WO2015040513A1

WO2015040513A1 - Photocurable polymer with a comb-like structure

Info

Publication number: WO2015040513A1
Application number: PCT/IB2014/063728
Authority: WO
Inventors: Irini GERGES; Cristina Lenardi; Federico MARTELLO; Valentina PISTIS; Margherita TAMPLENIZZA; Alessandro TOCCHIO
Original assignee: Fondazione Filarete Per Le Bioscienze E L'innovazione; Universita' Degli Studi Di Milano
Priority date: 2013-09-23
Filing date: 2014-08-06
Publication date: 2015-03-26
Also published as: ITMI20131560A1

Abstract

The present invention relates to a synthetic polymeric precursor with a comb-like or hyper- branched structure, which is capable of carrying out an irreversible in situ sol-gel transition by photo- cross-linking, under conditions which are compatible with a biological organism, to produce hydrogels, which is capable of acting as biocompatible and biodegradable tridimensional matrices suitable to the in vitro, and in vivo encapsulation and transport of: cells; drugs; peptides; proteins, and growth factors. In particular, the invention relates to a photocurable polymeric precursor, which can be administered in vivo by injection or minimally invasive surgical approaches, said- polymeric precursor being composed of: (i) an acrylic oligomer of formula (II) : which is obtained by random co-polymerization of an acrylic monomer of formula (III) : CH₂=C (R₂) -CO-OR-R₁-OH (III) wherein the functional group R₁ is a C₁-C₆ alkylene, preferably C₂ alkylene; wherein the R₂ functional group is H or a C₁-C₄ alkylene, preferably C₁ alkylene together with an acrylic co-monomer or acrylamide of formula ( IV) : CH₂=C(R₃) -CO-X-R₄-YH (IV) wherein the functional group R₃ is H or a C₁-C₄ alkylene, preferably C₁ alkylene; wherein the R₄ functional group is a C₁-C₆ alkylene, preferably C3 alkylene; X is 0 or NH; Y is NH or S; such co- polymerization occurs in the presence of a chain transfer agent, preferably 2-mercaptoethanol, on said acrylic oligomer being grafted (ii) side chains composed of linear poly (amidoamine) s, an end of which side chains is bounded to said acrylic oligomer, while the free end terminates with an acrylamide group.

Description

"PHOTOCURABLE POLYMER WITH A COMB-LIKE STRUCTURE"

The present invention relates to a synthetic polymeric precursor with a comb-like or hyper- branched structure, which is capable of carrying out an irreversible sol-gel transition in situ by photo- cross-linking, under photo-cross-linking conditions compatibility with a biological organism, to produce hydrogels, which is capable of acting as biocompatible and biodegradable tridimensional matrices suitable to the in vitro and in vivo encapsulation and transport of: cells; drugs; peptides; proteins, and growth factors. In particular, the invention relates to a photocurable polymeric precursor, which can be administered in vivo by injection or minimally invasive surgical approaches. In the last decade, the use of polymer carriers that can be injected and solidified in situ is drawing attention by the scientific and industrial community in both the biomedical and pharmaceutics fields. Particularly, in the development of carrier systems for the prolonged and controlled release of bioactives, such as drugs; hormones; peptides; proteins, and growth factors, in the pharmaceutics field, and in the transport of cells in the regenerative medicine. The main advantages are related to the patient's comfort and the times required for the post-surgical rehabilitation. To date, the most examined biomaterials for the formation of injectable and in situ jellifying matrices, particularly those suitable to the cell encapsulation and which are capable of obtaining a microenvironment that is appropriate to maintain the cell _. viability and · proliferation, are natural polymers, such as, for example: chemically modified gelatin, collagen, and chitosan.

The main limitations of the modified natural polymers are attributable to uncontrolled degradation kinetics, unsuitable mechanical properties, the stimulation of an undesired immune response, due to the presence of impurities and endotoxins, and the differences in the properties that can be found between a synthesis batch and another one during large-scale isolation procedures.

