WO2020152356A1

WO2020152356A1 - Cationic polymer

Info

Publication number: WO2020152356A1
Application number: PCT/EP2020/051826
Authority: WO
Inventors: Francesco Trotta
Original assignee: Francesco Trotta
Priority date: 2019-01-24
Filing date: 2020-01-24
Publication date: 2020-07-30
Also published as: IT201900001075A1

Abstract

The present invention relates to a cationic polymer comprising: - at least two cyclodextrin units; - at least one linker covalently bonding the at least two cyclodextrin units, wherein the at least one linker is selected from the group consisting of: dicarboxylic acid, dianhydride, carbonyldiimidazole, diphenylcarbonate, triphosgene, acylic dichloride, diisocyanate, a diepoxide, and a triepoxide; and - at least one substituent comprising at least one ammonium group. The present invention relates also to the preparation process of said cationic polymer and to its use as a vector for gene therapy.

Description

TITLE: CATIONIC POLYMER

DESCRIPTION

FIELD OF THE INVENTION The invention relates to a cationic polymer comprising cyclodextrin units, its preparation processes and uses thereof.

BACKGROUND

In recent years, a range of therapies involving the delivery of nucleic acid into a patient's cells as a drug to treat disease, collectively commonly known as gene therapy, has increasingly attracted attention.

Cellular gene expression is a process that can lead to the synthesis of proteins associated, sometimes, with the development of specific genetic pathologies. Through the insertion (transfection) within the cellular environment of appropriate molecules (Oligonucleotides, SiRNA, Micro RNA), conveyed in an appropriate manner, it is possible to block this course. This principle for example, is at the base of the so-called“anti-sense techniques”, inserted in the context of gene therapy.

Generally, a gene therapy formulation involves the use of a vector, in order to transport the genetic material (inside the cytosol) and to protect it from enzymatic or chemical degradation. The vectors are viruses, which may however lead to contraindications for the patient.

Non-viral vectors of gene delivery are therefore of extreme interest. The first task, and one of the most important ones too, of such a carrier is to form a stable complex with genetic material. For example, Samal S. K. et al. , Chem. Soc. Rev., 2012, 41 , 7147-7194 describes that cationic polymers are the subject of intense research as non-viral gene delivery systems, due to their flexible properties, facile synthesis, robustness and proven gene delivery efficiency.

SUMMARY OF INVENTION An object of the present invention is the provision of a new and improved delivery system suitable to be effective to complex genetic material and to succesfully protect it from enzymatic or chemical degradation.

Therefore, the present invention relates, in a first aspect, to a cationic polymer comprising:

- at least two cyclodextrin units;

- at least one linker covalently bonding the at least two cyclodextrin units, wherein the at least one linker is selected from the group consisting of: dicarboxylic acid, dianhydride, carbonyldiimidazole, diphenylcarbonate, triphosgene, acylic dichloride, diisocyanate, a diepoxide, and a triepoxide; and

- at least one substituent comprising at least one ammonium group.

The cationic polymer according to the invention, thanks to its structure comprising the combination of specific components, namely at least two cyclodextrin units, at least one specific type of linker covalently bonding the at least two cyclodextrin units and at least one substituent comprising at least one ammonium group, is highly effective to complex genetic material at the same time being also effective in protecting said genetic material from enzymatic or chemical degradation.

In particular, the cationic polymer according to the invention has shown to be effective to complex DNA and SiRNA and successfully protecting them from enzymatic or chemical degradation.

The cationic polymer according to the invention has also the advantage of being non-cytotoxic and furthermore of being based on a bio-based material generally biodegradable and biocompatible such as cyclodextrins, which can be easily obtained and transformed from a natural resource widely available such as starch. The non-cytotoxic nature of the cationic polymer according to the invention renders its use particularly advantageous when the contact with living organisms is foreseeable or even mandatory, such as in particular for gene delivery applications.

In a preferred embodiment, the at least one linker is selected from the group consisting of: 1 ,T-carbonyldiimidazole, a diepoxide, and a triepoxide. More preferably, the at least one linker is selected from butanediol diglycidyl ether and trimethylol propane triglycidyl ether.

In a further aspect, the present invention also relates to a process for preparing the cationic polymer according to the invention, said process comprising the steps of: - providing at least one cyclodextrin; - adding and reacting at least one linker compound selected from the group consisting of: dicarboxylic acid, dianhydride, carbonyldiimidazole, diphenylcarbonate, triphosgene, acylic dichloride, diisocyanate, a diepoxide, and a triepoxide; - adding and reacting at least one compound comprising at least one ammonium group or an ammonium group precursor; - obtaining the cationic polymer.

The process according to the present invention allows obtaining, starting from the aforesaid bio-based material, non-toxic and biocompatible cationic polymers with predetermined positive charge density, using a simple and efficient synthetic procedure that, advantageously, may not require the use of organic solvents. In another aspect, the present invention relates to a cationic polymer obtainable by said process according to the present invention.

Thanks to its structure, comprising the combination of specific components, namely at least two cyclodextrin units, at least one specific type of linker covalently bonding the at least two cyclodextrin units and at least one substituent comprising at least one ammonium group, the Applicant surprisingly found out that the cationic polymer according to the invention is also advantageously suitable for many other applications, comprising the purification of water.

In a further aspect, the present invention relates therefore to the use of the cationic polymer according to the invention for the purification of water. Waste waters may contain several contaminants that need to be removed. For example, in addition to anions that may have a negative effect on the health such as nitrate, chromate and dichromate anions, as well as in addition to contaminants of organic nature such as toluene, xylene, naproxene, waste waters may also contain colloidal particles that need to be removed. The Applicant has noted that said colloidal particles have prevalently negative charge and that the same can be removed by sedimentation once aggregated.

The Applicant has therefore observed that the cationic polymer according to the invention, due to its structure and to the high density of positive charges on its structure is effective for favoring the coagulation / flocculation process of such colloidal particles.

