Polyamidoamine-Carbon Nanodot Conjugates with Bioreducible Building Blocks: Smart Theranostic Platforms for Targeted siRNA Delivery.

This study focuses on designing hybrid theranostic nanosystems, utilizing gadolinium-doped carbon nanodots decorated with bioreducible amphoteric polyamidoamines (PAAs). The objective is to synergize the exceptional theranostic properties of gadolinium-doped carbon nanodots (CDs) with the siRNA complexation capabilities of PAAs. Linear copolymeric polyamidoamines, based on N,N'-bis(acryloyl)cystamine, arginine, and agmatine, were synthesized, resulting in three distinct amphoteric copolymers. Notably, sulfur bridges within the PAA repeating units confer pronounced susceptibility to glutathione-mediated degradation─a key attribute in the tumor microenvironment. This pathway enables controlled and stimuli-responsive siRNA release, theoretically providing precise spatiotemporal control over therapeutic interventions. The selected PAA, conjugated with CDs using the redox-sensitive spacer cystamine, formed the CDs-Cys-PAA conjugate with superior siRNA complexing capacity. Stable against polyanion exchange, the CDs-Cys-PAA/siRNA complex released siRNA in the presence of GSH. In vitro studies assessed cytocompatibility, internalization, and gene silencing efficacy on HeLa, MCF-7, and 16HBE cell lines.

TAT decorated siRNA polyplexes for inhalation delivery in anti-asthma therapy.

In this work, a novel protonable copolymer was designed to deliver siRNA through the inhalation route, as an innovative formulation for the management of asthma. This polycation was synthesized by derivatization of α,ß-poly(N-2-hydroxyethyl)D,L-aspartamide (PHEA) first with 1,2-Bis(3-aminopropylamino)ethane (bAPAE) and then with a proper amount of maleimide terminated poly(ethylene glycol) (PEG-MLB), with the aim to increase the superficial hydrophilicity of the system, allowing the diffusion trough the mucus layer. Once the complexation ability of the copolymer has been evaluated, obtaining nanosized polyplexes, polyplexes were functionalized on the surface with a thiolated TAT peptide, a cell-penetrating peptide (CPP), exploiting a thiol-ene reaction. TAT decorated polyplexes result to be highly cytocompatible and able to retain the siRNA with a suitable complexation weight ratio during the diffusion process through the mucus. Despite polyplexes establish weak bonds with the mucin chains, these can diffuse efficiently through the mucin layer and therefore potentially able to reach the bronchial epithelium. Furthermore, through cellular uptake studies, it was possible to observe how the obtained polyplexes penetrate effectively in the cytoplasm of bronchial epithelial cells, where they can reduce IL-8 gene expression, after LPS exposure. In the end, in order to obtain a formulation administrable as an inhalable dry powder, polyplexes were encapsulated in mannitol-based microparticles, by spray freeze drying, obtaining highly porous particles with proper technological characteristics that make them potentially administrable by inhalation route.

Sustained-Release Powders Based on Polymer Particles for Pulmonary Delivery of Beclomethasone Dipropionate in the Treatment of Lung Inflammation.

Inhaled corticosteroids are the mainstay in the management of lung inflammation associated to chronic lung diseases, such as asthma and chronic obstructive pulmonary disease (COPD). Nonetheless, available inhalation products are mostly short-acting formulations that require frequent administrations and do not always produce the desired anti-inflammatory effects. In this work, the production of inhalable beclomethasone dipropionate (BDP) dry powders based on polymeric particles was attempted. As starting material, the PHEA-g-RhB-g-PLA-g-PEG copolymer was chosen, obtained by grafting 0.6, 2.4 and 3.0 mol%, respectively, of rhodamine (RhB), polylactic acid (PLA) and polyethylene glycol 5000 (PEG) on alpha,beta-poly(N-2-hydroxyethyl)DL-aspartamide (PHEA). The drug was loaded into the polymeric particles (MP) as an inclusion complex (CI) with hydroxypropyl-cyclodextrin (HP-ß-Cyd) (at a stoichiometric ratio of 1:1) or as free form. The spray-drying (SD) process to produce MPs was optimized by keeping the polymer concentration (0.6 wt/vol%) constant in the liquid feed and by varying other parameters such as the drug concentration. The theoretical aerodynamic diameter (daer) values among the MPs are comparable and potentially suitable for inhalation, as confirmed also through evaluation of the experimental mass median aerodynamic diameter (MMADexp). BDP shows a controlled release profile from MPs that is significantly higher (more than tripled) than from Clenil®. In vitro tests on bronchial epithelial cells (16HBE) and adenocarcinomic human alveolar basal epithelial cells (A549) showed that all the MP samples (empty or drug-loaded) were highly biocompatible. None of the systems used induced apoptosis or necrosis. Moreover, the BDP loaded into the particles (BDP-Micro and CI-Micro) was more efficient than free BDP to counteract the effects of cigarette smoke and LPS on release of IL-6 and IL-8.