On the other hand, the currently available synthetic in situ jellifying polymers can yield a better mechanical efficiency compared to the polymers of natural origin, in addition to adjustable degradation kinetics, yet they are biologically insufficient in terms of cell adhesion and their ability to promote the cell viability and proliferation. In many cases, a chemical modification of the polymer with the tripeptide: Arg-Gly-Asp (or RGD) is necessary, in order to induce the adhesion of the cells onto such material.

K. Tanahashi and co-workers first improved the biologic properties of synthetic hydrogels based on Poly (propylene fumarate-co-ethylene glycol), by a co- polymerization with 4-aminobutyl guanidine (also referred to as Agmatine) . Tanahashi showed that the nature of the polymer-cell interaction is directly related to the Agmatine guanidine groups by virtue of its chemical structure, which is similar to the tripeptide RGD. In 2005, Ferruti and co-workers disclosed a class of polyamidoamines (PAA) that is biodegradable with good adhesion properties, obtained by a polyaddition reaction of the Michael type between 2, 2' -bis (acrylamide) acetic acid (BAC) and Agmatine. Furthermore, PAA-based hydrogels containing Agmatine showed a good ability in cell adhesion onto a surface, but their poor mechanical properties in terms of compression and traction resistance, besides their uncontrollable degradation kinetics, discouraged their use in tissue engineering. Therefore, it is apparent that there still is a need of providing a fully synthetic, injectable, in situ jellifying polymeric precursor which is capable of ensuring proper biological surroundings to the encapsulated cells, appropriate mechanical properties, and degradation kinetics that allow the material to provide the required mechanical and biological support, until the complete regeneration of the new tissue.

Therefore, an object of the present invention is to provide a synthetic, in situ jellifying, polymeric precursor that solves the problems associated to both the natural and the synthetic polymers of the prior art .

Such an object is achieved by a polymeric precursor as defined in the appended claims, which combines the excellent mechanical properties of the acrylic polymers to the good cell adhesion properties and degradability of PAAs containing Agmatine and which is capable of providing an irreversible sol-gel phase transition under photo-cross-linking conditions compatible with a biological organism.

Further characteristics and advantages of the present invention will be more apparent from the description of some embodiment examples, given herein below by way of illustrative, non-limiting example, with reference to the following Figures:

Fig. 1 represents the polymer with a comb-like structure of formula (I), which is the subject-matter of the invention;

Fig. 2 represents the synthesis scheme of the polymer with a comb-like structure: P (HEMA-co- APM) /OPAA;

Fig. 3 represents the overlapping of HNMR (D₂0) spectra of the backbone: P (HEMA-co-APM) (top) and the corresponding comb-like polymer: P (HEMA-co-APM) /OPAA;

Fig. 4 represents the MALDI TOF mass spectrum of the oligomer P (HEMA-co-APM) , obtained by using 2,5- dihydroxybenzoic acid (DHB) as the matrix;

Fig. 5 represents the load-strain profiles of the hydrogels obtained by photo-cross-linking the precursors: PAA (HEMA-co-APM)./OPAA and the control: PAAdiAc .

An object of the present invention is a polymeric precursor usable for obtaining a hydrogel suitable to the encapsulation of cells and the in situ release of cells, which combines the excellent mechanical properties of the acrylic polymers to the good cell adhesion properties and of degradability of PAAs containing Agmatine, and which is capable of carrying out an irreversible sol-gel phase transition under photo-cross-linking conditions compatible with a biological organism.

The polymeric precursor that is the object of the invention is characterized by a _. comb-like structure .

By the term "comb-like structure" is meant a hyper-branched structure having a main chain from which secondary chains originate. Such a comb-like structure is composed of:

(i) a main chain (herein below referred to as the "backbone") of formula (II):

(ID

composed of an acrylic oligomer which is obtained by random co-polymerization of an acrylic monomer of formula ( III ) :

CH₂=C (R₂) -CO-OR-Ri-OH (III) wherein the functional group Ri is a Ci-C₆ alkylene, preferably C₂ alkylene; wherein the functional group R₂ is H or a C1-C4 alkylene, preferably Ci alkylene, together with an acrylic co-monomer or acrylamide of formula (IV) :

CH₂=C (R₃) -CO-X-R4-YH (IV) wherein the functional group R₃ is H or a C₁-C4 alkylene, preferably Ci alkylene; wherein the functional group R₄ is a Ci-C₆ alkylene, preferably C₃ alkylene; X is 0 or NH; Y is NH or S;

A preferred example of a monomer of formula (III) is

2-hydroxyetilmethacrylate (defined by the acronym HEMA) .

