Novel cationic polyaspartamide with covalently linked carboxypropyl-trimethyl ammonium chloride as a candidate vector for gene delivery

EUROPEAN POLYMER JOURNAL

European Polymer Journal 42 (2006) 823–834

www.elsevier.com/locate/europolj

Novel cationic polyaspartamide with covalently linked carboxypropyl-trimethyl ammonium chloride as a candidate vector for gene delivery Mariano Licciardi, Monica Campisi, Gennara Cavallaro, Bianca Carlisi, Gaetano Giammona * Dipartimento di Chimica e Tecnologie Farmaceutiche, Via Archirafi 32, 90123 Palermo, Italy Received 20 July 2005; received in revised form 26 September 2005 Available online 23 November 2005

Abstract The non-viral gene vector properties of a protein-like polymer, the a,b-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) were investigated after its derivatization with 3-(carboxypropyl)trimethyl-ammonium chloride (CPTA) as molecule bearing cationic groups, in order to obtain stable polycations able to condense DNA. PHEA was firstly functionalized with hydrazide pendant groups by reaction with hydrazine monohydrate (HYD), obtaining the polyhydrazide a,b-poly(N2-hydroxyethyl/carbazate)-D,L-aspartamide (PHEA–HYD). In this paper we reported that polymer functionalization degree can be easily modulated by varying reaction conditions, so allowing us to produce two PHEA derivatives at different molar percentage of hydrazide groups. Subsequently, condensation reaction of PHEA–HYD copolymers with CPTA yielded a,b-poly(N-2-hydroxyethyl)-N-carbazate[N 0 -(3-trimethylammonium chloride)propylhydrazide]-D,L-aspartamide (PHEA–HYD–CPTA) polycation derivatives. In vitro studies were carried out to evaluate polycations ability to complex DNA and to protect it from nuclease degradation. Obtained results demonstrated the good efficiency of our new PHEApolycations derivatives, PHEA–HYD–CPTA, to complex and condense genomic material even at very low polycation/ DNA weight ratio. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Synthetic gene vectors; Polycation; Polyplexes, a,b-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA), 3-(carboxypropyl) trimethyl ammonium chloride

1. Introduction Gene therapy is an experimental treatment that involves the introduction of genetic material * Corresponding author. Tel.: +39 091 6236131; fax: +39 091 6236150. E-mail address: [email protected] (G. Giammona).

(DNA or RNA) into cells to fight disease. This therapeutic approach is being studied in clinical trials for many different types of diseases, such as treatment of cancer by replacing missing or altered genes with healthy genes [1,2]. Gene therapy is also used to stimulate the bodys natural ability to attack cancer cells, improving patient immune response against tumors; in another

0014-3057/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.eurpolymj.2005.10.005

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approach ‘‘suicide genes’’ could be introduced into cancer cells to destroy them. [1,2]. In general, due to its high instability in plasma, its high molecular weight and its polyanionic nature, a gene cannot be directly inserted into human cells: it must be delivered to the cell using a carrier or ‘‘gene vector’’. Gene delivery techniques can use viral and nonviral vectors [3]. For the first type, scientists alter the viruses to make them safe for humans and to increase their ability to deliver specific genes to patient cells. Depending on the type of virus and the goals of the research study, it may be possible to genetically inactivate viruses to prevent their pathogenicity, or to better introduce them into target cells [3]. Viruses have a unique ability to recognize certain cells and insert their DNA into the cells but their main problem is that they can infect different types of cells. Therefore, when viral vectors are used to carry genes into specific cells, they might infect healthy cells as well as target cells [4]. Other concerns include the possibility that transferred genes could be ‘‘over expressed’’, producing so much of the missing protein as to be harmful; moreover viral vectors could cause inflammation or an immune reaction [4]. For all these reasons, to effectively treat cancer and other diseases with gene therapy, researchers must develop vectors that can be successfully injected into the patient inserting the desired gene on the target cells located throughout the body [2]. For a number of years, a new and safer approach to deliver DNA, RNA or oligonucleotides for gene therapy is the use of non-viral vectors, such as cationic lipids [5], polymeric micelles [6], hydrophilic polycations [7–9], peptidic vectors [10]. Due to the improved safety profile and easy preparation and manipulation, non-viral techniques for gene delivery continue to be explored and optimized [11]. The ideal non-viral vector should be able to interact reversibly with DNA and to protect it against degradation during biodistribution; moreover they should be small enough to cross vessels and to enter into the cells, where they should deliver genes preferentially in the nucleus [9,12]. The aim of this work was to develop new polycationic derivatives that are able to interact electrostatically with DNA forming stable polyplexes [13]. Our research group has recently proposed the cationic derivatives of a protein-like polymer,

