BRPI0905347A2 - nanocapsules deformable polymeric capsules in bioactive encapsulation - Google Patents

Abstract

DEFORMABLE POLYMERIC NANOCAPSULES IN BIOACTIVE CAPSULATION. The present invention relates to a deformable nanocapsule comprising a poly (D, L-lactide) polymeric wall (PLA) and a retinyl palmitate oil core comprising one or more low deformation characteristic soluble lipophilic active ingredients. polydispersion, larger diameter compared to nanospheres, high encapsulation efficiency index, stable, elastic and low cytotoxicity. Thus, the object of the present invention may be of great interest for sustained release systems of pharmaceuticals or cosmetic actives by the pharmaceutical and cosmetic industries.

Description

DEFORMABLE POLYMERIC NANOCAPSULES IN BIOACTIVE CAPSULATION "

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FIELD OF INVENTION

The present invention is a deformation property nanocapsule system, which makes this system of great interest in the passage of biological barriers, for example, transepidermal permeation, in addition to the possibility of formation of uniform films. The nanocapsules were produced from a biocompatible and biodegradable polymer, poly (D, L-lactide) called PLA. The oily core of said nanocapsule system

comprises active substances.

Thus, this system may be of great interest to sustained release systems of pharmaceuticals or cosmetic actives by the pharmaceutical and cosmetic industries.

BACKGROUND OF THE INVENTION Nanoparticles

Nanoparticles as drug carriers have stood out in recent decades due to the possibility of reducing drug toxicity, sustained release, increased drug efficacy, decreasing the required therapeutic amounts, and the possibility of passage; by biological barriers (Panyman and Labhasetwar, Adv. Drug Del. Rev. 55, 329- 347, 2003; Vauthier, C. and Bouchemal, K. Pharm. Res., 26, 1025-1058, 2009.). In the context of skin permeation, nanocarriers have been noted for the possibility of increased bioactive permeation (Guterres et al., Drug Target Insights 2, 147-157, 2007; Hoet et al., J. Nanobiotech. 2, 12-26, 2004 ).

In pharmaceutical technology, nanoparticles are defined as submicron colloidal systems, generally in the range of 10 to 1000 nm (Soppimath et al., J. Control. King. 70, 1-20, 2001).

Pharmaceutical nanotechnology began in 1968 with liposomes as hydrophilic or lipophilic drug carriers (Sessa and Weissman, J. Lipid Res. 9, 310-315, 1968). In the late 70's, those of ρ

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polymeric nanoparticles carrying lipophilic drugs (Couvreur et al.

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J. Pharm. Pharmacol., 31, 331-332, 1979). In the 1990s, solid lipid nanoparticles (SLN), solid lipids at body temperature, were proposed as carriers of drugs and cosmetics (Schwarz et al., J. Control. Release, 30, 83-96, 1994), but had limitations as to premature expulsion of the drug. This problem was solved through the use of a lipid mixture including liquid lipids in the system, which system was called nanostructured lipid carriers (NLC) (Müller et al., Adv. Drug Del. Rev., 54, S131-S155, 2002). Compared to liposomes, polymeric nanoparticles exhibit greater stability in biological fluids and storage, and their preparation is easier to scale (Quintar-Guerrero et al., Drug Dev. Ind.

Pharm. 24, 1113-1128, 1998).

In polymeric nanoparticles, the therapeutic agent may be encapsulated in an oily core (nanocapsules) or dispersed in the matrix (nanospheres), and may be adsorbed or chemically bonded to the nanoparticle surface (Schaffazick et al., Chem. Nova 26, 726-737). , 2003).

One of the preparation methods commonly employed in obtaining these systems is the patented nanoprecipitation method (Hatem Fessi, Francis Puisieux and Jean-Philippe Devissaguet, 1987). by Fessi et al (Int. J. Pharm. 55, R1-R4, 1989). Such method, in the production of nanocapsules, consists of an organic phase containing a water miscible solvent, an oil, lipophilic active and optionally a lipophilic surfactant. This organic phase is added to an aqueous phase which may contain a hydrophilic surfactant. Subsequently, the organic solvent is removed, obtaining the aqueous suspension of nanoparticles. The advantages of this method are ease of scaling, the use of non-toxic solvents, and good reproducibility. In addition, it has been employed industrially since the patent by Fessi et al. was licensed Pr- 3/24 _

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by LOreaI for the production of polymeric nanocapsules in the early 1990s (Guterres et al., Drug Target Insights 2, 147-157, 2007). ~ ^ v

The polymers commonly employed in the preparation of polymeric nanoparticles are poly (D, L-lactide) (PLA) 1 poly (D, L-lactide-co-glycolide) (PLGA) and poly (s-caprolactone) (PCL), being biocompatible and biodegradable with non-toxic residues (Panyman and Labhasetwar1 Adv. Drug Del. Rev. 55, 329-347, 2003).