A preferred example of a monomer of formula (IV) is

3-aminopropylmetacrylamide (defined by the acronym APM) .

(ii) a variable number of side chains (herein below referred to as "pendant chains") composed of linear polyamidoamines (herein below referred to by the acronym OPAA) , an end of which side chains is linked to the backbone, while the fee end terminates with an acrylamide group.

The comb-like polymer of the invention has the structural formula (I) shown in Fig. 1. The comb-like polymer according to the invention can be obtained by a direct copolymerization of the Michael type between

a) a bis (acrylamide) of formula (V):

CH2=CH-CO-NH-R₅-NH-CO-CH=CH₂ (V)

or of formula (VI) :

CH₂=CH-CO-N-R₅-N-CO-CH=CH₂ (VI)

wherein R₅ is as defined below,

b) the monomer Agmatine, for the nucleophilic attachment of its amine groups, and

c) the backbone of formula (II), for the nucleophilic attachment of the amine or sulfhydryl groups belonging to the backbone deriving from the co- monomer of formula (IV) contained therein.

In the case of formula (V) , the R₅ functional group is selected from the following classes of functional groups :

i. aliphatic alkyl chains of formula -(CH₂)_n- where the value of n ranges between 1 and 10, preferably 1.

ii. alkoxy chains of formula - (CH₂-CH₂-0) n- , - (CH₂-CH (CH₃) -0)_n- and - (CH-CH₂-0) _n- (CH- CH (CH₃) -0) _m-, where the values of n and m range between 1 and 20, preferably 5. iii. carboxylic acids of formula -CH(-COOH)- or secondary alcohols of formula -CH(-OH)-. In the case of formula (VI), the R₅ functional group is an aliphatic heterocycle, preferably piperazine and 2-methylpiperazine .

The acrylic component, i.e., the backbone of the comb-like polymer that is the object of this invention, is the non-degradable portion of the polymeric precursor. For this reason the numeric and weight average molecular weights of the backbone were maintained below the filtration glomerular filtration limit threshold, so as to ensure a complete clearance of the polymer from the organism. The backbone average molecular weight is in the range of 1-100 kDa, preferably between 1 and 30 kDa, and it is adjusted by using 2-mercaptoethanol as the chain transfer agent, applying a Lewis and Mayo model (K. G Suddaby et al., Macromolecules , 30 (1997), 702-713) according to the following equation:

1 1 [S]

=- = =___- + C_s X Γ 7

DP_n DP_n0 [M] wherein DP_n and DP_no are the average degree of polymerization, in the presence and absence, respectively, of the chain transfer agent "S"; C_s is the chain transfer constant, defined as the ratio between the coefficients of chain transfer rate and the propagation speed, k_tr/k_p; [M] is the sum of the molar concentrations of the monomers (III) and (IV) .

The molar ratio between the monomers (III) and (IV) ranges between 10:1 and 1:1 preferably 10:3. The ratios between the co-monomeric reagents (III) and (IV) are kept in the resulting polymer (backbone), as shown by magnetic resonance (NMR) structural characterizations and MALDI TOF mass spectroscopy.

The polymeric precursor according to the invention is represented by the formula (I):

wherein the n/m ratio ranges between 10 and 1, preferably between 3 and 3.5; the values of n and m can be determined by NMR spectroscopy (Fig. 3) MALDI TOF spectrometry (Fig. 4); p ranges between 1 and 100, and more preferably between 1 and 20; R_i; R₂ and R₃ are as defined above; X is 0 or NH; Y is NH or S; R is a Ci-C₆ alkylene, preferably C₃ alkylene; R₅ is a C1-C6 alkylene or a C₅-C₆ cicloalkylene or CH-COOH or CH-COOR, where R is a Ci-C₄ alkyl, phenyl or benzyl; R₆ is a primary amine or a secondary diamine, preferably, the following monomeric unit:

The average molecular weight of the polymeric precursor according to the invention ranges between 2kDa and 400kDa, preferably between 2 kDa and 100 kDa.