Thanks to its structure incorporating positive charges, the cationic polymer according to the invention indeed shows improved properties in the removal of anions, such as nitrates, chromates and dichromates and of contaminants of organic nature such as toluene, xylene, naproxene, from waste waters as well as in promoting the flocculation/sedimentation of colloidal particles from the same, thus resulting to be effective for water purification processes.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the FTIR-ATR spectrum of the cationic polymer according to Example 1 ;

Figure 2 shows the TGA thermogram of the cationic polymer according to Example

1 ;

Figure 3 shows the FTIR-ATR spectrum of the cationic polymer according to Example 6; Figure 4 shows the TGA thermogram of the cationic polymer according to Example

6;

Figure 5 shows the FTIR-ATR spectrum of the cationic polymer according to Example 7;

Figure 6 shows the TGA thermogram of the cationic polymer according to Example 7;

Figure 7 shows the fluorescence microscopies of the FITC-Dextran sulfate complex with the cationic polymer according to Example 6, obtained by mixing fluorescein isothiocyanate (FITC)-dextran sulfate sodium salt (1 mg/ml) (Sigma-Aldrich) and a 0.9% w/v NaCI in distilled water solution of the cationic polymer according to Example 6 (2 mg/ml), respectively at a 1 :10 (image A) and 1 :50 (image B) cationic polymer/dextran sulfate solutions volume/volume ratio;

Figure 8 shows the banding pattern of the polyplexes of pDNA with the cationic polymer according to Example 6 in a gel retardation assay, at a pDNA/polymer volume/volume ratio of 1 :30 (image A), 1 :50 (image B) and 1 :200 (image C) compared with a reference solution of naked pDNA (“Naked”, in images A, B and C);

Figure 9 shows the banding pattern of the polyplexes of siRNA with the cationic polymer according to Example 6 in a gel retardation assay, at a siRNA/polymer N/P ratio of 1 :10 (image B), 1 :20 (image C) and 1 :30 (image D) compared with a reference solution of naked siRNA (image A);

Figure 10 shows the banding pattern of the obtained in the sodium dodecyl sulfate test for assessing the SDS concentration required to displace the pDNA from the pDNA/polymer polyplex;

Figure 11 shows the laser scanning confocal microscope image of a 20 pg/ml FITC- Dextran sulfate complex with the cationic polymer according to Example 6 internalized within the cell membrane of human fibroblast cells after a 60 minutes contact; Figure 12 shows the TGA thermogram of the cationic polymer according to Example 10;

Figure 13 shows the FTIR-ATR spectrum of the cationic polymer according to Example 10;

Figure 14 shows the results of the cytotoxicity MTT assay performed with the cationic polymer according to Example 6; and

Figure 15 shows the results of the cytotoxicity MTT assay performed with the cationic polymer according to Example 7.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates, in a first aspect, to a cationic polymer comprising:

- at least two cyclodextrin units;

- at least one substituent comprising at least one ammonium group.

Thanks to its structure comprising the combination of specific components, namely at least two cyclodextrin units, at least one specific type of linker covalently bonding the at least two cyclodextrin units and at least one substituent comprising at least one ammonium group, the cationic polymer according to the invention is highly effective to complex genetic material at the same time being also effective in protecting said genetic material from enzymatic or chemical degradation.

The cationic polymer according to the invention has also the advantage of being non-cytotoxic and furthermore of being based on a bio-based material generally biodegradable and biocompatible such as cyclodextrins.

The non-cytotoxic nature of the cationic polymer according to the invention renders its use particularly advantageous when the contact with living organisms is foreseeable or even mandatory, such as in particular for gene delivery applications.

Being based on a bio-based material generally biodegradable and biocompatible such as cyclodextrins, the cationic polymer according to the invention has also the advantage of being easily obtained and transformed from a natural resource widely available such as starch. Thanks to these properties, the cationic polymer according to the invention may be used without drawbacks linked to its final disposal or to the contact with the environment. Within the framework of the present description and in the subsequent claims, except where otherwise indicated, all the numerical entities expressing amounts, parameters, percentages, and so forth, are to be understood as being preceded in all instances by the term "about". Also, all ranges of numerical entities include all the possible combinations of the maximum and minimum values and include all the possible intermediate ranges, in addition to those specifically indicated herein below.

The present invention may present in one or more of the above aspects one or more of the characteristics disclosed hereinafter. Preferably, at least one cyclodextrin of the at least two cyclodextrin units of the cationic polymer according to the invention is selected from the group consisting of: a-cyclodextrin, b-cyclodextrin, g-cyclodextrin, or a derivative thereof.

Preferably, said derivative of said at least one cyclodextrin is selected from the group consisting of: hydroxypropyl- -cyclodextrin (HP- b -CD) and sulfobutyl ether- b -cyclodextrin (SBE- b-CD).

In a preferred embodiment, at least one cyclodextrin of the at least two cyclodextrin units of the cationic polymer according to the invention is b-cyclodextrin.

Preferably, in the cationic polymer according to the present invention the at least one linker is selected from the group consisting of: a dicarboxylic acid, a dianhydride, carbonyldiimidazole, a diisocyanate, a diepoxide, and a triepoxide.

Among the dicarboxylic acids, in the present invention the following diacids can be used: polyacrylic acid, butane tetracarboxylic acid, succinic acid, tartaric acid and citric acid. More preferably the at least one linker is citric acid. In an advantageous embodiment the cationic polymer comprises citric acid and tartaric acid as linkers. Among the dianhydrides, in the present invention the following dianhydrides can be used: diethylenetriaminepentaacetic dianhydride, ethylenediaminetetraacetic dianhydride, benzophenone-3,3',4,4'-tetracarboxylic dianhydride, and pyromellitic dianhydride. More preferably the at least one linker is pyromellitic dianhydride. Among the acylic chlorides, in the present invention the following acylic chlorides can be used: terephthaloyl chloride, sebacoil sebacoyl chloride, succinyl chloride. More preferably the at least one linker is terephthaloyl chloride.

