ITMI20121729A1 - POLYMERIC NANOPARTICLES BASED ON POLY (AMIDOAMINIC) MATERIALS BIODEGRADABLE CURLED BY MEANS OF FUNCTIONALITY COVALENT SUBJECT TO HYDROLYTIC ATTACK - Google Patents

Description

â € œ POLYMER NANOPARTICLES BASED ON BIODEGRADABLE RETICULATED POLYMARES (AMIDOAMINES) BY MEANS OF COVALENT FUNCTIONALITIES SUBJECT TO HYDROLYTIC ATTACK â €

The present invention relates to nanosystems based on biodegradable poly (amidoamine) matrices cross-linked by means of covalent functionalities susceptible to direct and / or enzyme-mediated hydrolytic attack and the process of preparation of the same.

State of the art

Poly (amidoamines) (PAA) are a class of synthetic polymers obtained by Michael-type polyaddition of primary monoamines or aliphatic secondary diamines with bisacrylamides (Scheme).

R1R1R1R1

H2C CH C N R2N C CH CH H N H CH2CH2C N R2N C CH2CH2N

O O R3 O OR3 <n>

R1R1R1R1

H2C CH C N R2N C CH CH H N R4NH <CH> 2 <CH> 2 <C> N R2N C CH2CH2N R4N O O R3R3O O R3 R3 <n> Scheme: Summary of PAA

PAAs are characterized by the presence of amide (a) and tertiary amine (b) groups regularly arranged along the polymer chain according to the sequences:

3⁄4a3⁄4a3⁄4b3⁄4

3⁄4a3⁄4a3⁄4b3⁄4b3⁄4

PAAs have properties that make them interesting for biomedical use, such as structural flexibility with the possibility of introducing additional functions in order to create â € œcustomizedâ € structures for every possible application, water solubility, biodegradability and biocompatibility, immunogenicity and the absence of toxicity of the degradation products. Furthermore, the acid-base characteristics can be modulated according to the structure of the starting amines and the ionic substituents present on one or both monomers, giving rise to PAA with basic or amphoteric characteristics. PAAs are capable of forming complexes with proteins or peptides of prevailing opposite polarity by ionic interaction. In the case of amphoteric PAAs it is possible to modulate the acid and basic strength in such a way that the polymer passes from a predominantly anionic state to a predominantly cationic one for modest pH variations, with consequent volume variation and release of any substances. A review of the biomedical applications of PAAs is reported in Macromol. Rapid Commun. 2002, 23, 332-355. PAAs have in particular been proposed as vehicles for drugs, in particular chemotherapeutic agents (WO 95/05200).

 € œdrug deliveryâ € technology is of increasing importance for the purpose of improving the effectiveness of pharmacological treatments. Several technologies are now available that can control the pharmacokinetic and release characteristics of drugs and improve their therapeutic index. Among these, one of the most promising is that of nano-structured materials based on biodegradable polymers, dendrimers, carbon nanotubes, micellar systems or hydrogels, liposomal systems, inorganic nanoparticles (noble metal nanoparticles, gold in particular). The variety of chemical structures of pharmacological agents (organic molecules, proteins, nucleic acids, immunoglobulins, metalloorganic complexes, carbohydrates, etc.) always poses new formulation problems and therefore the need for new â € œdrug systems is particularly felt. deliveryâ € that are adaptable to the different classes of drugs and that can at the same time be easy to prepare from easily available, non-toxic and inexpensive materials.

Description of the invention

A process has now been found for the preparation of nanoparticles based on polyamidoamines comprising the following steps:

- Preparation of an aqueous solution of polyamidoamines having functional groups capable of forming covalent bonds with bi- or poly-functional cross-linking agents;

- Conversion of solution a) into a colloidal system;

- Treatment of the colloidal system b) with a di- or polyfunctional cross-linking agent capable of forming covalent bonds with the functional groups present on the polyamidoamines;

- Recovery of the colloidal structures obtained at the end of the reaction. The nanoparticles thus obtained, useful in the biomedical and pharmaceutical fields, in particular for the loading and release of agents with biological activity, constitute a further object of the invention.

Detailed description of the invention

The polyamidoamines that can be used according to the invention can be both linear and branched, containing groups available for covalent crosslinking, such as amino, hydroxyl, thiol and carboxy groups. The structures, properties and methods of preparation of functionalized polyamidoamines are reported in Macromol. Rapid Commun. 2002, 23, 332-355 and in the references cited there.

Specific examples of functionalized polyamidoamines that can be used according to the invention are the polyamidoamines called ISA1 and AGMA1. The structures of these polyamidoamines are shown below:

O O

N N N

N N N

N N O O

1 / 2n1 / 2n H OH OISA1

O COOH O

NH NH N

n

NH

H N

NH2

AGMA1

The preparation of ISA1 is reported for example in J. Drug Targeting 1999, 6, 391-404.

AGMA1 is described in the Italian Patent Application MI2004A000435, 2004 and in WO 2010099962.

The conversion of the functionalized polyamidoamine aqueous solution to give colloidal particles occurs by adding a non-solvent or by variation of the environmental conditions, in particular temperature and pH.

Examples of non-solvents are acetone, ethyl ether, ethanol, methanol, chlorinated solvents.

