Gene therapy represents a promising strategy for the treatment of severe diseases through delivery of genetic material into target cells. Its clinical translation, however, remains limited by the need for safe and efficient vectors. β-Cyclodextrins (β-CDs), owing to their biocompatibility and structural tunability, represent an attractive platform. In this study, three novel cationic β-CD derivatives (CD1, CD2, and CD3), functionalized with amino groups and different lipophilic chains, were evaluated. Preformulation studies were conducted to optimize the composition and preparation of β-CD-based nanovectors (NVs), which were subsequently complexed with a GFP-encoding plasmid (pGFP). Sodium hyaluronate (HA) and polysialic acid (PSA), were incorporated at 10% w/w, to enhance loading capacity and biocompatibility. Physico-chemical characterization included size, polydispersity index (PdI), and zeta potential. Further insights concerning the architecture of NVs were obtained by TEM, Cryo-EM, and synchrotron SAXS, which indicated a hierarchical arrangement in the structure of the complexes, while electrophoresis verified efficient nucleic acid binding. Stability was assessed at 4 °C for one week and in culture medium for 24 h. MTT test was performed in HeLa cells and transfection efficiency was analysed by flow cytometry and fluorescence microscopy. NVs were then complexed with GFP-targeting-siRNA (N/P = 10), and silencing efficiency was assessed in HeLa-GFP cells. All formulations displayed suitable size (<200 nm) and PdI (<0.3). CD1 showed the highest biopharmaceutical performance, achieving GFP expression and silencing efficiency comparable to commercial Lipofectamine. Overall, CD1 emerged as the most promising candidate, supporting its potential as an effective and biocompatible NVs for gene delivery.

Uveal melanoma is the most common primary intraocular tumor in adults that appears mainly in the choroid, ciliary body, or iris. Standard non-surgical treatment consists of radiotherapy and chemotherapy by intravitreal injection of drugs, which is hampered by unpleasant side effects and high recurrence rate. As an alternative strategy, gene therapy has the capacity of targeting specific pathways in cancer cells by introducing tumor suppressor sequences that are rendered therapeutically effective by the use of suitable delivery vectors. In this paper, we describe the development of protamine nanocapsules as potential gene delivery carriers to the eye by its topical administration. These nanocapsules, composed of spherical oily nano-droplets surrounded by a protamine shell with small particle size (≤ 250 nm) and positive surface charge (+ 33 mV), can efficiently associate different nucleic acids such as pDNA and miRNA, and release them in biorelevant media. In vitro studies evidenced their low cytotoxicity and efficient internalization with 36

Poly(organo)phosphazenes (PPZs) are increasingly recognized as versatile biomaterials for drug delivery applications in nanomedicine. Their unique hybrid structure—featuring an inorganic backbone and highly tunable organic side chains—confers exceptional biocompatibility and adaptability. Through precise synthetic methodologies, PPZs can be engineered to exhibit a wide spectrum of functional properties, including the formation of multifunctional nanostructures tailored for specific therapeutic needs. These attributes enable PPZs to address several critical challenges associated with conventional drug delivery systems, such as poor pharmacokinetics and pharmacodynamics. By modulating solubility profiles, enhancing drug stability, enabling targeted delivery, and supporting controlled release, PPZs offer a robust platform for improving therapeutic efficacy and patient outcomes. This review explores the fundamental chemistry, biopharmaceutical characteristics, and biomedical applications of PPZs, particularly emphasizing their role in zero-dimensional nanotherapeutic systems, including various nanoparticle formulations. PPZ-based nanotherapeutics are further examined based on their drug-loading mechanisms, which include electrostatic complexation in polyelectrolytic systems, self-assembly in amphiphilic constructs, and covalent conjugation with active pharmaceutical agents. Together, these strategies underscore the potential of PPZs as a next-generation material for advanced drug delivery platforms.

