1. Nanoparticles comprising Berberine or its salt and polycaprolactone.

2. The nanoparticles as claimed in claim 1 wherein Berberine or its salt is present in an amount of 0.33%w/w and polycaprolactone is present in an amount of 0.33-0.60%w/w.

3. The nanoparticles as claimed in claim 1 wherein Berberine salt is selected from chloride, phosphate, diphosphate, acetate, sulfate or citrate salt.

4. The nanoparticles as claimed in claim 1 wherein the said nanoparticle has a particle size between of 100-150 nm.

5. A process for preparing nanoparticles as claimed in claim 1 said process comprising (i) dissolving polycaprolactone in a suitable organic solvent to obtain a solution; (ii) dissolving Berberine or its salt in a suitable organic solvent to obtain a solution; (iii) mixing the solution of step (i) and (ii) to obtain an organic phase; (iv) dissolving a stabilizer in water to obtain an aqueous phase; (v) adding the organic phase dropwise to the aqueous phase under continuous stirring and sonication; (vi) evaporating the organic phase to obtain a suspension; and (vii) sonicating the suspension to obtain the nanoparticles.

6. The process as claimed in claim 5 wherein the suitable organic solvent is selected from methanol, ethanol, chloroform, acetone, benzene, hexane, ethyl acetate, dichloromethane, toluene, acetonitrile, carbon tetrachloride, isopropanol, hexane or cyclohexane.

7. The process as claimed in claim 5 wherein the stabilizer is selected from Tween 80, tween 20, Span, Ethyl cellulose and Eudragit.

8. A composition comprising the nanoparticles as claimed in claim 1.

9. The composition as claimed in claim 8 wherein the said composition is an oral, parenteral, topical, transdermal or Transungual composition.

10. The composition as claimed in claim 8 wherein the said composition is a pharmaceutical, neutraceutical, personal care or cosmetic composition.

11. The composition as claimed in claim 8 wherein the said composition is a transungual composition.

12. The composition as claimed in claim 8 wherein the said cosmetic composition is a nail lacquer. Dated this the 5th day of November 2024.

Description:Field of the invention
The present invention relates to nanoparticles for enhanced delivery of active. In particular, the present invention provides nanoparticles for transungual delivery of Berberine and its salts. More particularly, the present invention provides Berberine loaded nanoparticulate nail lacquer formulation for enhanced delivery of active. The nanoparticles provide enhanced penetration, stability and antifungal activity.

Background of the invention
Berberine is a phytoconstituent of Berberis aristata belonging to family Berberidaceae. It possesses broad spectrum antimicrobial properties, and is effective against infection caused by Candida. It acts by binding with its cell membrane. It has been reported to have antimicrobial, antibacterial, antifungal activity and various approaches have been used to increase its bioavailability.

Mishra et al in Curr Drug Res Rev;. 2024;16(3):412-422 discloses oleic acid-containing berberine-hydrochloride-loaded transethosomal gel for effective treatment of dermatophytosis by Trichophyton rubrum. Berberine- hydrochloride-loaded transethosomal gels were fabricated using the hot homogenization method, followed by the incorporation of transethosomes into the gel-based system using carbopol 934. It exhibited controlled ex-vivo permeation of berberine-hydrochloride over 24 hr through excised rat skin, and CLSM confirmed deeper penetration into skin layers.

Ghareeb et al in BMC Pharmacology and Toxicology volume 24, Article number: 42 (2023) discloses Berberine-loaded albumin nanoparticles for reversing aflatoxin B1-induced liver hyperplasia. The nanoparticles were prepared by desolvation method.

IN511510 discloses berberine-loaded gelatin film for improvement of skin permeation The composition comprises i) gelatin in the range of 5 to 15%, ii) berberine in the range of 5 to 15 %, iii) solvent in the range of 50 to 60 % and iv) polymer in the range of 5 to 15%. . Preparation has been done by i) dissolving gelatin and polymer, separately in hot water with constant stirring to prepare homogeneous solutions, ii) blending the solutions with berberine to obtain a mixture, iii) casting the mixture onto the covered glass plate, followed by drying at room temperature to obtain a dried film, and iv) peeling off the dried film from the silicon cloth, followed by cutting into small pieces to obtain a berberine-loaded gelatin film.