the a,b-poly(asparthylhydrazide) (PAHy) bearing positive charge moieties, such as glycidyltrymethylammonium chloride (GTA) [7] or 3-carboxypropyltrimethylamonium chloride (CPTA) [8], as potential polymeric vectors for genomic materials. Now we are exploring the properties of another polyaminoacidic polymer, the a,b-poly(N-2-hydroxyethyl)D,L-aspartamide (PHEA) as starting polymer to obtain stable polycations, able to complex DNA. PHEA is a highly water soluble synthetic polymer with many interesting properties for its application in biomedical field. In fact it is biocompatible, not cytotoxic and haemolytic, not antigenic and not immunogenic [14]. Several terminal hydroxyl groups in PHEA backbone allow to link, via chemical bonds, high amount of molecules with different molecular weight, such as drugs or hydrophobic portions, thus obtaining macromolecular prodrugs [15–17], polymeric micelles [18], nanoparticles or hydrogels [19]. In this paper we describe the synthesis and the characterization of new PHEA polycationic derivatives realized by functionalization of PHEA backbone with positively charged groups, in such a way to easily modulate positive charge amount. The ability of these polycations to interact electrostatically with DNA and to neutralize its anionic charge was analysed by electrophoresis mobility studies on agarose gel [13]. Moreover, we investigated the dimensional distributions of the hydrated diameter and surface charge of the polyplexes in different media through light scattering and zeta potential analysis, respectively. Furthermore, DNA degradation studies of polyplexes formed by PHEA–HYD–CPTA polycations demonstrated, after incubation in presence of DNAse II, the polycation ability to protect condensed DNA against enzymatic hydrolysis [20]. 2. Experimental section 2.1. Materials and methods a,b-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) was prepared and purified according to the previously reported procedure [14]. Spectroscopic data (FT-IR and 1H NMR) were in agreement with attributed structure [14]: 1H NMR[D2O]: d 2.83 (m, 2H, –CH–CH2–CO–NH–), 3.37 (t, 2H, –NH– CH2–CH2–OH), 3.66 (t, 2H, –CH2–CH2–OH), 4.73 br (m, 1H, –NH–CH–CO–CH2–).

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PHEA average molecular weight was 45.0 kDa (Mw/Mn = 1.66) based on PEO/PEG standards, measured by size exclusion chromatography (SEC). 3-(Carboxypropyl)trimethyl-ammonium chloride (CPTA) was purchased from Aldrich (Milan Italy). Lambda DNA Hind III, calf thymus DNA sodium salt, deoxyribonuclease II HEPES and MEM media were all supplied by Sigma (UK). Hydrazine monohydrate, bis(4-nitrophenyl)carbonate (4-NPBC), N-(3-dimethylaminopropyl)-N 0 -ethyl-carbodiimide hydrochloride (EDC-HCl) and all other chemicals were purchased from Fluka (Switzerland). Infrared spectra were obtained using a PerkinElmer 1720 IR Fourier Transform Spectrophotometer in potassium bromide disks. The 1H NMR spectra were recorded in D2O (Aldrich) using a Bruker AC-250 spectrometer operating at 250.13 MHz. Centrifugations were performed using a Centra MP4R IEC centrifuge. 2.2. General procedure for synthesis of a,b-poly (N-2-hydroxyethylcarbazate)-D,L-aspartamide (PHEA–HYD) derivatives The synthesis of PHEA–HYD derivatives were performed as follows. A solution of PHEA (0.250 g, 1.57 mmol of repeating units) in 3 mL of anhydrous DMF was added dropwise to a solution of bis(4-nitrophenyl)carbonate (0.48 g, 1.57 mmol) in 4 mL of anhydrous DMF under stirring and the mixture was kept under argon at 40 °C for 1 h (sample a) or 4 h (sample b). After activation times, each solution of the activated polymer was added dropwise to a solution of hydrazine monohydrate (HYD) in anhydrous DMF (0.75 mL N2H4H2O, 15.72 mmol, in 0.75 mL DMF) keeping the temperature at 0 °C; after HYD addition the temperature was maintained at 20 ± 0.1 °C and each sample was stirred for 2 h under argon. At this point, reaction mixtures were precipitated into acetone and the suspension was centrifuged at 12,000g for 10 min at 4 °C and washed several times with the same solvent. The obtained solid residues (sample a and b) were solubilized in distilled water and purified by exhaustive dialysis using Visking Dialysis Tubing 18/3200 with molecular weight cut-off of 12,000– 14,000. After dialysis solutions were freeze-dried from water; the pure products of sample a and b were obtained in 90% yield (w/w) based on starting PHEA. Obtained copolymers were characterized by IR spectrophotometry and 1H NMR analysis.

