COMPOSITION COMPRISING NANOPARTICLES, METHOD FOR THE PREPARATION OF A COMPOSITION COMPRISING NANOPARTICLES AND USES OF THE COMPOSITION FOR DENTAL TREATMENT

Abstract

A composition comprising nanoparticles, a method for the preparation of a composition comprising nanoparticles and uses of the composition for dental treatment are provided. Each nanoparticle is made of polymeric material and includes an antimicrobial agent encapsulated therein. The polymeric material can include Poly(lactic-co-glycolic acid) (PLGA), or other polymers such as Polylactic (PLA), Polyglycolic (PGA), or Polycaprolactone (PCL). The active molecule is calcium hydroxide in the range of 0.1 to 5 mg/ml. The composition has a pH in the range of 8-13.

Claims

1. A composition comprising nanoparticles, the nanoparticles comprising a polymeric material and an antimicrobial agent comprising calcium hydroxide, wherein: the polymeric material is selected from the group consisting of: poly(L(+)-lactic-co-glycolic acid), PLGA; polylactic acid, PLA; polyglycolic acid, PGA; polycaprolactone, PCL; and/or combinations thereof; the antimicrobial agent being encapsulated in the nanoparticles, the antimicrobial agent comprising a concentration between 0.1 mg/ml to 5 mg/ml; and the composition comprising a pH between 8 and 13.

2. The composition according to claim 1, wherein the nanoparticles comprise a size lower than 300 nm.

3. The composition according to claim 1, wherein the polymeric material is PLGA comprising a concentration between 5 mg/ml to 20 mg/ml.

4. The composition according to claim 1, wherein the nanoparticles have a polydispersion index lower than 0.2 and a negative surface charge.

5. The composition according to claim 1, wherein the polymeric material is biodegradable.

6. The composition according to claim 1, wherein the composition is a gel, an aqueous solution or is in the form of dispersed particles.

7. A method for the preparation of a composition comprising nanoparticles, the method comprising the following steps: preparing an organic phase that comprises adding a first mixture to a second mixture, wherein the first mixture comprises calcium hydroxide and a first solvent and the second mixture comprises a polymeric material and a second solvent, the polymeric material is selected from the group consisting of: poly(L(+)-lactic-co-glycolic acid), PLGA; polylactic acid, PLA; polyglycolic acid, PGA; polycaprolactone, PCL; and/or combinations thereof; preparing an aqueous phase comprising a surfactant product and placing the aqueous phase under a magnetic stirring; adjusting a pH of the aqueous phase to a value above 10; adding the organic phase into the aqueous phase, drop by drop at a constant rate, and leaving a resulting mixture under the magnetic stirring; conducting an evaporation process at low pressure to remove the first solvent and second solvent; and obtaining the nanoparticles.

8. The method according to claim 7, further comprising lyophilizing the obtained nanoparticles.

9. The method according to claim 7, wherein the first solvent comprises dimethyl sulfoxide, DMSO, and the second solvent comprises acetone.

10. The method according to claim 9, wherein the second solvent comprises a volume that is 4 times the volume of the first solvent.

11. The method according to claim 7, wherein the polymeric material comprises PLGA, and wherein a quantity of the calcium hydroxide is double than the quantity of the organic phase relative to a concentration of the nanoparticles and a quantity of PLGA is double relative to the concentration of the nanoparticles.

12. The method according to claim 7, wherein the pH value is adjusted to a value that is between 11 and 12.

13. The method according to claim 7, wherein the drop rate of the organic phase into the aqueous phase is between 2 and 5 seconds.

14. The method according to claim 7, wherein each drop has a volume of 50 microliters.

15. The composition according to claim 1 for use in dental treatment as an antibacterial compound for its placement inside of a root canal, as a sealant of the root canal, for pulp capping, for repairing a dentin or dental-periodontal communication, as material for retrograding obturation in apical surgery and/or as indirect pulp capping and as cavitary base.

16. The composition according to claim 1, wherein the nanoparticles comprise a size that is lower than 150 nm.

17. The composition according to claim 1, wherein the polymeric material is PLGA comprising a concentration between 10 mg/ml to 12 mg/ml.