Cross-linking agents (with two or more functions) contain reactive groups such as epoxides, aldehydes, N-hydroxysuccinimide esters (NHS), imido esters, maleimide and isocyanates. Particularly preferred are 1,4 butanediol diglycidyl ether (BDGE), polyethylene glycol diglycidyl ether (PEGDE), disulfosuccinimidyl suberate, polyethylene glycol disuccinimide ester, dimethyl pimelimidate (DMP) and NHS-PEG-Maleimide.

The PAAs used have a molecular weight (MW) greater than 10kDa and preferably between 15 kDa and 30 kDa. PAA solutions with concentrations higher than 10mg / ml and preferably between 15 and 25mg are used. The amount of crosslinking agent and crosslinking conditions (temperature, pH and time) vary according to the chemical characteristics of the PAA (type and number of functional groups) and of the crosslinking agent used. In general, temperatures below 80 ° C (preferably between 50 ° C and 70 ° C), pH between 4 and 10 (preferably between 5 and 9) and reaction times of 2 hours are preferred, although in some cases may need to be extended up to 24 hours.

The recovery of the colloidal structures obtained at the end of the reaction can be carried out by removing the non-solvent or by restoring the starting aqueous conditions by evaporation of organic solvents or by resorting to processes of dialysis, diafiltration or the like.

The process of the invention has been successfully used with different types of polyamidoamines and crosslinking agents, resulting versatile and effective in terms of yield and insulation of nanosystems.

The nanoparticles thus prepared have an average diameter ranging from 80 to 500 nm (usually between 100 and 250 nm) with a polydispersity index (PI) between 0.11 and 0.25.

The nanoparticles of the invention can be advantageously used for loading and releasing agents with biological activity, in particular chemotherapeutic agents, proteins, peptides, DNA, RNA. The loading of the active agents can take place by electrostatic interaction, coacervation or by rehydration of the recovered nanoparticles.

The use of PAA as nano-structured matrix systems appears versatile and adaptable to the different routes of administration, with the possibility of dispersing the systems in vehicles for topical, inhalation applications or in the production of scaffolds for tissue engineering .

By means of the described technique it is possible to prepare devices which can be manufactured with substantially contained costs, both as regards the production costs and as regards the management costs, with a simple, safe and reliable procedure.

The invention is illustrated in greater detail in the following examples.

Example 1: Preparation of ISA1-based nanoparticles

A solution consisting of 30 mg of ISA1 in 2 ml of deionized water, added with 10 µl of 10N NaOH, was dropped using a syringe fitted with a 22G needle in 40 ml of acetone and 30 µl of 1.4 butanediol was added. diglycidyl ether (BDGE). After 15h under magnetic stirring at 25 ° C, 5 ml of deionized water were added and the acetone was removed by evaporation under a hood. The resulting nanoparticle suspension was analyzed by particle size in suspension with dynamic light scattering (DSL) technique to obtain the dimensional distribution in volume (mean diameter: 389 ± 197 nm, Figure 1A) and percentage number (diameter mean: 152 ± 80 nm, Figure 1B).

Example 2: Preparation of nanoparticles based on AGMA1 A solution consisting of 30 mg of AGMA1 in 2 ml of deionized water, added with 10 µl of 10N NaOH, was dropped using a syringe fitted with a 22G needle in 40 ml of acetone and 30 µl of 1,4 butanediol diglycidyl ether (BDGE) were added. After 15h under magnetic stirring at 25 ° C, 5 ml of deionized water were added and the acetone was removed by evaporation under a hood. The resulting nanoparticle suspension was filtered with a filter in mixed cellulose esters with 1.2 µm dimensional cut and analyzed by particle size in suspension with dynamic light scattering technique to obtain the dimensional distribution by volume (average diameter: 237 ± 114 nm, Figure 2A) and percentage number (mean diameter: 155 ± 51 nm, Figure 2B).

Claims (10)

CLAIMS 1. A process for the preparation of polyamidoamine-based nanoparticles comprising the following steps: - Preparation of an aqueous solution of polyamidoamines having functional groups capable of forming covalent bonds with polyfunctional cross-linking agents; - Conversion of solution a) into a colloidal system; - Treatment of the colloidal system b) with a di- or polyfunctional cross-linking agent capable of forming covalent bonds with the functional groups present on the polyamidoamines; - Recovery of the colloidal structures obtained at the end of the reaction. 2. A process according to claim 1 wherein the polyamidoamines comprise amino, hydroxy, thiol, carboxylic functional groups. A process according to claim 1 or 2 wherein the polyamidoamines are selected from ISA 1 and AGMA 1. 4. A process according to any one of claims 1 to 3 wherein the crosslinking agent is characterized by two or more epoxy, aldehyde and isocyanate groups. 5. A process according to one or more of claims 1 to 4 in which the conversion of solution a) in a colloidal system takes place by adding acetone as a non-solvent. 6. A process according to one or more of claims 1 to 5 in which recovery of the colloidal structures obtained at the end of the reaction with the crosslinking agent takes place by removing the non-solvent or restoring the starting aqueous conditions by evaporation, dialysis and / or diafiltration. 7. Nanoparticles based on colloidal cross-linked polyamidoamines obtained by the process of claims 1-6. 8. Use of the complexes of claims 6 for loading and releasing agents with biological activity. Use according to claim 8 wherein the charging is carried out by electrostatic interaction, coacervation or by rehydration of the recovered nanoparticles. Use according to claim 8 or 9 wherein the biological agents are chemotherapeutic agents, proteins, DNA, RNA. Milan, 12 October 2012