Composition comprising polyelectrolyte complexes, methods and uses thereof

Abstract

The present disclosure relates to a composition of at least one predominantly positively charged polyelectrolyte polymer and at least one predominantly negatively charged polyelectrolyte polymer, a preferred composition comprises poly-L-lysine and a gellan gum, preferably a methacrylate gellan gum. The present subject-matter further relates to methods for generating composition of the present disclosure and to uses of a mixture according to the disclosure for biomedical applications such as cellular and acellular systems for tissue engineering and regenerative medicine applications or as drug delivery systems, for the treatment of several diseases namely diabetes mellitus.

Claims

1. A spherical capsule composition comprising gellan gum and poly-L-lysine for use in veterinary or in human medicine, wherein the composition comprises: 0.05-0.1% (w/v) of poly-L-lysine, and 0.5-3% (w/v) of a methacrylated gellan gum.

2. The composition according to claim 1 wherein the methacrylated gellan gum comprises a methacrylation degree up to 10%.

3. The composition according to claim 2, wherein the methacrylation degree is between 1-2%.

4. The composition according to claim 1 wherein the methacrylated gellan gum molecular weight is between 510.sup.4 Da to 210.sup.6 Da.

5. The composition according to claim 1 wherein the poly-L-lysine molecular weight is between 30-500 kDa.

6. The composition according to claim 1 further comprising at least a second hydrogel or more hydrogel.

7. The composition according to claim 6 wherein the second, or more hydrogels, is selected from a list consisting of carbopol, hyaluronic acid, carboxymethylchitosan, dextran, alginate, collagen, and mixtures thereof.

8. The composition according to claim 1 further comprising a coupling agent.

9. The composition according to claim 8 wherein said coupling agent is selected from the group consisting of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, glutaraldehyde, epichlorohydrin, dianhydrides, diamines, and mixtures thereof.

10. A composition according to claim 1 for use in the treatment or prevention of diseases that involve the repair or regeneration of tissues.

11. A composition according to claim 1 for use as a drug delivery system.

12. The composition according to claim 1 for use in cell therapy or advanced-therapy medicinal product.

13. The composition according to claim 1 for use in the treatment or prevention of diabetes, obesity, ageing related-diseases, tumours or pancreatic diseases.

14. The composition according to claim 1 for use in the treatment or prevention of pancreatic cancer.

15. The composition according to claim 1 for the treatment or prevention of female infertility.

16. The composition according to claim 1 further comprising an anti-inflammatory agent, an antiseptic agent, an antipyretic agent, an anaesthetic agent, a therapeutic agent, a biological cell, a biological tissue and combinations thereof.

17. The composition according to claim 16 comprising an animal or human cell, or stem cell, or combinations thereof.

18. The composition according to claim 17 comprising an animal or human pancreatic -cell or androgen-sensitive human prostate adenocarcinoma cell.

19. The composition according to claim 16 comprising human tissue.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The following figures provide preferred embodiments for illustrating the description and should not be seen as limiting the scope of invention.

(2) FIG. 1: Schematic representation of a FTIR spectra of (A) GG/PLL complex, (B) GG and (C) PLL.

(3) FIG. 2: Size comparison of capsules before (black) and after (grey) immersion in distilled water, PBS and DMEM. Different formulations were used to produce the capsules, namely: A0.5% GG-LA 0.1% PLL; B0.5% GG-LA 0.05% PLL; C1% GG-LA 0.1% PLL; D1% GG-LA 0.05% PLL; E1% GG-MA 0.1% PLL; and FSize variation after immersion in distilled water, PBS and DMEM as compared to capsules measured before incubation.

(4) FIG. 3: Pictures of different formulations of GG/PLL capsules, before and after incubation with distilled water, PBS and DMEM. Scale bar=500 m.

(5) FIG. 4: SEM images of GG/PLL capsules at different magnifications. A0.5% GG-LA 0.1% PLL after one week in distilled water; B1% GG-LA 0.1% PLL after one week in distilled water; C1% GG-LA 0.1% PLL after one week in PBS; D1% GG-MA 0.1% PLL after one week in distilled water; E1% GG-MA 0.1% PLL after one week in DMEM.