CN103083249A discloses berberine chitosan drug-carrying microsphere. The microspheres are prepared by salting out process and have been reported for their effectiveness against bacterial infections such as bacillary dysentery and gastroenteritis.

WO2017027971A1 discloses transdermal formulations for delivery of berberine compounds, and their use in the treatment of berberine-responsive diseases and conditions such as Type I diabetes, Type 2 diabetes, pre-type I diabetes, pre-type 2 diabetes, hyperlipidemia.

WO2015044669A1 discloses Antifungal topical composition comprising a polymer and an antifungal agent. The polymer is polyhexamethylene biguanide and berberine is one of the antifungal agents.

Although the literature discloses many formulations of Berberine and various approaches have been reported to increase its bioavailability, considering the wide activity spectrum of berberine, still there is a need for development of more efficient formulations. In particular, there is need for more effective topical compositions. Since topical compositions, for example formulations, are subjected to regular washing, it is desired that the compositions should be water resistant so that the active present therein does not get leached out. Also, it should be stable and provide desired antibacterial activity for the desired treatment regime period.
Therefore, the objective of the present study is to develop berberine-loaded nanoparticles and incorporate them into a polymer-based lacquer formulation to enhance the transungual delivery of berberine.

Object of the invention
It is an object of the present invention to provide Berberine loaded nanoparticulate nail lacquer formulation for transungual delivery.

It is another object of the present invention to provide antimicrobial nanoparticles.

It is another object of the present invention to provide antimicrobial nanoparticle composition having enhanced antimicrobial efficacy.

It is another object of the present invention to provide a process for the preparation of antimicrobial nanoparticles.

It is another object of the present invention to provide antimicrobial nanoparticles composition suitable for transungual delivery.

It is another object of the present invention to provide antimicrobial nanoparticles composition wherein the composition is water resistant.

It is another object of the present invention to provide antimicrobial nanoparticles composition wherein the composition is stable.

Summary of the invention
Accordingly, the present invention provides nanoparticles comprising Berberine or its salt and polycaprolactone.

In one embodiment the present invention provides a process for preparing nanoparticles comprising Berberine or its salt and polycaprolactone.

In one embodiment the present invention provides an antimicrobial composition comprising nanoparticles of Berberine or its salt and polycaprolactone.

In yet another embodiment the present invention provides antimicrobial composition nanoparticles of Berberine or its salt and polycaprolactone wherein the said composition is effective for the treatment of Onychomycosis.

Brief description of drawings
Figure 1: Cumulative permeation profile of berberine chloride (BBR), berberine loaded nail lacquer (BNLT) and berberine nanoparticle loaded nail lacquer (BNNL) respectively.
Figure 2: (a) TEM micrograph of berberine chloride loaded nanoparticles (b) nanoparticles loaded in nail lacquer.
Figure 3: Zone of Inhibition of (a) vehicle control, (b) BNP (c) BNLT and (d) BNNL respectively.
Figure 4: Micrograph of histopathological studies of (a) blank (b) control (c) optimised nail lacquer and (d) nano nail lacquer respectively.

Detailed description of the invention with illustrating embodiments and examples
The present invention relates to Berberine nanoparticles and a topical composition comprising the same. The nanoparticles of the present invention provide high drug loading and have enhanced efficacy. When these nanoparticles are incorporated into a formulation, the formulation also provides enhanced efficacy and better bioavailability. The formulations of the present invention are effective against various pathogenic fungi, in particular, fungi infecting the nails. In order to achieve higher penetration of berberine into the lower layers of the nail plate and nail bed, berberine-loaded polycaprolactone nanoparticles of the present invention produce an antifungal reservoir in the upper dorsal surface of the nail plate. They are also retained at the nail surface for enough period of time to allow them to permeate through the nail plate. This enables deeper drug penetration into the nail plate, thereby improves the retention of drugs in the nail plate, and regulates drug release.

The present invention relates to Nanoparticles comprising Berberine or its salt and polycaprolactone. In one embodiment, the amount of Berberine or its salt is 0.35%w/w and the amount of polycaprolactone is 0.33-0.60%w/w.

The salt of berberine is selected from chloride, bromide phosphate, diphosphate, acetate, sulfate or citrate salt. In a preferred embodiment, chloride salt is used.

Further, the nanoparticles have an average particle size of 50-300 nm. In a preferred embodiment, the particles size is 100-150 nm.

