**1. Introduction**

Although it is not a deadly disease, asthma can be highly debilitating, resulting in irreversible lung damage [1]. The international guidelines of the Global Initiative for Asthma (GINA) define asthma as a pathological state characterized by chronic inflammation of the airways and reversible limitation of air flow. However, persistent lung inflammation could cause airway obstruction or hyper-reactivity, so that when the treatment is inadequate, airway re-modelling can occur and the obstruction becomes irreversible [1]. The inflammatory cascade in allergic asthma, which involves the lung infiltration of eosinophils, T lymphocytes, mast cells, and other inflammatory cells, is schematically summarized in Figure 1.

**Figure 1.** Schematic representation of the inflammatory cascade in allergic asthma.

The conventional drug therapy includes both bronchodilators (such as β-2 adrenergic agonists and anticholinergics) to control symptoms, and corticosteroids to reduce the inflammatory process. Inhaled corticosteroids represent an effective first-line treatment in mild asthma, but they have therapeutic limitations in patients with moderate to severe asthma due to several side effects, although the local administration allows to significantly reduce the administered dose [2,3].

The conventional therapies, therefore, are often associated with a series of limitations that have pushed the research towards the identification of new biological targets for treatment of the pathology. It has been seen that in individuals suffering from asthma there is overexpression of numerous genes and proteins, such as Signal Transducer and Activator of Transcription 6 (STAT6) [4], Plasminogen Activator Inhibitor-1 (PAI-1) [5], and Spleen Tyrosine Kinase (Syk) [6]. In particular, STAT6 regulates the T helper type 2 (Th2) immune response, PAI-1 is associated with asthma severity because of its role in airway remodeling, while Syk is implicated as central immune modulator promoting allergic airway inflammation; thus, their inhibition could be an effective therapeutic approach in asthma.

For these reasons, an alternative therapeutic approach of pathological states caused by an increase in the expression of some genes, as in the case of asthma, could be gene therapy, through the administration of small interference RNA (siRNA). These consists of small double-stranded RNA fragments capable of triggering the degradation of a specific mRNA [7] and therefore ultimately capable of blocking the synthesis of certain proteins. Recently, the true efficacy of siRNA directed against particular targets, such as STAT6, PAI-1, and Syk for the treatment of asthma was demonstrated through appropriate in vitro and in vivo studies [4–6].

However, despite the potential of this genetic material, it is well known that it cannot be administered as it is, also if given directly in situ, but requires the use of particular vectors capable of conveying it into the body [7,8]. The use of siRNA as therapeutic agents necessarily requires the use of a vector, which by neutralizing the negative charge may allow them to enter cells, as well as increasing their stability against enzymatic degradation by nucleases. Among gene vectors, polymeric materials have many advantages, as they can carry large quantities of genetic material and can be chemically derivatized to obtain systems specifically oriented towards particular target tissues [9,10]. The cationic character of such polymers, necessary for the establishment of interactions with negative-charged gene material to form polyplexes, is conferred by protonable amino groups at physiological or neutral pH. However, other peculiarities are often required of the polymeric material, therefore a starting material easily modifiable by simple chemical reactions is highly sought after.

Considering the above, the aim of the present experimental work was to realize a novel protonable polymeric derivative able to form a stable electrostatic complex with the chosen siRNA, and to delivery it, through the inhalation route, at the bronchial level for the realization of an innovative formulation for the management of asthma. We have chosen to work with the α,β-poly-N-2-hydroxyethyl-dl-aspartamide (PHEA) as the starting polymeric material [11], being a highly soluble in water, biocompatible, non-immunogenic, non-antigenic polymer, already used for the development of highly performing polymeric gene vectors [12,13], as well as many other drug carriers [14–17]. Moreover, as molecule to conjugate to the PHEA backbone, in order to give the cationic behavior, we have chosen an oligoamine, the 1,2-Bis(3-aminopropylamino)ethane (bAPAE), that could give a good complexing capability, an improvement of cell internalization, associated with a good cytocompatibility [18]. As genetic material has been chosen a therapeutic siRNA able to reduce the expression of STAT6, that is one of the most important transcription factors that regulate the production of Th2 cytokines and effector functions mediated by Th2 cytokines [19–22], which seems to have a major role in the mechanism that initiates an asthmatic attack.