(6) FIG. 5: Live/dead assay on encapsulated androgen-sensitive human prostate adenocarcinoma (LNCaP) cells after 24 hours and 3 days (from left to right).

(7) FIG. 6: Schematic representation of GG-PLL spheres, and method of obtaining thereof.

(8) FIG. 7: Characterization of an embodiment of GG microcapsules of the present disclosure, specifically 1% GG-MA 0.1% PLL. AMicrographs of 1% GG-MA 0.1% PLL capsules immersed in water, PBS or DMEM at 37 C. throughout 14 days; BSEM images of different magnification of 1% GG-MA 0.1% PLL capsules after 14 days of incubation in PBS (left) or DMEM (right); CSize of 1% GG-MA 0.1% PLL capsules along the 14 days of incubation. In water, capsules were disrupted after 14 days hampering their measuring; DSize variation of 1% GG-MA 0.1% PLL capsules as compared to initial capsules (before incubation).

(9) FIG. 8:Schematic representation of a controlled release of BSA-FITC or Methylene Blue from two different formulations, 1% GG-MA 0.1% PLL capsules and 1% GG-MA 0.05% PLL capsules.

(10) FIG. 9Pictures confirming that live cells are found inside the PEC capsules of the present disclosure. Live/dead assay and cytoskeleton analysis by DAPI/Phalloidin staining on human adipose derived stem cells.

DETAILED DESCRIPTION

(11) The present disclosure provides polyelectrolyte complexes in particular compositions comprising poly-L-lysine and gellan gum, their processing methods and use in the field of tissue engineering and regenerative medicine or drug delivery systems.

(12) Gellan gum is an anionic heteropolysaccharide that form hydrogels in the presence of mono-, di-, and tri-valent ions and exists in two different forms, the high-acyl gellan gum and the low-acyl gellan gum. In high-acyl gellan gum, the acyl residues are located on the periphery of the helix, obstructing the polymer chain association, resulting in soft, elastic and non-brittle gels. In contrast, low-acyl gellan gum produces firm, non-elastic, brittle gels since ions can easily link polymer chains and form a branched network.

(13) Poly-L-lysine is a cationic polymer, synthetically produced, commonly used as a charge enhancer and surface coating for adhesion purposes. It is available on formulations with different molecular weight that can vary from 30-70 kDa (lower molecular weight) to more than 300 kDa (high molecular weight).

(14) Due to the anionic nature of gellan gum, it can be conjugated with other positively charged polymers, in particular poly-L-lysine, forming stable PEC systems.

(15) PEC based on gellan gum and poly-L-lysine are formed by combination of the two compounds at controlled pH and temperature. By varying the components ratio, it was possible to obtain materials with different physicochemical properties. The materials are stable under physiological conditions due to the formed electrostatic bounds. The formation of PEC was verified by Fourier-transform Infra-red (FTIR) analysis performed before and after PEC formation. The biological properties of the obtained material were assessed in vitro, and using different cell types and cell agglomerates (see embodiments of FIGS. 1 and 6).

(16) The final physicochemical and biological properties, as well as the shape, of the PEC systems can be tailored by applying different formulations and processing conditions.

(17) An aspect of the disclosure is to improve both the mechanical performance of gellan gum hydrogel capsules and their permeability to nutrients and cell metabolites. Formulations can use different gellan gum forms and types of poly-L-lysine, i.e. with different degrees of acylation and molecular weight that self-assemble into PEC as a result of their opposite charge. This process allows the production of capsules with tuned physical properties (e.g., strength, softness, flexibility, degradability and permeability to solutes, bioactive molecules and cells) according to the desired use. Along with having adaptable properties, these systems can also provide the advantage of being straightforward, with no need of other chelating agents for capsule formation, and under physiological-like conditions (37 C. and pH 7.4).

(18) In an embodiment, the PEC systems of the present subject-matter used alone or together with cells and/or bioactive molecules, can provide a plethora of potential applications in tissue replacement and regenerative approaches, mostly for cell encapsulation technologies, namely pancreatic cells.

(19) The description of this disclosure is complemented through the following examples that are intended to provide a better understanding of the same, although these examples should not be addressed with a restrictive nature.