18. The method according to claim 7, wherein the drop rate of the organic phase into the aqueous phase is 3 seconds.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The foregoing and other characteristics and advantages will be more fully understood from the following detailed description of some exemplary embodiments, merely illustrative and non-limiting, with reference to the attached drawings, wherein:

[0034] FIG. 1 schematically depicts a method for preparing the nanoparticles, PLGA in this case, encapsulating calcium hydroxide as an active molecule or antimicrobial agent, according to an embodiment of the present invention.

[0035] FIG. 2 schematically depicts the process of filtration-centrifugation for quantifying non-encapsulated calcium hydroxide.

DETAILED DESCRIPTION OF THE INVENTION AND OF EXEMPLARY EMBODIMENTS

[0036] The present invention provides a composition, particularly for antibacterial dental treatment, comprising nanoparticles of biodegradable polymeric material, for example PLGA, PLA, PGA, PCL, or combinations thereof. In particular, the nanoparticles are of PLGA.

[0037] The nanoparticles are of nanospherical type with encapsulated calcium hydroxide. The composition comprising the nanoparticles has a pH value in the range of 8-13, preferably 9-13, more preferably 10-13.

[0038] Particularly, the nanoparticles are also enclosed by a surfactant material and comprise a certain quantity of non-encapsulated calcium hydroxide which is located around the surface of each nanoparticle. Subsequent to the method for preparing the nanoparticles, an additional quantity of Ca(OH).sub.2 is added as well as 5% of hydroxpropyl-β-cyclodextrin. Optionally, 15% of mannitol may also be added.

[0039] With reference to FIG. 1, therein an embodiment of the displacement method of the solvent used for elaborating the nanoparticles, PLGA in this case, is illustrated. To that end, in a first phase or organic phase 101, the method comprises separately preparing: [0040] calcium hydroxide in 1 ml of anhydrous DMSO; and [0041] poly(lactic-co-glycolic acid) or PLGA in 4 ml of acetone.

[0042] Subsequently, DMSO containing the calcium hydroxide is added to the 4 ml of acetone.

[0043] Thereafter, the second phase or aqueous phase 102 is prepared. According to this embodiment, this phase contains Poloxamer 188 (BASF Chemicals) as surfactant in a concentration of about 11 mg/ml. This aqueous phase is made of a buffer solution adjusted to pH 12. The aqueous phase is left under magnetic stirring adding a magnetic core at 400 r.p.m. per minute.

[0044] Next, the organic phase 101 is added to the aqueous phase 102, which is under magnetic stirring. The addition is carried out dropwise at a constant rate, adding, for example, a drop in the center of the stirring cone every 3 seconds. The total time this process takes is approximately 5 minutes.

[0045] Then, the mixture is left under magnetic stirring for another 10 minutes. Afterwards, it is evaporated at a reduced pressure 103 (Buchi rotavapor), 9 kPa, for 20 minutes, approximately, to remove the organic solvents used. 10 ml of the formulation of dispersed nanoparticles in water 104 are obtained.

[0046] In an embodiment, to achieve greater stability, the obtained nanoparticles are lyophilized. Lyophilizing consists in the extraction of water from the sample by means of a sublimation process. To that end, substances (cryoprotectants) which protect the nanoparticles against the aggression of the process are added and it consists of three steps: freezing, primary drying (here, the water of the nanoparticle is sublimated by using the low pressures), secondary drying (temperature is increased up to 20 degrees Celsius, leaving the pressure of the previous step constant, thereby also evaporating the structural water of the nanoparticles). Advantageously in the present invention it has been proved that after the lyophilization the nanoparticles maintain their initial physical-chemical characteristics. It has been found that by re-suspending the nanoparticles in a liquid medium after lyophilization, the original pH value is obtained, i.e. between 8 and 13.

[0047] The key parameters in the composition/formulation have been studied by creating a design space. To that end, the optimization has been carried out by means of a central factorial design created by the Statgraphics Centurion programme. Therein, the concentration parameters of the indicated compounds are optimized, as well as the pH of the aqueous phase 102. The influence in the average size, the polydispersity index, the Zeta potential (carried out by means of laser-Doppler electrophoresis with the M3 PALS system) and the calcium hydroxide association efficiency to the nanoparticles are studied. Thus, the tendencies the nanoparticles follow, as well as the corresponding response superficies, are obtained, which is necessary for achieving a definitive formulation guaranteeing that it is the best formulation that can be obtained.