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IR spectrum (KBr) of PHEA–HYD samples showed bands at: 3300, 3210, 3055 cm1 (–NH2, –NH–) belonging to PHEA; 1705 cm1 (attributed to uretanic carbonyl); 1657 cm1 (amide I) and 1532 cm1 (amide II) belonging to PHEA backbone. 1 H NMR of PHEA–HYD (D2O) reveals peaks at d 2.80 (m, 2H, –CO–CH–CH2–CO–NH–), 3.35 (m, 2H, –NH–CH2–CH2–OH), 3.48 (m, 2H, –NH– CH2–CH2–O(CO)NH–NH2), 3.65 (m, 2H, –NH– CH2–CH2–OH), 4.17 (m, 2H, –NH–CH2–CH2– O(CO)NH–NH2) 4.72 (m, 1H, –NH–CH(CO)CH2). 2.3. General procedure for synthesis of a,b-poly (N-2-hydroxyethyl-N-carbazate[N 0 -(3-trimethylammonium chloride)propylhydrazide]-D,L-aspartamide (PHEA–HYD–CPTA) derivatives PHEA–HYD derivatives (0.1 g, corresponding to 0.12 mmol for sample a and 0.25 mmol for sample b of pendant –NH–NH2 groups) were dissolved in 5 mL of distilled water followed by the addition of a proper amount of CPTA (44.32 mg, 0.244 mmol for sample a or 89.41 mg, 0.49 mmol for sample b). After adjusting pH to 4.5 with NaOH 0.1 N, EDC-HCl (70.16 mg, 0.37 mmol for sample a or 141.47 mg, 0.738 mmol for sample b) was added and reaction pH was kept constant using HCl 0.1 N (during the first hour pH gradually increases indicating that condensation reaction was proceeding). After stirring the reaction mixture for 1 h the solutions were left at room temperature for 4 h and purified by exhaustive dialysis using Visking Dialysis Tubing 18/32’’ with molecular weight cut-off of 12,000–14,000. After dialysis the solutions were freeze-dried from water; the pure products of CPTA-functionalized copolymers (sample a 0 and b 0 ) were obtained in 95% yield (w/ w) based on starting a and b PHEA–HYD derivatives, respectively). Obtained copolymers were characterized by IR spectroscopy and 1H NMR analysis. IR spectrum (KBr) of PHEA–HYD–CPTA samples showed bands at: 3300, 3210, 3055 cm1 (–NH2, –NH–) belonging to PHEA; 1708 cm1 (attributed to amidic and uretanic carbonyl); 1657 cm1 (amide I), and 1532 cm1 (amide II) belonging to PHEA backbone; 934 and 973 cm1 attributed to C–N stretching of CPTA. 1 H NMR of PHEA–HYD–CPTA (D2O) reveals peaks at d 2.18 (m, 2H, –CO–CH2–CH2–CH2– N+(CH3)3), 2.48 (m, 2H, –CO–CH2–CH2–CH2–

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N+(CH3)3), 2.84 (m, 2H, –CO–CH–CH2–CO–NH–), 3.19 (s, 9H, –C–N+(CH3)3), 3.42 (m, 4H, –NH– CH2–CH2–OH, –CO–CH2–CH2–CH2–N+(CH3)3), 3.55 (m, 2H, –NH–CH2–CH2–O(CO)NH–NH2), 3.69 (m, 2H, –NH–CH2–CH2–OH), 4.26 (m, 2H, –NH–CH2–CH2–O(CO)NH–NH2) 4.75 (m, 1H, –NH–CH(CO)CH2). 2.4. DNA–polycations interaction: gel retardation assay To evaluate the ability of these polycations to interact with DNA negative charges, all synthesized copolymers were solubilized in a sterile 0.9% w/v NaCl aqueous solution at room temperature. Lambda Hind III DNA solution was prepared in the same medium at 0.1 mg/mL concentration and stored at 4 °C. Complexation was performed by mixing known aliquots of DNA and polycation solutions at 37 °C, at different polycation/DNA weight ratios and left to stand for 30 min before analysis. For the assay we used an agarose gel (0.7%, w/v) containing ethidium bromide (0.25 lg/ mL) in tris-acetate /EDTA (TAE) buffer. The electrophoresis was performed at 80 V for 30 min and the pattern of banding was visualized by UV trans-illumination and photographed using a Polaroid land camera (667 film) [20]. DNA/well (500 ng) were loaded into agarose gel with a loading buffer (bromophenol blue 0.25% w/v, xylene cyanole 0.25% w/v, glycerol 30% in water). 2.5. DNase II degradation assay DNase II degradation of calf thymus DNA was assayed as elsewhere described [19]. Calf thymus DNA (100 lg/mL) or DNA/polycation complexes were incubated with DNase II (300 units/mL) in sodium acetate/acetic acid buffer (0.2 M, pH 5.5) containing potassium chloride (0.2 M) at 37 °C. Immediately two 0.5 mL samples were taken and used as blank. At various time up to 1 h, samples were precipitated with 10% (w/v) perchloric acid (0.5 mL). After keeping for 20 min at 4 °C, the samples were centrifuged at 12,000g for 20 min and the absorbance of the supernatant was measured at 265 nm. 2.6. Size exclusion chromatography (SEC) characterization The molecular weights and molecular weight distributions of PHEA, PHEA–HYD and PHEA–

