METHOD FOR THE PRODUCTION OF BIOCOMPATIBLE NANOMATERIALS WITH SELECTIVE RECOGNITION CAPABILITIES AND USES THEREOF

Abstract

A molecularly imprinted polymer in the form of nanoparticles, a method of preparation and uses thereof. Polymeric nanoparticles have recognition sites of at least one target molecule and are obtained by crosslinking of at least one polymer that is functionalized at least with polymerizable double bonds, in a liquid and in the presence of at least one target molecule as template molecule. Polymeric nanoparticles can be used for many applications, such as selective recognition of analytes, in vivo and in vitro targeting, labelling of biological molecules or in the preparation of molecular sensors.

Claims

1. Polymeric nanoparticles having recognition sites of a target molecule, said polymeric nanoparticles being obtained by crosslinking of a functionalized polymer at least with polymerizable double bonds, in a liquid and in the presence of a target molecule as a template molecule.

2. The polymeric nanoparticles according to claim 1, wherein the functionalized polymer comprises a biocompatible polymer optionally in a mixture with a synthetic polymer.

3. The polymeric nanoparticles according to claim 2, wherein the biocompatible polymer is selected from: polyamides, polysaccharides and combinations thereof.

4. The polymeric nanoparticles according to claim 1, wherein the functionalized polymer is a natural polyamide selected from: collagen, gelatin, silk fibroin, sericin, fibrinogen, fibrin, elastin, chitin, keratin and combinations thereof.

5. The polymeric nanoparticles according to claim 4, wherein said nanoparticles are obtained by crosslinking building elements formed by only natural functionalized polymers.

6. The polymeric nanoparticles according to claim 5, wherein said nanoparticles are obtained by crosslinking a single functionalized polymer which is silk fibroin.

7. The polymeric nanoparticles according to claim 2, wherein said synthetic polymer is selected from: polyethylene glycol, polyvinyl alcohol, polyhydroxyethyl methacrylate, poly-ϵ-caprolactone, polylactic acid, polyglycolic acid and copolymers thereof.

8. The polymeric nanoparticles according to claim 1, wherein said functionalized polymer is functionalized at least with polymerizable double bonds by a methacrylation reaction.

9. The polymeric nanoparticles according to claim 1, wherein said functionalized polymer with at least polymerizable double bonds comprises one or more functional groups selected from: fluorophore groups, reporter tags, chelating groups, hydrophilic groups, hydrophobic groups, electrically charged groups.

10. The polymeric nanoparticles according to claim 1, wherein said template molecule is at least one chosen from: peptides, proteins, enzymes, supramolecular complexes, cell portions, cells, bacteria, viruses and combinations thereof.

11. The polymeric nanoparticles according to claim 1, wherein said liquid is selected from: water, polar solvent, biocompatible polar solvent, non-polar solvent, biocompatible non-polar solvent.

12. A decorated cell comprising polymeric nanoparticles according to claim 1.

13. A method for preparing the polymeric nanoparticles according to claim 1, comprising: a. crosslinking the functionalized polymer at least with the polymerizable double bonds, in a liquid and in the presence of the target molecule as the template molecule to obtain polymer nanoparticles of a crosslinked polymer containing said template molecule; and b. removing said template molecule to obtain said polymeric nanoparticles having the recognition sites of said target molecule.

14. The method according to claim 13, wherein the crosslinking is carried out by photo-crosslinking or chemical crosslinking.

15. The method according to claim 13, wherein the size of the polymeric nanoparticles is adjusted by modulating the pH of the liquid.

16. The polymeric nanoparticles according to claim 1, which are suitable for at least one of: selective recognition of analytes in assays; in vivo or in vitro targeting of a biological molecule; labelling of biological molecules; administration of drugs and pharmacologically active ingredients to tissues or cells; growth and repair of tissues; as a recognition element in a sensor; or separation and enrichment of mixtures comprising said target molecule.

17. A molecularly imprinted polymer in the form of a nanoparticle having recognition sites of at least a portion of a target molecule, wherein said nanoparticle comprises a three-dimensional polymer structure formed by a set of crosslinked building elements, and wherein said set comprises: a first fraction of building elements consisting of a peptide and/or polypeptide functionalized with a first crosslinkable group; optionally, a second fraction of building elements consisting of a product having a structure other than a peptide or polypeptide, said product being functionalized with a second crosslinkable group; wherein said peptides and/or polypeptides are present in an amount higher than 10 mol % in relation to the total moles of said building elements of said first and second fraction.

