®09, respectively, induced comparable antigen-specific serum antibody titers. We found that antigen adjuvanted with L5N12-modified CAF®09 induced antigen-specific effector and memory CD4+ and CD8+T-cell responses in the spleen comparable to those induced when unmodified CAF®09 was used as adjuvant. However, incorporating L5N12 did not have a synergistic immunopotentiating effect on the antibody and T-cell responses induced by CAF®09. Moreover, vaccination with antigen adjuvanted with unmodified CAF®09, which was manufactured by using microfluidic mixing, induced significantly lower antigen-specific CD4+ and CD8+T-cell responses than vaccination with antigen adjuvanted with unmodified CAF®09, which was prepared by using the thin film method. These results show that the method of manufacturing affects CAF®09 liposome adjuvanted antigen-specific immune responses, which should be taken into consideration when evaluating immunogenicity of subunit protein vaccines.

Elucidating the nanostructure of small interfering RNA-loaded lipidoid-polymer hybrid nanoparticles.

We analyzed the structural and material properties of small interfering RNA (siRNA)-loaded lipid-polymer hybrid nanoparticles (LPNs) containing ionizable lipidoid and poly(dl-lactic-co-glycolic acid) (PLGA) using small-angle X-ray scattering, cryogenic transmission electron microscopy, polarized light microscopy, the Langmuir monolayer methodology, differential scanning calorimetry, and attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy. Scattering analyses showed that bulk lipidoid self-assemble into lamellar structures with a d-spacing of 38 Å, whereas lipidoid-siRNA lipoplexes display an in-plane lateral organization of siRNA in between lipidoid bilayers with a repeat distance of approximately 55 Å. The siRNA-loaded LPNs adopted a core-shell structure with an interaxial alignment of siRNA between lipidoid shell bilayers. Langmuir monolayer experiments showed a distinct interaction between the lipidoid headgroups and siRNA, which was dependent on buffer subphase pH. Thermal analyses suggested that PLGA and lipidoid interact, which was evident from a shift in the phase transition temperature of lipidoid, and the thermotropic phase behavior of lipidoid was affected by inclusion of siRNA. ATR-FTIR data confirmed the shift or disappearance of characteristic absorption bands of siRNA after lipidoid binding. In conclusion, siRNA-loaded LPNs display a core-shell structure, wherein the polymeric core functions as a colloid matrix support for siRNA-loaded lipidoid shell layers.

Pulmonary Administration of the Liposome-Based Adjuvant CAF01: Effect of Surface Charge on Mucosal Adjuvant Function.

Mucosal surfaces of the lungs represent a major site of entry for airborne pathogens, and pulmonary administration of vaccines is an attractive strategy to induce protective mucosal immunity in the airways. Recently, we demonstrated the potential of pulmonary vaccination with the tuberculosis subunit antigen H56 adjuvanted with the cationic liposomal adjuvant formulation CAF01, which consists of the cationic lipid dimethyldioctadecylammonium (DDA) bromide and the synthetic cord factor trehalose-6,6'-dibehenate. However, the cationic charge of DDA represents a major safety challenge. Hence, replacing DDA with a safer zwitterionic or anionic phospholipid is an attractive approach to improve vaccine safety, but the effect of liposomal surface charge on the induction of mucosal immunity after airway immunization is poorly understood. Here, we investigated the effect of surface charge by replacing the cationic DDA component of CAF01 with zwitterionic dipalmitoylphosphatidylcholine (DPPC) or anionic dipalmitoylphosphatidylglycerol (DPPG), and we show that charge modification enhances antigen-specific pulmonary T-cell responses against co-formulated H56. We systematically replaced DDA with either DPPC or DPPG and found that these modifications resulted in colloidally stable liposomes that have similar size and morphology to unmodified CAF01. DPPC- or DPPG-modified CAF01 displayed surface charge-dependent protein adsorption and induced slightly higher follicular helper T cells and germinal center B cells in the lung-draining lymph nodes than unmodified CAF01. In addition, modified CAF01 induced significantly higher levels of H56-specific Th17 cells and polyfunctional CD4+ T cells in the lungs, as compared to unmodified CAF01. However, the strong H56-specific humoral responses induced by CAF01 in the lungs and spleen were not influenced by surface charge. Hence, these results provide insights into the importance of surface charge for liposomal adjuvant function and can also guide the design of safe pulmonary subunit vaccines against other mucosal pathogens.

