Novel dual-flow perfusion bioreactor for in vitro pre-screening of nanoparticles delivery: design, characterization and testing.

An advanced dual-flow perfusion bioreactor with a simple and compact design was developed and evaluated as a potential apparatus to reduce the gap between animal testing and drug administration to human subjects in clinical trials. All the experimental tests were carried out using an ad hoc Poly Lactic Acid (PLLA) scaffold synthesized via Thermally Induced Phase Separation (TIPS). The bioreactor shows a tunable radial flow throughout the microporous matrix of the scaffold. The radial perfusion was quantified both with permeability tests and with a mathematical model, applying a combination of Darcy's Theory, Bernoulli's Equation, and Poiseuille's Law. Finally, a diffusion test allowed to investigate the efficacy of the radial flow using Polymeric Fluorescent Nanoparticles (FNPs) mimicking drug/colloidal carriers. These tests confirmed the ability of our bioreactor to create a uniform distribution of particles inside porous matrices. All the findings candidate our system as a potential tool for drug pre-screening testing with a cost and time reduction over animal models.

Development of New Targeted Inulin Complex Nanoaggregates for siRNA Delivery in Antitumor Therapy.

Here, a novel strategy of formulating efficient polymeric carriers based on the already described INU-IMI-DETA for gene material whose structural, functional, and biological properties can be modulated and improved was successfully investigated. In particular, two novel derivatives of INU-IMI-DETA graft copolymer were synthesized by chemical functionalisation with epidermal growth factor (EGF) or polyethylenglycol (PEG), named INU-IMI-DETA-EGF and INU-IMI-DETA-PEG, respectively, in order to improve the performance of already described "inulin complex nanoaggregates" (ICONs). The latter were thus prepared by appropriately mixing the two copolymers, by varying each component from 0 to 100 wt% on the total mixture, named EP-ICONs. It was seen that the ability of the INU-IMI-DETA-EGF/INU-IMI-DETA-PEG polymeric mixture to complex siGL3 increases with the increase in the EGF-based component in the EP-ICONs and, for each sample, with the increase in the copolymer:siRNA weight ratio (R). On the other hand, the susceptibility of loaded siRNA towards RNase decreases with the increase in the pegylated component in the polymeric mixture. At all R values, the average size and the zeta potential values are suitable for escaping from the RES system and suitable for prolonged intravenous circulation. By means of biological characterisation, it was shown that MCF-7 cells are able to internalize mainly the siRNA-loaded into EGF-decorated complexes, with a significant difference from ICONs, confirming its targeting function. The targeting effect of EGF on EP-ICONs was further demonstrated by a competitive cell uptake study, i.e., after cell pre-treatment with EGF. Finally, it was shown that the complexes containing both EGF and PEG are capable of promoting the internalisation and therefore the transfection of siSUR, a siRNA acting against surviving mRNA, and to increase the sensitivity to an anticancer agent, such as doxorubicin.

Correction to: Entrapment of an EGFR inhibitor into nanostructured lipid carriers (NLC) improves its antitumor activity against human hepatocarcinoma cells.

Following publication of our article [1], we became aware that Roberto Di Gesù had been omitted from the list of authors. The corrected author list and authors' contribution statement appear below. We apologize for any inconvenience this may have caused.

Nano into Micro Formulations of Tobramycin for the Treatment of Pseudomonas aeruginosa Infections in Cystic Fibrosis.

Here, nano into micro formulations (NiMs) of tobramycin for the treatment of Pseudomonas aeruginosa airway infections in cystic fibrosis (CF) are described. NiMs were produced by spray drying a solution containing polymers or sugars and a nanometric polyanion-tobramcyin complex (PTC), able to achieve a prolonged antibiotic release. NiMs properties were compared to TOBIPodhaler(Novartis), the only one commercially available dry powder inhalatory formulation based on porous microparticles. Produced NiMs showed adequate characteristics for pulmonary administration, as spherical shape, micrometric size, and high cytocompatibility toward human bronchial epithelial cells. Contrarily to TOBIPodhaler, some of produced NiMs, thanks to their specific chemical composition, are able to facilitate the drug diffusion through the mucus secretion, achieving, at the same time, a sustained tobramycin delivery. Moreover, NiMs showed pronounced antimicrobial activity against P. aeruginosa pathogens and their biofilm, if compared to free tobramycin and TOBIPodhaler, demonstrating the potential of obtained formulations as drug delivery systems for the treatment of pulmonary infections in CF patients.

Margination of Fluorescent Polylactic Acid-Polyaspartamide based Nanoparticles in Microcapillaries In Vitro: the Effect of Hematocrit and Pressure.