HYD–CPTA derivatives were determined by aqueous size exclusion chromatography (SEC). The standard SEC protocol involved using two Ultraydrogel ˚ ) (Milford, columns from Water (500 and 250 A MA, USA) connected to a Water 2410 refractive index detector. Phosphate buffer solution at pH 8 was used as eluent at 37 °C with a flux of 0.8 mL/ min, and poly(ethylene oxide) standards (range 145.0–1.5 kDa) were used for calibration. 2.7. Dynamic light scattering studies of PHEA– HYD–CPTA/DNA complexes Dynamic light scattering studies (DLS) were performed at 25 °C using a Malvern Zetasizer NanoZS instrument, fitted with a 532 nm laser at a fixed scattering angle of 90°. The PHEA–HYD–CPTA/DNA complexes were prepared using increasing polycation/DNA weight ratios, ranging from 2/1 up to 6/1 (naked DNA was used as reference), and size measurements were performed in three different media: double distilled water, 0.9% w/v NaCl solution and HEPES medium. The intensity-average hydrodynamic diameter (nm), and polydispersity index (PI) were obtained by cumulative analysis of the correlation function. 2.8. Zeta potential measurements Aqueous electrophoresis measurements were recorded at 25 °C using a Malvern Zetasizer NanoZS instrument. Complexes with DNA were prepared in double distilled water and MEM medium using increasing polycation/DNA weight ratios ranging from 1/1 up to 10/1 (naked DNA was used as reference). The zeta potential (mV) was calculated from the electrophoretic mobility using the Smoluchowsky relationship and assuming that k Æ a  1 (where k and a are the Debye-Hu¨ckel parameter and particle radius, respectively). 3. Results and discussion 3.1. Synthesis of PHEA–HYD–CPTA polycations Two new polycationic copolymers of PHEA containing different amounts of quaternary amine were synthesized in very high yields (95%). The activation reaction of hydroxyl groups of PHEA with bis(4nitrophenyl)carbonate (4-NPBC), followed by the reaction with hydrazine monohydrate (HYD),

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O O NH

NH O HN O

PHEA NH OH

β HO

α O2N

DMF

NO2

O

i)

O

( 40°C)

O

ii) NH 2-NH2H 2 O (0°C) O O

O

NH

NH

O

O

NH O

NH

HN

HN

O

O

NH

NH

OH

O

β

O HO

NH

O O

H2N

HN NH2

β1

α

PHEA-HYD

α1 Scheme 1. Synthesis of PHEA–HYD copolymer.

allowed the polymer chain derivatization with pendant hydrazide groups, obtaining the PHEA– HYD copolymers (as reported in Scheme 1). Different reaction attempts showed that activation time with 4-NPBC was the determining factor to varying PHEA derivatization degree with hydrazine groups. In fact, we synthesized two PHEA–HYD copolymers with an hydrazide molar derivatization degree proportional to activation time. Table 1, summarizes PHEA derivatization degrees with hydrazide groups as function of activation time and it is shown that samples a and b,

obtained using activation times of 1 and 4 h, respectively, present a significantly different percentage of hydrazide groups (20.2% and 40.6%, respectively). The introduction of hydrazide groups in the PHEA backbone was showed by FT-IR and 1H NMR spectra of purified PHEA–HYD copolymers. In particular, 1H NMR spectra allowed the quantification of pendant hydrazide groups in the polymer chain by comparing the integral of the peak related to protons at d 4.17 assignable to the protons –NH– CH2–CH2–O(CO)NH–NH2 (PHEA hydrazinederivatized side chain) with the integral of the peak

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Table 1 Summary of hydrazide functionalization degree for PHEA–HYD copolymers as function of activation time with 4-NPBC; CPTA derivatization degree for PHEA–HYD–CPTA polycations and molecular weight data of mentioned samples Copolymer composition

Activation time (h)

Derivatization degree (mol%)a

Mw (kDa)b

M bw Mn

PHEA–HYD (a) PHEA–HYD (b) PHEA–HYD–CPTA (a 0 ) PHEA–HYD–CPTA (b 0 )

1 4

20.2 40.6 20.0 40.0

40.0 40.5 46.0 47.0

1.81 1.78 1.69 1.66

a b

As determined by 1H NMR spectroscopy. As determined by aqueous SEC using poly(ethylene oxide) calibration standards.