Illustrative examples of polyamidoamines that can be used as the pendant chains in the comb-like polymer that is the object of the present invention are illustrated in Table (1) .

Table 1 - preferred examples of polyamidoamines which can be used as the pendant chains in the polymer with a comb-like structure that is the object of the present invention

Synthesis process of the polymeric precursor of formula ( I )

The polymeric precursor according to the invention can be obtained according to the scheme shown in Fig. 2 and providing for the following steps :

a) random co-polymerization of the co-monomer of formula (III) and the co-monomer of formula (IV) to obtain a random co-polymer of formula (II) in the presence of a thiol or a mercaptan as the chain transfer agent, more preferably 2-mercaptoethanol .

The control of the molecular weight of the backbone can further occur by using other thiols, for example, N-acetyl-l-cysteine or 1-cysteine, or aliphatic esters, such as methylbutanoate, diethylmalonate and diethyl 2-methylmalonate .

b) polymerization of the random co-polymer of formula (II) with a bisacrylamide of formula (V) or (VI), for example BAC, with a secondary diamine or a primary amine, for example Agmatine, to give the polymeric precursor represented in formula (I).

The step a) is carried out in the presence of a radical initiator, such as, for example, 2,2'- azobis (2-methylpropionitrile) (AIBN) , and at a temperature ranging between 60° C and 80° C, preferably at about 70° C. The reaction time ranges from 2 hours to 24 hours or longer, until reaching an average molecular weight ranging between 1 and 30 KDa. The monomers (III) and (IV) are reacted _. in a molar ratio ranging between 10:1 and 1:1, preferably 10: 3.

The step b) provides for a poliaddition of the Michael type, wherein the acrylic double bonds of a bisacrylamide: CH₂=CH-CO-NH-R₅-NH-CO-CH=CH₂, for example, BAC, react with the amine groups of Agmatine and the amine or sulfhydryl groups belonging to the backbone deriving from the monomer (IV) contained therein .

The step b) is carried out in a basic environment with a pH above 8.5, preferably of about 9. For example, a strong base, such as an alkaline or earth-alkaline metal hydroxide, will be able to be used in an aqueous solvent. In an embodiment, lithium hydroxide mono-hydrated can be used as the base. The reaction temperature preferably ranges between 20° C and 50° C, preferably between 25° C and 40° C.

The reaction time can range from one day to one or more weeks, so as to obtain an average molecular weight ranging between 2kDa and 200kDa. It is important that the polymerization reaction is carried out in a darkened environment, to avoid a photo-cross-linking of the polymeric precursor of the invention. The following preparation example further illustrates the invention, without for this limiting the scope thereof as defined in the appended claims.

Synthesis example

Step 1- Preparation of the copolymer P (HEMA-co-APM) of formula (II)

HEMA (10 g, 74.53 mmol) , 3-aminopropyl metacrylamide.HCl (APM) (4.07 g, 22.36 mmol), 2,2'- azobis (2-methylpropionitrile) (AIBN, 0.14 g, 0.84 mmol), 2-mercaptoethanol (1.16 ml, 16.42 mmol, 1.11 g/ml) and N, N' -dimethylformamide (DMF, 50.83 ml, 0.65 mol, 0,94 g/ml) were introduced under stirring in a 100-ml round flask, equipped with magnetic stirrer and nitrogen inlet. The reactor was degassed by repeated vacuum/nitrogen cycles, then put under an inert atmosphere at 70° C for 24 hours. During the polymerization process, the color of the reaction mixture gradually changed from colorless to yellow (after 12 hours) to orange (after 24 hours) . At the completion of the reaction, the mixture was brought to room temperature, the raw polymer was purified from the solvent, radical chain transfer agent and unreacted monomers in two steps:

a) precipitation in a four-fold higher volume of diethylether, b) ultrafiltration in distilled water through a semi-permeable membrane with a 1000 Da nominal molecular weight cut off.

