Microfluidic nanoparticle synthesis for oral solid dosage forms: A step toward clinical transition processes.

Nanomedicine provides various opportunities for addressing medical challenges associated with drug bioavailability, stability, and efficacy. In particular, oral nanoparticles (NPs) represent an alternative strategy to enhance the solubility and stability of active ingredients through the gastrointestinal tract. The nanocarriers could be used for both local and systemic targeting, enabling controlled release of encapsulated drugs. This approach allows more efficient therapies. In this work, we aim to develop reliable oral solid dosage forms incorporating NPs produced by either one pot synthesis or continuous production, following protocols that yield highly consistent outcomes, promoting their technology transfer and clinical use. Microfluidics technology was selected to allow an automated and highly productive synthetic approach suitable for the highly throughput production. In particular, innovative systems, which combine advantage of NPs and solid dosage formulation, were designed, developed, and characterized demonstrating the possibility to obtaining oral administration. The resulting NPs were thus carried on oral dosage forms, i.e., pellets and minitablets. NPs resulted stable after dosage forms manufacturing, leading to confidence also on protection of encapsulated drugs. Indomethacin was used as a tracer to test biopharmaceutical behaviour. Anti-inflammatories or cytotoxic chemotherapeutics could be vehiculated leading to a breakthrough in the treatment of severe diseases allowing the oral administration of these drugs. We believe that the advancement achieved with the results of our work paves the way for the progression of nanoproducts into clinical transition processes.

Co-processed materials testing as excipients to produce Orally Disintegrating Tablets (ODT) using binder jet 3D-printing technology.

The use of co-processed materials for Orally Disintegrating Tablets (ODT) preparation by direct compression is well consolidated. However, the evaluation of their potential for ODT preparation by 3D printing technology remains almost unexplored. The present study aimed to estimate the use of commercially available co-processed excipients, conventionally applied in compression protocols, for the preparation of ODTs with binder jetting-3D printing technology. The latter was selected among the 3D printing techniques because the deposition of multiple powder layers allows for obtaining highly porous and easily disintegrating dosage forms. The influence of some process parameters, including layer thickness, type of waveform and spread speed, on the physical and mechanical properties of the prototypes printed were evaluated. Our results suggested that binder jetting-3D printing technology could benefit from the co-processed excipients for the preparation of solid dosage forms. The process optimization conducted with the experiments reported in this work indicated that additional excipients were needed to improve the physical properties of the resulting ODTs.

Endotracheal nebulization of gold nanoparticles for noninvasive pulmonary drug delivery.

Background & aims: Gold nanoparticles (AuNPs) are useful tools for noninvasive drug delivery. AuNP nebulization has shown poor deposition results, and AuNP tracking postadministration has involved methods inapplicable to clinical settings. The authors propose an intratracheal delivery method for minimal AuNP loss and computed tomography scans for noninvasive tracking. Materials & methods: Through high-frequency and directed nebulization postendotracheal intubation, the authors treated rats with AuNPs. Results & conclusion: The study showed a dose-dependent and bilateral distribution of AuNPs causing no short-term distress to the animal or risk of airway inflammation. The study demonstrated that AuNPs do not deposit in abdominal organs and show targeted delivery to human lung fibroblasts, offering a specific and noninvasive strategy for respiratory diseases requiring long-term therapies.

This study presents an alternative method for drug delivery involving gold nanoparticle aerosolization directly into the major airways. Direct nebulization prevents particle loss and avoids drug administration through the blood. The particles can be detected successfully via upper body scans, which are noninvasive and allow for on-demand monitoring. Nanoparticles are flexible tools that can be modified to target specific cells of interest and can be excreted upon completion of their function. These results could represent an alternative method of drug administration in patients needing repeated cytotoxic therapies with known off-target effects.

Natural Products from Mangroves: An Overview of the Anticancer Potential of Avicennia marina.