Insights into the mechanisms of interaction between inhalable lipid-polymer hybrid nanoparticles and pulmonary surfactant.

Pulmonary delivery of small interfering RNA (siRNA) using nanoparticle-based delivery systems is promising for local treatment of respiratory diseases. We designed dry powder inhaler formulations of siRNA-loaded lipid-polymer hybrid nanoparticles (LPNs) with aerosolization properties optimized for inhalation therapy. Interactions between LPNs and pulmonary surfactant (PS) determine the fate of inhaled LPNs, but interaction mechanisms are unknown. Here we used surface-sensitive techniques to study how physicochemical properties and pathological microenvironments influence interactions between siRNA-loaded LPNs and supported PS layers. PS was deposited on SiO2 surfaces as single bilayer or multilayers and characterized using quartz crystal microbalance with dissipation monitoring and Fourier-transform infrared spectroscopy with attenuated total reflection. Immobilization of PS as multilayers, resembling the structural PS organization in the alveolar subphase, effectively reduced the relative importance of interactions between PS and the underlying surface. However, the binding affinity between PS and LPNs was identical in the two models. The physicochemical LPN properties influenced the translocation pathways and retention time of LPNs. Membrane fluidity and electrostatic interactions were decisive for the interaction strength between LPNs and PS. Experimental conditions reflecting pathological microenvironments promoted LPN deposition. Hence, these results shed new light on design criteria for LPN transport through the air-blood barrier.

Simultaneous quantification of multiple RNA cargos co-loaded into nanoparticle-based delivery systems.

Robust, sensitive, and versatile analytical methods are essential for quantification of RNA drug cargos loaded into nanoparticle-based delivery systems. However, simultaneous quantification of multiple RNA cargos co-loaded into nanoparticles remains a challenge. Here, we developed and validated the use of ion-pair reversed-phase high-performance liquid chromatography combined with UV detection (IP-RP-HPLC-UV) for simultaneous quantification of single- and double-stranded RNA cargos. Complete extraction of RNA cargo from the nanoparticle carrier was achieved using a phenol:chloroform:isoamyl alcohol mixture. Separations were performed using either a C18 or a PLRP-S column, eluted with 0.1 M triethylammonium acetate (TEAA) solution as ion-pairing reagent (eluent A), and 0.1 M TEAA containing 25 % (v/v) CH3CN as eluent B. These methods were applied to quantify mRNA and polyinosinic:polycytidylic acid co-loaded into lipid-polymer hybrid nanoparticles, and single-stranded oligodeoxynucleotide donors and Alt-R CRISPR single guide RNAs co-loaded into lipid nanoparticles. The developed methods were sensitive (limit of RNA quantification < 60 ng), linear (R2 > 0.997), and accurate (≈ 100 % recovery of RNA spiked in nanoparticles). Hence, the present study may facilitate convenient quantification of multiple RNA cargos co-loaded into nanoparticle-based delivery systems.

Leucine improves the aerosol performance of dry powder inhaler formulations of siRNA-loaded nanoparticles.

Thermostable dry powder inhaler (DPI) formulations with high aerosol performance are attractive inhalable solid dosage forms for local treatment of inflammatory lung diseases. We recently demonstrated that lipidoid-polymer hybrid nanoparticles (LPNs) loaded with small interfering RNA (siRNA) directed against tumor necrosis factor alpha (TNF-α) mediate efficient intracellular siRNA delivery and reduce inflammation in vivo. Here, we show that mixtures of the stabilizing excipients trehalose (Tre) and dextran (Dex), in combination with the shell-forming dispersion enhancer leucine (Leu), stabilize TNF-α siRNA-loaded LPNs during spray drying into nanocomposite microparticles, and result in DPI formulations with high aerosol performance. At low Leu content (0 to 10%, w/w), the DPI formulations were amorphous, and exhibited poor aerosol performance. When the Leu content was increased from 20 to 60% (w/w), the surface content of Leu increased from 39.2 to 68.1 mol%, and the flowability was significantly improved. Microscopy analysis suggest that the improved powder dispersibility is the result of a wrinkled surface morphology, which reduces the surface area available for interparticle interactions. Increasing the Leu content further (to above 10%, w/w) did not influence the aerosol performance, and the aerosol yield was maximal at 30-40% Leu (w/w). Formulations containing 40% Leu and a Tre:Dex ratio of 10:90 (w/w) displayed a high fine particle fraction and aerosol properties suitable for inhalation. The chemical integrity of TNF-α siRNA was preserved in the solid state, and biodistribution studies in mice showed that pulmonary administration of DPI formulations with high aerosol performance resulted in homogenous deep lung deposition. Our results demonstrate that at optimal ratios, ternary excipient mixtures of Leu, Tre and Dex protect TNF-α siRNA-loaded LPNs during spray drying. Hence, this study shows that microparticles with an amorphous Tre/Dex matrix and a crystalline Leu shell efficiently stabilize the nanocomposite LPNs in the solid state, and ensure aerosol properties suitable for inhalation.