In addition to complexing ability to the genetic material, the copolymer forming the siRNA complex should possess additional characteristics, after administration by the pulmonary route, to further diffuse trough the mucus layer present at the airway level, until reach the bronchial epithelial cells. The latter are the main target for the siRNA delivery. To confer penetrating mucus capacity to the system it is possible to increase the superficial hydrophilicity of the system, thus reducing interactions with the protein chains of the mucin; for this reason, the PHEA backbone was also conjugated with a proper amount of poly(ethyleneglycole) (PEG) [23,24]. Therefore the potential of this new polyaspartamide copolymers as material able to complex and delivery a specific siRNA for antiasthmatic therapy was tested.

#### **2. Materials and Methods**

#### *2.1. Materials*

Triethylamine (TEA), Bis(4-nitrophenyl)carbonate (BNPC), anhydrous *N,N'*-dimethylformamide (a-DMF), 1,2-Bis(3-aminopropylamino)ethane (bAPAE), *O*-(2-Aminoethyl)-*O'*-methyl poly(ethylene glycol) 2000 (H2N-PEG2000) (0.4 mmol NH2/g), disuccinimidylcarbonate (DSC), dichloromethane, aceton, diethylether, 2,4,6-Trinitrobenzenesulfonic acid (TNBS), agarose, ethidium bromide, mucine from porcin stomach were purchased from Sigma-Aldrich (Milan, Italy). All used reagents were of analytic grade.

Duplexed siRNA were purchased from Biomers.net (Ulm, Germany). The gene target sequences (5 →3 ) are: CAGUUCCGCCACUUGCCAA (sense), UUGGCAAUGGCGGAACUG (antisense).

α,β-poly(N-2-hydroxyethyl)-d,l-aspartamide (PHEA) was synthetized via polysuccinimide (PSI) reaction with ethanolamine in DMF solution, and purified according to a previously reported procedure [11].

1H-NMR (300 MHz, D2O, 25◦C, TMS): δ 2.71 (m, 2HPHEA, –COCHC**H2**CONH–), δ 3.24 (m, 2HPHEA, –NHC**H2**CH2O–), δ 3.55 (m, 2HPHEA, –NHCH2C**H2**OH), δ 4.59 [m, 1HPHEA, –NHC**H**(CO)CH2–].

#### *2.2. Copolymer Synthesis*

2.2.1. General Procedure for the Derivatization and Characterization of α,β-poly(*N*-2-hydroxyethyl)d,l-aspartamide with 1,2-Bis(3-aminopropylamino)ethane (PHEA*-g-*bAPAE)

Derivatization of PHEA with 1,2-Bis(3-aminopropylamino)ethane (bAPAE) was carried out by using Bis(4-nitrophenyl) carbonate (BNPC) as coupling agent. Two hundred milligrams of PHEA (1.26 mmol of repeating units (RU)) were dissolved in 4 mL of a-DMF; after complete solubilization, 230 mg of solid BNPC was added. The solution was stirred at 40 ◦C for 4 h. Simultaneously, 922.71 μL of bAPAE was dissolved in 7 mL of a-DMF. After activation time, the resulting polymeric solution was added dropwise and slowly to bAPAE solution. The reaction was carried out under and continuous stirring at 25 ◦C for 20 h. The amounts of each reagent were properly determined accordingly to

R1 = (mmol of BNPC/mmol of functionalizable RU on PHEA) = 0.6 and R2 = (mmol of bAPAE/mmol of functionalizable RU on PHEA) = 4.

After this time, the polymer was isolated from reaction mixture by precipitation in mixture 2:1 *v*/*v* diethyl ether/dichloromethane and the supernatant was removed by centrifugation at 4 ◦C for 8 min, at 9800 rpm. The obtained solid product was washed with acetone, until the pH of a mixture between the washing acetone with water (vol:vol 1:1) was neutral. Then, the obtained product was dried under vacuum. The solid residue was dissolved in double distilled water and then the solution was purified by dialysis (SpectraPor Dialysis Tubing, at MWCO 25 kDa), for two days against basic water (NaOH) and for other three days against bidistilled water, subsequently the solution was freeze-dried and stored for further characterization. PHEA*-g-*bAPAE graft copolymer was obtained with a yield of 80 wt % based on the starting PHEA.