Galactosylated Polymer/Gold Nanorods Nanocomposites for Sustained and Pulsed Chemo-Photothermal Treatments of Hepatocarcinoma.

In this paper, we propose a rational design of a hybrid nanosystem capable of locally delivering a high amount of hydrophobic anticancer drugs (sorafenib or lenvatinib) and heat (hyperthermia) in a remote-controlled manner. We combined in a unique nanosystem the excellent NIR photothermal conversion of gold nanorods (AuNRs) with the ability of a specially designed galactosylated amphiphilic graft copolymer (PHEA-g-BIB-pButMA-g-PEG-GAL) able to recognize hepatic cells overexpressing the asialoglycoprotein receptor (ASGPR) on their membranes, thus giving rise to a smart composite nanosystem for the NIR-triggered chemo-phototherapy of hepatocarcinoma. In order to allow the internalization of AuNRs in the hydrophobic core of polymeric nanoparticles, AuNRs were coated with a thiolated fatty acid (12-mercaptododecanoic acid). The drug-loaded hybrid nanoparticles were prepared by the nanoprecipitation method, obtaining nanoparticles of about 200 nm and drug loadings of 9.0 and 5.4% w/w for sorafenib and lenvatinib, respectively. These multifunctional nanosystems have shown to convert NIR radiation into heat and release charged drugs in a remote-controlled manner. Then, the biocompatibility and synergistic effects of a chemo-phototherapy combination, as well the receptor-mediated internalization, were evaluated by an in vitro test on HepG2, HuH7, and NHDF. The results indicate that the proposed nanoparticles can be considered to be virtuous candidates for an efficient and selective dual-mode therapy of hepatocarcinoma.

Development of a novel rapamycin loaded nano- into micro-formulation for treatment of lung inflammation.

It has recently emerged that drugs such as the mTOR inhibitor rapamycin (Rapa) may play a key role in the treatment of airway inflammation associated with lung diseases, such as chronic obstructive pulmonary disease, asthma, and cystic fibrosis. Nevertheless, Rapa clinical application is still prevented by its unfavorable chemical-physical properties, limited oral bioavailability, and adverse effects related to non-specific biodistribution. In this paper, the design and production of a novel formulation of Rapa based on nano into micro (NiM) particles are detailed. To achieve it, Rapa-loaded nanoparticles were produced by nanoprecipitation of an amphiphilic pegylated poly-ɛ-caprolactone/polyhydroxyethyl aspartamide graft copolymer. The obtained nanoparticles that showed a drug loading of 14.4 wt% (corresponding to an encapsulation efficiency of 82 wt%) did not interact with mucins and were able to release and protect Rapa from degradation in simulated lung and cell fluids. To allow their local administration to the lungs as a dry powder, particle engineering at micro-sized level was done by embedding nanoparticles into mannitol-based microparticles by spray drying. Obtained NiM particles had a mean diameter of about 2-µ, spherical shape and had good potential to be delivered to the lungs by a breath-activated dry powder inhalers. Rheological and turbidity experiments showed that these NiM particles can dissolve in lung simulated fluid and deliver the Rapa-loaded pegylated nanoparticles, which can diffuse through the mucus layer.

Hyaluronic acid dressing of hydrophobic carbon nanodots: A self-assembling strategy of hybrid nanocomposites with theranostic potential.

We propose a rational design of hyaluronic acid-dressed red-emissive carbon dots (CDs), with a well-structured hydrophobic core capable of locally delivering high amount doxorubicin (Doxo) (> 9% w/w) and heat (hyperthermia) in a light stimuli sensitive fashion. We combined in a unique micelle-like superstructure the peculiar optical properties of CDs (NIR photothermal conversion and red fluorescence) with the ability of hyaluronic acid (HA) shell of stabilizing nanomedicines in aqueous environment and recognizing cancer cells overexpressing CD44 receptors on their membranes, thus giving rise to smart theranostic agents useful in cancer imaging and NIR-triggered chemo-phototherapy of solid tumors. Hydrophobic CDs, named HCDs, were used as functional beads to self-assemble amphiphilic HA derivatives carrying polylactic acid side chains (HA-g-PLA), yielding to light-sensitive and biodegradable core-shell superstructures. We explored the biocompatibility and synergistic effects of chemo-phototherapy combination, together with fluorescence imaging, showing the huge potential of the proposed engineering strategy in improving efficacy. CHEMICAL COMPOUNDS.

Targeted delivery of siRNAs against hepatocellular carcinoma-related genes by a galactosylated polyaspartamide copolymer.