The last decade has seen the emergence of vascular-targeted drug delivery systems as a promising approach for the treatment of many diseases, such as cardiovascular diseases and cancer. In this field, one of the major challenges is carrier margination propensity (i.e., particle migration from blood flow to vessel walls); indeed, binding of these particles to targeted cells and tissues is only possible if there is direct carrier-wall interaction. Here, a microfluidic system mimicking the hydrodynamic conditions of human microcirculation in vitro is used to investigate the effect of red blood cells (RBCs) on a carrier margination in relation to RBC concentration (hematocrit) and pressure drop. As model drug carriers, fluorescent polymeric nanoparticles (FNPs) were chosen, which were obtained by using as starting material a pegylated polylactic acid-polyaspartamide copolymer. The latter was synthesized by derivatization of α,ß-poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA) with Rhodamine (RhB), polylactic acid (PLA) and then poly(ethyleneglycol) (PEG) chains. It was found that the carrier concentration near the wall increases with increasing pressure drop, independently of RBC concentration, and that the tendency for FNP margination decreases with increasing hematocrit. This work highlights the importance of taking into account RBC-drug carrier interactions and physiological conditions in microcirculation when planning a drug delivery strategy based on systemically administered carriers.

Pegylated Polyaspartamide-Polylactide-Based Nanoparticles Penetrating Cystic Fibrosis Artificial Mucus.

Here, the preparation of mucus-penetrating nanoparticles for pulmonary administration of ibuprofen in patients with cystic fibrosis is described. A fluorescent derivative of α,ß-poly(N-2-hydroxyethyl)-D,L-aspartamide is synthesized by derivatization with rhodamine, polylactide, and poly(ethylene glycol), to obtain polyaspartamide-polylactide derivatives with different degrees of pegylation. Starting from these copolymers, fluorescent nanoparticles with different poly(ethylene glycol) content, empty and loaded with ibuprofen, showed spherical shape, colloidal size, slightly negative ζ potential, and biocompatibility toward human bronchial epithelial cells. The high surface poly(ethylene glycol) density of fluorescent nanoparticles and poly(ethylene glycol) brush-like conformation assumed on their surface, conferred to pegylated nanoparticles the mucus-penetrating properties, properly demonstrated by assessing their ability to avoid interactions with mucus components and to penetrate cystic fibrosis artificial mucus. Finally, ibuprofen release profile and uptake capacity within human bronchial epithelial cells in the presence of cystic fibrosis artificial mucus showed how these mucus-penetrating nanoparticles could rapidly diffuse through the mucus barrier reaching the mucosal surface, where they could offer a sustained delivery of ibuprofen at the site of disease.

Improvements in Rational Design Strategies of Inulin Derivative Polycation for siRNA Delivery.

The advances of short interfering RNA (siRNA)-mediated therapy provide a powerful option for the treatment of many diseases, including cancer, by silencing the expression of targeted genes involved in the progression of the pathology. On this regard, a new pH-responsive polycation derived from inulin, Inulin-g-imidazole-g-diethylenetriamine (INU-IMI-DETA), was designed and employed to produce INU-IMI-DETA/siRNA "Inulin COmplex Nanoaggregates" (ICONs). The experimental results showed that INU-IMI-DETA exhibited strong cationic characteristics and high solubility in the pH range 3-5 and self-aggregation triggered by pH increase and physiological salt concentration. INU-IMI-DETA showed as well a high buffering capacity in the endosomal pH range of 7.4-5.1. In the concentration range between 25 and 1000 µg/mL INU-IMI-DETA had no cytotoxic effect on breast cancer cells (MCF-7) and no lytic effect on human red blood cells. ICONs were prepared by two-step procedure involving complexation and precipitation into DPBS buffer (pH 7.4) to produce siRNA-loaded nanoaggregates with minimized surface charge and suitable size for parenteral administration. Bafilomycin A1 inhibited transfection on MCF-7 cells, indicating that the protonation of the imidazole groups in the endolysosome pathway favors the escape of the system from endolysosomal compartment, increasing the amount of siRNA that can reach the cytoplasm.

Nanocomplexes for gene therapy of respiratory diseases: Targeting and overcoming the mucus barrier.

Gene therapy, i.e. the delivery and expression of therapeutic genes, holds great promise for congenital and acquired respiratory diseases. Non-viral vectors are less toxic and immunogenic than viral vectors, although they are characterized by lower efficiency. However, they have to overcome many barriers, including inflammatory and immune mediators and cells. The respiratory and airway epithelial cells, the main target of these vectors, are coated with a layer of mucus, which hampers the effective reaching of gene therapy vectors carrying either plasmid DNA or small interfering RNA. This barrier is thicker in many lung diseases, such as cystic fibrosis. This review summarizes the most important advancements in the field of non-viral vectors that have been achieved with the use of nanoparticulate (NP) systems, composed either of polymers or lipids, in the lung gene delivery. In particular, different strategies of targeting of respiratory and airway lung cells will be described. Then, we will focus on the two approaches that attempt to overcome the mucus barrier: coating of the nanoparticulate system with poly(ethylene glycol) and treatment with mucolytics. Our conclusions are: 1) Ligand and physical targeting can direct therapeutic gene expression in specific cell types in the respiratory tract; 2) Mucopenetrating NPs are endowed with promising features to be useful in treating respiratory diseases and should be now advanced in pre-clinical trials. Finally, we discuss the development of such polymer- and lipid-based NPs in the context of in vitro and in vivo disease models, such as lung cancer, as well as in clinical trials.