18. The polymeric nanoparticles according to claim 1, wherein the functionalized polymer comprises a biocompatible polymer of natural origin in a mixture with a synthetic polymer.

19. The polymeric nanoparticles according to claim 4, wherein said nanoparticles are obtained by crosslinking building elements formed by only natural functionalized polymers selected from natural polyamides.

20. The polymeric nanoparticles according to claim 1, wherein said template molecule is bovine serum albumin.

Description

[0111] In the following examples, reference will be made to the accompanying figures, which illustrate:

[0112] FIG. 1, Size distribution of nanoparticles of selective biocompatible nanomaterials according to the invention determined by dynamic light scattering measurements;

[0113] FIG. 2, Fluorescent signal of selective biocompatible nanomaterials of free target molecules and bound to the imprinted cavity;

[0114] FIG. 3, Proof of selectivity of nanomaterials according to the invention;

[0115] FIG. 4, Proof of non-toxicity of nanomaterials according to the invention;

[0116] FIG. 5, Fluorescence images of 3T3 cells decorated with nanomaterials according to the invention: (A) cytoskeleton; (B) rhodamine-tagged nanomaterials.

Example 1. SELECTIVE FIBROIN-BASED NANOMATERIALS

[0117] By way of example, molecularly imprinted silk fibroin nanoparticles are produced, i.e. a molecular imprinted polymer obtained using silk fibroin as the sole building block. Silk fibroin offers interesting characteristics, such as its natural origin, which gives it biocompatibility, and makes it suitable for numerous uses, including but not limited to tissue engineering, in vivo and in vitro cell targeting, drug delivery (Altman G G. et al. Biomaterials 2003, 24:401, Nazarov R. et al. Biomacromolecules 2004, 5: 718, Mottaghitalab F. et al. J. Control. Release 2015, 206:161). Silk fibroin has special mechanical properties (Lawrence B D. et al. J. Mater. Sci. 2008, 4:6967; Sofia S. et al. J. Biomed. Mater. Res. 2001, 54:139; Meinel L. et al. Bone 2006, 39: 922; Jin H.-J. et al. Biomacromol. 2002, 3:1233), biological properties (Santin M. et al. J. Biomed. Mater. Res. 1999, 46:382; Pritchard E. M. et al. J.Control. Release 2010, 144:159) and optical properties (Perry H. et al. Adv. Mater. 2008, 20:3070; Lawrence B. D. et al. Biomacromol. 2008, 9:1214) indicated for use in the biomedical, optical, opto-electronic field (Perotto G. et al. Appl. Phys. Lett. 2017, 111:103702; Bay H. H. et al. Nano Lett. 2019, 19:2620; US2015368417A1).

[0118] To obtain silk fibre, Bombyx mori silk cocoons are cut into small pieces and placed in a high-temperature thermostatic bath in the presence of 0.01 M sodium carbonate (Na.sub.2CO.sub.3) for 1 hour. This is followed by a second bath in sodium carbonate at a concentration of 0.003 M for 1 hour. The resulting silk fibre is thoroughly rinsed three times using ultra-pure water and then dried for 2 days.

[0119] The resulting fibroin was then functionalized by methacrylation. For this purpose, 20 g of scoured silk fibre is dispersed in 100 mL of a 9.3 M aqueous solution of lithium bromide (LiBr) at 60° C. for 4 hours in an oven. Next, 10 mL of glycidyl methacrylate (GMA) is added to the suspension, which is then shaken at 65° C. for 4 hours to allow the conjugation reaction to take place. To remove the salt and unreacted GMA, the resulting methacrylate fibroin suspension is dialyzed for 4 days against water using a dialysis system with a molecular cut-off corresponding to 3.5 kDa, filtered through a 50 μm glass filter and then stored at 4° C. until use.