Due to the increase in the surface area of the polymeric nanoparticles due to the reduction in diameter, there is an increase in the surface tension of the system, which favors the coalescence of the nanoparticles. Thus, the use of surfactants, typically between 0.2 and 2.0% (w / w), is commonly required to increase the physical stability of the particles (Legrand et al., S. P. Pharm. Sci. 9, 411-418, 1999; Quintar-Guerrero et al., Drug Dev. Ind. Pharm. 24, 1113-1128, 1998). Among the most used stabilizers, polyvinyl alcohol (PVA) stands out, which, however, has its use restricted by the Food and Drug Administration (FDA), and cannot have intravenous application. Polaxamers, also known as Pluronics, are widely used because they have low cytotoxicity. Polysorbates (Tweens and Spans), permitted by the FDA, are nonionic surfactants and are in this class surfactants that can be solubilized in the aqueous or organic phase (Quintar-Guerrero et al., Drug Dev. Ind. Pharm. 24, 1113- 1128, 1998). Oil cores

Nanocapsules have as their main advantage a greater amount of drug incorporated over nanospheres. The oil core is formed by an oil solubilizing the generally inert active, such as Miglyol 810 or Miglyol 812® (Guterres et al., Drug Target Insights 2, 147-157, 2007; Schaffazick et al., Quim Nova 26, 726 -737, 2003). In principle, other oils may be employed in the oil core as long as the nanocapsule wall polymer is not soluble in such oil. Retinyl palmitate oil is the most stable form of storage.

of vitamin A that plays an important role in cell differentiation as well as prevention of epithelial cancer (Kurlandsky et al., J. Biol. Chem. 271,6,6 ''

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use of retinyl palmitate as a photoprotector, demonstrating the prevention of erythema, transepidermal water loss and sunburn in hairless mice (Antille et al., J. Invest. Dermatol. 121, 1163-1167, 2003). Thus, retinyl palmitate is widely used in cosmetic formulations, especially in anti-wrinkle creams. However, the application of retinyl palmitate is not restricted to cosmetic applications as it has important biological functions, and can act in synergistic effect when combined with other bioactive, such as flavonoids. However, this oil, retinyl palmitate, was not observed in nanocapsule application using PLA polymer.

Skin Permeation

Regarding the cutaneous application, the skin in its outermost layer, horny layer, has a cement-brick structure, where the bricks are the keratinocyte cells and the cement the lipids that join these cells. The permeation mechanism can therefore be intercellular (through cement) or intracellular (through bricks). Another important mechanism is via appendages, which is basically formed by pilosebaceous follicles that represent only around 1% of the skin surface, besides permeation through thin pores averaging 30 nm, influenced by transepidermal pressure gradient, ie, without occlusion (Trotta et al., Int. J. Pharm. 2002, 241, 319-327; Wertz and Downing, Stratum Corneum: biological and biochemistry consideration. In: Hadgraft, J., Guy (Eds), Transdermal Drug Delivery: Development Issues and Research Initiatives (Mareei Dekker: New York, 1989).

The mechanisms by which nanocarriers increase skin permeation are not fully elucidated, but some properties, besides size, such as surface charge and lipidicity are important in the permeation profile (Hoet et al., J. Nanobiotech. 2, 12-26, 2004). With regard to charge, positive, negative and neutral latex nanoparticles of size 50, 100, 200 and 500 nm were studied at skin permeation and only negatively charged 50 and 500 nm nanoparticles completely permeated the skin and were detected in the solution. experimental recipient using Franz vertical diffusion cells (Kohli and Alpar1 Int. J. Pharm. 275, -St - 13-17, 2004). The authors suggested a limit load that should be achieved to allow the passage of particles through time channels formed by repulsion between the skin lipids and the particles, leading to permeation.In another permeation study by confocal laser scanning microscopy (CLSM) technique, the images revealed that the Nile red dye encapsulated in PCL (250 nm) reached deeper layers of the skin when compared to the unencapsulated dye (Alvarez-Román et al., Pharm. Res. 21, 1818-1825, 2004). In a subsequent study Fluorescent polystyrene particles between 20 and 200 nm were used to verify the distribution in the skin, with a larger accumulation of particles in the follicles instead of uniform permeation. Based on this work the authors suggested that in their previous work (with Nile red dye) nanoparticles did not permeate the skin but allowed uniform release of the lipophilic model (Alvarez-Román et al., J. Contr. Rei. 99, 53 -62, 2004). Polymeric nanoparticles of polyesters, in principle, for not having ions do not have a formal charge. However, the nanocapsules, object of this invention, as well as the other polyesters reported in the literature present slightly negative zeta potential (charge in the hydrodynamic radius of the particle) due to hydrolysed groups of the polyesters located on the surface of the nanoparticles (Ahlin et al., Int. J. Pharm., 239, 113-120,2002).

PLGA nanoparticles have been investigated by increasing flufenamic acid permeation. Only the drug presented higher permeation, and the nanoparticles were retained in the outermost layer of the skin. In addition, pH influenced the concentration gradient of this drug from the outermost layers (corneal layer) toward the dermis (Luengo et al., Skin Pharmacol. Physiol. 9, 190-197, 2006). Thus, the results of the literature indicate that the increase in drug permeation using polymeric nanoparticles occurs due to the formation of a film on the skin, resulting from the retention of nanoparticles, which decreases the transepidermal water loss and favors the active release evenly. The advantage of nanocapsules now presented in relation to other systems

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PLGA1 is a higher permeation in biological barriers due to their deformability. The aforementioned PLGA particles, for example, do not permeate the skin and are trapped on the surface. The advantage of nanocapsules, object of this invention and that does not use PLGA1 is that of greater permeation of the active nanocarrier, allowing the release in even deeper layers of the skin.

are known to permeate the skin evenly allowing transdermal application (Trotta et al., Int. J. Pharm. 241, 319-327, 2002; Bouwstra and Honeywell-Nyguyen, Adv. Drug Deliv. Rev. 54, S41-S55, 2002). When compared to liposomes, the polymeric nanocapsules, object of this invention, present greater stability in biological fluids and storage, besides being easier to prepare.

large scale production.

Recently, polymerosomes (polycaprolactone-polyethylene glycol-polycaprolactone block copolymer vesicles) have been reported for increased skin permeation due to the particle deformation capacity observed by transmission electron microscopy (TEM) due to the possibility of carrier passage through the pores of the skin (Rastogi et al., Colloid Surf. B-Biointerfaces, 72, 161-166, 2009). This system presents aqueous nucleus, therefore, more efficient in the storage of hydrophilic molecules, whereas the present invention proposes the encapsulation of hydrophobic molecules by comprising oily nucleus.