(20) Synthesis of PEC system as capsulesIn an embodiment, the PEC capsules were prepared by reacting gellan gum with poly-L-lysine. Two commercially available gellan gum were used, namely: 1) low-acyl gellan gum (Sigma, St. Louis, Mo., USA) and 2) methacrylated gellan gum (Mimsys G, Irisbiosciences, Portugal). For that, gellan gum solutions, with a final concentration varying from 0.5 to 3% (w/v), were prepared by dissolving the material in distilled water under constant stirring. For low-acyl gellan gum, the solutions were heated until 90 C. to obtain a homogenous dispersion. Then, solutions were cooled down 10 C. above the respective setting temperature.

(21) In an embodiment, PEC capsules were produced as follows: the well-dispersed gellan gum solution was extruded drop-wise from a 30G needle into a poly-L-lysine bath (Mw from 30,000 to 300,000, Sigma, St. Louis, Mo., USA) using a peristaltic pump to control the flow rate. The formed capsules were maintained on poly-L-lysine solution to allow the formation of a complete PEC membrane, and then transferred to PBS (Phosphate-buffered saline).

(22) Synthesis of PEC system as particlesIn an embodiment, the PEC capsules were prepared by reacting gellan gum with poly-L-lysine. Two commercially available gellan gum were used, namely: 1) low-acyl gellan gum (Sigma, St. Louis, Mo., USA); and 2) methacrylated gellan gum (Mimsys G, Irisbiosciences, Portugal). For that, gellan gum solutions, with a final concentration varying from 0.5 to 3% (w/v), were prepared by dissolving the material in distilled water under constant stirring. For low-acyl gellan gum, the solutions were heated until 90 C. to obtain a homogenous dispersion. Then, solutions were cooled down 10 C. above the respective setting temperature.

(23) The well-dispersed gellan gum solution can be then extruded drop-wise from a 30G needle into a poly-L-lysine bath (Mw from 30,000 to 300,000, Sigma, St. Louis, Mo., USA) using a peristaltic pump to control the flow rate. The formed capsules were maintained on poly-L-lysine solution to allow the formation of a complete PEC membrane.

(24) In an embodiment, to obtain particles, as-prepared capsules can be transferred into a 3% CaCl.sub.2 bath to allow the ionic cross-linking of the inner hydrogel. At last, particles were immersed in PBS until further use.

(25) PEC system with encapsulated cellsIn an embodiment, the PEC capsules with encapsulated cells were prepared by reacting gellan gum with poly-L-lysine. Two commercially available gellan gum were used, namely: 1) low-acyl gellan gum (Sigma, St. Louis, Mo., USA); and 2) methacrylated gellan gum (Mimsys G, Irisbiosciences, Portugal). For that, gellan gum solutions, with a final concentration varying from 0.5 to 3% (w/v), were prepared by dissolving the material in distilled water under constant stirring. For low-acyl gellan gum, the solutions were heated until 90 C. to obtain a homogenous dispersion. Then, solutions were cooled down 10 C. above the respective setting temperature.

(26) In an embodiment, cells were carefully mixed with the different gellan gum suspensions and the mix was extruded drop-wise from a 30G needle into a poly-L-lysine bath (Mw from 30,000 to 300,000, Sigma, St. Louis, Mo., USA) using a peristaltic pump to form capsules. The formed capsules were maintained on poly-L-lysine solution for 10 minutes, to allow the formation of a complete membrane and the resulting PEC capsules with loaded cells.

(27) In an embodiment, PEC capsules can be subsequently cultured together with the previously described growth medium and kept at 37 C. with 5% CO.sub.2 in a standard tissue culture incubator.

(28) In an embodiment, the FTIR spectra of GG/PLL complex of the present disclosure, are present in FIG. 1 herein (a) is the FTIR spectra of GG/PLL complex of the present disclosure, (b) is the FTIR spectra of GG and (c) is the FTIR spectra of PLL.

(29) An embodiment for measuring the size and morphology of CapsulesCapsules of the present disclosure were placed either in water, PBS (Phosphate-buffered saline) or DMEM (Dulbecco's Modified Eagle Medium) for 1 week. Particles size was measured before and after the environmental change.