Among the diisocyanates, in the present invention the following diisocyanates can be used: toluenediisocyanate, isophorone diisocyanate, 1 ,4-phenylene diisocyanate, poly(hexamethylene diisocyanate), and hexamethylene diisocyanate. More preferably the at least one linker is hexamethylene diisocyanate.

More preferably, the at least one linker is selected from the group consisting of: pyromellitic dianhydride, 1 ,1’-carbonyldiimidazole, hexamethylene diisocyanate, citric acid, and tartaric acid, a diepoxide, and a triepoxide.

In a preferred embodiment, the at least one linker is selected from the group consisting of: 1 ,1’-carbonyldiimidazole, a diepoxide, and a triepoxide. More preferably, the at least one linker is selected from butanediol diglycidyl ether and trimethylol propane triglycidyl ether. The cationic polymer according to the invention comprises at least one substituent comprising at least one ammonium group.

Preferably, the at least one ammonium group is a quaternary ammonium group.

Preferably, the at least one substituent comprising at least one ammonium group is covalently bonded to at least one cyclodextrin unit. More preferably, the at least one substituent comprising at least one ammonium group is covalently bonded to a hydroxyl group of the at least one cyclodextrin unit. Even more preferably, the at least one substituent comprising at least one ammonium group is covalently bonded to a primary hydroxyl group of the at least one cyclodextrin unit.

Preferably, the at least one substituent comprising at least one ammonium group is selected from the group consisting of:

wherein

- Ri is a C1-C3 alkylene moiety, optionally substituted with a group selected from OH and C1-C3 alkyl; and

- R2, R3, R4 are indepentently selected from the group consisting of: H, CH3, CH2-CH3, CH2-CH2-CH3, and CH(CH₃)2.

More preferably, the at least one substituent comprising at least one ammonium group is selected from the group consisting of:

Preferably, in the cationic polymer according to the invention the molar ratio between the at least one linker and the at least one substituent comprising at least one ammonium group is from 0.5 to 5.

Depending on the molar ratio between the at least one linker and the at least one substituent comprising at least one ammonium group, the cationic polymer according to the invention may show a broad range of degrees of branching and charge densities.

In a preferred embodiment, the molar ratio between the at least one linker and the at least one substituent comprising at least one ammonium group is from 0.5 to 0.8. In this way, the cationic polymer according to the invention is a hyperbranched polymer, at the same time being a polymer soluble in water. In the context of the present invention, the expression“hyperbranched polymer” means that the polymer has a structure in which the amount of linker is lower than the amount of the substituent comprising at least one ammonium group, and the expression“polymer soluble in water” means a polymer having a solubility higher than 50% by weight in water, said solubility being measured by immersing a sample of the polymer in water at 25 °C and at concentration of 0.005 g/ml and maintaining the sample under stirring for a time of at most 12 hours.

In a further preferred embodiment, the molar ratio between the at least one linker and the at least one substituent comprising at least one ammonium group is from 1 to 5. In this way, the cationic polymer according to the invention shows a cross- linked structure. Polymers having such a structure are referred to also as “nanosponges”.

Preferably, the cationic polymer according to the invention further comprises: - at least one maltodextrin.

The expression“maltodextrin” classically refers to the starchy material obtained by acid and/or enzymatic hydrolysis of starch.

Preferably, the maltodextrin useful to the invention has a DE chosen in the range of 2 to 50, preferably of 5 to 50, preferably of 10 to 40, preferably of 15 to 35, preferably of 15 to 30, preferably of 15 to 20. This DE is for instance equal to 2 or to 17.

Preferably, the maltodextrin useful to the invention is derived from starch comprising 25 to 50 % of amylose, expressed as dry weight relative to the total dry weight of said starch.

This amylose content can be classically determined by the person skilled in the art by way of potentiometric analysis of iodine absorbed by amylose to form a complex. Preferably, the maltodextrin useful to the invention is derived from a starch exhibiting an amylose content chosen within the range of 25 to 50 %, preferably of 30 to 45 %, preferably of 35 to 40 %; these percentages being expressed in dry weight of amylose with respect to the total dry weight of starch. It is reminded that the expression“starch” classically refers to the starch isolated from any suitable botanical source, by any technique well known to those skilled in the art. Isolated starch typically contains no more than 3 % of impurities; said percentage being expressed in dry weight of impurities with respect to the total dry weight of isolated starch. These impurities typically comprise proteins, colloidal matters and fibrous residues. Suitable botanical source includes for instance legumes, cereals, and tubers. In this regard, the starch of the invention is preferably a legume starch, even more preferably a pea starch, even more preferably a smooth pea starch.

Preferably, the maltodextrin useful to the invention has a weight average molecular weight chosen within the range of 1 000 to 300.000 daltons (Da), 5 000 to 100.000

Da, preferably of 10 000 to 15 000 Da, preferably of 10 000 to 14 000, for instance equal to 12 000 Da.

This weight average molecular can in particular be determined by the person skilled in the art by liquid chromatography with detection by differential refractometer, preferably by using pullulan standards.

The maltodextrin useful to the invention is obtained by hydrolysis of starch, but might has undergone other chemical and/or physical modification, as long as it does not interfere with the desired properties, notably in term of safety and efficiency of the final cross-linked maltodextrin. However, and because it appears that it is not necessary in the present invention, the maltodextrin useful to the invention is preferably no further modified.

Suitable maltodextrins are commercially available, for instance those marketed under the name KLEPTOSE® Linecaps (ROQUETTE), Glucidex® (ROQUETTE), Stabilys® (ROQUETTE) e Tackidex® (ROQUETTE). Preferably, the nitrogen content of the cationic polymer is from 0.5 to 8.0 wt%. Said nitrogen content may advantageosly be determined by elemental analysis technique with combustion method, preferably a flash dynamic combustion method. The elemental analysis for determining the nitrogen content of the cationic polymer according to the invention may be for example performed using a Thermo Fisher FlashEA 1112 Series

In a further aspect, the present invention also relates to a process for preparing the cationic polymer according to the invention, said process comprising the steps of: - providing at least one cyclodextrin; - adding and reacting at least one linker compound selected from the group consisting of: dicarboxylic acid, dianhydride, carbonyldiimidazole, diphenylcarbonate, triphosgene, acylic dichloride, diisocyanate, and diepoxide; - adding and reacting at least one compound comprising at least one ammonium group or an ammonium group precursor; - obtaining the cationic polymer.