The possibility of using a polymeric nanoparticle in which the carrier can present deformation capacity, similar to elastic liposomes, is extremely important in the permeation of biological barriers, especially with regard to transdermal applications. One of the possible advantages of the polymeric nanocapsules, presented here, over elastic liposomes is a slower release of the active. Liposomes, being self-organized systems, present reorganization

Other nanocarreders, however, such as elastic liposomes of the very dynamic bilipid layers favoring active expulsion.

US7498045 (Chang, Thomas MS; Yu, Wei-Ping; Powanda, Douglas. Polymeric nanocapsules and uses thereof. Filing Date: 08/28/2002) relates to biodegradable poly (D, L - lactide) polyethylene glycol (PLA-PEG) prepared by encapsulating hemoglobin as an example of macromolecule. The main purpose of this technology is an increase in nanocapsule circulation time due to the presence of PEG. It includes a more complex method of preparation ranging from copolymer synthesis to the subsequent increase in hemoglobin residence time by the addition of crosslinking agents. The nanocapsules of this system are not deformable, use various formulation materials other than those employed in the present invention, having only PLA as a common reagent and not proposing use in dermal application. Using multiple steps and multiple reagents, in addition to increasing the cost of the process, increases the possibility of failed steps, for example, inactivation of the encapsulated asset due to the use of

cross-linking reagent.

Strain characteristics are important both for increasing uniform film forming capacity and for increasing permeation in biological barriers such as skin permeation. Elastic characteristic in polymeric systems has previously been observed only in polycaprolactone-polyethylene glycol-polycaprolactone micelles, which differ from the present invention by the type of material employed and the structure (self-organized system), and has no oily core (Rastogi et al. Colloid Surf, B-Biointerfaces, 72, 161-166, 2009). Other systems having elastic characteristics are some liposomes (Cevic and Blume, Biochim. Biophys. Acta-Biomembr. 1104, 226-232, 1992) and SLN (Müller et al., Eur. J. Pharm. Biopharm., 50, 161 -177). The PLA nanocapsules, object of this invention, have deformation characteristics, which give them the possibility of reaching deeper layers of the skin and retinyl palmitate as an oil core in an 8/24 nanocapsule. <S »V Vi

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EP1342471-1 (Simonnet, Jean-Thierry; Guerin, Gilles. Nanocapsules based on polyol polyester, and cosmetic and dermatological compositions containing same. Filing date: 09/10/2003) discloses nanocapsules containing a polyester polyol polymer wall obtained by polycondensation for the encapsulation of retinol or its esters in nanocapsules for cosmetic or dermatological use. As an oily core, it proposes the use of conventional oils such as jojoba oil and castor oil. The inventors have suggested an increase in skin permeation of nanocapsules based only on submicron size (less than 1 μητι), but no embodiment has been performed demonstrating skin penetration let alone increased permeation. In addition, nanocapsules are not deformable, so the nanocapsules of the present invention are more promising for skin permeation.

EP1316301-A (Duranton, Albert; Renault, Beatrice; Mahe, Yann; Marion, Catherine; Catroux, Philippe. Cosmetic or dermatological composition containing a retinoid and / or a carotenoid and acexamic acid. Filing date: 04/06/06 2004) describes the use of retinyl palmitate in combination with acexamic acid in a cosmetic formulation, which has the advantage of improving skin reepithelialization. However, it differs from the present invention because it is not nanocarriers and the formulation differs from the composition of the present invention. , having as common component only retinyl palmitate. The formulation of the present invention has as its main advantage in relation to this work the targeting of retinyl palmitate to deeper layers of the skin, which means increasing the effectiveness of retinyl palmitate.

EP0557489 (Ribier, Alain; Richart, Pascal; Handjani, Rose-Marie. Composition Providing a Lasting Cosmetic and / or Pharmaceutical Treatment of the Upper Epidermal Layers by Topical Application on the Skin. Filing Date: 03/01/1992) employs a non-biodegradable polymer at 9/24% A r— ί »

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Obtaining polymeric nanocapsules justified by the increased time to release the drug. However, the release profile can be either diffusion or degradation, and in general diffusion is faster than degradation, ie such an invention does not increase the residence time of all assets (due to diffusion). It also creates a problem that is the use of a non-degradable plastic that is unknown if and when it will be disposed of if absorbed.

In view of the state of the art, the present invention advantageously provides a proposal for polymeric PLA nanocapsules and retinyl palmitate oil core with deformation characteristic, low polydispersion index, larger diameter compared with nanospheres, high encapsulation efficiency index, stable, elastic, low cytotoxicity among other attributions described later. It is an additional object of this invention to be able to add one or more retinyl palmitate liposoluble active ingredients, selected from benzophenoma and baicalein, but without restriction. Thus, the nanocapsules presented here are of great interest to the pharmaceutical and cosmetic industries.

BRIEF DESCRIPTION OF THE INVENTION

Deformable nanocapsules comprising a PLA polymeric wall and a retinyl palmitate oily core containing an oil / polymer ratio preferably ranging from 2.5 / 1.0 to 3.5 / 1.0 m / m, an oily core containing a or more acceptable lipophilic active ingredients, such as, for example benzophenone and baicalein, in at least one reagent selected from the group: water-miscible organic solvent, water-immiscible organic solvent, surfactant and mixture thereof, a low polydispersion index, a mean particle diameter ranging from 182.7 to 285.8 nm, elastic, an analytically acceptable range of retinyl palmitate recovery from the initial amount, an encapsulation efficiency of 99.9% and a negative zeta potential. Thus, the object of the present invention may be

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BRIEF DESCRIPTION OF THE FIGURES Figure 1: shows the chemical structures of the main components.

system: retinyl palmitate (PR) and poly (D, L-lactide) (PLA).

