SOLID LIPID NANOPARTICLE FOR INTRACELLULAR RELEASE OF ACTIVE SUBSTANCES AND METHOD FOR PRODUCTION THE SAME

Abstract

The invention relates to solid lipid nanoparticle for intracellular release of active substances, can be used in the pharmaceutical industry, in the medicine, cosmetics, as well as for food supplements. Solid lipid nanoparticle has spherical shape with a diameter of 15 - 100 nm, the lipid is a solid lipid selected from natural plant wax or its synthetic analogue, as the surface acting agent is used TMDSC. The particles of solid lipid nanoparticle is characterized with high melting point, high lipophilicity and low (or lack) of in-vitro dissolution profile. The system is lipase-resistant and is capable to freely penetrate through cell membranes into cells where to release the active substance(s) due to an intracellular digestion with controllable depo-effect. In a second aspect, the invention relates to a method of production of the solid lipid nanoparticle. The preferred technology for production of the compositions is a Phase Inversion Temperature method.

Claims

1. A method for intracellular administration of an active substance, said method comprising administering to a target tissue a solid lipid nanoparticle comprising a lipid, a surface acting agent, water and an active compound, wherein: said solid lipid nanoparticle is a solid lipid nanoparticle with spherical shape with a diameter of 15-100 nm; said lipid is natural or synthetic carnauba wax mixed with a liquid lipid with 30% tocotrienol content relative to total amount of said liquid lipid, where the liquid lipid is in an amount up to 20 weight percentage of total amount of said lipid; said surface acting agent is d-α-Tocopheryl polyethylene glycol 1000 succinate in combination with polysorbate; said active compound is incorporated in said nanoparticles; and components of said solid lipid nanoparticle are in the following quantitative ratios in w/w parts, relative to 100 parts, wherein 100 parts is total weight of said solid lipid nanoparticle: from 1-5 parts carnauba wax, from 0.2-1 parts liquid lipid, from 0.5 to 2.5 parts d-α-Tocopheryl polyethylene glycol 1000 succinate, from 0.7 to 3.5 parts polysorbate, from 0.00001 to 10 parts active substance, and water in the amount up to 100 parts.

2. A method for intracellular release of active substances according to claim 1, wherein the liquid lipid is selected from the group consisting of natural or synthetic red palm oil, rice bran oil, wheat germ oil or animal oils.

3. A method for intracellular release of active substances according to claim 2, wherein the liquid lipid comprises mono- or mix from isomers of Tocotrienol.

4. A method for intracellular release of active substances according to claim 1, wherein the polysorbate is selected from polysorbate 20, polysorbate 40, polysorbate 60 or polysorbate 80.

5. A method for intracellular release of active substances according to claim 2, wherein the polysorbate is polysorbate 40.

6. A method for intracellular release of active substances according to claim 1, wherein the active substance is drug substances, diagnostic agents, biological products, food supplements, cosmetic products or medical devices.

7. Method of production of solid lipid nanoparticle, according to claim 1, characterized by that, the carnauba vax, is mixed with a liquid lipid, d-α-Tocopheryl polyethylene glycol 1000 succinate, with a high content of tocotrienol, a polysorbate and an active substance the mixture obtained is melted by heating to 90o ± 2oC under stirred to receiving a homogeneous clear mixture, to which by stirring is added dropwise water, heated in advance to the same temperature and after that the dispersion obtained is cooled down gradually under stirring to 20o ± 2oC to forming the nanoparticle dispersion.

8. A method for intracellular release of active substances according to claim 1, wherein the in-vitro release for 24 h is less than 5%, and it occurs completely only after cellular internalization and subsequent in-place particle digestion.

9. A method for intracellular release of active substances according to claim 2, wherein the in-vitro release for 24 h is less than 5%, and it occurs completely only after cellular internalization and subsequent in-place particle digestion.

10. A method for intracellular release of active substances according to claim 3, wherein the in-vitro release for 24 h is less than 5%, and it occurs completely only after cellular internalization and subsequent in-place particle digestion.

11. A method for intracellular release of active substances according to claim 4, wherein the in-vitro release for 24 h is less than 5%, and it occurs completely only after cellular internalization and subsequent in-place particle digestion.

12. A method according to claim 1, wherein said target tissue is a nasal mucosa.

13. A method according to claim 1, wherein 4.1% or less of said active compound is released within 24 hours.

14. A method according to claim 1, wherein said liquid lipid comprises red palm oil with 30% tocotrienol, in an amount up to 20% of the total lipid.

15. A method according to claim 1, wherein said lipid nanoparticle is a solid lipid nanoparticle with spherical shape with a diameter of 15-35 nm.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0064] FIG. 1 is a XRD spectra of Menthol (1); carnauba wax (2); TPGS (3); of the composition from example 1B (B) and example 1C (C), which indicate the crystallinity of raw carnauba wax and loaded compositions, changes of its crystallinity but still preserving the ordered state.

[0065] FIG. 2 is a XRD spectra of placebo particles, of the composition from example 1A (A), and the particles, loaded with 0.1% Mometasone - example 1D (D), which indicate decrease of crystallinity of carnauba wax in placebo and loaded compositions.

[0066] FIG. 3 shows a TEM micrograph of placebo particles, composition from example 1A, which show spherical shape of particles with narrow size distribution in the range between 15 and 35 nm and their position in the space with no tendency to form aggregates.

[0067] FIG. 4 shows a TEM micrograph of particles, loaded with 0.01% Mometasone furoate- example 1D, which shows spherical shape of particles with narrow size distribution in the range between 15 and 35 nm and their position in the space with no tendency to form aggregates.

[0068] FIG. 5 shows diffraction of particles, loaded with 0.01% Mometasone furoate- example 1D, which corresponds to an amorphous structure. The halo around the bright spot in the center indicates that the electrons are diffracted randomly which is inherent for the amorphous materials. However, a limited degree of crystalline structure was proved in another test with XRD analysis of the SLNs.

[0069] FIG. 6 shows AFM micrograph of placebo particles, according to example 1A, which indicates that particles have spherical shape with narrow size distribution in the range between 15 and 35 nm. The particles are dispersed separately with no tendency to form aggregates.