[0048] The optimized nanoparticles present an average size lower than 300 nm, a polydispersity index lower than 0.2, characteristic of the unimodal systems, and a negative surface charge (approximately of −20.0 mV). The polydispersity index is analyzed with a zetasizer model Zetasizer Nano ZS by means of the dynamic light scattering technique. Additionally, the purpose is to encapsulate the maximum quantity possible of calcium hydroxide within the nanoparticles. The encapsulation efficiency (EE) of the nanoparticles is measured indirectly by means of the separation of the encapsulation-free drug by means of filtration-centrifugation (FIG. 2). The fraction of free calcium hydroxide is quantified and, using the following equation, the encapsulated quantity is derived:

[00001]$\mathrm{EE}\left(\%\right)=\frac{\begin{array}{c}\mathrm{Initial}\mathrm{calcium}\mathrm{hydroxide}\mathrm{concentration}-\\ F\stackrel{\_}{\mathrm{ree}\mathrm{calcium}\mathrm{hydrox}}\mathrm{ide}\mathrm{concentration}\end{array}}{\mathrm{Initial}\mathrm{calcium}\mathrm{hydroxide}\mathrm{concentration}}.Math.100$

[0049] For the characterization of the nanoparticles, the drug-polymer interactions have been studied using various analysis techniques such as infrared spectroscopy (FTIR), differential scanning calorimetry (DSC) and X-ray diffraction of the freeze-dried samples. Additionally, the liquid samples have been visualized by means of transmission electronic microscopy (TEM) after a negative staining.

[0050] Likewise, in some embodiments, it has been proven by confocal microscopy that the nanoparticles penetrate more than the free drug.

Example: pH Stability

[0051] To test pH stability of the composition according to the present invention, nanoparticles comprising calcium hydroxide and PLGA were studied. An aqueous buffer at pH 12 was prepared, by preparing 100 mL of 0.05 M Na.sub.2HPO.sub.4, 53.8 mL of 0.1 M NaOH and adding water up to 200 mL. Poloxamer 188 was solved in 10 mL of the previously mentioned buffer obtaining a concentration of 11 mg/mL. The organic phase was prepared by mixing Calcium hydroxide with 1 mL of DMSO and PLGA with 4 ml of acetone. Afterwards, both were mixed obtaining an organic phase. This organic phase was added dropwise on the water phase under magnetic stirring. Afterwards, the organic solvents were evaporated under reduced pressure. The final concentration of calcium hydroxide was 1.7 mg/mL and 11.5 mg/mL of PLGA.

[0052] The aqueous buffer had an initial pH of 12, and pH of the composition according to the invention was measured using a pH meter for a total of 18 days, yielding values over 7.5 during the entire measurement scope. The following table shows the values of the composition from 0 to 432 hours.

TABLE-US-00001 Time (hours) pH 0 9.65 0.5 9.49 1 9.45 2 9.38 3 9.38 5 9.20 24 9.12 27 9.05 48 9.72 72 9.80 168 8.72 192 8.38 216 8.25 240 8.17 288 (12 days) 8.05 336 (14 days) 7.92 384 (16 days) 7.78 432 (18 days) 7.77

[0053] Such as they are used in this document, the terms “about” and/or “approximately”, when they refer to a value or characteristic, are meant to cover variations of ±10% of some embodiment, ±5% of some embodiment, relative to the specified value or characteristic, as said variations are appropriate to carry out the described composition and method/uses.

[0054] Although in this document the embodiments of the present invention have been described with reference to various specific features, it will be obvious for a skilled person to carry out the invention with modifications. All of these modifications are considered to be within the scope of the claims. Likewise, the claims are intended to cover all the generic and specific characteristics of the described exemplary embodiments and all the statements of the scope of protection which, by language reasons, may be said to be therewithin.

[0055] The scope of the present invention is set forth in the attached claims.