Figure 2 shows examples of the normal and accelerated stability of nanocapsules with and without antioxidants as a function of the percentage of nanoencapsulated retinyl palmitate over a given storage time in relation to the content of the same active ingredient at initial time. NC corresponds to nanocapsules without stabilizers; NCEST corresponds to BHT and Tinogard Q nanocapsules.

Figure 3 shows the exemplary embodiment of nanocapsule stability with respect to the pH of the present invention as a function of time. NC corresponds to nanocapsules without stabilizers; NCEST corresponds to BHT and Tinogard Q nanocapsules.

Figure 4: presents the measured zeta potential values of nanocapsules as a function of time. NC corresponds to nanocapsules without stabilizers; NCEST corresponds to nanocapsules with BHT and Tinogard Q. Figure 5: shows the diameter distribution of nanocapsules of

retinyl palmitate in relation to time. NC corresponds to nanocapsules without stabilizers; NCEST corresponds to BHT and Tinogard Q nanocapsules.

Figure 6: shows the cytotoxicity assays of retinyl palmitate nanocapsules in main cell cultures found in the skin: fibroblasts and keratinocytes. The cell lines used were mouse embryo (BALB / c 3T3) and human keratinocyte (HaCaT) fibroblasts.

Figures 7A-D: Transmission electron microscopy (TEM) images of: (A) niosomes (Span 60), (B) nanospheres, (C) nanoemulsion relative to (D) nanocapsules of the present invention. «Al ρ ,. ν

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Figure 9 shows the hysteresis curves of the rheological behavior of nanocapsules (NC) incorporated in the gel compared to the gel without the addition of nanocapsules.

Figure 10 shows permeation tests during application of the composition comprising retinyl palmitate nanocapsules, object of this invention in human abdominal skin after 24 h of permeation. Figure 11: Shows permeation results of encapsulated BZ3

in retinyl palmitate (NC) nanocapsules and nanospheres (NS) in human abdominal skin after 24 h of permeation in Franz vertical diffusion cell.

Figure 12: shows a representative confocal laser scanning microscopy (CLSM) imaging scheme in human skin permeation assays in Franz vertical diffusion cell for 4 hours, analyzed by optical sectioning of the microscope at the depth axis of the skin (XjZ). From the original image, skin corresponds to the absence of fluorescence under the conditions employed (black); retinyl palmitate corresponds to green image; retinyl palmitate plus PLA-Nile blue corresponds to redder green, respectively corresponds to orange image.

Figure 13: Release profile of free benzophenone (BZ3) incorporated into nanoemulsions (NEBZ3), nanocapsules (NCBZ3) and nanospheres (NSBZ3).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a deformable nanocapsule comprising a PLA polymeric wall and an oil retinyl palmitate oil core. A further object of the present invention is a deformable nanocapsule as described above comprising an oil core containing one or more retinyl palmitate soluble lipophilic active ingredients. For the purposes of the present invention.

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Active ingredients were selected from benzophenoma and baicalein, but without restriction.

The object of the present invention depicts the use of deformable nanocapsules comprising a PLA polymeric wall and a retinyl palmitate oily core containing an oil / polymer ratio preferably ranging from 2.5 / 1.0 to 3.5 / 1.0. m / m, an oily core containing one or more acceptable lipophilic active ingredients in an organic solvent and a surfactant, a polydispersion index preferably ranging from 0.07 to 0.19, an average particle diameter ranging from 182.7 at 285,8 nm, a yield value comprising 31,8 ± 6,8, a recovery range for retinyl palmitate from the initial amount between 90 and 110%, an encapsulation efficiency of 99,6 to 99, 9% and a zeta potential ranging from -32.0 ± 4.4 mV to -7.2 ± 5.2 mV.

A further object of the present invention depicts the use of deformable nanocapsules comprising a PLA polymeric wall and a benzophenone-containing retinyl palmitate oily core in the preparation of a cosmetic for use as sunscreen for dermal application and use of deformable nanocapsules comprising a wall. PLA polymer and an oily retinyl palmitate core containing an active ingredient selected from the group of flavonoids preferably baicalein in the preparation of an antiinflammatory and antioxidant drug for dermal or parenteral application.

Figure 1 shows the chemical structure of the nanocapsule of the present invention.

In the present invention the following definitions are considered: poly (D, L-

lactide) labeled PLA, retinyl palmitate labeled PR, baicalein labeled BAI, benzophenone-3 labeled BZ3, nanocapsules labeled NC, nanospheres labeled NS, nanoemulsions labeled NE, 3,5-di-tert-butyl-4-hydroxy toluene labeled BHT , PBS saline phosphate buffer, polyoxyethylene glycol 8-lauryl PEG-8L, mouse embryo fibroblast cell line named BALB / c 3T3, human keratinocyte cell line named HacaT, neutral red v »8Í ρ 13/24

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VN1 dicyclohexylcarbodiimine DCC, nanoparticles / solid lipids denominated SLN1 nanostructured lipid carriers ^ / - t® "NLC1 poly (lactide-co-glycolide) denominated PLGA1 ροΙί (ε-caprolactone) polyvinylpeptide Food-grade PCL, administration named FDA, ultraviolet UV, confocal laser scanning microscopy CLSM1 transmission electron microscopy TEM1 high performance liquid chromatography HPLC1 dynamic light scattering DLS1

polydispersion called PDI. The process of preparing nanocapsule compositions is

Known in the prior art, an oil, preferably retinyl palmitate and optionally an acceptable lipophilic active agent in a water miscible organic solvent (acetone), with or without the addition of a surfactant is solubilized. This organic phase is added to an aqueous phase under magnetic stirring, in which a surfactant of 0.2% (w / v) or greater is added. The organic solvent is then removed and the aqueous suspension concentrated under evaporation under reduced pressure. In this method, nanocapsules are formed by precipitation of their components in nanocapsule form, from the insolubility achieved in the diffusion of the organic solvent to the aqueous phase. Water-immiscible organic solvents (such as chloroform) may also be employed, but in this case another production method is used as emulsification in which submicron-sized organic water droplets are formed from high energy methods (Utraturrax®, for example), wherein the nanocapsules are formed during solvent evaporation.