[0070] FIG. 7 shows AFM micrograph of particles loaded with 0.03% Mometasone furoate, according to example 1E, which indicates that Mometasone furoate loaded particles have spherical shape with narrow size distribution in the range between 15 and 35 nm. The particles are dispersed separately with no tendency to form aggregates.

[0071] FIG. 8 shows AFM micrograph of particles loaded with 0.01% Mometasone furoate, according to example 1D, which indicate that Mometasone furoate loaded particles show spherical shape with narrow size distribution in the range between 15 and 35 nm. The particles are dispersed separately with no tendency to form aggregates.

[0072] FIG. 9 shows TMDSC of row carnauba wax and TPGS, according to example 1 with their characteristic isotherms.

[0073] FIG. 10 shows TMDSC of row Menthol (A) Loratadine (B) and Mometasone furoate (C), according to example 1 with their characteristic isotherms.

[0074] FIG. 11 is TMDSC of 0.1% Menthol lipid particles, according to example 1. The results show that after loading on the SLNs in appropriate concentrations, active substances’ melting peaks disappear. This is thought to be related either with their amorphization in result of molecule dispersion within the lipid matrix.

[0075] FIG. 12 is TMDSC of lipid particles loaded with 0.01% Mometasone furoate and 0.1% Loratadine, according to example 1F. The results show that after loading on the SLNs in appropriate concentrations, active substances’ melting peaks disappear. This is thought to be related either with their amorphization in result of molecule dispersion within the lipid matrix or dissolution of Loratadine in the liquefied lipid at temperatures above 82° C.

[0076] FIG. 13 represents TMDSC of physical mixture of the particle composition, according to example 1G. The heat flow shows lack of characteristic Loratadine isotherm. This is thought to be related with dissolution of Loratadine in the liquefied lipid at temperatures above 82° C.

[0077] FIG. 14 DLS graph of particle size distribution of composition 1A.

[0078] FIG. 15 shows DLS graph of particle size distribution of composition 1A′.

[0079] FIG. 16 shows DLS graph of particle size distribution of composition 1D.

[0080] FIG. 17 shows DLS graph of particle size distribution of composition 1D′.

[0081] FIG. 18 is a plot of added amount Mometasone furoate to the SLNs against measured absorption with determination of the maximum amount of the active substance in % that can be incorporated into the nanoparticles (i.e. loading capacity).

[0082] FIG. 19 represents Mometasone furoate release from SLNs. The kinetic study shows extremely low release profile for both the tested compositions. The lipase treated composition shows slightly higher but still neglible release.

[0083] FIG. 20 shows absorption of the dye in methanol and TE buffer (conc of the dye 1.10.sup.-5 M, conc of the nucleic acids 4 mkM).

[0084] FIG. 21 shows fluorescence spectra of Thiazole orange analog free in TE buffer, in presence of DNA and in presence of RNA.

[0085] FIG. 22 shows a viability cell count (HaCaT) after 30 min incubation with different formulations: TPGS 5%; TPGS 1 %( example 1A); compositions 1A; 3A and 1G.

[0086] FIG. 23 shows light photomicrographs of cross-sections from biopsy samples of treatment groups A, B and C., FIGS. (21a), (21b), (21c) from the right (R) and left (L) nasal vestibules, demonstrating the presence of normal stratified squamous epithelium. Respectively 21a L x400; 21a R x260; 21b L x260; 21b R x360; 21c L x360 and 21c R x300; bar 1 cm; H&E.

[0087] FIG. 24 shows a scheme of the wheal/treatment zones. HR - Head Right; HL - Head Left; MR -Middle Right; ML - Middle Left; TR - Tail Right; TL - Tail Left.

[0088] FIG. 25 shows evaluation of the effect of topical application of SLNs loaded with Loratadine, Fenistil gel and placebo SLNs on histamine induced wheals (mean, n=6).

[0089] FIG. 26 shows evaluation of the effect of intradermal injection of placebo SLNs on histamine induced wheals (mean ± SD, n=6)

[0090] FIG. 27 and FIG. 27A illustrate of cell internalization of SLNs loaded with fluorescence dye. Micrographs of Saccharomyces cerevisiae cells treated with dye solubilizate (2A) and dye loaded in SLNs (2B) according to example 2

EXAMPLES

[0091] Hereinafter, the present invention is described in more detail and specifically with reference to the Examples, which however are not intended to limit the present invention.

Examples 1. Preparation of Lipid Nanoparticles in Variants With and Without Active Substances, According to the Invention

A. Preparation of Placebo 1% Lipid Nanoparticles

[0092] For the preparation of placebo 1% lipid nanoparticles are used the following compounds:

TABLE-US-00002 Compounds Amount in w/w parts Carnauba wax 1.00 Red palm oil concentrate (30% tocotrienols) 0.2 d-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) 0.5 Polysorbate 40 0.7 Water up to 100

[0093] For the preparation of lipid nanoparticles are mixed Carnauba wax, Red palm oil concentrate, d-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) and Polysorbate 40. The mixture is heated up to 90° C. +/-2° C. to melt and stirred until homogeneous clear mixture is obtained. The needed amount of the water is heated up to 90° C. +/-2° C. and it is added dropwise to the homogeneous mixture obtained under stirring.

[0094] The obtained dispersion is cooled down under stirring to 20° C. +/-2° C. to give the nanoparticle dispersion.

A′. Preparation of Placebo 1% Lipid Nanoparticles With Pancreatic Lipase 200 UI/g

[0095] For the preparation of placebo 1% lipid nanoparticles with Pancreatic lipase 200 UI/g are used the following compounds:

TABLE-US-00003 Compounds Amount in w/w parts Carnauba wax 1.00 Red palm oil concentrate (30% tocotrienols) 0.2 d-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) 0.5 Polysorbate 40 0.7 Pancreatic lipase 200 UI/g Water up to 100

[0096] The lipid nanoparticles are obtained as per the procedure described in Example 1A. The calculated amount of pancreatic lipase is added into the cooled nanoparticle dispersion under steering.