The nanoparticles of the present invention are prepared by nanoprecipitation technique. In one embodiment, the present invention provides a process for preparing Berberine loaded nanoparticles, said process comprising:

(i) dissolving polycaprolactone in a suitable organic solvent to obtain a solution;
(ii) dissolving Berberine or its salt in a suitable organic solvent to obtain a solution;
(iii) mixing the solution of step (i) and (ii) to obtain an organic phase;
(iv) dissolving a stabilizer in water to obtain an aqueous phase;
(v) adding the organic phase dropwise to the aqueous phase under continuous stirring;
(vi) evaporating the organic phase to obtain a suspension; and
(vii) sonicating the suspension to obtain the nanoparticles.

The suitable organic solvent is selected from methanol, ethanol, chloroform, acetone, benzene, hexane, ethyl acetate, dichloromethane, toluene, acetonitrile, carbon tetrachloride, isopropanol, hexane or cyclohexane.

In a preferred embodiment the solvent used at step (i) is chloroform.

In another preferred embodiment the solvent at step (ii) is ethanol.

The stabilizer is selected from tween 80, tween 20, Span, Ethyl cellulose and Eudragit. In a preferred embodiment the stabilizer is Tween 80.
The present invention also provides a composition comprising berberine loaded polycaprolactone nanoparticles. The composition is selected from an oral, parenteral, topical, transdermal or Transungual composition. In a preferred embodiment the composition is a transungual composition.

The composition is in the form of various personal care and color cosmetic end use formulations. The formulations can be rinse off or leave-on. Examples are lotion, tablet, capsule, dusting powder, paper soap stripes, liquid handwash, color cosmetics, nail lacquer, lipsticks, lip balm and nail paint. In a preferred embodiment the formulation is a nail lacquer.

The end use formulation of the present invention are water resistant and retain the active, i.e., berberine nanoparticles in the leave on formulation for a longer period of time so that upon repeated washing also the activity is retained.

The composition of the present invention are suitable for the treatment of various bacterial and fungal infections caused by S. epidermidis, S. hominis subsp. hominis, S. warneri, S. saprophyticus, S. capitis subsp. capitis, S. intermedius, Herpes simplex, Trichophyton rubrum, Candida, Candida Albicans.

In a particular embodiment the composition of the present invention is effective against Candida and it efficient in the treatment of onychomycosis.

Examples:

Examples 1: Preparation of berberine chloride- loaded polycaprolactone nanoparticles Polycaprolactone (PCL) loaded nanoparticles were prepared by nanoprecipitation technique. Briefly, an organic phase comprising of PCL (0.33-0.60% w/v) and berberine chloride (0.33% w/v) was prepared by dissolving PCL in chloroform (6ml) and berberine chloride in ethanol (4ml) and then mixing the two solutions. The organic phase (10ml) was added drop by drop in the aqueous phase (25ml) containing Tween 80 (1-2.5 %), as a stabilizer over a period of 5 min under probe sonication at an amplitude of 60 %, 40 W output power, Branson Sonifier) for 5 min at 4°C. The organic phase was evaporated using a rotary vacuum evaporator at 55ºC and a pressure of 100 mbar and dispersion was concentrated to 7.5 ml. Further, the prepared suspension was probe sonicated at an amplitude of 40 % and 40 W output power for 10 min.

Examples 2: Preparation of optimized nano-loaded nail lacquer
The optimised batch of berberine chloride loaded polycaprolactone nanoparticles were loaded in nail lacquer vehicle. The nail lacquer was prepared by mixing the HPMC E15 (0.55% w/w, 35ml) solution and Eudragit RL 100 (0.45% w/w, 65 ml dissolved in the ethanolic solution). To the above solution, PEG 400 (10% w/v) as a plasticizer and thioglycolic acid (5% w/v) as permeation enhancers were added. The solution was continuously stirred at 400 rpm for 1 hr or until a clear solution was formed. To the nail lacquer formulation so obtained 7.5 ml of optimized formulation of nanoparticles was added under continuous stirring for 15min at 400 rpm on a magnetic stirrer to obtain a formulation of berberine chloride-loaded polycaprolactone nanoparticulate nail lacquer.