Gene therapy presents promising opportunities to target critical pathways in complex cancers like glioblastoma multiforme, though it necessitates the use of efficient delivery vectors. Polyphosphazenes (PPZs) are highly flexible materials that lead to biodegradable, high-performance materials in various applications, including gene delivery.In this work, we synthesized various PPZ derivatives, incorporating primary amines, secondary amines, hydrophilic, and hydrophobic groups, and evaluated their gene transfection capabilities in combination with an anionic polyphosphazene (6MHA-PPZ) that acts as a charge quencher and transfection enhancer. Combining 6MHA-PPZ with a hydrophobic polymer demonstrated the highest gene delivery efficiency and safety, significantly surpassing previous benchmarks.Using these optimized nanoparticles, we delivered a BMP4-expressing plasmid (pBMP4) in glioblastoma models. The pBMP4 nanoparticles, when combined with the chemotherapeutic agent temozolomide (Tz), resulted in significant reductions in tumor volume, improved survival rates in preclinical models, and normalized the expression of drug resistance markers, providing a synergistic antitumoral effect with Tz.This study highlights the potential of PPZ-based nanoparticles for gene delivery and suggests that the combination of pBMP4-NPs and Tz could offer a promising therapeutic strategy for treating glioblastoma.

The use of oral antibiotic therapy for the treatment of respiratory diseases as tuberculosis has promoted the appearance of side effects as well as resistance to these treatments. The low solubility, high metabolism, and degradation of drugs as rifabutin, have led to the use of combined and prolonged therapies, which difficult patient compliance. In this work, we develop inhalable formulations from biomaterials such as protamine to improve the therapeutic effect. Rifabutin-loaded protamine nanocapsules (NCs) were prepared by solvent displacement method and were physico-chemically characterized and evaluated for their dissolution, perme-ability, stability, cytotoxicity, hemocompatibility, internalization, and aerodynamic characteristics after a spray-drying procedure. Protamine NCs presented a size of around 200 nm, positive surface charge, and drug asso-ciation up to 54%. They were stable as suspension under storage, as well as in biological media and as a dry powder after lyophilization in the presence of mannitol. Nanocapsules showed a good safety profile and cellular uptake with no tolerogenic effect on macrophages and showed good compatibility with red blood cells. More-over, the aerodynamic evaluation showed a fine particle fraction deposition up to 30% and a mass median aerodynamic diameter of about 5 mu m, suitable for the pulmonary delivery of therapeutics.

Pneumococcal conjugate vaccines offer an excellent safety profile and high protection against the serotypes comprised in the vaccine. However, inclusion of protein antigens fromStreptococcus pneumoniaecombined with potent adjuvants and a suitable delivery system are expected to both extend protection to serotype strains not represented in the formulation and stimulate a broader immune response, thus more effective in young children, elderly, and immunocompromised populations. Along this line, nanoparticle (NP) delivery systems can enhance the immunogenicity of antigens by protecting them from degradation and increasing their uptake by antigen-presenting cells, as well as offering co-delivery with adjuvants. We report herein the encapsulation of a semisynthetic glycoconjugate (GC) composed of a synthetic tetrasaccharide mimicking theS. pneumoniae serotype 14 capsular polysaccharide (CP14) linked to the Pneumococcal surface protein A (PsaA) using chitosan NPs (CNPs). These GC-loaded chitosan nanoparticles (GC-CNPs) were not toxic to human monocyte-derived dendritic cells (MoDCs), showed enhanced uptake, and displayed better immunostimulatory properties in comparison to the naked GC. A comparative study was carried out in mice to evaluate the immune response elicited by the glycoconjugate-administered subcutaneously (SC), where the GC-CNPs displayed 100-fold higher IgG response as compared with the group treated with nonencapsulated GC. Overall, the study demonstrates the potential of this chitosan-based nanovaccine for efficient delivery of glycoconjugate antigens.