Fig. 1. Assigned 1H NMR (D2O) spectrum for PHEA–HYD copolymer (sample b as an example) and starting PHEA.

at d 2.80 assignable to –CH–CH2–CO–NH– of polymer backbone. This point is illustrated in Fig. 1, which shows a 1H NMR spectrum of a

PHEA–HYD copolymer (sample b as an example) compared with a typical 1H NMR spectrum of starting PHEA.

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CPTA polycation (sample b 0 as an example) is illustrated in Fig. 2. The average molecular weights of synthesized PHEA–HYD and PHEA–HYD–CPTA derivatives, determined by Size Exclusion Chromatography (SEC), ranged from 40 to 40.5 kDa for PHEA– HYD copolymers (samples a and b, respectively) and from 46 to 47 kDa for PHEA–HYD–CPTA polycations (samples a 0 and b 0 , respectively), as summarized in Table 1. The increment of molecular weight and the presence of only one peak in the SEC chromatogram, confirmed that positive charge moieties (CPTA) were linked to the polymer chain, and were not present as small molecule contaminants.

Quantitative 2,4,6-trinitrobenzenesulfonic acid (TNBS) assay, performed as elsewhere described [21], confirmed the reliability of 1H NMR estimation of hydrazide derivatization degree in PHEA– HYD copolymers, (data not reported). The pendant hydrazide groups of PHEA–HYD copolymers easily reacted with 3-(carboxypropyl)trimethylammonium chloride (CPTA) in aqueous medium at pH 4.75 in the presence of EDC as activating agent, yielding functionalization of hydrazide groups with the carboxylic moieties of CPTA (see Scheme 2). After purification, the obtained PHEA–HYD– CPTA polycations were characterized by FT-IR and 1H NMR analysis. Spectra confirmed not only the introduction of the charged groups in the copolymers, but also (by 1H NMR analysis) the quantitative derivatization of hydrazide groups, by comparing the integral of the peak related to trimethyl-ammonium groups [–N+(CH3)3] of CPTA molecules linked to PHEA (d 3.19), with the integral of the peak related to protons at d 2.84 assignable to –CH–CH2–CO–NH– of polymer backbone. The assigned 1H NMR spectrum of a PHEA–HYD–

3.2. DNA complexing studies The interactions between PHEA–HYD–CPTA copolymers and DNA were investigated by retardation of the DNA electrophoretic mobility [12]. The complexes were formed in NaCl 0.9% w/v sterile aqueous solution for almost half an hour, mixing fixed DNA amounts with increasing amounts of PHEA–HYD–CPTA at different derivatization

PHEA-HYD

H2O

O

CH3 CH3 N+ - CH3 Cl

EDC-HCl HO

O

O

O

O

NH

NH

O

NH

NH

HN

O O

HN

NH

O

OH

NH

β

O HO

O NH

O

α1 H3C H3C

CH3 +N

Cl -

O

HN

HN NH

O

β1

α

CH3 CH3 N+ Cl CH3

O

Scheme 2. Synthesis of PHEA–HYD–CPTA polycation.

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M. Licciardi et al. / European Polymer Journal 42 (2006) 823–834

Fig. 2. Assigned 1H NMR (D2O) spectrum for PHEA–HYD–CPTA polycations (sample b 0 as an example).

degrees in such a way to have polycation/DNA weight ratios ranging from 0.5/1 to 4/1. As shown in Fig. 3a and b all PHEA–HYD– CPTA polycations have a great capacity to retard DNA migration in agarose gel. In particular PHEA–HYD–CPTA with 40 mol% of positive moieties (sample b 0 ) shows no DNA migration at polycation/DNA weight ratio of 2/1 (corresponding to a polycation/DNA charge molar ratio of 1/1); at 3/1 ratio a slight exclusion of ethidium bromide was also evident, as illustrated in Fig. 3a. PHEA– HYD–CPTA with 20 mol% of positive charges (sample a 0 ) showed inhibition of DNA migration at polycation/DNA weight ratio of 3/1, as depicted in Fig. 3b. Moreover the complexing ability of PHEA– HYD–CPTA copolymer b 0 compared with that of DEAE-dextran [22,23], used as positive control, showed that the retardation ability of this polyca-

tion was slightly higher than that of DEAE-dextran, starting from a polymer/DNA weight ratio of 2/1, under the same experimental conditions (see Fig. 4). The interaction with DNA was investigated also for PHEA–HYD copolymers. Unlike polycations, PHEA–HYD copolymers did not show ability to complex DNA under the used experimental conditions. These results confirm the good capacity of our new PHEA–HYD-CPTA polycations to complex DNA and neutralize its anionic charges; in particular sample b 0 was very efficient to complex and compact the genomic material, at polycation/DNA weight ratios ranging from 1/1 to 3/1. 3.3. Biophysical properties The biophysical properties of PHEA–HYD– CPTA/DNA polyplexes were determined via DLS