Exploring the potential of natural extracts for pharmaceutical applications in the treatment of different diseases is an emerging field of medical research, owing to the tremendous advantages that they can offer. These include compound sustainability due to the natural origin and virtually unlimited availability. In addition, they contribute to promoting the countries in which they are extracted and manufactured. For this reason, wild active compounds derived from plants are attracting increasing interest due to their beneficial properties. Among them, Avicennia marina has been recently recognized as a potential source of natural substances with therapeutic activities for anti-cancer treatment. A. marina beneficially supplies different chemical compounds, including cyclic triterpenoids, flavonoids, iridoids, naphtaquinones, polyphenols, polysaccharides, and steroids, most of them exhibiting potent antitumor activity. The in vivo and in vitro studies on different models of solid tumors demonstrated its dose-dependent activity. Moreover, the possibility to formulate the A. marina extracted molecules in nanoparticles allowed researchers to ameliorate the therapeutic outcome of treatments exploiting improved selectivity toward cancer cells, thus reducing the side effects due to nonspecific spread.

Conjugation of gold nanoparticles with multidentate surfactants for enhanced stability and biological properties.

This work originated from the need to functionalize surfactant-coated inorganic nanoparticles for biomedical applications, a process that is limited by excess unbound surfactant. These limitations are connected to the bioconjugation of targeting molecules that are often in equilibrium between the free aliquot in solution and that which binds the surface of the nanoparticles. The excess in solution can play a role in the biocompatability in vitro and in vivo of the final nanoparticles stock. For this purpose, we tested the ability of common surfactants - monothiolated polyethylene glycol and amphiphilic polymers - to colloidally stabilize nanoparticles as excess surfactant is removed and compared them to newly appearing multidentate surfactants endowed with high avidity for inorganic nanoparticles. Our results showed that monothiolated polyethylene glycol or amphiphilic polymers have an insufficient affinity to the nanoparticles and as the excess surfactant is removed the colloidal stability is lost, while multidentate high-avidity surfactants excel in the same regard, possibly allowing improvement in an array of nanoparticle applications, especially in those stated.

Saporin Toxin Delivered by Engineered Colloidal Nanoparticles Is Strongly Effective against Cancer Cells.

Ribosome-inactivating proteins, including Saporin toxin, have found application in the search for innovative alternative cancer therapies to conventional chemo- and radiotherapy. Saporin's main mechanism of action involves the inhibition of cytoplasmic protein synthesis. Its strong theoretical efficacy is counterbalanced by negligible cell uptake and diffusion into the cytosol. In this work, we demonstrate that by immobilizing Saporin on iron oxide nanoparticles coated with an amphiphilic polymer, which promotes nanoconjugate endosomal escape, a strong cytotoxic effect mediated by ribosomal functional inactivation can be achieved. Cancer cell death was mediated by apoptosis dependent on nanoparticle concentration but independent of surface ligand density. The cytotoxic activity of Saporin-conjugated colloidal nanoparticles proved to be selective against three different cancer cell lines in comparison with healthy fibroblasts.

Impact of Tuning the Surface Charge Distribution on Colloidal Iron Oxide Nanoparticle Toxicity Investigated in Caenorhabditis elegans.

Assessing the toxic effect in living organisms remains a major issue for the development of safe nanomedicines and exposure of researchers involved in the synthesis, handling and manipulation of nanoparticles. In this study, we demonstrate that Caenorhabditis elegans could represent an in vivo model alternative to superior mammalians for the collection of several physiological functionality parameters associated to both short-term and long-term effects of colloidally stable nanoparticles even in absence of microbial feeding, usually reported to be necessary to ensure appropriate intake. Contextually, we investigated the impact of surface charge on toxicity of superparamagnetic iron oxide coated with a wrapping polymeric envelop that confers them optimal colloidal stability. By finely tuning the functional group composition of this shallow polymer-obtaining totally anionic, partially pegylated, partially anionic and partially cationic, respectively-we showed that the ideal surface charge organization to optimize safety of colloidal nanoparticles is the one containing both cationic and anionic groups. Our results are in accordance with previous evidence that zwitterionic nanoparticles allow long circulation, favorable distribution in the tumor area and optimal tumor penetration and thus support the hypothesis that zwitterionic iron oxide nanoparticles could be an excellent solution for diagnostic imaging and therapeutic applications in nanooncology.