Adsorption of protein antigen to the cationic liposome adjuvant CAF®01 is required for induction of Th1 and Th17 responses but not for antibody induction.

The degree of antigen adsorption to adjuvants in subunit vaccines may significantly influence the immune responses they induce upon vaccination. Commonly used approaches for studying how the level of adsorption affects the induction of antigen-specific immune responses include (i) using adjuvants with different abilities to adsorb antigens, and (ii) comparing different antigens selected based on their ability to adsorb to the adjuvant. A weakness of these approaches is that not only the antigen adsorption level is varied, but also other important functional factors such as adjuvant composition and/or the B/T cell epitopes, which may affect immunogenicity. Hence, we investigated how changing the adsorption capabilities of a single antigen to an adjuvant influenced the vaccine-induced immune responses. The model antigen lysozyme, which displays a positive net charge at physiological pH due to an isoelectric point (pI) of 11, was succinylated to different extents, resulting in a reduction of the pI value to 4.4-5.9, depending on the degree of succinylation. A pronounced inverse correlation was found between the pI value of the succinylated lysozyme analogues and the degree of adsorption to a cationic liposomal adjuvant consisting of dimethyldioctadecylammonium bromide (DDA) and trehalose dibehenate (TDB) (CAF®01). Furthermore, increased adsorption to this adjuvant correlated directly with the magnitude of lysozyme-specific Th1/Th17 immune responses induced by the vaccine in mice, while there was an inverse correlation with antibody induction. However, high lysozyme-specific antibody titers were induced with an increased antigen dose, even upon vaccination with a strongly adsorbed succinylated lysozyme analogue. Hence, these data illustrate that the degree of lysozyme adsorption to CAF®01 strongly affects the quality of the resulting immune responses.

Delivery of oligonucleotide-based therapeutics: challenges and opportunities.

Nucleic acid-based therapeutics that regulate gene expression have been developed towards clinical use at a steady pace for several decades, but in recent years the field has been accelerating. To date, there are 11 marketed products based on antisense oligonucleotides, aptamers and small interfering RNAs, and many others are in the pipeline for both academia and industry. A major technology trigger for this development has been progress in oligonucleotide chemistry to improve the drug properties and reduce cost of goods, but the main hurdle for the application to a wider range of disorders is delivery to target tissues. The adoption of delivery technologies, such as conjugates or nanoparticles, has been a game changer for many therapeutic indications, but many others are still awaiting their eureka moment. Here, we cover the variety of methods developed to deliver nucleic acid-based therapeutics across biological barriers and the model systems used to test them. We discuss important safety considerations and regulatory requirements for synthetic oligonucleotide chemistries and the hurdles for translating laboratory breakthroughs to the clinic. Recent advances in the delivery of nucleic acid-based therapeutics and in the development of model systems, as well as safety considerations and regulatory requirements for synthetic oligonucleotide chemistries are discussed in this review on oligonucleotide-based therapeutics.

Engineering of Solid Dosage Forms of siRNA-Loaded Lipidoid-Polymer Hybrid Nanoparticles Using a Quality-by-Design Approach.