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Fig. 3. (a) Gel electrophoresis of PHEA–HYD (sample b) and PHEA–HYD–CPTA (sample b 0 ) at 0.5/1, 0.8/1, 1/1, 2/1 and 3/1 polycation/DNA weight ratios, compared with naked DNA (k). (b) Gel electrophoresis of PHEA–HYD (sample a) and PHEA–HYD– CPTA (sample a 0 ) at 0.8/1, 1/1, 2/1, 3/1 and 10/1 polycation/DNA weight ratios, compared with naked DNA (k).

Fig. 4. Gel electrophoresis of PHEA-HYD-CPTA (sample b 0 ) and DEAE dextran (sample d) at 0.5/1, 0.8/1, 1/1, 2/1 and 3/1 polycation/ DNA weight ratios, compared with naked DNA (k).

and zeta-potential measurements. Table 2 shows values of hydrodynamic diameter (nm) and polydis-

persity index (PI) in different media (double distilled water, 0.9% w/v NaCl aqueous solution and

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Table 2 Hydrodynamic diameter and polydispersity index (PI) of PHEA–HYD–CPTA polyplexes measured after 30 min of incubation in different aqueous media, related to polycation/DNA weight and charge molar ratios Polycation

Size (nm) 30 0

Polycation/DNA charge molar ratios

Medium

PHEA–HYD–CPTA 2/1 40 mol% (b 0 ) 40 mol% (b 0 ) 4/1 40 mol% (b 0 ) 6/1 40 mol% (b 0 ) 2/1 4/1 40 mol% (b 0 ) 40 mol% (b 0 ) 6/1 40 mol% (b 0 ) 2/1 4/1 40 mol% (b 0 ) 40 mol% (b 0 ) 6/1

1/1 2/1 3/1 1/1 2/1 3/1 1/1 2/1 3/1

H2O H2O H2O NaCl 0.9% NaCl 0.9% NaCl 0.9% HEPES HEPES HEPES

231.9 162.4 132.9 265.6 248.0 211.0 201.5 184.8 175.6

0.10 0.20 0.17 0.29 0.33 0.50 0.31 0.32 0.29

PHEA–HYD–CPTA 2/1 20 mol% (a 0 ) 20 mol% (a 0 ) 4/1 20 mol% (a 0 ) 6/1 20 mol% (a 0 ) 2/1 4/1 20 mol% (a 0 ) 20 mol% (a 0 ) 6/1 20 mol% (a 0 ) 2/1 4/1 20 mol% (a 0 ) 20 mol% (a 0 ) 6/1

0.5/1 1/1 1.5/1 0.5/1 1/1 1.5/1 0.5/1 1/1 1.5/1

H2O H2O H2O NaCl 0.9% NaCl 0.9% NaCl 0.9% HEPES HEPES HEPES

205.8 202.3 200.9 316.3 308.0 241.3 370.5 304.2 231.7

0.30 0.27 0.23 0.58 0.47 0.44 0.21 0.24 0.22

H2O NaCl 0.9% HEPES

648.5 1824.0 371.9

0.67 0.98 0.42

DNA DNA DNA

Polycation/DNA weight ratios

– – –

HEPES medium) of PHEA–HYD–CPTA/DNA polyplexes by using either of the two polycations (samples a 0 or b 0 ) and different polycation/DNA weight ratios ranging from 2/1 to 6/1 corresponding to the a charge molar ratio in the range between 1/1 and 3/1 for b 0 and between 0.5/1 and 1.5/1 for a 0 . In the case of polycation b 0 , the polyplex average diameter was around 230 nm in all used media and it decreased with increasing polycation/DNA weight (or charge) ratio. On the contrary, the polyplexes formed by polycation a 0 were larger than that formed by polycation b 0 , but similarly with the latter, linearly correlated with the increment of the polycation/DNA weight (or charge) ratio. These results confirmed that the ionic strength of complex incubation medium strongly affected the size of resulting polyplexes. Considering the critical role of polyplex size to obtain an effective in vivo transfection, the choice of the proper medium for formation and administration of polyplexes is a crucial step in the preformulation studies of gene therapy. The zeta potential trends of polyplexes in water, illustrated in Fig. 5a and b, showed that increasing the polycation weight amount in the polyplex formation, zeta potential values decreased, starting