(30) As it is possible to observe in FIG. 3, when in contact with PBS and DMEM particles size decreases for all conditions. Regarding the presence of water, particles size increases for all conditions except when GG-MA (methacrylated gellan gum) was used. A morphological difference is also noticed when capsules were observed using a magnifying glass (FIG. 3) and SEM (FIG. 4).

(31) The observed shrinkage can be a consequence of ionic crosslinking of inner GG-based polymers, that changes the conformation of gellan. Such modification may cause changes on PEC membrane, including the crease-like structures observed.

(32) An embodiment of release studiesThe albumin-fluorescein isothiocyanate conjugate (albumin-FITC) was used as a model drug to study the release profile from GG/PLL capsules. Briefly, 1% low acyl gellan gum (GG-LA) solution was heated until 90 C. and then cooled to 37 C. At this point, albumin-FITC (100 g/mL) was added to the hydrogel and the solution was stirred until complete dissolution, under dark conditions. The mixture was then extruded to a 0.1% (w/v) PLL bath using a 30G needle. The resulted capsules were distributed by three different wells, where each well had three capsules immersed in 1.5 mL of PBS. The release of albumin-FITC was tested at 0.5, 1, 2, 3, 4, 5 and 6 hours. At each time point, the supernatant was removed and equal volume of fresh PBS was added. The fluorescence intensity of 100 L supernatant of the removed PBS was read by a microplate reader (excitation wavelength at 485/20 nm and the emission wavelength at 528/20) to quantify the albumin-FITC released. The total amount of albumin-FITC incorporated inside the capsules was estimated through their volume.

(33) As depicted in FIG. 5, during the experiment albumin-FITC was slowly released from de capsules. At last, almost 50% of the albumin present in each well was released.

(34) Live Dead AssayIn an embodiment to evaluate the biocompatibility of GG/PLL capsules, androgen-sensitive human prostate adenocarcinoma cells (LNCaP) cells were mixed with 1% GG-MA, and then extruded to a 0.1% PLL solution. After corn plexation, particles were washed with PBS and then cultured for three days in RPMI 1640 media, supplemented with 10% FBS and 1% antibiotic/antimycotic.

(35) As depicted in FIG. 5, the material did not significantly affect cell viability through the three days of experiment. It is also possible to observe a uniform cell distribution within the capsule.

(36) The present disclosure also describes a biomaterial to encapsulate pancreatic islet cells for type I diabetes treatment. The chemical structure of gellan gum (GG), based on a linear anionic heteropolysaccharide, and was exploited to encapsulate cells through its bio-adhesive and thermo-reversible gel properties. Methacrylate, and low acyl forms of GG were studied to obtain gels with different mechanical properties and permeability to biomacromolecules.

(37) At the end, the microcapsule should be able to fully protect the encapsulated cells from the host immune system while allowing the free diffusion of small molecules such as nutrients and oxygen.

(38) In an embodiment, capsules were formed by gravitational dripping, as illustrated in FIG. 6. Drops with different GG concentrations (0.5, 1 and 1.5% w/v of low acyl GG; 1 and 1.5% GG methacrylate (GG-MA)) were extruded from a 30G syringe into a Poly-L-lysine bath (PLL) with two different concentrations: 0.1 and 0.05% (w/v). This results in the formation of spheres due to the interaction between free carboxylic groups of the GG and the PLL amines, forming polyelectrolyte complexes (PEC). Among the tested conditions, 1% w/v GG-MA with 0.1% PLL was the most reproducible and easy to manipulate. Therefore, the following experiments were performed using this condition.

(39) In an embodiment morphology of GG microcapsules of the present disclosure, was assessed using both scanning electron microscopy (SEM) and micrographs (FIG. 8A).

(40) In an embodiment, the diameter of the spheresGG microcapsules of the present disclosure was, on average, 2.30.145 mm after production (FIG. 7C). Then, particles were incubated in water, PBS and DMEM for 14 days at 37 C. to assess the effect of each solution on capsule's size. After the incubation time, it was possible to observe a decrease on spheres diameter of 11% and 14% on capsules incubated respectively with PBS and DMEM (FIG. 7D). This can be due to the diffusion of ions, present in PBS and DMEM, into the capsules, leading to GG gelation. Furthermore, it was possible to observe the formation of a creases on the surface of these capsules that may result from the tightening of the GG chains as result of the ionic cross-linking. SEM images confirm the presence of the crease-like structures only on the surface of capsules incubated with PBS and DMEM.