[0120] The nanoparticles are obtained by high-dilution polymerization. In order to obtain the nanometric fibroin suspension, the concentration of methacrylate fibroin was then adjusted in the range of 0.01% to 0.5% w/v in aqueous buffer, specifically to the value of 0.03% w/v and the value of 0.3% w/v, in the presence of the template molecule, whether it is a small molecule, a peptide, or a protein. In the example provided here the use of bovine serum albumin (BSA) is shown, added at a final concentration of 0.1 mg/mL. A photoinitiator (e.g. lithium phenyl-2,4,6-trimethyl benzoyl phosphinate) is then added at a final concentration of 0.2% or 0.02% w/v and the suspension is photo-polymerized under UV light for 10 minutes to allow crosslinking. A population of nanometric particles is formed suitable for recognizing the template molecule, or a part thereof, or a chimera thereof. At the end of the crosslinking process, the template molecule is removed from the formed nanoparticles by several washes (subsequent dialysis against 4×3 L aqueous solutions, or ultrafiltration on 100 KDa molecular sized membranes with 3 L of aqueous solution). If the imprinted molecule is a protein, it is removed by adding trypsin enzyme to the material for 1 hour at room temperature and pH 8.0, followed by acidification of the solution, while the removal of the template protein from the nanomaterial is confirmed by SDS-PAGE electrophoresis.

[0121] The size of the imprinted biocompatible nanomaterials, estimated by dynamic light scattering, is in the range of 30-200 nm, the most represented hydrodynamic sizes in the prepared nanoparticle populations being 50 nm and 100 nm (FIG. 1). In FIG. 1, the two curves with maximum intensity at approximately 50 nm refer to the fibroin suspension at a concentration of 0.03% w/v (measurements made on two aliquots of the same sample), while the three curves with maximum intensity at approximately 100 nm refer to the fibroin suspension at a concentration of 0.3% w/v (measurements made on three aliquots of the same sample).

[0122] The binding properties of the imprinted biocompatible nanomaterials are tested by incubating the nanomaterial (0.1 mg/mL) in the presence of the modified template molecule with a fluorescent tag to form a fluorescent peptide (0.2 and 2 nmol) and monitoring the fluorescent signal over time. During binding of the fluo-peptide to the imprinted cavity, a decrease in the fluorescent signal is observed, because the amount of free fluo-peptide in solution decreases, while the amount of fluo-peptide bound to the imprinted cavities increases (FIG. 2).

[0123] The selectivity of the imprinted biocompatible nanomaterials is demonstrated by a 30-minute incubation of the nanomaterials (0.1 mg/mL) in the presence of either fluo-peptide alone (500 pmol), or alternatively by incubating the same amount of nanomaterial in the presence of a mixture consisting of the fluo-peptide (500 pmol) and one of the following competitor molecules: the same peptide used as the template molecule, but not fluorescently tagged (100 nmol); a biopeptide with a sequence unrelated to the peptide used as the template molecule, angiotensin (6 nmol); a protein with a sequence unrelated to the template peptide, namely cytochrome (6 nmol); an extremely abundant serum protein with a sequence unrelated to the template peptide, namely human serum albumin (6 nmol). The results demonstrate the preferential binding of the selective biocompatible nanomaterial to the template molecule. Competition is only observed when the untagged template molecule (the peptide) is present (FIG. 3).

[0124] The non-cytotoxicity of the prepared biocompatible selective nanomaterial is tested according to ISO 10993, using the NIH 3T3 cell line expanded with the respective standard medium and evaluated at a confluence of approximately 70%. The percentage of cell death was evaluated by measuring the amount of lactate dehydrogenase released into the medium from cells fed with medium containing a concentration of 0.25 and 1.5 mg/ml of nanoparticle suspension, then compared with the negative control (untreated cells, as reference for non-cytotoxic material) and positive control (all dead cells, as reference for totally cytotoxic material) (FIG. 4).

Example 2. Selective Fibroin-Based Nanomaterials Functionalized with Fluorescent Tags

[0125] The preparation of functionalized selective biocompatible nanomaterials occurs as in Example 1, but includes the addition of polymerizable fluorescent tags (such as, for example: methacryloxyethyl thiocarbamoyl rhodamine B; 9-anthracenylmethyl acrylate; fluorescein o-acrylate) as additional building elements, used in the concentration range of 0.0002% to 0.02% w/v with respect to the concentration of the fibroin suspension. These biocompatible fluorescent nanomaterials are used to decorate cells (FIG. 5).