The process according to the present invention allows obtaining, starting from the aforesaid bio-based material, non-toxic and biocompatible cationic polymers with controllable positive charge density, using a simple synthetic procedure that advantageously may not require the use of organic solvents.

When in the process according to the present invention a compound comprising at least one ammonium group precursor is used, the ammonium group is formed during the reaction.

Advantageously, the process according to the invention provides for adding and reacting the at least one linker compound together with the at least one compound comprising at least one ammonium group or an ammonium group precursor.

Preferably, the molar ratio between the at least one cyclodextrin and the at least one linker compound is of from 0.1 to 1.

Preferably, the molar ratio between the at least one cyclodextrin and the at least one compound comprising at least one ammonium group or an ammonium group precursor is of from 0.05 to 2. Preferably, the molar ratio between the at least one linker compound and the at least one compound comprising at least one ammonium group or an ammonium group precursor is of from 0.5 to 5.

Preferably, the at least one compound comprising at least one ammonium group or an ammonium group precursor is selected from the group consisting of:

diazabiciclo[2.2.2]octane (DABCO)

wherein

- Ri is a C1 -C3 alkylene moiety, optionally substituted with a group selected from OH and C1 -C3 alkyl; - R2, R3, R4 are indepentently selected from the group consisting of: H, CH3, CH2-CH3, CH2-CH2-CH3, and CH(CH₃)₂;

- X is a monovalent anion, preferably selected from the group consisting of Cl, Br, I or OH. More preferably, the at least one compound comprising at least one ammonium group or an ammonium group precursor is selected from the group consisting of:

diazabiciclo[2.2.2]octane (DABCO)

wherein

X is a monovalent anion, preferably selected from the group consisting of Cl, Br, I or OH. In a preferred embodiment, in the process according to the invention, at least one compound comprising at least one ammonium group precursor is used. Preferably, said compound comprising at least one ammonium group precursor is diazabiciclo[2.2.2]octane (DABCO). When in the process according to the present invention DABCO is used as ammonium group precursor, the inventors surprisingly found out that the ammomium group is formed during the reaction.

Preferably, the at least one compound comprising at least one ammonium group is selected from the group consisting of:

Preferably, the process according to the invention comprises also the step of: - adding and reacting at least one nucleophilic compound selected from the group consisting of: tosylate, amine, halogen compound.

Preferably, said step of adding and reacting at least one nucleophilic compound is carried out before the step of adding and reacting at least one compound comprising at least one ammonium group or an ammonium group precursor.

Preferably, the process according to the invention comprises also the step of: - adding and reacting at least one maltodextrin. Advantageously, the process according to the invention provides for adding and reacting the at least one maltodextrin together with the at least one cyclodextrin.

In a first preferred embodiment, the process according to the invention comprises the steps of : a1. providing at least one cyclodextrin; b1. adding to and reacting with the at least one cyclodextrin of step a1. the at least one compound comprising at least one ammonium group or an ammonium group precursor together with at least one linker compound; c1. obtaining the cationic polymer from step b1. In a second preferred embodiment, the process according to the invention comprises the steps of : a2. providing at least one cyclodextrin; b2. adding to and reacting with the at least one cyclodextrin of step a2. the at least one compound comprising at least one ammonium group or an ammonium group precursor, to obtain a cyclodextrin derivative, said cyclodextrin derivative being substituted with at least one substituent comprising at least one ammonium group or an ammonium group precursor; c2. adding to and reacting with the cyclodextrin derivative of step b2. the at least one linker compound; d2. obtaining the cationic polymer from step c2.

In a third preferred embodiment, the process according to the invention comprises the steps of: a3. providing at least one cyclodextrin; b3. adding to and reacting with the at least one cyclodextrin of step a3. at least one nucleophilic compound selected from the group consisting of: tosylate, amine, halogen compound, to obtain a first cyclodextrin derivative; c3. adding to and reacting with the first cyclodextrin derivative of step b3. the at least one compound comprising at least one ammonium group or an ammonium group precursor, to obtain a second cyclodextrin derivative, said second cyclodextrin derivative being substituted with at least one substituent comprising at least one ammonium group or an ammonium group precursor; d3. adding to and reacting with the second cyclodextrin derivative of step c3. the at least one linker compound; e3. obtaining the cationic polymer from step d3.

In a fourth a preferred embodiment, the process according to the invention comprises the steps of: a4. providing at least one cyclodextrin; b4. adding to and reacting with the at least one cyclodextrin of step a4. at least one nucleophilic compound selected from the group consisting of: tosylate, amine, halogen compound, to obtain a first cyclodextrin derivative; c4. adding to and reacting with the first cyclodextrin derivative of step b4. the at least one compound comprising at least one ammonium group or an ammonium group precursor together with the at least one linker compound; d4. obtaining the cationic polymer from step c4.

In a fifth preferred embodiment, the process according to the invention comprises the steps of: a5. providing at least one cyclodextrin; b5. adding to and reacting with the at least one cyclodextrin of step a5. at least one nucleophilic compound selected from the group consisting of: tosylate, amine, halogen compound, to obtain a first cyclodextrin derivative; c5. adding to and reacting with the first cyclodextrin derivative of step b5. the at least one linker compound, to obtain a cyclodextrin polymer; d5. adding to and reacting with the cyclodextrin polymer of step c5. the at least one compound comprising at least one ammonium group or an ammonium group precursor; e5. obtaining the cationic polymer from step d5. In another aspect, the present invention relates to a cationic polymer obtainable by said process according to the present invention.

In a further aspect, the present invention relates also to the cationic polymer according to the invention for use as a medicament.