B. Preparation of Placebo 1% Lipid Nanoparticles With 0.1% Oleic Acid

[0097] For the preparation of placebo 1% lipid nanoparticles with oleic acid are used the following compounds:

TABLE-US-00004 Compounds Amount in w/w parts Carnauba wax 1.00 Red palm oil concentrate (30% tocotrienols) 0.2 d-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) 0.5 Polysorbate 40 0.7 Oleic acid 0.1 Water up to 100

[0098] The lipid nanoparticles are obtained as per the procedure described in Example 1A. The calculated amount of oleic acid is added into the cooled nanoparticle dispersion under steering.

C. Preparation of 1% Lipid Nanoparticles With 0.1% Menthol

[0099] For the preparation of 1% lipid nanoparticles with Menthol are used the following compounds:

TABLE-US-00005 Compounds Amount in w/w parts Carnauba wax 1.00 Red palm oil concentrate (30% tocotrienols) 0.2 d-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) 0.5 Polysorbate 40 0.7 Menthol 0.1 Water up to 100

[0100] The lipid nanoparticles are obtained as per the procedure described in Example 1A. The calculated amount of Menthol is added into the cooled nanoparticle dispersion under steering.

D. Preparation of 1% Lipid Nanoparticles With 0.01 % Mometasone Furoate (Mometasone Furoate/Lipid = 0.83/100)

[0101] For the preparation of 1% lipid nanoparticles with Mometasone furoate are used the following compounds:

TABLE-US-00006 Compounds Amount in w/w parts Carnauba wax 1.00 Red palm oil concentrate (30% tocotrienols) 0.2 d-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) 0.5 Polysorbate 40 0.7 Mometasone furoate 0.01 Water up to 100

[0102] The lipid nanoparticles are obtained as per the procedure described in Example 1. The calculated amount of Mometasone furoate is added into the cooled nanoparticle dispersion under steering. D′. Preparation of 1% lipid nanoparticles with 0.01% Mometasone furoate and Pancreatic lipase 200UI/g

[0103] For the preparation of 1% lipid nanoparticles with Mometasone furoate and Pancreatic lipase are used the following compounds:

TABLE-US-00007 Compounds Amount in w/w parts Carnauba wax 1.00 Red palm oil concentrate (30% tocotrienols) 0.2 d-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) 0.5 Polysorbate 40 0.7 Mometasone furoate 0.01 Pancreatic lipase 200 UI/g Water up to 100

[0104] The lipid nanoparticles are obtained as per the procedure described in Example 1D.

E. Preparation of Lipid Nanoparticles With 0.03% Mometasone Furoate (Mometasone Furoate/Lipid = 0.83/100)

[0105] For the preparation of lipid nanoparticles with Mometasone furoate are used the following compounds:

TABLE-US-00008 Compounds Amount in w/w parts Carnauba wax 3.00 Red palm oil concentrate (30% tocotrienols) 0.60 d-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) 1.50 Polysorbate 40 2.7 Mometasone furoate 0.03 Water up to 100

[0106] The lipid nanoparticles are obtained as per the procedure described in Example 1D.

F. Preparation of Lipid Nanoparticles With 0.01% Mometasone Furoate and 0.1% Loratadine

[0107] For the preparation of lipid nanoparticles with Mometasone furoate and Loratadin are used the following compounds:

TABLE-US-00009 Compounds Amount in w/w parts Carnauba wax 1.00 Red palm oil concentrate (30% tocotrienols) 0.20 d-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) 0.50 Polysorbate 40 0.7 Mometasone furoate 0.01 Loratadin 0.10 Water up to 100

[0108] The lipid nanoparticles are obtained as per the procedure described in Example 1D.

G. Preparation of Lipid Nanoparticles With 0.1% Loratadine (Loratadine/Lipid = 8.3%)

[0109] For the preparation of a lipid nanoparticles with 0.1% Loratadine are used the following compounds:

TABLE-US-00010 Compounds Amount in w/w parts Carnauba wax 1.00 Red palm oil concentrate (30% tocotrienols) 0.20 d-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) 0.50 Polysorbate 40 0.7 Loratadin 0.10 Water up to 100

[0110] The lipid nanoparticles are obtained as per the procedure described in Example 1D.

Test Analyses of the Solid Lipid Nanoparticles, According to the Invention

Analysis of the Particle Size Structure and Morphology

[0111] This analysis has been performed by X-ray powder diffraction (XRD). Microstructural studies were performed using XRD with Cu-Kα radiation on a Bruker D8 Advance diffractometer with Cu-Kα radiation in θ-2θ geometry. The tests have been performed of 1 ml samples, which were freeze-dried and desiccated until testing.

[0112] Results from XRD analysis of ingredients of the placebo composition 1A are shown in FIG. 1, representing overplayed spectra of: methanol (1), carnauba wax (2); TPGS (3); composition of the solid lipid nanoparticles from example 1B (B); composition of the solid lipid nanoparticles example 1C (C).

[0113] Analyses of the particles structure and morphology has been performed also on placebo particles, 1A (A), and particles, loaded with 0.1% Mometasone, example 1D (D) and shown in FIG. 2.

[0114] Analysis of the obtained XRD spectra indicate that the crystallinity of raw carnauba wax is reduced markedly in placebo and loaded compositions, still preserving the ordered state. The crystallinity corresponding to carnauba component in particles decreases with the decrease in melting temperature of the added active substance (Mometasone furoate, mt 218-220° C.; Menthol, mt 31° C.; oleic acid, mt 13-14° C.).

Tests of Local Structure and Morphology

[0115] The tests are performed by:

[0116] A. Transmission electron microscopy (TEM). JEOL 2100 transmission electron microscope and a JEOL 2100 XEDS: Oxford Instruments, X-MAX.sup.N 80T CCD Camera ORIUS 1000, 11 Mp, GATAN at accelerating voltage of 200 kV. The analysis was carried out using the Digital Micrograph software.

[0117] Selected particle suspensions according to example 1 were diluted 500 x and 20 .Math.l were applied on standard holey carbon/Cu grids then left in a desiccator for 72 hours.

[0118] The tested samples of placebo (example 1A, FIG. 3) and 0.01% Mometasone furoate loaded particles (example 1D, FIG. 4) show spherical particles with narrow size distribution in the range between 15 and 35 nm. The particles are spread separately with no tendency to form aggregates.