Therapy based on RNA interference (RNAi), which can be mediated by exogenous small interfering RNA (siRNA), has potential for the management of diseases at the genetic level by silencing gene function(s). In all eukaryotic cells, RNAi is an endogenous regulatory mechanism, where messenger RNA (mRNA) is degraded, preventing its translation into protein. A significant advantage of RNAi therapy is that siRNA is very potent and gene silencing is highly specific, ensuring few off-target effects. However, the delivery of exogenous siRNA to the RNAi pathway in the cytosol is a challenge, and there is a need for development of advanced delivery systems to ensure safe and effective delivery of siRNA to the intracellular target site. Recently, we demonstrated the ability of lipid-polymer hybrid nanoparticles (LPNs) composed of cationic lipidoid 5 (L5) and the biodegradable polymer poly(DL-lactic-co-glycolic acid) to effectively deliver siRNA directed against tumor necrosis factor alpha (TNF-α) intracellularly to macrophages. L5 is a novel lipid-like material consisting of a tetraamine backbone linked to five C12 alkyl chains. Here, we describe a systematic quality-by-design (QbD) approach including risk assessment and design of experiments to investigate the influence of critical formulation parameters (i.e., L5 content and L5:TNF-α siRNA ratio (w/w)) on the physicochemical properties and the TNF-α gene silencing ability of TNF-α siRNA-loaded LPNs, prepared by using a double emulsion solvent evaporation method. We then detail protocols for the manufacturing of more stable solid dosage forms of LPNs using freeze drying and spray drying processes, respectively. We also provide protocols for characterization of the physicochemical properties of the nanocomposite dry powders, including (1) process yield, (2) aerodynamic particle size, (3) surface morphology, (4) moisture content, and (5) solid state properties. General considerations are provided that emphasize the advantages and disadvantages of applying QbD approaches for optimizing nanoparticulate formulations.

Inhaled RNA Therapeutics for Obstructive Airway Diseases: Recent Advances and Future Prospects.

Obstructive airway diseases, e.g., chronic obstructive pulmonary disease (COPD) and asthma, represent leading causes of morbidity and mortality worldwide. However, the efficacy of currently available inhaled therapeutics is not sufficient for arresting disease progression and decreasing mortality, hence providing an urgent need for development of novel therapeutics. Local delivery to the airways via inhalation is promising for novel drugs, because it allows for delivery directly to the target site of action and minimizes systemic drug exposure. In addition, novel drug modalities like RNA therapeutics provide entirely new opportunities for highly specific treatment of airway diseases. Here, we review state of the art of conventional inhaled drugs used for the treatment of COPD and asthma with focus on quality attributes of inhaled medicines, and we outline the therapeutic potential and safety of novel drugs. Subsequently, we present recent advances in manufacturing of thermostable solid dosage forms for pulmonary administration, important quality attributes of inhalable dry powder formulations, and obstacles for the translation of inhalable solid dosage forms to the clinic. Delivery challenges for inhaled RNA therapeutics and delivery technologies used to overcome them are also discussed. Finally, we present future prospects of novel inhaled RNA-based therapeutics for treatment of obstructive airways diseases, and highlight major knowledge gaps, which require further investigation to advance RNA-based medicine towards the bedside.

Thermo-Mechanical Fractional Injury Enhances Skin Surface- and Epidermis- Protoporphyrin IX Fluorescence: Comparison of 5-Aminolevulinic Acid in Cream and Gel Vehicles.

BACKGROUND AND OBJECTIVES: Thermo-mechanical fractional injury (TMFI) impacts the skin barrier and may increase cutaneous drug uptake. This study investigated the potential of TMFI in combination with 5-aminolevulinic acid (ALA) cream and gel formulations to enhance Protoporphyrin IX (PpIX) fluorescence at the skin surface and in the skin. STUDY DESIGN/MATERIALS AND METHODS: In healthy volunteers (n = 12) a total of 144 test areas were demarcated on the upper back. Test areas were randomized to (i) TMFI (6 milliseconds, 400 µm at a single pass) or no pretreatment and (ii) 20% ALA in cream or gel formulations. Skin surface PpIX fluorescence was quantified by PpIX fluorescence photography and photometry in 30-minute intervals until 3 hours. PpIX fluorescence microscopy quantified separate PpIX fluorescence in the epidermis, and in superficial-, mid-, and deep- dermis from punch biopsies sampled after 3 hours of ALA incubation. Local skin reactions (LSR) and pain intensities (numerical rating scale 0-10) were evaluated immediately, at 3 hours and 14 days after the intervention. RESULTS: TMFI exposure before photosensitizer application significantly increased skin surface PpIX fluorescence, both for ALA cream (TMFI-ALA-cream 7848 arbitrary units [AU] vs. ALA-cream 5441 AU, 3 hours, P < 0.001) and ALA gel (TMFI + ALA-gel 4591 AU vs. ALA-gel 3723 AU, 3 hours, P < 0.001). The TMFI-mediated increase in PpIX fluorescence was similar for ALA-cream and -gel formulations (P = 0.470) at the skin surface. In the epidermis, PpIX fluorescence intensities increased from combination treatment with TMFI and ALA-cream (TMFI + ALA-cream 421 AU vs. ALA-cream 293 AU, P = 0.034) but not from combination with TMFI and ALA-gel (TMI + ALA-gel 264 AU vs. ALA-gel 261 AU, P = 0.791). Dermal fluorescence intensities (superficial-, mid-, or deep dermis) were unaffected by TMFI pretreatment in both ALA-cream and ALA-gel exposed skin (P = 0.339). ALA-cream generally induced higher PpIX fluorescence intensities than ALA-gel (skin surface P < 0.001 and epidermis P < 0.03). TMFI induced low pain intensities (median 3) and mild LSR that were resolved at 14 days follow-up. CONCLUSION: Given the present study design, TMFI, in combination with the standardized application of 20% ALA cream and gel formulations, significantly enhanced skin surface PpIX fluorescence compared to no pretreatment. Additionally, TMFI increased epidermal PpIX fluorescence combined with 20% ALA cream vehicle. Thus, TMFI pretreatment and formulation characteristics exert influence on PpIX fluorescence intensities in normal skin. Lasers Surg. Med. © 2020 Wiley Periodicals LLC.