1H-NMR (300 MHz, D2O pD 5, 25 ◦C, TMS): δ 1.70–2.20 (m, 4HbAPAE, –NHCH2CH2CH2NHCH2CH2NHCH2CH2CH2NH–), δ 2.73 (m, 2HPHEA, –COCHCH2CONH–), δ 3,12 (m, 8HbAPAE, –NHCH2CH2CH2NHCH2CH2NHCH2CH2CH2NH2), δ 3.23 (m, 2HPHEA, –NHCH2CH2O–), 3,38 (m, 4HbAPAE, –CONHCH2CH2–, –CH2CH2CH2NH2), δ3.54 (m, 2HPHEA, –NHCH2CH2OH), δ 3.60 (m, 4HPEG, –[OCH2CH2O]44–), δ 4.02 (m, 2HPHEA, –NHCH2CH2OCO–), δ 4.62 (m, 1HPHEA, –NHCH(CO)CH2–). The content of amine was also determined by TNBS assay [12].

2.2.2. General Procedure for the Derivatization and Characterization of PHEA with methoxy polyethylene glycol amine (H3CO-PEG-NH2)

Derivatization of PHEA with different amount of H3CO-PEG-NH2, was carried out by using N,N'-disuccinimidyl carbonate (DSC) as coupling agent [23]. Five hundred milligrams of PHEA (6.32 mmol of RU) was dissolved in 10 mL of a-DMF at 40 ◦C and then a proper amount of triethylamine (TEA), as catalyst, and DSC were added; subsequently, the reaction mixture was left at 40 ◦C for 4 h.

After the activation time, the latter dispersion of DSC-activated PHEA was added drop-wise to increasing volumes of H3CO-PEG-NH2 dispersions in a-DMF, at a concentration of 50 mg/mL. Then, the obtained mixture reactions were left at 25 ◦C for 18 h. The amounts of TEA, DSC and PEG were added according to the following moles ratios, as reported in Table 1.


**Table 1.** Molar ratio and mL of poly(ethyleneglycole) (PEG) solution (50 mg/mL) used for synthesis.

R3 = (mmol of aminoPEG/mmol of functionalizable RU on PHEA)

R4 = (mmol of DSC/mmol of functionalizable RU on PHEA)

R5 = (mmol of TEA/mmol of DSC)

After this time, each polymer was isolated from reaction mixture by precipitation diethyl ether and the supernatant was removed by centrifugation at 4 ◦C for 8 min, at 9800 rpm. The obtained solid product was washed with acetone one time and then, the obtained product was dried under vacuum. The solid residue was dissolved in double distilled water and then the solution was purified by dialysis (SpectraPor Dialysis Tubing, at MWCO 25 kDa), subsequently freeze-dried and stored for further characterization. PHEA*-g-*PEG graft copolymers were obtained with a yield of 220 wt % based on the starting PHEA. Three different PHEA*-g-*PEG graft copolymers in terms of PEG grafted on the PHEA backbone, that were named PHEA*-g-*PEG(A) and PHEA*-g-*PEG(B) and PHEA*-g-*PEG(C).

1H-NMR (300 MHz, D2O, 25 ◦C, TMS): δ 2.71 (m, 2HPHEA, –COCHCH2CONH–), δ 3.24 (m, 2HPHEA, –NHCH2CH2O–), δ3.55 (m, 2HPHEA, –NHCH2CH2OH), δ 3.60 (m, 4HPEG, –[OCH2CH2O]44–), δ 4.59 (m, 1HPHEA, –NHCH(CO)CH2–).

#### 2.2.3. General Procedure for the Derivatization and Characterization of PHEA*-g-*PEG with bAPAE

Derivatization of PHEA*-g-*PEG with bAPAE was carried out by using BNPC as coupling agent. 232 mg of PHEA*-g-*PEG(A), 278 mg of PHEA*-g-*PEG(B), or 329 mg of PHEA*-g-*PEG(C) (corresponding to 1.26 mmol of functionalizable RU) was dissolved in 4 mL of a-DMF; after complete solubilization, 230 mg of solid BNPC was added. The solution was stirred at 40 ◦C for 4 h. Simultaneously, 922.71 μL of bAPAE was dissolved in 7 mL of a-DMF. The reagents were added accordingly to R1 = (mmol of BNPC/mmol of functionalizable RU on PHEAPEG) = 0.6 and R7 = (mmol of bAPAE/mmol of functionalizable RU on PHEAPEG) = 4.