[0119] The corresponding electron diffraction pattern is shown on FIG. 5. The diffraction pattern corresponds to an amorphous structure. The halo around the bright spot in the center indicates that the electrons are diffracted randomly which is inherent for the amorphous materials. However, a limited degree of crystalline structure was proved with XRD analysis of the SLNs.

B. Atomic Force Microscopy (AFM)

[0120] AFM imaging was performed on the NanoScope V system (Bruker Ltd, Germany) operating in tapping mode in air at room temperature.

[0121] Selected particle suspensions according to example 1 were diluted 100 x and 100 .Math.L were applied on mica support and spin-coated on Precision Spin Coater Model KW-4A (West Chester, PA, USA).

[0122] The tested samples of placebo (example 1A, FIG. 6), 0.01% (example 1D, FIG. 7) and 0.03% (example 1E, FIG. 8) Mometasone furoate loaded particles show spherical shape with narrow size distribution in the range between 15 and 35 nm. The particles are dispersed separately with no tendency to form aggregates.

[0123] C. Temperature modulated differential scanning calorimetry (TMDSC) was performed on DSC apparatus Q200, TA instruments, USA. The temperature calibration was performed with sapphire disc supplied by TA instruments in Tzero aluminum pans (TA instruments) in the desired temperature interval.

[0124] Row ingredients and compositions 1C, 1F and 1G were scanned (FIGS. 9-13). The samples were freeze-dried and desiccated until testing. The samples were tested in the Tzero pans from 20° C. to 250° C. with 2° C./min heating rate, modulation amplitude 1° C. and a period of 60 seconds under nitrogen flow (50 mL/min).

[0125] The results indicate that after loading on the SLNs in appropriate concentrations, active substances’ melting peaks disappear. This is thought to be related either with their amorphization in result of molecule dispersion within the lipid matrix or dissolution of Loratadine in the liquefied lipid at temperatures above 82° C.

Study of Z Average and Polydispersity by Dynamic Light Scattering (DLS)

[0126] The DLS analyses were performed on Zetasizer Nano ZSP,

[0127] Malvern Instruments, England and Malvern 4700 C, Malvern Instruments, England.

A. Analysis of Freshly Prepared Samples

[0128] The analysis was performed on fresh not filtered samples from compositions 1A, 1B, 1C, 1D. The particle size of the tested samples varies between 29.4 and 32.8. The results of size, polydispersity and z- potential of tested SLNs dispersions received are indicated in the Table 2.

TABLE-US-00011 Size, polydispersity and z-potential of tested SLNs dipsersions Measured components 1A 1A′ 1B 1C 1D 1D′ Size (nm) 32.8 30.8 29.4 33.6 30.9 27.8 Polydispersity 0.399 0.452 0.255 0.321 0.398 0.375 Z potential (mV) -18.5 - -33.5 -30.4 - - “-” - mean “not tested”

[0129] The size and % distribution of tested SLNs dispersions of the laboratory technology produces particles with bimodal size distribution are indicated in Table 3. The yield of the particles with size range between 20 nm and 30 nm (peak 1) is over 95%.

TABLE-US-00012 The size and % distribution of tested SLNs dispersions of the laboratory technology produces particles with bimodal size distribution Sample Size distribution % distribution 1A Peak 1:20.3 nm Peak 2:87.6 nm Peak 1:97.7 Peak 2:2.3 1D Peak 1:21.1 nm Peak 2:90.1 nm Peak 1:96.9 Peak 2:3.1

B. Analysis of Samples After Filtration

[0130] Samples from compositions from Examples 1A, 1B and 1C were analysis after filtration trough filter 0.22 .Math.m. Filtered samples have narrower size distributed in monomodal pattern.

[0131] The results of the analysis of size and polydispersity after filtration trough 0.22 .Math.m filter are indicated in the Table 4.

TABLE-US-00013 The results of the analysis of size and polydispersity after filtration trough 0.22 .Math.m filter Measured parameters 1A 1B 1C 1D Size(nm) 19.3 22.2 22.6 18.5 Polydispersity 0.0117 0.0045 0.0088 0.0108

C. Analysis of Samples After Incubation With Pancreatic Lipase

[0132] Samples 1A, 1A′, 1D and 1D′ were compared. After preparation samples 1A′ and 1D′ were incubated at 37° C. for 24 h before testing.

[0133] A slight size reduction was observed in 1A′ and 1D′ when compared with the corresponding sizes of 1A and 1D (see Table 2 and FIGS. 14-17). A lack of the second peak at both samples 1A′ and 1D′ was also detected. Probable explanation for the noted differenc l es can be found in the difference in produced lot quantities: 1A and 1D were prepared each in 50.00 g dose; 1A′ and 1D′ were prepared each in 200.00 g dose, and had undergone slower cooling of the hot microemulsion at stage C of their production.

D. Formation of Protein Corona

[0134] The test was conducted to study the possible affinity of the SLNs of the present invention to form protein corona with bovine serum albumin (BSA). Formation of protein corona is a sign of affinity of particles to adsorb body proteins. Such complexes possess different ability to move through membranes due to increased size and changed surface structure and charge. Also, particles with protein corona can become immunogenic. Lipid nanoparticles generally have the affinity to form protein corona with soluble body proteins.

Methodology

[0135] The experiment was conducted with sample 1A. 5 ml of dispersion 1A was added to 25 ml volumetric flasks inscribed “1A” and another 5 ml of dispersion 1A was added to a 25 ml volumetric flask inscribed “1A+BSA” respectively. Flask 1A was diluted to the mark with ultrapure water and homogenized; 5% w/v solution of bovine serum albumin in ultrapure water was added to the mark of the flask 1A+BSA, homogenized and incubated at 37° C. for 2 hours before the test.

[0136] The results are shown in table 6. No protein corona was detected. The slight increase in particle size in sample 1A+BSA with less than 1 nm here is thought to be influenced by the slight increase in viscosity of the 1A+BSA. Thus, the determination based on the DLS analysis result in a bigger hydrodynamic diameter corresponding to the slower fluctuations of the particles in the more viscous medium.