The Role of Polymeric Coatings for a Safe-by-Design Development of Biomedical Gold Nanoparticles Assessed in Zebrafish Embryo.

In the biomedical field, gold nanoparticles (GNPs) have attracted the attention of the scientific community thanks to their high potential in both diagnostic and therapeutic applications. The extensive use of GNPs led researchers to investigate their toxicity, identifying stability, size, shape, and surface charge as key properties determining their impact on biological systems, with possible strategies defined to reduce it according to a Safe-by-Design (SbD) approach. The purpose of the present work was to analyze the toxicity of GNPs of various sizes and with different coating polymers on the developing vertebrate model, zebrafish. In particular, increasing concentrations (from 0.001 to 1 nM) of 6 or 15 nm poly-(isobutylene-alt-maleic anhydride)-graft-dodecyl polymer (PMA)- or polyethylene glycol (PEG)-coated GNPs were tested on zebrafish embryos using the fish embryo test (FET). While GNP@PMA did not exert significant toxicity on zebrafish embryos, GNP@PEG induced a significant inhibition of embryo viability, a delay of hatching (with the smaller size NPs), and a higher incidence of malformations, in terms of tail morphology and eye development. Transmission electron microscope analysis evidenced that the more negatively charged GNP@PMA was sequestered by the positive charges of chorion proteins, with a consequent reduction in the amount of NPs able to reach the developing embryo and exert toxicological activity. The mild toxic response observed on embryos directly exposed to GNP@PMA suggest that these NPs are promising in terms of SbD development of gold-based biomedical nanodevices. On the other hand, the almost neutral GNP@PEG, which did not interact with the chorion surface and was free to cross chorion pores, significantly impacted the developing zebrafish. The present study raises concerns about the safety of PEGylated gold nanoparticles and contributes to the debated issue of the free use of this nanotool in medicine and nano-biotechnologies.

Loading Imatinib inside targeted nanoparticles to prevent Bronchiolitis Obliterans Syndrome.

Bronchiolitis Obliterans Syndrome seriously reduces long-term survival of lung transplanted patients. Up to now there is no effective therapy once BOS is established. Nanomedicine introduces the possibility to administer drugs locally into lungs increasing drug accumulation in alveola reducing side effects. Imatinib was loaded in gold nanoparticles (GNP) functionalized with antibody against CD44 (GNP-HCIm). Lung fibroblasts (LFs) were derived from bronchoalveolar lavage of BOS patients. GNP-HCIm cytotoxicity was evaluated by MTT assay, apoptosis/necrosis and phosphorylated-cAbl (cAbl-p). Heterotopic tracheal transplantation (HTT) mouse model was used to evaluate the effect of local GNP-HCIm administration by Alzet pump. GNP-HCIm decreased LFs viability compared to Imatinib (44.4 ± 1.8% vs. 91.8 ± 3.2%, p < 0.001), inducing higher apoptosis (22.68 ± 4.3% vs. 6.43 ± 0.29; p < 0.001) and necrosis (18.65 ± 5.19%; p < 0.01). GNP-HCIm reduced cAbl-p (0.41 GNP-HCIm, 0.24 Imatinib vs. to control; p < 0.001). GNP-HCIm in HTT mouse model by Alzet pump significantly reduced tracheal lumen obliteration (p < 0.05), decreasing apoptosis (p < 0.05) and TGF-ß-positive signal (p < 0.05) in surrounding tissue. GNP-HCIm treatment significantly reduced lymphocytic and neutrophil infiltration and mast cells degranulation (p < 0.05). Encapsulation of Imatinib into targeted nanoparticles could be considered a new option to inhibit the onset of allograft rejection acting on BOS specific features.

Modeling the interaction of amphiphilic polymer nanoparticles with biomembranes to Guide rational design of drug delivery systems.