Tuberculosis (TB) is a life-threatening disease and a main cause of death worldwide. It mainly affects the lungs, and it is attributed to the infection with Mycobacterium tuberculosis (MTB). Current treatments consist of the oral administration of combinations of antibiotics including rifabutin, in high doses and for long periods of time. These therapeutic regimens are associated with many side effects and high rates of drug resistance. To overcome these problems, this study aims at developing a nanosystem for the improved delivery of antibiotics, with potential application in pulmonary delivery. Chitosan-based nanomaterials are widely used in biomedical applications, due to their biodegradability and biocompatibility, as well as their potential antimicrobial effects and lack of toxicity. In addition, this polymer is particularly attractive for mucosal delivery due to its bioadhesive properties. Therefore, the structure of the proposed nanocarrier consists of a chitosan shell and a lipid core with a combination of different oils and surfactants to allow optimal association of the hydrophobic drug rifabutin. These nanocapsules were characterized in terms of size, polydispersity index, surface charge, morphology, encapsulation efficiency and biological stability. The release kinetics of the drug-loaded nanostructures was evaluated in simulated lung media. Moreover, in vitro studies in different cell models (A549 and Raw 264.7 cells) demonstrated the safety of the nanocapsules as well as their efficient internalization. An antimicrobial susceptibility test was performed to evaluate the efficacy of the rifabutin-loaded nanocapsules against Mycobacterium phlei. This study indicated complete inhibition for antibiotic concentrations within the expected susceptibility range of Mycobacterium (& LE; 0.25-16 mg/L).

Crossing the Blood Brain Barrier constitutes a challenge in drug administration to the brain. In this context, nose-to-brain delivery is explored as an alternative route in the treatment of central nervous system disorders, and nanotechnology constitutes a promising tool for drug delivery to the brain. In this work, we explored niosomes and chitosan-coated niosomes (chitosomes) as possible tools for nose-to-brain delivery of clonazepam. The formulations have been optimised using different chitosan concentrations and different preparation methods as Thin Layer Evaporation-paddle (TLE-P), Evaporation (E), and Solvent Displacement Technique (SDT). The most suitable formulations were loaded with clonazepam (CLZ) and a full physicochemical characterization was performed. Chitosomes presented a size of around 200 nm, PDI < 0.3, a positive surface charge, spherical shape and a CLZ encapsulation above 60%. Chitosomes were stable for 12 weeks under storage conditions at 4oC, in simulated nasal fluid for 24 h as well as after a lyophilization-sonication process. A CLZ release of 50% was also achieved after 4 h in this media. The mucoadhesive properties of chitosomes were also confirmed, with a 1.5-fold reduction of CLZ toxicity after encapsulation and a 10-fold increase of its permeability.

Integrating peptide epitopes in self-assembling materials is a successful strategy to obtain nanovaccines with high antigen density and improved efficacy. In this study, self-assembling peptides containingMAGE-A3/PADRE epitopes were designed to generate functional therapeutic nanovaccines. To achieve higher stability, peptide/polymer hybrid nanoparticles were formulated by controlled self-assembly of the engineered peptides. The nanoparticles showed good biocompatibility to both human red blood- and dendritic cells. Incubation of the nanoparticles with immature dendritic cells triggered immune effects that ultimately activated CD8+ cells. The antigen-specific and IgG antibody responses of healthy C57BL/6 mice vaccinated with the nanoparticles were analyzed. The in vivo results indicate a specific response to the nanovaccines, mainly mediated through a cellular pathway. This research indicates that the immunogenicity of peptide epitope vaccines can be effectively enhanced by developing self-assembled peptide-polymer hybrid nanostructures.