The polymer was obtained as a yellowish powder after freeze-drying . The reaction yield was 51%, Mn=6700, Mw=7800, P.D. index=l,16 (determined by volume size exclusion chromatography using polystyrene standards) .

H-NMR (D₂0) : 0.83 (s, 3H, CH₃-C- "APM") ; 1.02 (s, 3H, CH₃-C- "HEMA" ) ; 1.79 (s, 2H, -CH₂-CCH₃ "APM") ; 1.91 (s, 2H, CH₂-CCH₃ "HEMA"); 2.94 (m, 2H, -CH₂-CH₂-NH₂HCI) ; 3.13 (m, 2H, CH₂-NH-C=OR) ; 3.62 (m, 2H, -CH₂-NH₂HCI) ; 3.76 (m, 2H, -CH₂-OH) ; 4.03 (m, 2 H, CH₂-OR-C=OR) .

Step 2 - Preparation of the polymeric precursor PAA (HEMA-co-APM) /OPAA of formula (I)

In a two-neck round flask, equipped with magnetic stirrer and nitrogen inlet, 2.0 g P(HEMA- co-APM) of formula (II) obtained according to the step 1 was dissolved in 1.267 ml distilled water. 3.8 g 2 , 2' -bis (acrylamide) acetic acid and 0.9 g lithium hydroxide mono-hydrated were added to the P (HEMA-co-APM) solution, which was kept under magnetic stirring. Finally, 2.65 g 4-aminobutyl guanidine (Agmatine) sulfate is added to the reaction mixture. The initial pH of the mixture was 9.5, but it reached a value of 8 after 2 days. The reaction was carried out protected from light, in an inert atmosphere, and it was left to react for 7 days at 35° C. The polymeric precursor with a comblike structure P (HEMA-co-APM) /OPAA of formula (I) was purified by ultrafiltration through a semipermeable membrane with a 3000 Da cut off. After freeze-drying, a yellowish powder product was obtained, with 60% yield.

H NMR (D20) : 0.82; (s, 3H, CH₃-C- "APM" ) ; 1.02; (s, 3H, CH₃-C- "HEMA"); 1.56 (s, 2H, -CH2-CH2-NH-CNHNH2 ) ; 1.70 (s, 2H, -N-CH2-CH2-) ; 1.83 (s, 2H, -CH₂-CCH₃ "APM") , 1.93-1.97 (m, 2H, CH₂-CCH₃ "HEMA"); 2.71 (s, 2H, -CH2-CONH) ; 3.07 (s, 2H, -CH2-NH-CNHNH2 ) ; 3.15 (t, 2H, -N-CH2-); 3.34 (s, 2H, -N-CH₂-CH2- CONH) ; 3.62 (m, 2H, -NH-CH₂- (CH2) 2-NHCO-) ; 3.76 (s, 2H, -CH₂-OH) ; 4.02 (m, 2 H, - COO-CH2-CH2-OH) ; 5.50-5.70 (m, 2H, CH2=CH-CONH-) ; 6.16(m, 1H, CH2=CH-CONH- ) The synthesis scheme of the polymer with a comb-like structure, the synthesis of which has been set forth in the example above, herein below referred to as PAA (HEMA-co-APM) /OPAA, is set forth in Fig. 2.

Step 3- Formation of a hydrogel for photo- cross-linking of the polymeric precursor of formula (I) in the presence of a cell culture

A hydrogel for the encapsulation of cells can be prepared according to methods known in the field, by a photo-cross-linking (UV irradiation) of the polymeric precursor of formula (I) in the presence of a cross-linking photoinitiator and a cell suspension in a culture medium.

The photoinitiator can be, for example, l-[4- ( 2-hydroxyethoxy) phenyl ] -2-hydroxy-2-methyl-1- propan-l-one, for example, commercially available by Ciba Specialty Chemicals under the trade name Irgacure^® 2959. However, other photoinitiators will be able to be used, such as, for example, bis- acylphosphine oxides (Irgacure 819); 1 , 5-diphenyl- 1 , 4-dien-3-one (Diinone) ; phenanthrenequinone ; 4'- phenoxyacetophenone and thioxanthen-9-one .