Given the lack of effective treatments for Hepatocellular carcinoma (HCC), the development of novel therapeutic approaches is very urgent. Here, siRNAs were delivered to HCC cells by a synthetic polymer containing α,ß-poly-(N-2-hydroxyethyl)-D,L-aspartamide-(PHEA) derivatized with diethylene triamine (DETA) and bearing in the side chain galactose (GAL) linked via a polyethylene glycol (PEG) to obtain (PHEA-DETA-PEG-GAL, PDPG). The GAL residue allows the targeting to the asialo-glycoprotein receptor (ASGPR), overexpressed in HCC cells compared to normal hepatocytes. Uptake studies performed using a model siRNA or a siRNA targeted against the enhanced green fluorescence protein, demonstrated the PDPG specific delivery of siRNA to HuH7 cells, a human cellular model of HCC. GAL-free copolymer (PHEA-DETA-PEG-NH2, PDP) or the chemical block of ASGPR, impaired PDPG targeting effectiveness in vitro. The specificity of PDPG delivery was confirmed in vivo in a mouse dorsal skinfold window chamber assay. Functional studies using siRNAs targeting the mRNAs of HCC-related genes (eEF1A1, eEF1A2 and E2F1) delivered by PDPG, significantly decreased HuH7 vitality/number and down regulated the expression of the target genes. Only minor effectiveness was in contrast observed for PDP. In IHH, a human model of normal hepatocytes with reduced ASGPR expression, PDPG barely reduced cell vitality. In a subcutaneous xenograft mouse model of HCC, PDPG-siRNAs reduced HCC tumor growth compared to controls without significant toxic effects. In conclusion, our study demonstrates the valuable potentials of PDPG for the specific delivery of siRNAs targeting HCC-related genes.

Carbon Nanodots as Functional Excipient to Develop Highly Stable and Smart PLGA Nanoparticles Useful in Cancer Theranostics.

Theranostic systems have attracted considerable attention for their multifunctional approach to cancer. Among these, carbon nanodots (CDs) emerged as luminescent nanomaterials due to their exceptional chemical properties, synthetic ease, biocompatibility, and for their photothermal and fluorescent properties useful in cancer photothermal therapy. However, premature renal excretion due to the small size of these particles limits their biomedical application. To overcome these limitations, here, hybrid poly(lactic-co-glycolic acid) (PLGA-CDs) nanoparticles with suitable size distribution and stability have been developed. CDs were decisive in the preparation of polymeric nanoparticles, not only conferring them photothermal and fluorescent properties, needed in theranostics, but also having a strategic role in the stabilization of the system in aqueous media. In fact, CDs provide stable PLGA-based nanoparticles in aqueous media and sufficient cryoprotection in combination with 1% PVP. While PLGA nanoparticles required at least 5% of sucrose. Comparing nanosystems with different CDs content, it is also evident how these positively impinge on the loading and release of the drug, favoring high drug loading (~4.5%) and a sustained drug release over 48 h. The therapeutic and imaging potentials were finally confirmed through in vitro studies on a breast cancer cell line (MDA-MB-231) using fluorescence imaging and the MTS cell viability assay.

Design of New Polyaspartamide Copolymers for siRNA Delivery in Antiasthmatic Therapy.

Here, a novel protonable copolymer was realized for the production of polyplexes with a siRNA (inhibitor of STAT6 expression in asthma), with the aim of a pulmonary administration. The polycation was synthesized by derivatization of α,ß-poly(N-2-hydroxyethyl)d,l-aspartamide (PHEA) with 1,2-Bis(3-aminopropylamino)ethane (bAPAE) in proper conditions to obtain a PHEA-g-bAPAE graft copolymer with a derivatization degree in amine (DDbAPAE%) equal to 35 mol%. The copolymer showed a proper buffering behavior, i.e., ranging between pH 5 and 7.4, to potentially give the endosomal escape of the obtained polycations. In effect, an in vitro experiment demonstrated the effect on biological membranes of the copolymer on bronchial epithelial cells (16-HBE) strongly dependent on the pH of the medium, i.e., higher at pH 5. bAPAE-based copolymers were further obtained with an increasing pegylation degree, i.e., equal to 1.9, 2.7, and 4.4 mol%, respectively. All the obtained copolymers were able to complex siRNA at a N/P ratio that decreases as the pegylation degree increases. At the same time, the tendency of polyplexes to aggregate and the capability to interact with mucin also decreases as the pegylation in the copolymer increases. Gene silencing experiments on 16-HBE showed that these copolymers have a significant role in improving the intracellular transport of naked siRNA, where the presence of PEG does not seem to hinder the cellular uptake of polyplexes. The latter obtained at polymer/siRNA weight ratio (R) equal to 10 with PHEA-g-PEG(C)-g-bAPAE also seems to be not susceptible to the presence of mucin, avoiding the polyanionic exchange of complexed siRNA, thus showing adequate behavior to be used as an effective vector for siRNA.