In a still further aspect, the cationic polymer according to the invention is for use as a vector in gene therapy.

The advantages of this use have been already outlined with reference to the cationic polymer according to the invention and are not repeated herewith.

Thanks to its structure, comprising the combination of specific components, namely at least two cyclodextrin units, at least one specific type of linker covalently bonding the at least two cyclodextrin units and at least one substituent comprising at least one ammonium group, the cationic polymer according to the invention is also advantageously suitable for many other applications, comprising the purification of water.

The cationic polymer according to the invention shows indeed improved properties in the removal of anions, such as nitrates, chromates and dichromates, as well as in promoting the flocculation/sedimentation of colloidal particles from the same, thus resulting to be effective for water purification processes.

Further features and advantages of the invention will appear more clearly from the following description of some preferred embodiments thereof, made hereinafter by way of a non-limiting example with reference to the following exemplary examples.

EXPERIMENTAL PART

Example 1 (nanosponge)

In a 50 ml flask containing 24 ml of anhydrous dimethylformamide, 4.00 grams (0.0035 moles) of anhydrous b-cyclodextrin were solubilized. Then 0.98 grams (0.007 moles) of anhydrous choline chloride and 2.28 grams (0.014 moles) of 1 ,1’- carbonyldiimidazole were added. The solution thus obtained was kept under stirring at 90 °C for 1 hour until a gel was obtained and then left cool to ambient temperature. The reaction product was recovered from the flask and subsequently washed with water, followed by a further washing with acetone, so as to remove any non reacted reagent and solvent. At the end of the washing step, the product was dried and then ground with a ball mill, thus obtaining a white powder. Figure 1 shows the FTIR-ATR spectrum of the cationic polymer thus obtained, and Figure 2 shows the TGA thermogram of the same, obtained according to the following methods.

FTIR-ATR All the spectra were collected in the 650-4000 cm-1 wavenumber range, at room temperature, with a resolution of 4 cm-1 and 8 scans/spectrum. A Perkin Elmer Spectrum 100 FT-IR Spectrometer equipped with an Universal ATR Sampling Accessory was used. TGA

Thermogravimetric analysis were carried out using a TA Instruments Q500 TGA, from 50 to 700 °C, under nitrogen flow, with an heating rate of 10 °C/min.

Example 2 (nanosponge)

In a 250 ml flask containing 100 ml of anhydrous dimethylformamide, 4.00 grams (0.0035 moles) of anhydrous b-cyclodextrin were solubilized. Then 0.98 grams

(0.007 moles) of anhydrous choline chloride and 3 grams (0.014 moles) of diphenlycarbonate were added. The solution thus obtained was kept under stirring at 90 °C for 5 hour until a gel was obtained and then left cool to ambient temperature. The reaction product was recovered from the flask by filtration and subsequently washed with water, followed by a further washing with acetone, so as to remove any non reacted reagent and solvent. At the end of the washing step, the product was dried and then ground with a ball mill, thus obtaining a white powder.

Example 3 (nanosponge)

In a 50 ml flask containing 8 ml of an aqueous NaOH solution (0.2M), 2.00 grams (0.00017 moles) of anhydrous b-cyclodextrin were solubilized. Then 0.98 grams

(0.007 moles) of anhydrous choline chloride and 2.64 grams (0.014 moles) of 1 ,4- butanediol diglycidyl ether were added. The solution thus obtained was kept under stirring at 90 °C for 1 hour until a gel was obtained and then left cool to ambient temperature. The reaction product was recovered from the flask and subsequently washed with water, followed by a further washing with acetone, so as to remove any non reacted reagent and solvent. At the end of the washing step, the product was dried and then ground with a ball mill, thus obtaining a white powder.

Example 4 (nanosponge) In a 100 ml flask containing 32 ml of anhydrous dimethyl sulfoxide, 3.60 grams (0.0031 moles) of anhydrous b-cyclodextrin and 0.40 grams of anhydrous mono-6- deoxy-6-amino-p-cyclodextrin (0.00035 moles) were solubilized. Then 3.9 ml (0.028 moles) of triethylamine and 3.07 grams (0.014 moles) of pyromellitic anhydride were added. The solution thus obtained was kept under stirring at 25 °C for 2 hour until a gel was obtained. The reaction product was recovered from the flask and subsequently washed with water, followed by a further washing with acetone, so as to remove any non reacted reagent and solvent. At the end of the washing step, the product was dried and then ground with a ball mill, thus obtaining a pale yellow powder.

Example 5 (nanosponge)

In a 100 ml flask containing 32 ml of anhydrous dimethyl sulfoxide, 3.60 grams (0.0031 moles) of anhydrous b-cyclodextrin and 0.40 grams of anhydrous mono-6- deoxy-6-amino^-cyclodextrin (0.00035 moles) were solubilized. Then 1.58 grams (0.028 moles) of 1 ,4-diazabicyclo[2.2.2]octane and 2.25 ml (0.014 moles) of hexamethylenediisocyanate were added. The solution thus obtained was kept under stirring at 25 °C for 2 hour until a gel was obtained. The reaction product was recovered from the flask and subsequently washed with water, followed by a further washing with acetone, so as to remove any non reacted reagent and solvent. At the end of the washing step, the product was dried and then ground with a ball mill, thus obtaining a pale powder.

Example 6 (hyperbranched polymer)

In a 50 ml flask containing 24 ml of anhydrous dimethylformamide, 4.00 grams (0.0035 moles) of anhydrous b-cyclodextrin were solubilized. Then 4.43 grams (0.032 moles) of anhydrous choline chloride and 4.57 grams (0.028 moles) of 1 ,1’- carbonyldiimidazole were added. The solution thus obtained was kept under stirring at 90 °C for 1 hour showing an increase in viscosity and then left cool to ambient temperature. The reaction product was dispersed and washed in acetone twice and then filtered and left to dry. The product is then solubilized in deionized water and filterd with a membrane (cut off 3 kDA), so as to remove any non reacted reagent and low molecular weight by-products. The product was then liophylized. A white powder was obtained. Figure 3 shows the FTIR-ATR spectrum of the cationic polymer thus obtained, and Figure 4 shows the TGA thermogram of the same, obtained according to the method disclosed in Example 1.