The final nanocapsule suspension can be dried by spray-drying methods or used directly to obtain the final formulation as semi-solids for topical use.

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Example 1: Deformable nanocapsule comprising a PLA polymeric wall and an oily core of retinyl palmitate.

comprising a PLA polymeric wall and an oily retinyl palmitate core was performed by conventional techniques reported above.

Example 2: Deformable nanocapsule comprising a PLA polymeric wall and a benzophenone-containing retinyl palmitate oil core.

The benzophenone-3 (BZ3) UV filter was encapsulated in the deformable nanocapsule system by solubilizing the bioactive in the organic phase in the preparation of the nanocapsules as described above.

Example 3: Deformable Nanocapsule comprising a polymeric wall

of PLA and an oily nucleus of retinyl palmitate containing baicalein.

Baicalein flavonoid (BAI) was encapsulated in the deformable nanocapsule system by solubilizing the bioactive in the organic phase in the preparation of nanocapsules as described above.

Physicochemical characterization of nanocapsule compositions

In the preparation of nanocapsules of the present invention, formulations having an oil / polymer ratio of 2.5 / 1.0 to 3.5 / 1.0 (m / m) were tested. Nanocapsia formation occurred in all systems, which is indicated by the low polydispersion index (P.D.I. <0.2), in addition to the larger diameter in relation to the nanospheres (Table 1, measured by dynamic light scattering, DLS).

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Table 1. Average particle (d) and polydispersity (P.D.I.) diameters of the NS1 NC1, NC2 and NC3 preparations.

Preparations Diameters

d / nm P.D.I.

NS Nanospheres (without added oil) 125.7 0.154

NC1 Nanocapsules 2.5 / 1.0 238.3 0.112

NC2 (oil / polymer 3.0 / 1.0 261.5 0.104

NC3 m / m) 3.5 / 1.0 265.9 0.075

The results of Table 1 refer to raw materials of the same

lot. When the polymer batch is varied, for example, there are changes in diameters and zeta potentials, in which case NC samples with diameters from 182.7 nm to 285.8 nm were found, polydispersity indexes between 0.07 and 0.19 and zeta potential between -32.0 ± 4.4 mV and -7.2 ± 5.2 mV.

The encapsulation efficiency for this formulation was determined by the Millipore Microcon 100 kDa membrane centrifugation / filtration method. The filtrate was injected by HPLC analytical method, quantifying the "free" (unencapsulated) active. The total amount of retinyl palmitate in the formulation was determined after dissolving 100 μΙ_ suspension in 10 mL acetonitrile. The amount of retinyl palmitate was determined to be between 90 and 110% of the initial amount (acceptable recovery range by the analytical method), indicating that no active losses occur during preparation. Encapsulation efficiency was determined by Free Asset Amount / Total Asset Amount (x100). The encapsulation efficiency was 99.9%.

The zeta potential (measurement of particle shear surface charge in the hydrodynamic radius) was determined by measuring electrophoretic mobility in ZetaSizer NanoZs equipment. Approximately -20 mV zeta potential was obtained. The negative zeta potential refers to the charge formed by hydrolysis of the PLA polymer (polyester) that generates carboxylic groups. The non-ionic surfactants employed, Polaxamer 188 or. "ν ·· 'V, 16/24

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Polysorbate 80, do not significantly influence negative values, and the main mechanism of stabilization of the nanocapsules, nefref ψ case, besides stabilization by charge, is by steric effects (osmotic effect) and hydration layer (increase of the distance between particles), conferred by the stabilizers.

The type of surfactant employed did not significantly influence particle diameter distribution, obtaining effective diameters of 219.7 nm and 219.6 nm using polysorbate 80 or polaxamer 188 (1% w / v), respectively. In addition, obtaining nanocapsules with the same diameter distribution was possible without the addition of water soluble surfactant. However, this suspension coalesce approximately one hour after preparation. In fact, the presence of surfactants is not a major factor in the formation of colloidal systems. They provide greater stability by preventing aggregation over time (kinetic stability).

Regarding chemical stability (Figure 2) evaluated by HPLC, it was observed that, even encapsulated, retinyl palmitate shows a rapid degradation as a function of time. It was possible, however, to stabilize the suspensions formed by the addition of a fat soluble stabilizer, di-tert-butyl-4-hydroxy toluene (BHT), and a sunscreen, Tinogard Q (tris citrate (tetramethyl hydropiperidinol, which reduces the potential for degradation reaction by minimizing the average life of intermediate excited states), both at 0.1% (m / V). Only in the "accelerated" stability assessment, ie at 42 ° C, was observed loss of palmitate within one month, equivalent to 25% of the initial amount, and within 3 months (120 days), less than 10% retinyl palmitate is observed in the 42 ° C assay.