Polyaspartamide-Polylactide Graft Copolymers with Tunable Properties for the Realization of Fluorescent Nanoparticles for Imaging.

Here, the synthesis and the characterization of novel amphiphilic graft copolymers with tunable properties, useful in obtaining polymeric fluorescent nanoparticles for application in imaging, are described. These copolymers are obtained by chemical conjugation of rhodamine B (RhB) moieties, polylactic acid (PLA), and O-(2-aminoethyl)-O'-methyl poly(ethylene glycol) (PEG) on α,ß-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA). In particular, PHEA is first functionalized with RhB to obtain PHEA-RhB with a derivatization degree in RhB (DDRhB ) equal to 0.55 mol%. By varying the reaction conditions, different amounts of PLA are grafted on PHEA-RhB to obtain PHEA-RhB-PLA with DDPLA equal to 1.9, 4.0, and 6.2 mol%. Then, PEG chains are grafted on PHEA-RhB-PLA derivatives to obtain PHEA-RhB-PLA-PEG graft copolymers. The preparation of polymeric fluorescent nanoparticles with tunable properties and spherical shape is described by using PHEA-RhB-PLA-PEG with DD in PLA and PEG equal to 4.0 and 4.9 mol%, by following easily scaling up processes, such as emulsion-solvent evaporation and high pressure homogenization (HPH)-solvent evaporation techniques.

Entrapment of an EGFR inhibitor into nanostructured lipid carriers (NLC) improves its antitumor activity against human hepatocarcinoma cells.

BACKGROUND: In hepatocellular carcinoma (HCC), different signaling pathways are de-regulated, and among them, the expression of the epidermal growth factor receptor (EGFR). Tyrphostin AG-1478 is a lipophilic low molecular weight inhibitor of EGFR, preferentially acting on liver tumor cells. In order to overcome its poor drug solubility and thus improving its anticancer activity, it was entrapped into nanostructured lipid carriers (NLC) by using safe ingredients for parenteral delivery. RESULTS: Nanostructured lipid carriers (NLC) carrying tyrphostin AG-1478 were prepared by using the nanoprecipitation method and different matrix compositions. The best system in terms of mean size, PDI, zeta potential, drug loading and release profile was chosen to evaluate the anti-proliferative effect of drug-loaded NLC versus free drug on human hepatocellular carcinoma HA22T/VGH cells. CONCLUSIONS: Thanks to the entrapment into NLC systems, tyrphostin AG-1478 shows an enhanced in vitro anti-tumor activity compared to free drug. These finding raises hope of future drug delivery strategy of tyrphostin AG-1478 -loaded NLC targeted to the liver for the HCC treatment.

Rapamycin-based inhaled therapy for potential treatment of COPD-related inflammation: production and characterization of aerosolizable nano into micro (NiM) particles.

Our paper describes the production and characterization of inhalable microparticles loaded with nanoparticles for the lung administration of rapamycin (Rapa). In detail, core-shell lipid/polymer hybrid nanoparticles loaded with Rapa (Rapa@Man-LPHNPs) were produced with mean size of about 128 nm and slightly negative ζ potential (-13.8 mV). A fluorescent graft polyaspartamide-poly(lactic-co-glycolic acid) copolymer (PHEA-g-RhB-g-PLGA) for use as the polymeric core was obtained by nanoprecipitation, while an appropriate mixture of DPPC and mannosylated phospholipid (DSPE-PEG2000-Man) was used to provide the macrophage-targeting lipid shell. The successful formation of Rapa@Man-LPHNPs was confirmed by TEM and DSC analyses. The loaded drug (4.3 wt% of the total weight) was slowly released from the polymeric core and protected from hydrolysis, with the amount of intact drug after 24 h of incubation in the medium being equal to 74 wt% (compared to 40% when the drug is freely incubated at the same concentration). To obtain a formulation administrable by inhalation, Rapa@Man-LPHNPs were entrapped inside PVA : LEU microparticles by using the nano into micro (NiM) strategy, specifically by spray drying (SD) in the presence of a pore-forming agent. In this way, NiM particles with geometric and theoretical aerodynamic diameters equal to 4.52 µm and 3.26 µm, respectively, were obtained. Furthermore, these particles showed optimal nebulization performance, having an FPF and an MMAD equal to 27.5% and 4.3 µm, respectively.