The culture medium of the cells can be any cell culture medium suitable or adapted to the growth and maintenance of viability of the selected cells. For example, a DMEM (Dulbecco Modified Eagle's Medium) integrated with serum and other factors, such as a standard cell culture, will be able to be used. There are several DMEM formulations, which can be selected according to the needs. The conventional DMEM comprises D-glucose, inorganic salts, amino acids, and vitamins in suitable ratios. Other culture media can be, for example, RPMI 1640, Medium 199 and basic culture media or adapted to the differentiation of mesenchymal stromal cells.

Cross-linking conditions are selected so as to obtain a photo-cross-linking under mild conditions. For example, the polymeric precursor with a comblike structure is initially dissolved into an aqueous solution or a cell culture medium or in a cell suspension in a cell culture medium at a weight/weight concentration ranging between 5 and 25%, best between 10 and 15%. The cross-linking of the polymer solution can occur by UV-ray exposure at a wavelength of 365 nm, for exposure times ranging between 1 and 5 minutes, preferably about 2 minutes. Alternatively, the photo-polymerization can occur by exposing the polymer solution to a blue light, commonly used in the dentistry field, with a wavelength ranging between 400 and 500 nm, preferably about 460 nm.

Mechanical characterizations of the polymer PAA (HEMA-co-APM) /OPAA obtained according to the example set forth above

Static compression tests were conducted on the hydrogel obtained by photo-cross-linking the polymer P (HEMA-co-APM) /OPAA in bi-distilled water. Analyses were performed at room temperature, about 25° C. In order to investigate the role of the backbone P (HEMA-co-APM) in improving the hydrogel mechanical properties, analyses were performed comparatively with respect to a control hydrogel obtained by photo-cross-linking .diacrylamide-terminated poly (amidoamine) oligomers, herein below referred to as PAAdiAcs. The P (HEMA-co-APM) /OPAA and PAAdiAc photo-cross-linking conditions were kept identical in terms of: polymer concentration in water (13.5% weight/volume) ; UV light exposure time (2 minutes) and load application rate ( 5%/minute) . The load- strain profiles of the hydrogels obtained by photo- cross-linking P (HEMA-co-APM) /OPAA and the control polymer PAAdiAc are graphically illustrated in Fig. 5. The values of compression elastic modulus and compressive strength are set forth in Table 2.

Table 2 - Modulus, compressive strength at a 30% strain and compressive strength of hydrogels obtained by photo-cross-linking precursors PAA (HEMA- co-APM) /OPAA and control PAAdiAc

From the results obtained and set forth in Fig. 5, and Table 2, it can be noticed that the compression resistance of the hydrogel obtained by photo-cross- linking P (HEMA-co-APM) /OPAA is more than double the comparative PAAdiAc polymer, and that the superior mechanical performance of the hydrogel obtained from the precursor P (HEMA-co-APM) /OPAA is due to the backbone: P (HEMA-co-APM) .

The hydrogel made of the polymeric precursor of the invention can be used to encapsulate a wide variety of cell types, which will be able to be used for several purposes, including the regenerative medicine, or targeting in a target organ of cells secreting enzymes, proteins, or bioactives.

The applications and uses of the polymeric precursor that is the object of this invention are further extended to the biomedical and pharmaceutics fields. A list of the applications and uses of the comb-like polymer according to this patent is set for the herein below:

• Photocurable polymer for in vitro cell assays, in the biological, biomedical, and pharmaceutical field.

• Photocurable polymer for cell encapsulation for in vitro studies in the biological, biomedical, and pharmaceutical field.

• Injectable, in-situ photocurable polymer and for a controlled or prolonged release of drugs.

• Injectable, in-situ photocurable polymer and for a controlled or prolonged release of enzymes, hormones, peptides, proteins, or genetic material, for example, RNA or DNA.

• Injectable, in-situ photocurable polymer as a connective tissue filler in the cosmetic field and regenerative medicine.

• Injectable, in-situ photocurable polymer for the stem cells release for experimental or clinical treatments of neurodegenerative diseases .