The Long Road Toward COVID-19 Herd Immunity: Vaccine Platform Technologies and Mass Immunization Strategies.

There is an urgent need for effective countermeasures against the current emergence and accelerating expansion of coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Induction of herd immunity by mass vaccination has been a very successful strategy for preventing the spread of many infectious diseases, hence protecting the most vulnerable population groups unable to develop immunity, for example individuals with immunodeficiencies or a weakened immune system due to underlying medical or debilitating conditions. Therefore, vaccination represents one of the most promising counter-pandemic measures to COVID-19. However, to date, no licensed vaccine exists, neither for SARS-CoV-2 nor for the closely related SARS-CoV or Middle East respiratory syndrome-CoV. In addition, a few vaccine candidates have only recently entered human clinical trials, which hampers the progress in tackling COVID-19 infection. Here, we discuss potential prophylactic interventions for SARS-CoV-2 with a focus on the challenges existing for vaccine development, and we review pre-clinical progress and ongoing human clinical trials of COVID-19 vaccine candidates. Although COVID-19 vaccine development is currently accelerated via so-called fast-track programs, vaccines may not be timely available to have an impact on the first wave of the ongoing COVID-19 pandemic. Nevertheless, COVID-19 vaccines will be essential in the future for reducing morbidity and mortality and inducing herd immunity, if SARS-CoV-2 becomes established in the population like for example influenza virus.

Treatment of acute lung inflammation by pulmonary delivery of anti-TNF-α siRNA with PAMAM dendrimers in a murine model.

To improve the efficacy of nucleic acid-based therapeutics, e.g., small interfering RNA (siRNA), transfection agents are needed for efficient delivery into cells. Several classes of dendrimers have been found useful as transfection agents for the delivery of siRNA because their surface can readily be functionalized, and the size of the dendriplexes they form with siRNA is within the range of conventional nanomedicine. In this study, commercially available generation 3 poly(amidoamine) (PAMAM) dendrimer was investigated for pulmonary delivery of siRNA directed against tumor necrosis factor (TNF) α for the treatment of acute lung inflammation. Delivery efficiency was assessed in vitro in the RAW264.7 macrophage cell line activated with lipopolysaccharide (LPS), and efficacy was evaluated in vivo in a murine model of LPS-induced lung inflammation upon pre-treatment with TNF-α siRNA. The PAMAM dendrimer-siRNA complexes (dendriplexes) displayed strong siRNA condensation and high cellular uptake in macrophages compared with non-complexed siRNA. Q-PCR analyses showed that the dendriplexes mediated efficient and specific TNF-α silencing in vitro, as compared to non-complexed siRNA and dendriplexes with negative control siRNA. Also in vivo, the PAMAM dendriplexes induced efficacious TNF-α siRNA inhibition, as compared to non-complexed siRNA, upon pulmonary administration to mice with LPS-induced lung inflammation. Hence, these data suggest that PAMAM dendrimers are promising for the local delivery of TNF-α siRNA in the treatment of lung inflammation via pulmonary administration.