Regarding the pH of the suspensions (Figure 3), it is observed that the hydrophilic stabilizer raises the pH of the suspensions initially and protects the suspension. Without stabilizer there is a decrease in the pH of the suspensions and with higher temperature degradation occurs. The PLA Mygliol 810 nanocapsule system also showed a decrease in the pH of L ·

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as a function of time by polymer degradation (Guterres et al., Int. J. Pharma. 113, 57-63, 1995).

The physical stability of the particles was evaluated for mean diameters and zeta potential. Potential zeta data show a decrease in function of time in the first month (Figure 4), which indicates the presence of carboxylic acids generated on the nanoparticle surface inducing negative charge on the shear surface (hydrodynamic radius).

Regarding the average diameters (Figure 5), it is observed that over time at least up to 1 month of analysis there are no large variations in size with the exception of samples stored in the refrigerator, and nanocapsules without stabilizers increase in size. in the early times. Such measurements were made for samples containing polaxamer 188. It should be noted that for samples containing Tween 80, the diameters remain constant for at least 4 months of preparation. Cytotoxicity

Figure 6 shows the results of cytotoxicity assays performed on HaCaT human keratinocyte lineages and BALB / c 3T3 mouse embryonic fibroblasts. Cells were seeded in 96-well plates and a concentration of 3.0 x 10 5 cells / ml and incubated at 37 ° C under Cima atmosphere containing 5% CO 2 for 24 h. After 24h incubation, cells were treated with different surfactant concentrations and incubated for 24h. Cell proliferation was then assessed by neutral red uptake (VN: 3-amino-7-dimethylamino-2-methylphenazine hydrochloride). Results were expressed relative to control (100%) and represent mean ± standard deviation of 2 independent experiments performed on sextuplicate. Nanocapsule formulates showed cytotoxicity (46% cell viability) only at the highest concentration tested (1140 μηιοΙ L · 1) in keratinocyte lining. Since the tested linages correlate well with the keratinocytes and fibroblasts present in human skin, it may be suggested that the nanocapsules are safe for dermal application. 18/24

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Morphological Characterization

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For the purpose of comparing nanocapsules with other structures, nanospheres, niosomes and nanoemulsions were also prepared by the same method of preparation, and the nanospheres are obtained by omission of the oil, nanoemulsion is not used in the polymer, and niosomes are omitted. ο oil and ο polymer, having only structures formed from surfactants.

Figure 7 shows the transmission electron microscopy (TEM) image of PLA (non-oil white) nanospheres, nanoemulsion (polymer free), niosomes (polymer and oil free) and nanocapsules obtained. In this case, the deformation mechanism is shown in Figure 8, in which it is observed that only the nearby dry particles (Figure 8-A) show deformation while isolated dry spheres remain spherical (Figure 8-B). Unlike nanospheres and nanoemulsion, nanocapsules have deformation characteristics, and when drying on the sample holder, the capillary effect, according to the Young-Laplace equation, allows them to deform without particle coalescence occurs (Adamson, Physical chemistry of surfaces, 6th ed .; John Wiley & Sons: New York, 1997). Passage through synthetic polycarbonate membranes

Human skin has average diameter pores of 30 nm, with an external pressure of 2.5 atm. Thus, in the literature, elasticity of elastic liposomes has been determined by skin pore mimicking (Trotta et al., Int. J. Pharm. 241 '319-327, 2002; Bouwstra and Honeywell-Nyguyen, Adv. Drug Deliv. Rev. 54, S41-S55, 2002). The method consists of extruding the particles into two overlapping polycarbonate membranes under transepidermal pressure. Two overlapping polycarbonate membranes (50 nm in diameter, Osmonics Inc.) were used to simulate skin tortuosity at two different pressures 2.5 and 6.0 atm generated by N2 (g). The nanocapsules, although deformable (as observed in the TEM images of Figures 7 and 8), did not pass through the membrane. To increase the wettability of this system, polyoxyethylene glycol 8-lauryl 19/24 β "■ <

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(PEG-8L) at (0.2% m / V), which is commonly employed in the production

Elastic liposomes (Cevc et al., BBA-Biomembranes, 1564, 21-30, 2002)

Thus, the passage of nanocapsules was possible, and the

MATERIALS OF MEMBERS THAT PERCHED THE Membranes

relation to the initial suspensions and what was retained in the membranes are

Table 2. Both experiments, under 2.5 atm and 6.0 atm,

resulted in the same permeation profile with flows of 20 g / h / cm2 and 38

g / h / cm 2 respectively. The elasticity value comprises 31.8 土 6.8.

Table 2. Diameter and polydispersity data (P.D.I.) in permeation study on synthetic membranes.

Average Diameter / nm (PDI) Extruded Suspensions Retained in early membranes NC 227, (0.12) (undeterminable) 231.6 (0.16) NC + PEG-8L 2.5 atm 220.0 (0.11 ) 296.0 (0.54) 604.3 (0.61) NC + PEG-8L 6.0 atm 215.7 (0.07) 295.7 (0.27) 608.1 (0.55)

Critical micellar concentration is not detected by DLS when filtered due to further dilution of the filtrate for analysis, ie the particles measured in the suspension are actually nanocapsules. Despite the elasticity of nanocapsules, there is a need for PEG-8L for passage through synthetic pores, which may possibly be related to capillary repulsion phenomena due to the low wettability of particles to pass through smaller pores in the absence of this surfactant.

The data from Figures 7 and 8 and Table 2 suggest that the nanocapsule system can be classified as elastic, given that the pressure difference caused by the droplet reduction during the particle drying process on the sample holder (force? Capillary Adhesion, ΔΡ), apart from the transepidermal pressure in the membrane tests, is sufficient to deform the particles. Incorporation in semi-solid formulation

The possibility of using suspensions in

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semi-solid formations. The hysteresis curves of rheological behavior 20/24, 身 \ —J Fk. Jf 食

of the semi-solid formulations are shown in Figure 9 ， where the ΓΤ —W incorporation of nanoparticles did not significantly alter the rheological characteristics of the system. Carbopol 940 gel employed exhibited thixotropic viscosity behavior, ie, increasing the torque employed decreases viscosity (P. M. Alves et al., Pharmazie 60, 900-904, 2005). This rheological behavior is of great importance for products during topical application and can be classified as easy to apply.