Gravimetric analysis confirmed a synthesis yield of 80 %%, calculated by considering the weight of the product with respect to the theoretical weight, equal to the sum of the reactants

The cationic polymer was then characterized to determine its elemental composition, its solubility in water, dimethyl sulfoxide, in ethanol and in hexane.

Elemental composition The elemental composition has been determined by means of Flash Dynamic Combustion Method using a Thermo Fisher FlashEA 1112 Series.

Table 1 shows the elemental analysis of the polymer.

TABLE 1

Solubility test

For the solubility test, a sample of the polymer was immersed in the solvent at 25 °C and at concentration of 0.005 g/ml.

The solubility and swelling of the polymer was determined either immediately after immersion (time = 0) and after 12 hours, during which the system was maintained under stirring (time =12 hours).

The polymer was considered soluble when at least 50% by weight of the same was found to be dissolved, and swellable when, at the time of the observation, it changed in linear dimensions or through volumetric change. Table 2 shows the solubility in water, dimethyl sulfoxide (DMSO), in ethanol and in hexane. In Table 2,“YES” and“NO” respectively means that the polymer was, or was not, soluble or swellable according to this definition.

TABLE 2

Example 7 (hyperbranched polymer)

In a 50 ml flask containing 8 ml of an aqueous NaOH solution (0.2M), 2.00 grams (0.0017 moles) of anhydrous b-cyclodextrin were solubilized. Then 0.49 grams (0.0035 moles) of anhydrous choline chloride and 1.32 grams (0.007 moles) of 1 ,4- butanediol diglycidyl ether were added. The solution thus obtained was kept under stirring at 90 °C for 1 hour showing an increase in its viscosity and then left cool to ambient temperature. The reaction product was dispersed and washed in acetone twice and then filtered and left to dry. The product is then solubilized in deionized water and filtered with a membrane (cut off 3 kDA), so as to remove any non reacted reagent and low molecular weight by-products. The product was then liophylized. A white powder was obtained. Figure 5 shows the FTIR-ATR spectrum of the cationic polymer thus obtained, and Figure 6 shows the TGA thermogram of the same, obtained according to the method disclosed in Example 1.

The cationic polymer was then characterized to determine its elemental composition, its solubility in water, dimethyl sulfoxide, in ethanol and in hexane according to the methods disclosed in Example 6.

Table 3 shows the elemental analysis of the polymer. TABLE 3

Table 4 shows the solubility in water, dimethyl sulfoxide (DMSO), in ethanol and in hexane. TABLE 4

Example 8

In order to investigate the use of the cationic polymer according to the invention as vector of genetic material, the polymer according to Example 6 was used to perform complexation tests with dextrane sulfate, with DNA, with siRNA.

Preparation of the complexes

1 ) Preparation of a FITC-Dextran sulfate/polymer complex using the cationic polymer according to Example 6

The FITC-Dextran sulfate/polymer complex was prepared by adding together different aliquots of a distilled water solution of fluorescein isothiocyanate (FITC)- dextran sulfate sodium salt (1 mg/ml) (Sigma-Aldrich) and a 0.9% w/v NaCI in distilled water solution of the cationic polymer according to Example 6 (2 mg/ml), respectively using a 1 :10 and 1 :50 cationic polymer/dextran sulfate solutions volume/volume ratio. 2) Preparation of pDNA/polymer complexes (polyplexes) using the cationic polymer according to Example 6

Three pDNA/polymer polyplexes were prepared by mixing different aliquots of a solution of pDNA (60 pg/ml) and a 0.9% w/v NaCI distilled water solution of the cationic polymer according to Example 6 (2 mg/ml), respectively using a pDNA/polymer volume/volume ratio of 1 :30, 1 :50 and 1 :200.

3) Preparation of siRNA/polymer complexes (polyplexes) using the cationic polymer according to Example 6

Three siRNA/polymer polyplexes were prepared by mixing different aliquots of a solution of siRNA (6 pg/ml) and a 0.9% w/v NaCI distilled water solution of the cationic polymer according to Example 6 (2 mg/ml), respectively using a siRNA/polymer N/P ratio (i.e. the ratio of moles of amine groups of cationic polymer to those of the phosphate ones of the DNA) of 10, 20 and 30.

Characterization of the complexes - Fluorescent microscopy

The FITC-Dextran sulfate/polymer complex prepared with the cationic polymer according to Example 6 was analyzed with a Leica DM 2500 equipped with ebq-100 ISOLATED mercury xenon discharge lamp fluorescent microscope.

Figure 7 shows the fluorescence microscopies of FITC-Dextran sulfate complex with the cationic polymer according to Example 6. In said figure, image A on the left shows the 1 :10 and image B on the right shows 1 :50 cationic polymer/dextran sulfate solutions volume/volume ratio, respectively.

The interaction between the cationic polymer according to Example 6 and FITC- Dextran sulfate (Figure 7) was detected by the formation of globular nanoparticles (Figure 7A, 7B)

- Gel Electrophoresis Assay

A gel electrophoresis assay was employed to evaluate the formation of the pDNA/polymer and siRNA/polymer polyplexes prepared with the cationic polymer according to Example 6. The polyplexes were subjected to electrophoresis on (1 % w/v for pDNA and 3% w/v for siRNA) agarose gel run in (0.7% w/v) TAE buffer 1 % (40 mM Tris base, 20 mM acetic acid and 1 mM EDTA; pH 8.0) for 1 h at 100 V to confirm the complexation. The gel was stained with ethidium bromide (0.25 pg/ml). The banding pattern was obtained using an ultraviolet (UV) transilluminator and photographed with a Kodak EDAS 290 camera.