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.

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.

Inhalable polymeric microparticles as pharmaceutical porous powder for drug administration.

In this work, the production of inhalable polymeric microparticles with modulable porosity is described. The starting polymeric material was the PHEA-g-RhB-g-PLA graft copolymer, which was suitably processed by spray drying (SD). Thanks to the addition of AB (weight percentage equal to 10 and 20 % with respect to the polymer) in the liquid feed, three biocompatible matrices were obtained with an increasing porosity in terms of pore volume (from 0.015 to 0.024 cc/g) and pore average diameter (from 1.942 to 3.060 nm), a decreasing tapped density values (from 0.75 to 0.50), and favorable aerosolization characteristics. These differences were highlighted also by a significant increase in the release of Rapamycin from the sample which showed the higher porosity (31.0 wt% after 24 hrs incubation) than the sample with the lowest porosity (14.9 wt%) in simulated lung fluid.

mPEG-PLGA Nanoparticles Labelled with Loaded or Conjugated Rhodamine-B for Potential Nose-to-Brain Delivery.

Nowdays, neurodegenerative diseases represent a great challenge from both the therapeutic and diagnostic points of view. Indeed, several physiological barriers of the body, including the blood brain barrier (BBB), nasal, dermal, and intestinal barriers, interpose between the development of new drugs and their effective administration to reach the target organ or target cells at therapeutic concentrations. Currently, the nose-to-brain delivery with nanoformulations specifically designed for intranasal administration is a strategy widely investigated with the goal to reach the brain while bypassing the BBB. To produce nanosystems suitable to study both in vitro and/or in vivo cells trafficking for potential nose-to-brain delivery route, we prepared and characterized two types of fluorescent poly(ethylene glycol)-methyl-ether-block-poly(lactide-co-glycolide) (PLGA-PEG) nanoparticles (PNPs), i.e., Rhodamine B (RhB) dye loaded- and grafted- PNPs, respectively. The latter were produced by blending into the PLGA-PEG matrix a RhB-labeled polyaspartamide/polylactide graft copolymer to ensure a stable fluorescence during the time of analysis. Photon correlation spectroscopy (PCS), UV-visible (UV-vis) spectroscopies, differential scanning calorimetry (DSC), atomic force microscopy (AFM) were used to characterize the RhB-loaded and RhB-grafted PNPs. To assess their potential use for brain targeting, cytotoxicity tests were carried out on olfactory ensheathing cells (OECs) and neuron-like differentiated PC12 cells. Both PNP types showed mean sizes suitable for nose-to-brain delivery (<200 nm, PDI < 0.3) and were not cytotoxic toward OECs in the concentration range tested, while a reduction in the viability on PC12 cells was found when higher concentrations of nanomedicines were used. Both the RhB-labelled NPs are suitable drug carrier models for exploring cellular trafficking in nose-to-brain delivery for short-time or long-term studies.

Rapamycin-Loaded Polymeric Nanoparticles as an Advanced Formulation for Macrophage Targeting in Atherosclerosis.

Recently, rapamycin (Rapa) represents a potential drug treatment to induce regression of atherosclerotic plaques; however, its use requires site-specific accumulation in the vessels involved in the formation of the plaques to avoid the systemic effects resulting from its indiscriminate biodistribution. In this work, a stable pharmaceutical formulation for Rapa was realized as a dried powder to be dispersed extemporaneously before administration. The latter was constituted by mannitol (Man) as an excipient and a Rapa-loaded polymeric nanoparticle carrier. These nanoparticles were obtained by nanoprecipitation and using as a starting polymeric material a polycaprolactone (PCL)/α,ß-poly(N-2-hydroxyethyl)-dl-aspartamide (PHEA) graft copolymer. To obtain nanoparticles targeted to macrophages, an oxidized phospholipid with a high affinity for the CD36 receptor of macrophages, the 1-(palmitoyl)-2-(5-keto-6-octene-dioyl) phosphatidylcholine (KOdia-PC), was added to the starting organic phase. The chemical-physical and technological characterization of the obtained nanoparticles demonstrated that: both the drug loading (DL%) and the entrapment efficiency (EE%) entrapped drug are high; the entrapped drug is in the amorphous state, protected from degradation and slowly released from the polymeric matrix; and the KOdia-PC is on the nanoparticle surface (KP-Nano). The biological characterization demonstrated that both systems are quickly internalized by macrophages while maintaining the activity of the drug. In vitro studies demonstrated that the effect of KP-Nano Rapa-loaded, in reducing the amount of the Phospo-Ser757-ULK1 protein through the inhibition of the mammalian target of rapamycin (mTOR), is comparable to that of the free drug.

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.

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.