[0137] The determination of frequency shifts and ensuing calculation to obtain particle size in the DLS analysis is based on the Stokes-Einstein equation: D.sub.H = kT/3πηD [0138] D.sub.H = hydrodynamic diameter [0139] k = Boltzmann constant [0140] T= absolute Temperature [0141] η = dynamic viscosity [0142] D = Diffusion coefficient

[0143] The lack of protein corona in spite of the negative charge of the particles can be explained with the high lipophilicity of the SLNs and steric stabilization by the surfactants (TPGS; polysorbate 40).

E. Stability Studies

[0144] Stability was studied with respect to tendency of aggregates formation. Changes in particle size were studied over a period of 24 months. Samples from compositions 1A and 1D were prepared and to each were added 0.5% EDTA disodium salt as a microbial preservative. Samples were filtered through 0.22 .Math.m filter and filled in brown glass bottles and stored in dark place at room temperature. Samples were tested on the 0.sup.th, 12.sup.th and 24.sup.th month.

[0145] The results from Size and polydispersity index of tested SLNs dispersions are indicated in the Table 5.

TABLE-US-00014 The results from Size (polydispersity) of tested SLNs dispersions Sample 0.sup.th month 12.sup.th month 24.sup.th month 1A 19.3 (0.352) 19.9 (0.364) 21.4 (0.373) 1D 18.5 (346) 19.4 (0.355) 21.3 (0.358)

[0146] The results indicate neglible increase in particle size in both tested samples with time. No aggregates were formed during the test period.

Entrapment Efficiency and In-Vitro Dissolution Kinetics

1. Entrapment Efficiency

[0147] A. The Entrapment efficiency relates to the % drug that is successfully entrapped into nanoparticles. It is calculated as follows: %EE = [(Drug added - Free “unentrapped drug”)/Drug added] *100.

Methodology

[0148] The test is conducted with freshly prepared, not filtered composition 4D. 5 ml of the tested dispersion were placed in a Dialysis Membrane Tubing 3500 Dalton MWCO 10 mm diameter and 80 mm long, pre-hydrated for 24 h. The membrane is permeable for the active substance, but not for the particles. The membrane is gently folded and placed into 15ml centrifuge tube and pinched with the polypropylene cap. The samples are centrifuged at 2000x rpm for 20 min. Aliquots from the filtrate were analysis on Cary UV-Vis spectrophotometer, Agilent Technologies for Mometasone furoate content according to a developed analytical method.

[0149] The %EE is 98.8.

B. Active Substance Loading

[0150] The drug loading relates to the maximum amount of the active substance, % that can be incorporated into the nanoparticles.

Methodology

[0151] Cary UV-Vis spectrophotometer, Agilent Technologies was used for determination of Mometasone furoate content according to a developed analytical method.

[0152] Series of compositions derived from composition 1D with concentration of the loaded active substance ranging from 0.0025% w/w/ to 0.0200% w/w were used for the experiment.

TABLE-US-00015 Compounds 1D1 1D2 1D3 1D 1D4 1D5 1D6 1D7 Carnauba wax 1.00 Red palm oil concentrate (30% tocotrienols) 0.2 d-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) 0.5 Polysorbate 40 0.7 Mometasone furoate 0.0025 0.0050 0.0075 0.01 0.0125 0.0150 0.0175 0.0200 Water to 100

[0153] Mometasone furoate have water solubility 0.00523 mg/mL, which makes it practically insoluble. The compositions were prepared according to the technology, described in example 2. After obtaining the samples were stored for 24 h in refrigerator at 4° C. to allow equilibrium between dissolved/undissolved states of Mometasone furoate to be reached. Before analysis the samples were filtered through 0.45 .Math.m filter. The samples were diluted appropriately if necessary and the absorption spectra were taken. The used concentrations were plotted on a graph against the measured absorptions (FIG. 18). The system reaches equilibrium at 1.34×10.sup.-2% which well corresponds with the visual change of the dispersions with the appearance of sediment in concentrations of 1.5 ×10.sup.-2% and above.

2. Dissolution of 0.01% Mometasone Furoate Loaded Particles According to 1D

[0154] This example gives another demonstration of the ability of SLNs of the present invention to keep the encapsulated substance “locked” without releasing it in an in-vitro dissolution experiment. In a separate experiment, the influence of added pancreatic lipase on the drug release was investigated. The tests were designed for the purpose and consist of a donor phase of the particle suspension loaded with active substance and acceptor phase, separated with a dialysis membrane.

Methodology

[0155] In separate experiments the test was conducted with freshly prepared not filtered compositions 1D and 1D′. 5 ml of the tested dispersion were placed in a Dialysis Membrane Tubing 3500 Dalton MWCO 10 mm diameter and 80 mm long, pre-hydrated for 24 h. The membrane is permeable for the active substance, but not for the particles and lipase. The membrane is sealed both sides and submerged in a beaker glass with 250 ml ultrapure water with 0.1% Polysorbate 40 under constant steering and thermostated at 37° C. Samples were collected at 0 min, 15 min, 30 min, 60 min, 120 min, 240 min, 480 min, 960 min, and 1440 min. The samples were evaporated under nitrogen and the residue dissolved in methanol. The obtained solutions were analysis on Cary UV-Vis spectrophotometer, Agilent Technologies.

[0156] The kinetic study (FIG. 19) shows extremely low release profile for both the tested compositions. The lipase treated composition shows slightly higher release. At the 24 h released Mometasone from 1D and 1D′ was 2.2% resp. 4.1%. These values are thought to origin both from the very low dissolution profile of the particles and from the free/unincluded Mometasone furoate.

Example 2. Particles Internalization Into Cells

[0157] This experiment was built on the hypothesis that if the particle does not release the dye trough passive diffusion or leak, the only way the dye to meat and form a fluorescent complex with cell RNA is by particle enzymatic erosion and free dye release.

[0158] To do this experiment the yeast Saccharomyces cerevisiae (S. cerevisiae) has been chosen as one of the most widely used eukaryotic model organisms.

[0159] Dye Thiazole orange (TO) derivative with high affinity to form fluorescent complexes with RNA was synthesized for the purpose. The dye is highly lipophilic and once loaded in the SLNs, or solubilized, it’s is not fluorescent. Due to its positive charge the dye is not permeable through the cell wall. The only way the dye can enter the cell is to be loaded on a transport carrier, such as SLNs. The ways of SLNs internalization into the cells is by diffusion through the cell pores by endocytosis.