Figure 8 shows the banding pattern of the polyplexes of pDNA with the cationic polymer according to Example 6 in a gel retardation assay, at a pDNA/polymer volume/volume ratio of 1 :30 (image A), 1 :50 (image B) and 1 :200 (image C) compared with a reference solution of naked pDNA (“Naked”, in images A, B and C).

The test confirmed the formation of the polyplexes as the bands at 1 :30, 1 :50, and 1 :200 pDNA/polymer volume/volume ratio for the polyplexes (indicated by the upward arrow) moved significantly less than the respective bands of the naked pDNA (indicated by the right-pointing arrow) towards the anode.

The test showed that the retention of pDNA was strictly related to the capability of the cationic polymer according to Example 6 to form polyplexes, thus masking the pDNA negative charge.

Figure 9 shows the banding pattern of the polyplexes of siRNA with the cationic polymer according to Example 6 in a gel retardation assay, at a N/P ratio 10 (image B), 20 (image C) and 30 (image D) compared with a reference solution of naked siRNA (image A).

The test confirmed the formation of the polyplexes as it was possible to observe the displacement from the starting hole of the free siRNA control band (A), indicated by the arrow, while the bands in position B, C and D (indicated by the arrow) were still in the starting positions. This result demonstrated the capability of the cationic polymer according to Example 6 to form polyplexes also with siRNA samples.

- Sodium Dodecyl Sulfate Test

A sodium dodecyl sulfate (SDS) test was carried out to examine the stability of the electrostatic interactions between pDNA and the cationic polymer according to Example 6. In particular, a gel electrophoresis assay was used to assess the SDS concentration required to displace the pDNA from the pDNA/polymer polyplex.

For the (SDS) test, 5 solutions were prepared:

Solution A: a solution of naked pDNA (60 pg/ml), used as negative control; Solution B: a solution of a polyplex between Poly L-Lys (2 mg/I) and pDNA (60 pg/ml)

(pDNA/poly L-Lys volume/volume ratio of 1/30), used as positive control;

Solution C: a solution of a pDNA/polymer polyplex between the cationic polymer according to Example 6 and pDNA (pDNA (60 pg/ml)//polymer (2 mg/I) volume/volume ratio of 1/30), used as positive control;

Solution D: a solution of a pDNA/polymer polyplex between the cationic polymer according to Example 6 and pDNA (pDNA (60 pg/ml)/polymer (2 mg/I) volume/volume ratio of 1/30) and 2% w/v of SDS, prepared incubating for 30 minutes the pDNA/polymer polyplex in a 2% w/v of SDS solution; and

Solution E: a solution of a pDNA/polymer polyplex between the cationic polymer according to Example 6 and pDNA (pDNA (60 pg/ml)/polymer (2 mg/I volume/volume ratio of 1/30) and 5% w/v of SDS, prepared incubating for 30 minutes the pDNA/polymer polyplex in a 5% w/v of SDS solution; The solutions were subjected to electrophoresis on 1 % w/v agarose gel run in (0.7% w/v) TAE buffer 1 % (40 mM Tris base, 20 mM acetic acid and 1 mM EDTA; pH 8.0) for 1 h at 100 V. The gel was stained with ethidium bromide (0.25 pg/ml). The banding pattern was obtained using an ultraviolet (UV) transilluminator and photographed with a Kodak EDAS 290 camera. Figure 10 shows the banding pattern of the 5 solutions, in which the band corresponding to the solutions is indicated by the arrow, respectively in image A, B, C, D and E. The test showed that a 5% w/v SDS solution was required to completely displace the pDNA from the polyplex, as reported in image E. In this case, indeed, the band clearly moved from the starting hole, meaning that the pDNA was again free to move towards the anode.

Example 9

In order to investigate the use of the cationic polymer according to the invention as vector of genetic material, the polymer according to Example 6 was used to perform internalization experiments on Human Fibroblast cells.

In this experiment, Human Fibroblast cells were grown in DMEM (Dulbecco’s Modified Eagle Medium) High Glucose supplemented with 10% w/v Fetal Bovine Serum, 2 mM L-glutamine and 2% w/v of antibiotics (penicillin and streptomycin). The cells were then transferred in 6-well plates at 5 ^c 10⁵ cells per well and incubated at 37 °C in a 5% v/v carbon dioxide’s atmosphere overnight.

The FITC-dextran sulfate sodium salt/Polymer (Example 6) complexes were prepared by mixing FITC-dextran sulfate sodium salt 1 mg/I water solution and Polymer (Example 6) water solution 2mg/l with a volume/volume ratio of 1 :50. Then the prepared complexes were preincubated in a 2 mg/ml NaCI solution overnight at 5 °C. Before internalization, cell medium was replaced with serum-free medium. The complexes were added to the culture medium for 60 minutes at 20 pg/ml.

Post incubation, the cells were washed with PBS (Phosphate Buffered Saline, Sigma-Aldrich), fixed with 4% w/v PAF (Paraformaldehyde, Merck) for 12 minutes and made permeable with 0.25% w/v saponin solution. Then, to perform an immunofluorescent assay they were incubated with Early Endosome Autoantigen 1 (EEA1 ) primary antibody, and aGoat (Alexa Fluor 555) secondary antibody. Each one was applied during 1 hour at 25°C. Subsequently they were washed three times with PBS. They were stained with phalloidin (Alexa Fluor 647) and 4’,6-diamidine- 2-phenylindole (DAPI). Finally, these samples were analyzed with a confocal laser scanning microscope (Leica TCS SP2 AOBS) equipped with 63X/1.40 HCX Plan- Apochromat oil-immersion objective. Confocal images are maximum projections of a z section of ~3 pm.

Figure 11 shows the laser scanning confocal microscope image at 63X of a 20 pg/ml solution of the FITC-Dextran sulfate complex with the cationic polymer according to Example 6 internalized within the cell membrane of human fibroblast cells after a 60 minutes contact. The image shown represents a x-y view at a given z and it is the overlap of three fields/pictures, in which it was possible to observe the presence of the FITC-Dextran sulfate complex within the cell membrane (see the light-grey areas, indicated by the arrows), showing that the internalization was successfully occurred.