PI

from a value of 40 mV of naked plasmid, and became positive at polycation/DNA weight ratio of 2 for polycation b 0 and 4 for polycation a 0 , corresponding for both polycations to a polycation/ DNA charge molar ratio of 1/1. A different electrophoretic behaviour was observed when polyplexes were formed in a medium at higher ionic strength than water, such as MEM. In this medium, using the polycation at the higher CPTA derivatization degree (sample b 0 ), neutral polyplexes were obtained only at a minimum polycation/DNA weight ratio of 6 (see Fig. 5). 3.4. DNase II degradation assay To allow the successful transfection of a gene, its necessary to avoid degradations of gene material by serum enzymes or by nucleases [24]; for this reason an effective polymeric vector should be able not only to complex genomic material, but also to protect DNA decreasing its degradation rate. To detect the capability of our polymers to protect DNA, we used the method described by Barret and Heath [20]. The DNase II was selected as model enzyme for degradation studies. It is known that this enzyme is able to cleave both strands of DNA leaving the 3 0 phosphate

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833

25 15

15 -5

0

1

2

3

4

-25

Zeta potential (mV)

Zeta potential (mV)

35

5 -5 0

1

2

3

4

5

6

7

8

-15 -25 -35 -45

-45

(a)

(b)

Polycation/DNA (w/w)

Polycation/DNA (w/w)

Zeta potential (mV)

15 10 5 0 0

2

4

6

8

10

12

-5 -10 -15 -20

(c)

Polycation/DNA (w/w)

Fig. 5. (a) Zeta potential values of PHEA–HYD–CPTA b 0 /DNA polyplexes measured in distilled water, as function of polycation/DNA weight ratio. (b) Zeta potential values of PHEA–HYD–CPTA a 0 /DNA polyplexes measured in distilled water, as function of polycation/ DNA weight ratio. (c) Zeta potential values of PHEA–HYD–CPTA b 0 measured in MEM medium, as function of polycation/DNA weight ratio.

end group associated to the nucleotide [20]. We tested polyplexes formed by the PHEA–HYD– CPTA polycations at different CPTA derivatization degrees, a 0 and b 0 , using a polycation/DNA weight ratio of 2/1 for sample b 0 and of 4/1 for sample a 0 .

Absorbance 265 nm

0.6 0.5 0.4 0.3

This difference was reasonably used according with the lower ability of polycation a 0 (20%) to complex DNA than its analogous at higher CPTA derivatization degree (40%; sample b 0 ). Fig. 6 shows the protection effect of PHEA–HYD–CPTA on plasmid DNA, in comparison with naked DNA, after incubation in presence of DNAse II. It was evident that degradation rate of DNA (expressed as the absorbance of cleaved nucleotides in solution) was significantly reduced when it was complexed with PHEA–HYD–CPTA polycations; moreover the protection effect of the two polycations, a 0 and b 0 , were quite similar at the used polycation/DNA weight ratios (4/1 and 2/1, respectively).

0.2

4. Conclusions

0.1 0 0

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40

50

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70

Time (min)

Fig. 6. UV absorbance of cleaved nucleotides after incubation of PHEA–HYD–CPTA b 0 (j) and PHEA–HYD–CPTA a 0 (m) polyplexes, at polycation/DNA weight ratios of 2/1 and 4/1, respectively, in the presence of DNAse II, in comparison with naked DNA (d).

The use of bis(4-nitrophenyl)carbonate as activating agent for PHEA hydroxyl groups, resulted in a very efficient method to easily functionalize polymeric backbone of PHEA with pendant hydrazide groups in the side chain; furthermore this activating agent allowed copolymers with two different derivatization degrees to be obtained simply by varying the activation times from 1 to 4 h. After