(41) In an embodiment, the drug release ability of the PEC capsules was assessed using two model molecules. BSA-FITC, with a molecular weight of approximately 66 kDa, was used as a model of large molecules while Methylene Blue, with a molecular weight of 319.85 Da, was used as a small molecule model.

(42) Both compounds were mixed with the GG solutions before the dripping process. After processing, capsules were incubated in PBS at 37 C. Solutions with defined concentrations of BSA-FITC and Methylene Blue were also incubated under the same conditions of the samples, to be used as controls. Furthermore, nine as-prepared capsules were randomly separated into three different eppendorfs, and stored at 4 C. with PBS, to be analysed as t=0 h samples. At each time point, a sample of the supernatant was retrieved and the same volume of fresh PBS was added.

(43) The results, presented in FIG. 8, have shown a controlled release of BSA-FITC (FIG. 8A). Moreover, it was possible to tune capsules' permeability by changing the concentration of PLL. For 0.1% PLL (w/v), capsules have released 37% of the incorporated BSA-FITC while capsules produced using 0.05% PLL (w/v) released 49% of their content. On the other hand, Methylene Blue was rapidly release from the capsules, with all the material being released within 1 h (FIG. 9B). These results show that PEC capsules have a selective permeability that can be useful for several TE applications.

(44) In an embodiment at last, biocompatibility was also evaluated using human adipose stem cells (hASC). For that, cells were properly mixed in GG-MA solution solutions, at a concentration of 110.sup.6 cell/mL, and microparticles were produced as stated before. Then, cell viability was studied using a Live/Dead staining, followed by fluorescent microscope observation, after defined timepoints.

(45) As depicted in FIG. 9, live cells were found inside the PEC capsules after the experimental timeframe.

(46) The results herein presented show the effect of GG-MA and PLL particles disclosed in the present subject-matter for cell encapsulation strategies. These capsules are easy to produce, using one-step only instead of the commonly used alginate-PLL-alginate system. The spheres are stable on culture media (DMEM) and PBS for at least 14 days and are compatible with hASC, since live cells were found after 7 days of culture.

(47) All references recited in this document are incorporated herein in their entirety by reference, as if each and every reference had been incorporated by reference individually.

(48) Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. The scope of the present invention is not intended to be limited to the above description, but rather is as set forth in the appended claims.

(49) Where singular forms of elements or features are used in the specification of the claims, the plural form is also included, and vice versa, if not specifically excluded. For example, the term a cell or the cell also includes the plural forms cells or the cells, and vice versa. In the claims articles such as a, an, and the may mean one or more than one unless indicated to the contrary or otherwise evident from the context. Claims or descriptions that include or between one or more members of a group are considered satisfied if one, more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process unless indicated to the contrary or otherwise evident from the context. The invention includes embodiments in which exactly one member of the group is present in, employed in, or otherwise relevant to a given product or process. The invention also includes embodiments in which more than one, or all of the group members are present in, employed in, or otherwise relevant to a given product or process.

(50) Furthermore, it is to be understood that the invention encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc., from one or more of the claims or from relevant portions of the description is introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. Furthermore, where the claims recite a composition, it is to be understood that methods of using the composition for any of the purposes disclosed herein are included, and methods of making the composition according to any of the methods of making disclosed herein or other methods known in the art are included, unless otherwise indicated or unless it would be evident to one of ordinary skill in the art that a contradiction or inconsistency would arise.

(51) Where ranges are given, endpoints are included. Furthermore, it is to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value within the stated ranges in different embodiments of the invention, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. It is also to be understood that unless otherwise indicated or otherwise evident from the context and/or the understanding of one of ordinary skill in the art, values expressed as ranges can assume any subrange within the given range, wherein the endpoints of the subrange are expressed to the same degree of accuracy as the tenth of the unit of the lower limit of the range.

(52) The above described embodiments are combinable.

(53) The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities to modifications thereof.

(54) The following claims further set out particular embodiments of the disclosure.