Example 10

In a 50 ml flask containing 10 ml of an aqueous NaOH solution (0.2M), 0.86 grams (0.0009 moles) of anhydrous a-cyclodextrin, 1.0 grams (0.0009 moles) of anhydrous b-cyclodextrin, 1.14 grams (0.0009 moles) of anhydrous g-cyclodextrin were solubilized. Then 0.54 (0.005 moles) grams of diazabiciclo[2.2.2]octane (DABCO) and 1.94 ml (0.01 moles) of 1 ,4-butanediol diglycidyl ether (BDE) were added. The solution thus obtained was kept under stirring at 90 °C for 1 hour until a gel was obtained and then left cool to ambient temperature. The reaction product was recovered from the flask and subsequently washed with water, so as to remove any unreacted reagent and solvent. At the end of the washing step, the product was dried and then ground with a ball mill, thus obtaining a brown powder.

Figure 12 shows the TGA thermogram of the cationic polymer thus obtained, and Figure 13 shows the FTIR-ATR spectrum of the same, obtained according to the method disclosed in Example 1.

Gravimetric analysis confirmed a synthesis mass balance of 90 %, calculated by considering the weight of the product with respect to the theoretical weight, equal to the sum of the reactants.

The cationic polymer was then characterized to determine its elemental composition, zeta-potential, thermal stability, its solubility in water, dimethyl sulphoxide, in ethanol and in hexane. Furthermore, also its capability to remove toluene, xylene, naproxen and sodium dichromate from water solutions has been tested.

Elemental composition The elemental composition was determined according to the method disclosed in Example 6 by means of Flash Dynamic Combustion Method using a Thermo Fisher FlashEA 1 1 12 Series.

Table 5 shows the elemental analysis of the polymer TABLE 5

Zeta-potential

The zeta-potential was determined by means of a Malvern Zetasizer Nano - ZS. Table 6 shows the elemental analysis of the polymer

TABLE 6

Solubility test

Table 7 shows the solubility in water, dimethyl sulphoxide, in ethanol and in hexane, carried out according to the method disclosed in Example 6. TABLE 7

Water purification test

In order to investigate the use of the cationic polymer according to the invention for the purification of water, the cationic polymer was also used to perform removal test from samples of water containing different amounts of contaminants, such as toluene, xylene, naproxene and potassium dichromate. The removal was evaluated after 24 hours on three tested aqueous solutions after separation of the cationic polymer, by using a Metrohm 883 Basic IC plus ion chromatography system (for the potassium dichromate), and by UV-Vis absorbance for toluene, xylene and naproxen (Perkin-Elemer Lamda 25) .

Table 8 shows the adsorption of toluene, xylene, naproxene and potassium dichromate from water solutions.

Tested aqueous solutions: 10 ml of saturated xylene and naproxen solutions with 10 mg of the polymer;

10 ml of 2.5 ppm naproxen solution with 10 mg of the polymer;

10 ml of 25 ppm K2q2q7 solution with 200 mg of the polymer.

TABLE 8

The cationic polymer according to the invention resulted very effective in removing all the tested contaminants.

Example 11 In order to investigate the cytotoxicity of the cationic polymer according to the invention, the polymers according to Example 6 and 7 were subjected to a test.

The effect of the two cationic polymers was determined in HUVEC cells through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) assay. This colorimetric assay, able to determine the level of metabolic activity in cells, may be interpreted as a measure of both cell viability and cell proliferation (Sylvester 2011 ). Cells were seeded (1.500 cells/well) in 96-well plates with 200 pi of serum-supplemented medium, and were allowed to attach for 24 h. Then, cells were treated with increasing concentrations of cationic polymer. The MTT assay was performed at 72h from the beginning of the treatment.

Figure 14 shows the results of the tests performed with the cationic polymer according to Example 6, whereas Figure 15 shows the results of the tests performed with the cationic polymer according to Example 7.

The tests showed that the cationic polymer according to the invention is not cytotoxic at the tested concentrations.

Claims

1 . A cationic polymer comprising:

- at least two cyclodextrin units;

- at least one substituent comprising at least one ammonium group.

2. Cationic polymer according to claim 1 , wherein the at least one linker is selected from the group consisting of: 1 , 1’-carbonyldiimidazole, a diepoxide, and a triepoxide.

3. Cationic polymer according to claim 1 or 2, wherein the at least one substituent comprising at least one ammonium group is selected from the group consisting of:

wherein - Ri is a C1 -C3 alkylene moiety, optionally substituted with a group selected from OH and C1 -C3 alkyl; and

4. Cationic polymer according to claim 3, wherein the at least one substituent comprising at least one ammonium group is selected from the group consisting of:

5. Cationic polymer according to any of claims from 1 to 4, wherein the molar ratio between the at least one linker and the at least one substituent comprising at least one ammonium group is from 0.5 to 5.

6. Cationic polymer according to any of claims from 1 to 5, wherein the at least one substituent comprising at least one ammonium group is covalently bonded to at least one cyclodextrin unit.

7. Cationic polymer according to any one of claims from 1 to 6, wherein the nitrogen content of the cationic polymer is from 0.5 to 8.0 wt%.

8. A process for preparing the cationic polymer according to any one of claims from 1 to 7, said process comprising the steps of:

- providing at least one cyclodextrin;

- adding and reacting at least one linker compound selected from the group consisting of: dicarboxylic acid, dianhydride, carbonyldiimidazole, diphenylcarbonate, triphosgene, acylic dichloride, diisocyanate, a diepoxide, and a triepoxide;

- adding and reacting at least one compound comprising at least one ammonium group or an ammonium group precursor; - obtaining the cationic polymer.

9. A cationic polymer obtainable by means of the process according to claim 8.

10. Cationic polymer according to any one of claims from 1 to 7 or 9, for use as a vector in gene therapy.

11. Use of the cationic polymer according to any one of claims from 1 to 7 or 9, for the purification of water.