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PHEA functionalization with hydrazine (PHEA– HYD), two new cationic derivatives of PHEA have been synthesized by coupling the pendant hydrazide groups of this copolymer with the carboxylic moiety of CPTA. The obtained PHEA–HYD–CPTA polycations have shown to be very effective to complex DNA and to reduce its degradation rate by DNAse II at low weight ratios. Interestingly, PHEA–HYD– CPTA at higher CPTA derivatization degree (40 mol%) showed the ability to block DNA migration at a polycation/DNA weight ratio of 2/1 and to form polyplexes with hydrodynamic diameter, in various media, suitable for gene delivery purposes. In particular, at the highest investigated polycation/DNA weight ratio (6/1), polyplexes size ranged from about 130 to 241 nm. Moreover, aqueous electrophoresis studies of these polyplexes confirmed the ability of PHEA–HYD–CPTA polycations to neutralize the negative charge on the DNA, which is a prerequisite for its cell membrane permeation. Acknowledgement Authors thank MIUR for funding. References [1] El-Aneed A. Current strategies in cancer gene therapy. Eur J Pharmacol 2004;498:1–8. [2] El-Aneed A. An overview of current delivery systems in cancer gene therapy. J Control Release 2004;94:1–14. [3] Boulikas T. Status on gene therapy in 1997: molecular mechanisms disease targets and clinical applications. Gene Ther Mol Biol 1998;1:1–172. [4] McTaggart S, Al-Rubeai M. Retroviral vectors for human gene delivery. Biotechnol Adv 2002;20:1–31. [5] Yi SW, Yune TY, Kim TW, Chung H, Choi YW, Kwon IC, et al. A cationic lipid emulsion/DNA complex as a physically stable and serum-resistant gene delivery system. Pharm Res 2000;17:314–20. [6] Kakizawa Y, Kataoka K. Block copolymer micelles for delivery of gene and related compounds. Adv Drug Deliv Rev 2002;54:203–22. [7] Pedone E, Cavallaro G, Richardson SCW, Duncan R, Giammona G. a,b-poly(asparthylhydrazide)-glycidyltrimethylammonium chloride copolymers (PAHy-GTA): novel polymers with potential for DNA delivery. J Control Release 2001;77:139–53. [8] Cavallaro G, Palumbo FS, Licciardi M, Giammona G. Novel cationic copolymers of a polyasparthylhydrazide: synthesis and characterization. Drug Delivery 2005;12:377–84.

[9] Oupicky D, Konak C, Ulbrich K, Wolfert MA, Seymour LW. DNA delivery systems based on complexes of DNA with synthetic polycations and their copolymers. J Control Release 2000;65:149–71. [10] Haines AM, Irvine AS, Mountain A, Charlesworth J, Farrow NA, Husain RD, et al. CL22—a novel cationic peptide for efficient transfection of mammalian cells. Gene Ther 2001;8:99–110. [11] Rolland A. Gene medicines: the end of the beginning? Adv Drug Deliv Rev 2005;57:669–73. [12] Gosselin MA, Guo W, Lee RJ. Efficient gene transfer using reversibly cross-linked low molecular weight polyethylenimine. Bioconj Chem 2001;12:989–94. [13] Kabanov AV, Kabanov VA. DNA complexes with polycations for the delivery of genetic material into cells. Bioconj Chem 1995;6:7–20. [14] Giammona G, Carlisi B, Palazzo S. Reaction of a,b-poly(Nhydroxyethyl)-D,L-aspartamide with derivatives of carboxylic acids. J Polym Sci : Polym Chem Ed 1987;25:2813–8. [15] Cavallaro G, Licciardi M, Giammona G, Caliceti P, Salmaso S. Synthesis, physico-chemical and biological characterization of a paclitaxel macromolecular prodrug. Euro J Pharma Biopharm 2004;58(1):151–9. [16] Giammona G, Puglisi G, Carlisi B, Pignatello R, Spadaro A, Caruso A. Polymeric prodrugs: a,b-poly(N-2-hydroxyethyl)D,L-aspartamide as macromolecular for some non-steroidal anti-inflammatory agents. Int J Pharm 1989;57:55–62. [17] Cavallaro G, Pitarresi G, Licciardi M, Giammona G. Polymeric prodrug for release of an antitumoral agent by specific enzymes. Bioconj Chem 2001;12:143–51. [18] Mendichi R, Giacometti Schieroni A, Cavallaro G, Licciardi M, Giammona G. Molecular Characterization of a,bpoly(N-2-hydroxyethyl)-D,L-aspartamide derivatives as potential self-assembling copolymers forming polymeric micelles. Polymer 2003;44:4871–9. [19] Mandracchia D, Pitarresi G, Palumbo FS, Carlisi B, Giammona G. pH-sensitive hydrogel based on a novel photocross-linkable copolymer. Biomacromolecules 2004;5:1973–82. [20] Barret AJ, Heath MF. Lysosomal enzymes. In: Dingle JT, editor. Lysosomes: a laboratory handbook. second ed. Amsterdam, New York and Oxford: North Holland publishing Company; 1977. p. 19–147. [21] Maniatis T, Fritsch EF, Sambrook J. Molecular cloning: a laboratory manual. second ed. New York: Cold Spring Harbour Press; 1986. p. 18.24–5. [22] Gaur RK, Gupta KC. A spectrophotometric method for the estimation of amino groups on polymer supports. Anal Biochem 1989;180:253–8. [23] Richardson SCW, Kolbe HVJ, Duncan R. Potential low molecular mass chitosans as DNA delivery system: biocompatibility, body distribution and ability to complex and to protect DNA. Int J Pharm 1999;178:231–43. [24] Hashida M, Mahado RJ, Kawabata K, Myao M, Nishikawa Y, Tokamura Y. Pharmacokinetics and targeted delivery of proteins and genes. J Control Release 1996;41:91–7.