Peptide drugs and biologics provide opportunities for treatments of many diseases. However, due to their poor stability and permeability in the gastrointestinal tract, the oral bioavailability of peptide drugs is negligible. Nanoparticle formulations have been proposed to circumvent these hurdles, but systemic exposure of orally administered peptide drugs has remained elusive. In this study, we investigated the absorption mechanisms of four insulin-loaded arginine-rich nanoparticles displaying differing composition and surface characteristics, developed within the pan-European consortium TRANS-INT. The transport mechanisms and major barriers to nanoparticle permeability were investigated in freshly isolated human jejunal tissue. Cytokine release profiles and standard toxicity markers indicated that the nanoparticles were nontoxic. Three out of four nanoparticles displayed pronounced binding to the mucus layer and did not reach the epithelium. One nanoparticle composed of a mucus inert shell and cell-penetrating octarginine (ENCP), showed significant uptake by the intestinal epithelium corresponding to 28 ± 9% of the administered nanoparticle dose, as determined by super-resolution microscopy. Only a small fraction of nanoparticles taken up by epithelia went on to be transcytosed via a dynamin-dependent process. In situ studies in intact rat jejunal loops confirmed the results from human tissue regarding mucus binding, epithelial uptake, and negligible insulin bioavailability. In conclusion, while none of the four arginine-rich nanoparticles supported systemic insulin delivery, ENCP displayed a consistently high uptake along the intestinal villi. It is proposed that ENCP should be further investigated for local delivery of therapeutics to the intestinal mucosa.

Marketed dosage forms fail to deliver anti-tubercular drugs directly to the lungs in pulmonary Tuberculosis (TB). Therefore, nanomediated isoniazid (INH)-loaded dry powder for inhalation (Nano-DPI) was developed for macrophage-targeted delivery in TB. Mannosylated chitosan (MC) and hyaluronic acid (HA) with an affinity for the surface mannose and CD44 receptors of macrophages were used in conjugation to prepare hybrid nanosuspension by ionic gelation method using cross-linker, sodium tri-polyphosphate (TPP) followed by freeze-drying to obtain a dry powder composed of nanoparticles (INH-MC/HA NPs). Nanoformulations were evaluated for aerodynamic characteristics, cytotoxicity, hemocompatibility, macrophage phenotype analysis, and immune regulation. Cellular uptake imaging was also conducted to evaluate the uptake of NPs. The nanopowders did not pose any significant toxicity to the cells, along with good compatibility with red blood cells (RBCs). The pro-inflammatory costimulatory markers were upregulated, demonstrating the activation of T-cell response. Moreover, the NPs did not show any tolerogenic effect on the macrophages. Furthermore, confocal imaging exhibited the translocation of NPs in the cells. Altogether, the findings present that nano-DPI was found to be a promising vehicle for targeting macrophages.

Pollen-based microcapsules for pulmonary delivery of anti-tuberculotic drugs Sandra Robla, Jose Manuel Ageitos, Rita Ambrus, Noemi Csaba 1 University of Santiago de Compostela, Center for Research in Molecular Medicine and Chronic Diseases (CiMUS) and Department of Pharmacology, Pharmacy and Pharmaceutical Technology, Santiago de Compostela, Spain 2 University of Szeged, Faculty of Pharmacy, Institute of Pharmaceutical Technology and Regulatory Affairs, Szeged, Hungary

Mannosylated chitosan-based pulmonary drug delivery system for targeting macrophages Mahwash Mukhtar, Noemi Csaba, Sandra Robla, Rita Ambrus 1 University of Szeged, Faculty of Pharmacy, Institute of Pharmaceutical Technology and Regulatory Affairs, Szeged, Hungary 2 University of Santiago de Compostela, Center for Research in Molecular Medicine and Chronic Diseases (CiMUS) and Department of Pharmacology, Pharmacy and Pharmaceutical Technology, Santiago de Compostela, Spain

In this work, it is described a novel micro-nanoplatform for pulmonary administration of active ingredients (AIs), including chitosan (CS)-based nanocapsules (NCs) with/without hyaluronic acid (HA), to treat lung diseases. Specifically, CS NCs and HA/CS NCs were microencapsulated in mannitol microspheres (Ma MS) by a simple spray-drying technique that gave as result dry powders of suitable characteristics for inhalation. Ma MS showed spherical morphology and sizes between 1 and 5 μm, with homogeneous distribution of NCs inside them. In aqueous media, the powders released rapidly the NCs, maintaining approximately their physicochemical characteristics. In vitro studies in A549 cells showed high cell viabilities after contact with both NCs (>90%) and Ma (≥99%); being, approximately, a 100% of cells internalized with NCs. For first time, it is exhaustively described the adjustment of the spray-drying process of NCs, resulting in a suitable and safe micro-nanoplatform of great potential for pulmonary administration.