[0160] The experiment is also informative for possible leak or passive diffusion of the dye from the SLNs. Such pre-internalization release should result in fluorescence incidents outside the cells with RNA secreted with exosomes. Secretion of extracellular vesicles is part of the physiology of Saccharomyces cerevisiae.

[0161] TO analogues do not fluoresce in the free state in solution. Fluorescence arises when rotation about the monomethine bridge between the two heterocyclic moieties is somehow restricted [Carlsson, C.; Larsson, A.; Jonsson, M.; Albinsson, B.; Norden, B. The Journal of Physical Chemistry, 1994, 98, 10313-21.]. Such a restriction occurs when TO derivatives bind to nucleic acids by intercalation between the base pairs [Netzel, T. L.; Nafisi, K.; Zhao, M.; Lenhard, J. R.; Johnson, I. The Journal of Physical Chemistry, 1995, 99, 17936-47;Nygren, J.; Svanvik, N.; Kubista, M. Biopolymers, 1998, 46, 39-51.] or presumably between individual bases in single-stranded nucleic acids and in both cases a dramatic increase of the fluorescence is observed.

##STR00001##

[0162] For the present test Thiazole orange analogue was used were R.sup.1, R.sup.2, R.sup.3 and R.sup.4 are alkyl or substituted alkyl.

[0163] To understand the reason for the observed fluorescence in the cell’s cytoplasm an in vitro experiment was made with the TO analog in the absence and in the presence of salmon sperm double stranded DNA (dsDNA) and transfer RNA (tRNA) from bovine liver (Sigma-Aldich).

[0164] The absorption of the dye in methanol and TE buffer (conc. of the dye 1.10.sup.-5 M, conc of the nucleic acids 4 mkM) is indicated in FIG. 19 and the fluorescence spectra of Thiazole orange analog free in TE buffer, in presence of DNA and in presence of RNA is indicated in FIG. 20

[0165] It was demonstrated that the dye is highly specific to RNA.

Tested Compositions

[0166] Particles loaded with fluorescence dye in the following compositions:

TABLE-US-00016 A Solubilizate of dye Compound Amount (w/w parts) Polysorbate 40 0.70 DYE 0.00001 Water to 100.00 B Particles, loaded with dye Compound Amount (w/w parts) Carnauba wax 1.00 Red palm oil concentrate (30% tocotrienols) 0.20 d-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) 0.50 Polysorbate 40 0.70 DYE 0.00001 Water to 100.00

Methodology

[0167] The particles were obtained according to the procedure according to Example 1.

[0168] Saccharomyces Cerevisiae cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 100 units/ml penicillin and 100 .Math.g/ml streptomycin at 37° C. and 5% CO2. For micro-irradiation and image acquisition cells were plated in MatTek glass bottom dishes (~40% confluence), then cultured for 24 h, washed with PBS and supplemented with 2 ml FluoroBrite DMEM medium containing 10% FBS and 2 mM GlutaMAX. Before imaging 100 .Math.l lipid particles was added. Image acquisition was performed on an Andor Revolution system using Nikon 60x (NA1.2) water immersion objective and iXon897 EMCCD camera. Images were acquired in two channels - 488 nm laser excitation in 7 Z planes with 0.5 .Math.m plane spacing and single plane in bright-field DIC.

[0169] The dye has been synthesized for the purpose and possesses high lipophilicity and strong emission after binding to nucleic acids. The captures from defined time points up to the end of the experiment (13.sup.thh) are presented in FIG. 27 and FIG. 27A

[0170] The dye solubilizate gives very low diffuse emission with no detected emission spots within the examined cells. The absence of emission is illustrative for inability of the dye to enter the cell in spite of the presence of polysorbate 80, known solubilizer and absorption enhancer (see picture set 2A). Solubilizate also doesn’t give extracellular fluorescent complexes with RNA most probably because of molecule associations within surfactant micelles rather than free incorporated molecules. As can be observed from the picture set in column 2B, 60 min after the addition of nanoparticle suspension, the fluorescence within cells progressively increased until the end of the experiment (13 hours). As far the emission time is limited (less than millisecond) in result of the fluorescence decay the increasing intensity of the recorded multiple light spots (incidents) within the Saccharomyces cerevisiae cells and the lack of fluorescence outside the cell walls can be explained with slow particle enzymatic degradation and subsequent free dye release. No pre-internalization dye release was detected.

Example 3. Safety and Toxicity Evaluations

1. Toxicity on Cells

[0171] The spontaneously immortalized human HaCaT keratinocyte cell line was maintained in a 70 % monolayer culture in a Dulbecco’s Modified Eagle’s Medium (DMEM-F12; Lonza) supplemented with 10% fetal bovine serum (FBS; Lonza). Both cell lines were maintained in a humidified atmosphere containing 5 ± 1%CO2 in air (standard culture conditions) at 37° C.

[0172] Samples 1G, 2A and 2B have been tested. The cytotoxicity of the lipid nanoparticles was assessed following 30 min exposure and staining with Trypan blue using a Countess Automated cell counter (Invitrogen).

[0173] All tested samples showed no observable effects on viability of HaCaT cells. Interestingly, the increase in lipid concentration from 1% to 5% (1A to 2A) led to a decrease in toxicity on HaCaT cells. A possible explanation can be related with antioxidant and nutritive value of the lipid composition.

[0174] Additional tested simple have the following compositions:

TABLE-US-00017 3A Placebo 5% lipid phase) Compounds Amount (w/w parts) Carnauba wax 5.00 Red palm oil concentrate (30% tocotrienols) 1.00 d-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) 2.50 Polysorbate 40 3.50 Water To 100.00

TABLE-US-00018 3B 0.1% Loratadine (6% lipid phase) Compounds Amount (w/w parts) Carnauba wax 5.00 Red palm oil concentrate (30% tocotrienols) 1.00 d-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) 2.50 Polysorbate 40 3.50 Loratadine 0.1 Water To 100.00

[0175] Viability cell count (HaCaT) after 30 min incubation with different formulations are indicated in FIG. 20.