• Injectable, in-situ photocurable polymer for loading inorganic fillers, for example: hydroxyapatite, brucite, tricalcium phosphate, beta-tricalcium phosphate, alumina, calcium oxide, titanium oxide, zinc oxide, etc., for the regeneration of bone tissue, cartilaginous tissue, in the maxillofacial surgery field, and in the dentistry field.

The hydrogels for cell encapsulation, made by photo- cross-linking the polymeric precursor of the invention as described above, have been found efficient in both promoting cell adhesion and creating optimum surroundings for cell viability. It is apparent that only some particular embodiments of the present invention have been described, to which those skilled in the art will be able to make all the modifications which are required for the adaptation thereof to particular applications, without for this departing from the protection scope of the present invention.

Claims

1. 1. A photocurable polymeric precursor, composed

(ID

Wherein the n/m ratio ranges between 10 and 1, preferably between 3 and 3.5;

Ri is a C1-C6 alkylene, preferably C2 alkylene;

R2 is H or a C1-C4 alkylene, preferably CI alkylene;

R3 is H or a C1-C4 alkylene, preferably CI alkylene;

R₄ is a C1-C6 alkylene, preferably C3 alkylene;

X is 0 or NH;

Y is NH or S;

which is obtained by random co-polymerization of an ^" acrylic monomer of formula (III):

CH₂=C (R₂) -CO-OR-Rx-OH (III) together with an acrylic co-monomer or acrylamide of formula ( IV) :

CH₂=C (R₃) -CO-X-R4-YH (IV) wherein Ri, R₂, R₃, R4, X and Y are as defined above, in the presence of a chain transfer agent, preferably 2-mercaptoethanol, on said acrylic oligomer being grafted

(ii) side chains composed of linear polyamidoamines , an end of which side chains is bounded to said acrylic oligomer, while the free end terminates with an acrylamide group.

2. The polymeric precursor according to claim 1, being obtainable by a direct copolymerization of the Michael type between

a) a bis (acrylamide ) of formula (V):

CH₂=CH-CO-NH- ₅-NH-CO-CH=CH₂ (V)

or of formula (VI) :

CH₂=CH-CO-N-R₅-N-CO-CH=CH₂ (VI)

wherein,

in the case of formula (V) , the R5 functional group is selected from the following classes of functional groups :

i. aliphatic alkyl chains of formula - (CH₂)_n-, where the value of n ranges between 1 and 10, preferably 1/

i. alkoxide chains of formula - (CH2-CH2-O) n-, -(CH₂- CH(CH₃)-0)_n- and - (CH-CH₂-0) _n- (CH-CH (CH₃) -0) _m-, where the values of n and m range between 1 and 20, preferably 5; iii. carboxylic acids of formula -CH(-COOH)- or secondary alcohols of formula -CH(-OH)-,

and wherein,

in the case of formula (VI), the R₅ functional group is an aliphatic heterocycle, preferably piperazine or 2-methylpiperazine,

b) a primary amine or a secondary diamine, preferably the monomer Agmatine, and

c) the acrylic oligomer of formula (II).

3. The polymeric precursor according to claim 1 or 2, wherein the average molecular weight of the acrylic oligomer of formula^' (II) is in the range of 1-100 kDa, preferably between 1 and 30 kDa .

4. The polymeric precursor according to any of the claims 1 to 3, wherein the molar ratio between the monomers (III) and (IV) ranges between 10:1 and 1:1, preferably 10:3.

5. The polymeric precursor according to any of the claims 1 to 4, having the structural formula (I) :

wherein the n/m ratio ranges between 10 and 1, preferably between 3 and 3.5; p ranges between 1 and 100, and more preferably between 1 and 20; Ri, R₂, R₃, and R are as defined in claim 1; X is 0 e- or NH; Y is NH or S; R₅ is as defined in claim 2; R6 is a primary amine or a secondary amine, preferably the following monomeric unit:

6. The polymeric precursor according to any of the claims 1 to 5, having an average molecular weight ranging between 2 kDa and 400 kDa, preferably between 2 kDa and 100 kDa.