Nanoparticle assisted drug delivery to the cytoplasm is limited by sequestration of nanoparticles in endosomes. Endosomal escape through polymer-induced membrane destabilization is one of a few well characterized mechanisms to overcome it. Aiming to utilize this method in vivo, it is necessary to understand how modulating the structural and chemical features of the polymer and the presence of proteins in biological fluids can affect this activity. Here, as a model for the endosomal membrane, we use the membrane of red blood cells to evaluate the membrane destabilization ability of a model amphiphilic polymer in the presence of blood plasma using a hemolysis assay. This allows determination of red blood cells membrane permeabilization through the quantification of hemoglobin leakage. Our results showed a strong inhibitory effect of plasma, and that hemolytic activity can be improved by chemical modification of the polymeric micelle, reducing its interaction with plasma proteins. Finally, a second mechanism of pH-induced direct diffusion is proposed and tested using an oil/water partitioning model. These results offer a body of knowledge to improve delivery of drugs across biological membranes using carefully designed polymeric nanocarriers.

Imatinib-loaded gold nanoparticles inhibit proliferation of fibroblasts and macrophages from systemic sclerosis patients and ameliorate experimental bleomycin-induced lung fibrosis.

Interstitial lung involvement in Systemic Sclerosis (SSc-ILD) is a complication with high morbidity and mortality. Specifically, engineered gold nanoparticles (GNPs) are proposed as targeted delivery system increasing efficacy of drugs with antifibrotic effect, such as tyrosine kinases. We aimed to test in vitro and in vivo the activity of targeted Imatinib (Im)-loaded GNP on SSc-ILD patients derived cells and in experimental model of lung fibrosis. GNPs functionalized with anti-CD44 and loaded with Im (GNP-HCIm) were synthesized. Lung fibroblasts (LFs) and alveolar macrophages from bronchoalveolar lavage fluids of SSc-ILD patients were cultured in presence of nanoparticles. GNP-HCIm significantly inhibited proliferation and viability inducing apoptosis of LFs and effectively reduced IL-8 release, viability and M2 polarization in alveolar macrophages. Anti-fibrotic effect of tracheal instilled GNP-HCIm was evaluated on bleomycin lung fibrosis mouse model comparing effect with common route of Im administration. GNP-HCIm were able to reduce significantly lung fibrotic changes and collagen deposition. Finally, electron microscopy revealed the presence of GNPs inside alveolar macrophages. These data support the use of GNPs locally administered in the development of new therapeutic approaches to SSc-ILD.

Half-Chain Cetuximab Nanoconjugates Allow Multitarget Therapy of Triple Negative Breast Cancer.

The use of therapeutic monoclonal antibodies (mAbs) has revolutionized cancer treatment. The conjugation of mAbs to nanoparticles has been broadly exploited to improve the targeting efficiency of drug nanocarriers taking advantage of high binding efficacy and target selectivity of antibodies for specific cell receptors. However, the therapeutic implications of nanoconjugation have been poorly considered. In this study, half-chain fragments of the anti-EGFR mAb cetuximab were conjugated to colloidal nanoparticles originating stable nanoconjugates that were investigated as surrogates of therapeutic mAbs in triple negative breast cancer (TNBC). Three TNBC cell lines were selected according to EGFR expression, which regulates activation of MAPK/ERK and PI3K/Akt pathways, and to distinctive molecular profiling including KRAS, PTEN, and BRCA1 mutations normally associated with diverse sensitivity to treatment with cetuximab. The molecular mechanisms of action of nanoconjugated half-chain mAb, including cell targeting, interference with downstream signaling pathways, proliferation, cell cycle, and apoptosis, along with triggering of ADCC response, were investigated in detail in sensitive and resistant TNBC cells. We found that half-chain mAb nanoconjugation was able to enhance the therapeutic efficacy and improve the target selectivity against sensitive, but unexpectedly also resistant, TNBC cells. Viability assays and signaling transduction modulation suggested a role of BRCA1 mutation in TNBC resistance to cetuximab alone, whereas its effect could be circumvented using half-chain cetuximab nanoconjugates, suggesting that nanoconjugation not only improved the antibody activity but also exerted different mechanisms of action. Our results provide robust evidence of the potential of half-chain antibody nanoconjugates in the treatment of TNBC, which could offer a new paradigm for therapeutic antibody administration, potentially allowing improved curative efficiency and reduced minimal effective dosages in both sensitive and resistant tumors.