Example 4. Evaluation of Nasal Epithelium Safety/Toxicity

[0176] As far as all the ingredients in the composition of the particle core of the present invention are Pharmacopoeial (EP, USP) and/or GRASS listed by FDA, the possible toxicity and/or safety concerns can arise from the particle size itself and from the expected high intracellular drug concentrations. Nasal mucosa has been chosen as a potential place for application of the SLNs of the current invention and for its delicate structure.

Animals

[0177] The use of animals in example 4 was authorized by the Local Animal Ethics Committee at veterinary faculty of Trakia University, Stara Zagora, Bulgaria, with protocol 38/05.12.2013. Ten male and ten female healthy New Zealand rabbits were selected. The temperature in the room was kept in the range 20° C. +/- 2° C. The animals were fed on standardized normal diet, water ad libitum and kept in individual cages. Seven days for adaptation were given to the animals after their receiving. At the beginning of the tests the weight of males was 3.248 ± 0.321 kg and the weight of the females 3.452 ± 0.483 kg.

[0178] The animals were divided in three groups: [0179] group A (4 males and 3 females) was treated with Allergodil nasal spray; [0180] group B (4 females and 3 males) with the test formulation LORNP; [0181] group C (3 males and 3 females) was treated with sodium chloride sol 0.9 %.

[0182] 100 .Math.l of the respective formulation was sprayed daily into the left nostril for 30 days. The right nostril was left untreated.

[0183] At the day of the last administration all animals were anaesthetized with i.m. injection of ketamine (35 mg/kg) and xylazine (5 mg/kg).

[0184] Biopsy samples from the left and right nostrils were obtained using a 2 mm punch biopsy needle from the lateral wall of the rostral portion of the dorsal nasal meatus, which corresponds to the approximate area of the sprayed drug.

[0185] The preparations were fixed in 10% neutral buffered formalin. The tissue samples were then processed routinely and imbedded in paraffin blocks. Tree-micron-thick sections were sliced, mounted on glass slides and stained with hematoxylin and eosin (H&E). The biopsy samples from the untreated right nostrils served as control in each experimental group. The slides were examined under light microscope. Each slide was inspected for integrity and presence of pathological alterations associated with irritation and toxicity the epithelial and sub-epithelial layers confined in the samples.

[0186] Microscopy evaluation of nasal biopsy samples from treatment groups A, B and C in the samples from each group were presented from normal wavy and slightly keratinized squamous stratified epithelium (FIG. 21) and small portions of transitional pseudo stratified ciliated epithelium (not shown). The sub-epithelial layer consisted of loose connective tissue with occurrence of numerous cavernous veins and tubular serous glands supported by a hyaline cartilage.

[0187] The microscopic examination of biopsies showed no pathological changes in the nasal epithelial membrane and its adjacent layers in any sample. When the samples were compared no significant difference was detected in the findings between treatment groups.

[0188] Light photomicrographs of cross-sections from biopsy samples of treatment groups A, B and C., FIGS. (21a), (21b), (21c) from the right (R) and left (L) nasal vestibules, demonstrating the presence normal stratified squamous epithelium. Respectively 21a L x400; 21a R x260; 21b L 260 x; 21b R360; 21c L x360 and 21c R x300; bar 1 cm; H&E.

Example 5. Evaluation of Eye Safety/Toxicity

[0189] The eye test was conducted to test the safety of the SLNs on eye tissues integrity. The Draize test was used as well established for evaluation of toxicity of chemical agents. The specificity of the current experiment is that all the ingredients of the compositions are well studied for local toxicity, they are Pharmacopoeial (EP, USP) and/or Grass listed by FDA, and so the test aims to evaluate the safety of the particle size.

A. Draize Test

[0190] Draize test is internationally recognized for estimation the local toxicity and uses standardized protocol for instilling agents onto the cornea and conjunctiva of laboratory animals. A sum of ordinal-scale items of the outer eye gives an index of ocular morbidity. The test serves to study the integrity and condition of the eye after exposure to examined agent and is based on “activation” of fluorescein by damaged tissue.

[0191] 24 h before the experiment the condition of eyes of all animals were inspected.

[0192] Two males and one female animal received single dose of 100 .Math.l particle dispersion according to example 3B in the right eye. Both eyes of the animals were inspected at 24, 48 and 72 hours intervals and rated according to the scale in table 6.

[0193] No observable changes in rabbit cornea, conjunctiva or iris have been observed.

B. Modified Draize Test

[0194] 24 h before the experiment the condition of eyes of all animals were inspected.

[0195] Three male and three female animals received 100 .Math.l particle dispersion, according to example 3B in the right eye on a daily manner for 14 days. Both eyes were inspected at 24, 48 and 72 hours intervals after the last treatment and rated according to the scale in table 6.

[0196] No observable changes in rabbit cornea, conjunctiva or iris have been observed.

TABLE-US-00019 Evaluation of symptom score according to the “Scale of Weighted Scores for Grading the Severity of Ocular Lesions” * Group Single treatment 14 days treatment Animal 1 2 3 4 5 6 7 8 9 24h after last administration I. Cornea A: 0 0 0 0 0 0 0 0 0 B: 0 0 0 0 0 0 0 0 0 II. Iris A: 0 0 0 0 0 0 0 0 0 III. Conjunctiva A: 0 0 0 1 0 0 0 1 0 B: 0 0 0 0 0 0 0 0 0 C: 0 0 0 0 0 0 0 0 0 48h after last administration I. Cornea A: 0 0 0 0 0 0 0 0 0 B: 0 0 0 0 0 0 0 0 0 II. Iris A: 0 0 0 0 0 0 0 0 0 III. Conjunctiva: A: 0 0 0 1 0 0 0 0 0 B: 0 0 0 0 0 0 0 0 0 C: 0 0 0 0 0 0 0 0 0 72h after last administration I. Cornea: A: 0 0 0 0 0 0 0 0 0 B: 0 0 0 0 0 0 0 0 0 II. Iris A: 0 0 0 0 0 0 0 0 0 III. Conjunctiva A: 0 0 0 0 0 0 0 0 0 B: 0 0 0 0 0 0 0 0 0 C: 0 0 0 0 0 0 0 0 0