7. The polymeric precursor according to any of the claims 1 to 6, wherein polyamidoamine is selected from the following structures: ^^^-^{' '}^ L i A A

8. A synthesis process of the polymeric precursor according to any of the claims 1 to 7, comprising the following steps:

a) random co-polymerization of the acrylic monomer CH₂=C (R₂) -CO-OR-Ri-OH of formula (III) and the acrylic co-monomer or acrylamide CH₂=CH (R₃) -CO-X-R4-YH of formula (IV) to obtain a random co-polymer of formula (II) in the presence of a thiol or a mercaptan as the chain transfer agent, more preferably 2- mercaptoethanol ;

b) polymerization of the random co-polymer of formula (II) with a bisacrylamide of formula (V):

CH₂=CH-CO-NH-R₅-NH-CO-CH=CH₂ (V) , for example, BAC, or of formula (VI) :

CH₂=CH-CO-N-R₅-N-CO-CH=CH₂ (VI)

wherein R₅ is as defined in claim 2,

with a secondary diamine or a primary amine, for example, Agmatine, to give the polymeric precursor of formula ( I ) .

9. The process according to claim 8, wherein the step a) is carried out in the presence of a radical initiator, such as, for example, 2, 2 ' -azobis (2- methylpropionitrile) , and at a temperature ranging between 60° C and 80° C, preferably at about 70° C, for a reaction time ranging from 2 hours to 24 hours or longer, until reaching an average molecular weight ranging between 1 and 30 KDa ; and wherein the monomers CH₂=C (R₂) -CO-O-Ri-OH of formula (III) and CH₂=CH (R₃) -CO-X-R4-YH of formula (IV) are reacted in a molar ration ranging between 10:1 and 1 :1, preferably 10:3.

10. The process according to claim 8 or 9, wherein the step b) is carried out in a basic environment, with a pH above 8.5, preferably of about 9, at a reaction temperature preferably ranging between 20° C and 50° C, more preferably between 25° C and 40° C.

11. The process according to any of the claims 8 to 10, wherein in the step b) the reaction time ranges between one day and one or more weeks, so as to obtain an average molecular weight ranging between 2kDa and 200kDa, and the polymerization reaction is carried out in a dark environment.

12. A hydrogel obtainable by photocuring the polymeric precursor according to any of the claims 1 to 7 in the presence of a photocuring initiator in an aqueous solution or a cell suspension in a culture medium.

13. A process for preparing a hydrogel according to claim 12, wherein the photo-initiator is selected from 1- [4- { 2-hydroxyethoxy) phenyl] -2-hydroxy-2- methyl-l-propan-l-one, bisacylphosphine oxides, 1,5- diphenyl-1 , 4-dien-3-one, phenanthrenequinone, 4'- phenoxyacetophenone and thioxanthen-9-one .

14. The process according to claim 13, wherein the polymer precursor with a comb-like structure is initially dissolved into an aqueous solution or a cell culture medium or in a cell suspension in a cell culture medium at a weight/weight concentration ranging between 5 and 25%, preferably 10 and 15%, and wherein the polymer solution is exposed to UV rays at a wavelength of 365 nm, for exposure times ranging between 1 and 5 minutes, preferably about 2 minutes, or the polymer solution is exposed to a blue light having a wavelength ranging between 400 and 500 nm, preferably about 460 nm.

15. The polymeric precursor according to any of the claims 1 to 7, for use in medicine.

16. The polymeric precursor according to claim 15, for use in forming a hydrogel for the controlled or prolonged release of drugs, enzymes, hormones, peptides, inorganic substances, proteins, or genetic material, for example, DNA or RNA, and as a connective tissue filler in the regenerative and/or reconstructive medicine.

17. Cosmetic use of a polymer precursor according to any of the claims 1 to 7, for the formation of a •hydrogel for filling connective tissue.

18. The polymeric precursor according to claim 15, for use in stem cells release for the treatment of neurodegenerative diseases.

19. The polymeric precursor according to claim 15, for use as a carrier of inorganic fillers, such as, hydroxyapatite , brucite, tricalcium phosphate, beta- tricalcium phosphate, alumina, calcium oxide, titanium oxide, zinc oxide, for the regeneration of bone tissue, cartilaginous tissue, in the maxillofacial surgery field, and■ in the dentistry field.