Use of nanocapsules for bioactive encapsulation Two bioactive models were encapsulated in the

Deformable nanocapsules: ο benzophenone-3 UV filter (BZ3) and ο baicalein flavonoid (BAI). The nanocapsule preparation method consisted of solubilizing the bioactive in the organic phase in the preparation of the nanocapsules.

Table 3 shows the main physicochemical characteristics of the system (size and zeta potential) and the incorporation of bioactive with high encapsulation efficiency.

The main advantage of nanocapsules is greater incorporation of the drug over nanospheres. Incorporation of the drug did not significantly change particle diameters and zeta potential. Table 3. Encapsulation efficiency, Diameter distribution and zeta potential for retinyl palmitate nanosphere (NS) and nanocapsule (NC) systems incorporating baicalein (BAI) and benzophenone-3 (BZ3) bioactives.

Sample Embedded Quantity /% (maturity / mp0 | [mere * 100) Encapsulation Efficiency% Suspension Diameters / nm (PDI) Potential Zeta / mV NCBZ3 10 99.7 210.7 (0.057) -17.0 ± 1.0 NSBZ 3 1 99.8 112.2 (0.045) -27.5 ± 2.1 NCBAI 10 99.6 226.2 (0.143) -26.7 土 6.6 NSBAI 1 95.6 154.7 (0.041) - 11.1 ± 5.1

These results show that the nanocapsule system is quite

efficient as a bioactive reservoir system. An additional advantage of 21/24 <^ 1¾. t

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nanocapsule system compared to nanospheres (without oil) and possibility of a larger amount of drug incorporated. Abdominal human skin permeation tests

Biological membrane permeation experiments were performed on human abdominal skin donated from plastic surgery in Franz vertical diffusion cell. Adipose tissues were removed immediately after surgery and the skin was stored at -20 ° C for up to 3 months before use. The Franz diffusion cell employed has an area of 2.01 cm 2 and a receiver compartment capacity of 7 mL. The epidermis was exposed to the donor cell, while the dermis was kept in contact with phosphate buffer (PBS), pH 7.4, containing 5% Tween 80 in the recipient cell. In the donor cell, 0.5% (w / w) Carbopol 940 ® gel was used with the nanoparticles. The system was ground under magnetic stirring in the recipient cell at 37 ° C for 24 h. After the incubation period. The skin surface was carefully washed with distilled water and dried with cotton. The quantification of the active in the corneal layer was performed and removed by the technique of applying successive tapes. Extraction of the active was performed at 40 ° C in 5 mL of methanol, alternating between Vortex and Ultrasound for 30 minutes. Extraction of the active from the remainder of the epidermis and dermis was performed by the same extraction procedure. The samples were filtered through 0.45 μΓΠ filter and analyzed by HPLC. Results represent mean ± standard deviation of 6 Franz cells for each formulation.

Figure 10 shows the results of Franz vertical diffusion cell permeation studies of Carbopol gel-embedded nanocapsules after 24 h of experiment. Nanocapsules reach in high concentration the viable epidermis and dermis, in addition to the receptor solution, indicating that nanocapsules have very efficient skin permeation.

Skin is a very effective barrier to permeation of various substances, and the corneal layer (anucleated keratinocyte cells) is the main barrier to permeation of assets (ABURJAI and NATSHET, Phytother. Res., 17, 987 -1000, 2003). Once deeper layers of the epidermis or dermis are reached, the active may 22/24 f 《η 、

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easily reach the bloodstream (in this case mimicked by the receiving cell). When permeation is uniform and is non-toxic active, the amount in which the bloodstream is reached may be negligible. In addition, depending on the desired action of dermatological or cosmetic products, it is necessary that it is active to reach viable skin cells (keratinocytes in the epidermis and / or fibroblasts in the dermis).

The permeation of assets in the retinyl palmitate nanocapsule system was evaluated using BZ3 as a model molecule in both the nanosphere and nanocapsule system, object presented here. Figure 11 presents the results of the analyzes. It is observed that nanocapsules allow a higher permeation of the active relative to nanospheres, which may be due to higher carrier permeation (due to the flexibility of nanocapsules) according to the data observed by TEM. It should be mentioned that BZ3 was not detected in the recipient cell due to the low concentration used. In the case of palmitate and retinyl (Figure 10), it can be detected in the recipient cell because it is in a higher concentration. in the formulation (15 times by mass).

Evaluation of permeation in human abdominal skin by CLSM

In visualizing the depth of the skin particles, permeation experiments were analyzed by confocal laser scanning microscopy (CLSM). The experiment consisted in the formulation of the particle suspension formulation in the donor cell. In the receptor solution, phosphate buffer (PBS) pH 7.4 was employed. The system was kept under magnetic stirring in the receiving solution at 37 ° C for 4 h. After this time the suspensions were removed and the skin washed with PBS pH 7.4 and dried with cotton.

Retinyl palmitate shows green fluorescence when excited with UV. For visualization of the PLA polymer, a functionalization of the Nile blue dye polymer chain terminations was performed. The functionalization reaction was performed by activating the dicyclohexylcarbodiimine (DCC) 1 polymeric terminal carboxylic groups in which reaction occurs between the polymer's dye and carboxylic groups, having an amide-like covalent ligation (Pearson and Roush (editors ), Handbook of. ^^ 各 、 23/24

Reagents for Organic Synthesis: Activating Agents and Protecting Groups, Joh Wiley & Sons, Chichester, UK, 2000). After purification of the polymer, its characterization was performed by fluorescence spectroscopy, with excitement and red emission observed.