After activation time, the resulting polymeric solution was added dropwise and slowly to bAPAE solution. The reaction was carried out under continuous stirring at 25 ◦C for 20 h. After this time, the polymer was isolated from reaction mixture by precipitation in mixture 2:1 *v*/*v* diethyl ether/dichloromethane and the supernatant was removed by centrifugation at 4 ◦C for 8 min, at 9800 rpm. The obtained solid product was washed with acetone, until the pH of the washing surnatant was neutral. Then, the obtained product was dried under vacuum. The solid residue was dissolved in double distilled water and then the solution was purified by dialysis (SpectraPor Dialysis Tubing, at MWCO 25 kDa), subsequently the solution was freeze-dried and stored for further characterization. PHEA*-g-*PEG*-g-*bAPAE graft copolymers were obtained with a yield of 80 wt% based on the starting PHEA*-g-*PEG.

1H-NMR (300 MHz, D2O pD 5, 25 ◦C, TMS): δ 1.70–2.20 (m, 4HbAPAE, –NHCH2CH2CH2NHCH2CH2NHCH2CH2CH2NH–), δ 2.73 (m, 2HPHEA, –COCHCH2CONH–), δ 3.12 (m, 8HbAPAE, –NHCH2CH2CH2NHCH2CH2NHCH2CH2CH2NH2), δ 3.23 (m, 2HPHEA, –NHCH2CH2O–), 3,38 (m, 4HbAPAE, –CONHCH2CH2–, –CH2CH2CH2NH2), δ3.54 (m, 2HPHEA, –NHCH2CH2OH), δ 3.60 (m, 4HPEG, –[OCH2CH2O]44–), δ 4.02 (m, 2HPHEA, –NHCH2CH2OCO–), δ 4.62 (m, 1HPHEA, –NHCH(CO)CH2–).

#### *2.3. Determination of the Amine Content*

The content of amine-terminated side chains was also determined by TNBS assay. A stock solution of PHEA*-g-*bAPAE or PHEA*-g-*PEG(A)*-g-*bAPAE or PHEA*-g-*PEG(B)*-g-*bAPAE or PHEA*-g-*PEG(C)*-g-*bAPAE (5 mg/mL) was prepared in a borate buffer (0.1M Na2B4O7·H2O, pH 9.3). An aliquot of this solution (50 μL) was added to a cuvette containing 900 μL of borate buffer and 50 μL of 0.03 M TNBSA solution. After 120 min incubation, absorbance at λ 500 nm was measured and compared with that estimated for the reaction of H2N-PEG-OCH3 (–NH2 in the range between 0.01 and 0.001 mmol/mL) with TNBSA.

#### *2.4. Size Exclusion Chromatography*

Weight-average molecular weight (Mw), polydispersity index (Mw/Mn), of each copolymer was determined by a size exclusion chromatography (SEC) analysis, performed using Tosho Bioscience TSK-Gel G4000 PWXL and G3000 PWXL columns(Sursee, Switzerland) connected to an Agilent 1260 Infinity Multi-Detector GPC/SEC system(Santa Clara, United States), and a refractive index detector. Analyses were performed with buffer citrate/phosphate 0.15 M + 0.1 M NaCl pH 5 as eluent with a flow of 1 mL/min and poly(ethylene oxide) standard (40 kDa) to obtain the calibration curve. The column temperature was set at 30 ◦C.

#### *2.5. Potentiometric Titration of PHEA-g-bAPAE Graft Copolymer*

#### 2.5.1. Qualitative Titration of PHEA*-g-*bAPAE Copolymer

To determine the relative buffering capacity of PHEA*-g-*bAPAE copolymer, potentiometric acid−base titrations were performed. Typically, 6 mg of copolymer was dissolved in 30 mL in 0.1 N NaCl, used as ionic strength stabilizer, and the pH was adjusted to nearly 10.0 using 0.1 N sodium hydroxide. Then the mixture was titrated by gradually adding 20 μL of 0.1 N HCl until reaching pH 3. Titrations of comparable amounts of PHEA, and bAPAE, calculated considering the derivatization degree of the PHEA*-g-*bAPAE copolymer, at the same concentration present in PHEA-bAPAE, were also studied.