Chitosan-based nanosystems have been described as interesting tools for antigen delivery and for enhancing the immunogenicity of nasally administered vaccines. As a possible vaccine delivery method, the chemical conjugation of chitosan nanocapsules with the Streptococcus pneumoniae cell membrane protein PsaA (pneumococcal surface adhesin A) is suggested here. The antigen PsaA, common to all pneumococcus serotypes, is expected to improve its uptake by immune cells and to activate specific T cells, generating an adaptive immune response against pneumococcus. With this aim, chitosan nanocapsules with thiol-maleimide conjugation between the polymer (chitosan) and the antigen (PsaA) were designed to enable the surface presentation of PsaA for immune cell recognition. Spherical-shaped particles, with a size of 266 ± 32 nm, positive charge of +30 ± 1 mV, and good stability profiles in simulated nasal fluids (up to 24 h) were achieved. PsaA association rates were three times higher compared with nanocapsules without covalent polymer-protein conjugation. Cytotoxicity studies in cell culture media showed non-toxic effect under 150 µg/mL concentration of nanocapsules, and subsequent studies on the maturation of immature dendritic cells in the presence of antigen-conjugated nanocapsules displayed peripheral blood mononuclear cell activation and lymphocyte differentiation after their presentation by dendritic cells. Secretion of TNFα following exposure to nanocapsules and the ability of nanocapsules to activate CD4 and CD8 T lymphocytes had also been studied. Antigen loaded nanocarrier uptake and presentation by professional presenting cells.

Pollen grains are natural microcapsules comprised of the biopolymer sporopollenin. The uniformity and special tridimensional architecture of these sporopollenin structures confer them attractive properties such as high resistance and improved bioadhesion. However, natural pollen can be a source of allergens, hindering its biomedical applicability. Several methods have been developed to remove internal components and allergenic compounds, usually involving long and laborious processes, which often cannot be extended to other pollen types. In this work, we propose an abridged protocol to produce stable and pristine hollow pollen microcapsules, together with a complete physicochemical and morphological characterization of the intermediate and final products. The optimized procedure has been validated for different pollen samples, also producing sporopollenin microcapsules from Matricaria species for the first time. Pollen microcapsules obtained through this protocol presented low protein content (4.4%), preserved ornamented morphology with a nanoporous surface, and low product density (0.14 g/cm3). These features make them interesting candidates from a pharmaceutical perspective due to the versatility of this biomaterial as a drug delivery platform.

Tuberculosis (TB) is an infectious disease that causes a great number of deaths in the world (1.5 million people per year). This disease is currently treated by administering high doses of various oral anti-TB drugs for prolonged periods (up to 2 years). While this regimen is normally effective when taken as prescribed, many people with TB experience difficulties in complying with their medication schedule. Furthermore, the oral administration of standard anti-TB drugs causes severe side effects and widespread resistances. Recently, we proposed an original platform for pulmonary TB treatment consisting of mannitol microspheres (Ma MS) containing iron (III) trimesate metal–organic framework (MOF) MIL-100 nanoparticles (NPs). In the present work, we loaded this system with the first-line anti-TB drug isoniazid (INH) and evaluated both the viability and safety of the drug vehicle components, as well as the cell internalization of the formulation in alveolar A549 cells. Results show that INH-loaded MOF (INH@MIL-100) NPs were efficiently microencapsulated in Ma MS, which displayed suitable aerodynamic characteristics for pulmonary administration and non-toxicity. MIL-100 and INH@MIL-100 NPs were efficiently internalized by A549 cells, mainly localized in the cytoplasm. In conclusion, the proposed micro-nanosystem is a good candidate for the pulmonary administration of anti-TB drugs.