Figure 12 shows the CLSM image of nanocapsules (emission

red and green, with excitation at 543 nm and 360 nm, respectively). Retinyl palmitate (PR) permeates the corneal layer, corroborating the HPLC result. It is noteworthy that it was not possible to observe the placement of green and red, and the retinyl palmitate (green) forms the core of the nanocapsules and the Nile PLA-blue (red), the polymeric wall. Under the experimental conditions used, palmitate is present in high concentration, presenting the phenomenon of quenching. Therefore, the green marking can be attributed mainly to free palmitate (0.1% unencapsulated). In this way, nanocapsules are better characterized by red permeation. It is observed that part of the possibly "free" retinyl palmitate remains on the surface, while nanocapsules (with encapsulated and possibly free retinyl palmitate from release) permeate deeper layers, in which case they are characterized by red of the polymeric wall. This indicates that part of the free (perhaps release) or encapsulated palmitate permeates together with the polymer, it may be suggested that the main mechanism of nanocapsule permeation is intercellularly and through thin pores of the skin, as permeation is present. quite uniform.

The results presented show that this system is of great

bioactive or even as a storage form of fat-soluble vitamin for sustained release. Its main potentiality is suggested by the tests performed and in transcutaneous application in which a uniform carrier permeation is desired. Moreover, these particles show a higher film forming capacity compared to rigid particles.

In vitro release 24/24 Z 〜 -c -ο 、 知.

In addition to the possibility of association with other actives, it has been investigated), also an additional advantage of nanocapsules over nanoemulsions which supposedly consists of a slower release of active nanocapsules due to the presence of the polymeric wall. The in vitro release profile (through spectraPor® 3.5 kDa regenerated cellulose dialysis membrane) under conditions of unsaturation of the active medium is shown in Figure 13. Nanocapsules have a slower release profile whereas the nanospheres have a slow profile at the beginning, but at the end of the period the release increases approximately 100% of the active.

Slower release of active nanocapsules over

nanoemulsions may be due to the presence of the polymeric wall. Regarding the nanospheres, the differences observed are attributed to different interactions

oil-active and polymer-active.

Claims (19)

Deformable nanocapsules characterized in that they comprise the polymeric wall of PLA and an oily retinyl palmitate core. Deformable nanocapsules according to claim 1, characterized in that the oil / polymer ratio ranges from 1.0 / 1.0 to 4.0 / 1.0 m / m. Deformable nanocapsules according to claim 2, characterized in that the oil / polymer ratio preferably ranges from 2.5 / 1.0 to 3.5 / 1.0 m / m. Deformable nanocapsules according to claim 2, characterized in that the oily core contains one or more soluble lipid-active ingredients. Deformable nanocapsules according to Claim 4, characterized in that said active ingredient is soluble in at least one reagent selected from the group: water-miscible organic solvent, water-immiscible organic solvent, surfactant and a mixture thereof. Deformable nanocapsules according to claim 5, characterized in that said active ingredient is soluble in acetone and polysorbate. Deformable nanocapsules according to claim 2 or 4, characterized in that they comprise a polydispersity index of 0.2 or less. Deformable nanocapsules according to claim 7, characterized in that the polydispersity index preferably ranges from 0.07 to 0.19. Deformable nanocapsules according to claim 7, characterized in that they comprise an average particle diameter ranging from 182.7 to 285.8 nm. Deformable nanocapsules according to claim 9, characterized in that the elasticity value comprises 31.8 土 6.8. Deformable nanocapsules according to any one of the preceding claims, characterized in that they comprise a range of in: retinyl palmitate recovery with respect to the initial amount between 90 and 110%. Deformable nanocapsules according to claim 11, characterized in that they comprise an encapsulation efficiency of 99.6 to 99.9%. Deformable nanocapsules according to claims 3, 6, 8 and 12, characterized in that they comprise a zeta potential ranging from -32µ0 ± 4'4mVe-7.2 ± 5.2mV. Deformable nanocapsules according to any one of the preceding claims, characterized in that they comprise a cytotoxicity of 46% cell viability at a concentration of 1140 μΓηοΙ L_1 within 24 hours of incubation. Use of deformable nanocapsules comprising a PLA polymeric wall and an oily retinyl palmitate core containing an oil / polymer ratio preferably ranging from 2.5 / 1.0 to 3.5 / 1.0 m / m , an oily article containing one or more acceptable lipophilic active ingredients in an organic solvent and a surfactant, a polydispersity index preferably ranging from 0.07 to 0.19, an average particle diameter ranging from 182.7 to 285, 8 nm, an elasticity value comprising 31.8 土 6.8 'a retinyl palmitate recovery range over the initial amount between 90 and 110%, an encapsulation efficiency of 99.6 to 99.9% and a zeta potential ranging from -32.0 土 4.4 mV to -7.2 ± 5.2 mV. Use of deformable nanocapsules comprising a PLA polymer wall and a benzophenone-containing retinyl palmitate oil core in the preparation of a cosmetic for use as a sunscreen. Use of deformable nanocapsules according to claim 16, characterized in that they comprise dermal application. Use of deformable nanocapsules characterized in that they comprise a PLA polymeric network and a retinyl immitate oily core containing an active ingredient selected from the group of flavonoids preferably baicalein in the preparation of an antiinflammatory and antioxidant medicament. Use of deformable nanocapsules according to claim 18, characterized in that they comprise dermal or parenteral application.