TABLE-US-00020 Scale of Weighted Scores for Grading the Severity of Ocular Lesions *(Draize J, Woodard G, Calvery H. 1944. Methods for the study of irritation and toxicity of substances applied topically to the skin and mucous membranes. J Pharm Exp Ther 82:377-390.) Cornea Lesion Score 1 A. Opacity - Degree of density (area which is most dense is taken for reading) Scattered or diffuse area - details of iris clearly visible 1 Easily discernible translucent areas, details of iris slightly obscured 2 Opalescent areas, no details of iris visible, size of pupil barely discernible 3 Opaque, iris invisible 4 B. Area of cornea involved One quarter (or less) but not zero 1 Greater than one quarter but less than one half 2 Greater than one half but less than three quarters 3 Greater than three quarters up to whole area 4 Score equals A x B x 5 Total maximum = 80 Iris Lesion Score 1 A. Values Folds above normal, congestion, swelling, circumcorneal injection (any one or all of these orcombination of any thereof), iris still reacting to light (sluggish reaction is positive) 1 No reaction to light, hemorrhage; gross destruction (any one or all of these) 2 Score equals A x 5 Total possible maximum = 10 Conjunctiva Lesion Score 1 A. Redness (refers to palpebral conjunctiva only) Vessels definitely injected above normal 1 More diffuse, deeper crimson red, individual vessels not easily discernible 2 Diffuse beefy red 3 B.Chemosis Any swelling above normal (includes nictitating membrane) 1 Obvious swelling with partial eversion of the lids 2 Swelling with lids about half closed 3 Swelling with lids about half closed to completely closed 4 C. Discharge Any amount different from normal (does not include small amount observed in inner canthus of normal animals 1 Discharge with moistening of the lids and hairs just adjacent to the lids 2 Discharge with moistening of the lids and considerable area around the eye 3 .sup.1 The maximum total score is the sum of all scores obtained for the cornea, iris and conjunctiva. Scores of 0 are assigned for each parameter if the cornea, iris, or conjunctiva is normal.

[0197] No toxicity or irritation was observed after single and 14-day-application. No difference between the treated and untreated eyes of all animals was detected. Very low score in the Draize test (practically zero) was established for both 5A and 5B tests.

Example 6. Efficacy of Particle Dispersion, Loaded With 0.1% Loratadine on Histamine Induced Wheals

A. Evaluation of the Effect of Topical Application of SLNs Loaded With Loratadine on Histamine Induced Wheals

[0198] Rabbits weighting 4.183 ± 0.421 kg (males) and 4.661 ± 0.542 kg (females) were used for the test. The day before the test a zone sized 10 × 7 cm was shaved on the back of each rabbit with electric razor. At the day of the test rabbits with skin defects were excluded.

[0199] Six zones each sized 30×30 mm were sketched on the shaved area (FIG. 22).

[0200] Four males and two females were pretreated with the test formulations.

[0201] Zone HL was treated with 100 .Math.l Fenistil gel, zone TL was treated with 100 .Math.l 3A, zone TR was treated with 100 .Math.l 3B and the other zones were left untreated. After 15 minutes the formulations were gently cleaned with cotton and water and intradermal injections of 40 .Math.l histamine dihydrochloride solution (1 mg/ml) were applied to zones HL(Fenistil gel), HR (untreated control), TL(3A) and TR (3B). Zone MR was injected with 40 .Math.l sodium chloride 0.9% and zone ML was only pricked.

[0202] The reaction towards histamine was photographed with digital camera at 1.sup.th, 5.sup.th, 10.sup.th, 15.sup.th, 20.sup.th, 25.sup.th, 30.sup.th and 60.sup.th minute post injections of histamine solution. The size of the wheals was determined on the obtained images with the use of ImageJ software. The results are presented as ratio between the size of the control from zone HR to the size of the wheal from tested zone (FIG. 25).

[0203] The test SLNs dispersion loaded with 0.1% Loratadine (3A) showed faster and stronger antihistamine effect compared to the marketed product Fenistil gel. Surprisingly, the placebo treated wheals were bigger in diameter compared to the control. Still, placebo treated wheals were sensibly softer and diffuse then the control treated ones which leads to the assumption that placebo SLNs alone contribute for faster resolution of the wheals. After the application of histamine injection, the size of wheals, pretreated with Fenistil gel, grew in a slower manner within the time before the 5.sup.th min, compared to composition 3A. The probable reason can be finding in the full molecular availability compared to the “locked” Loratadine within the SLNs. However, after the 1.sup.st min. the composition 3A expressed a sharp incline and on the 5.sup.th min showed twice the effect of Fenistil gel.

B. Effect of Intradermal Injection of Placebo SLNs on Histamine Induced Wheals

[0204] Studying the effect of topical application of placebo on the size and texture of the wheals, further co-administration experiment was conducted of placebo SLNs to the histamine intra dermal injection.

[0205] Rabbits weighting 4.183 ± 0.421 kg (males) and 4.661 ± 0.542 kg (females) were used for the test. The day before the test a zone sized 10 × 7 cm was shaved on the back of each rabbit with electric razor. At the day of the test rabbits with skin defects were excluded.

[0206] Six zones each sized 30×30 mm were sketched on the shaved area (FIG. 24).

[0207] Five males and one female were selected for the experiment. Zones HR and TR were treated with intra dermal injection of mixture 1:1 of histamine solution and sodium chloride. Zone HL was treated with mixture of 1:1 of histamine solution (2 mg/ml):3A.

[0208] The reaction towards histamine was photographed with digital camera at 5th, 10th, 15th, 20th, 25th, 30th and 60th minute post injections of histamine solution. The size of the wheals was determined on the obtained images with the use of Image J software. The results were presented as ratio between the sizes of the control from zone HR to the size of the wheal from tested zone (FIG. 26).

[0209] Surprisingly, the wheals were smaller in the group treated with histamine solution/3A. This comes to show that placebo SLNs doesn’t increase and in fact decrease the effect of injected histamine, thus ensuring their safety use as carriers. It is likely that the combination of particle ingredients, namely, carnauba fatty acid esters and acids, tocotrienols and tocopherols possess notable antiinflammatory effects.