Examples 3: Evaluation of berberine chloride nano-loaded nail lacquer formulation

The optimised batch of berberine chloride-loaded PCL nanoparticles (BNP) was incorporated into the optimised batch of nail lacquer formulation (NLT) as it would be easier to retain the nanoparticles at the site of action. The berberine chloride nano-loaded nail lacquer (BNNL) was evaluated for drying time, viscosity, film adhesion, non-volatile content, water resistance, blush test, ex vivo permeation studies, transmission electron microscopy, FTIR, X-ray diffraction, Raman spectroscopy, antifungal and histopathology studies.

Examples 3 (a) Drying time:
The drying time for nail lacquer formulation was observed as 84.46±0.51s while 81.41± 0.63s for BNNL. The drying time is a crucial parameter for the evaluation of nail lacquer. Rapid drying leads to steaky and uneven application on the nail surface which makes the film lustreless. It will reduce the residence time of lacquer application through lacquer brush which makes it difficult to apply on the surface of the nail. The delayed drying makes the lacquer film sticky.

Examples 3 (b) Viscosity
Viscosity is an important parameter for the evaluation of nail lacquer formulations. The formulations bearing low viscosity flow away from the surface of the nail, while the formulations having a viscosity of more than 75 cps are difficult to apply on the nail. Therefore, an optimum viscosity is essential for nail lacquer formulation to remain on the surface of the nail. The optimum viscosity of nail lacquer was found to be 44.45 cps while the BNNL exhibited 48.26 cps. The incorporation of nanoparticles in the nail lacquer lead to an increase in the viscosity of the nail lacquer but was within the range.

Examples 3 (c) Film adhesion studies
Nail lacquer must adhere well to the nail plate to form a uniform film over the nail plate requiring virtuous adhesive properties. The film adhesion properties of formulated lacquer should not be more than 10%. The optimised formulations of nano-nail lacquer showed good adhesion properties. Hence, the choice of film-forming polymers for the preparation of nano-loaded nail lacquer was advantageous. Observed film adhesion for nail lacquer was 5.86± 0.98 % while 4.93± 0.63 % for BNNL. No significant change in film adhesion was observed after incorporation of nanoparticles in nail lacquer.

Examples 3 (d) Non-volatile content
The amount of solid content present on the surface of the nail plate after solvent evaporation is termed as non-volatile content. According to the Bureau of Indian Standards, the amount of non-volatile content in the formulation remained on the plate should not be less than 20% w/w. The berberine chloride nail lacquer and BNNL showed an optimum non-volatile content i.e., 41.69± 1.06 % and 46.67± 1.6 % respectively.

Examples 3 (e) Water resistance
The water resistance of the film determines its resistance to aqueous permeability. This was assessed by applying a continuous layer on a flat surface and submerging it in water. The film's weight was measured before and after submersion, and the difference in weight was recorded. Film absorbs more water it led to increase in film weight that indicated lower water resistance. The water resistance for BNNL and berberine chloride nail lacquer was 6.77±0.31% and 6.07±0.38% respectively. The results of water resistance test confirmed that optimised formulations provided adequate water resistance as it was not more than 10% of the total weight.

Examples 3 (f) Blush test
Blush test was carried out to analyse the effect of exposure to water on the surface of nail lacquer film. It was observed that film showed little dullness after submerging film in water during blush test. The presence of hydrophilic polymers might had contributed to dullness of film on the contrary hydrophobic polymer in lacquer film resisted leaching as it was insoluble in water. No blistering and peeling off was seen in the film. It indicated that combination of hydrophilic and hydrophobic polymers maintained the lustre and consistency of the film. Thus, BNNL and berberine chloride lacquer films passed the blush test.

Examples 3 (g) Ex-vivo permeation studies
The ex vivo permeation was studied by placing nail lacquer loaded berberine chloride nanoparticles (BNNL) and nail lacquer (BNLT) on the dorsal surface of the bovine hoof, affixed between the donor and receptor compartment of the Franz diffusion cell. Figure 1 showed the ex vivo permeation profile of berberine chloride (BBR), BNLT and BNNL across the nail plate. Permeation of BBR was observed as 90.53±1.93%, in 8 h while permeation of BNLT and BNNL was observed as 77.32±0.97% and 43.56±1.65% respectively in 24 hr. The permeation of pure drug i.e. BBR was found highest when compared to BNNL and BNLT. The fact behind fast release of BBR is absence of formulation barriers. Another factor attributed to highest and fastest release of BBR is the increased surface area due to its solution form. The permeation of berberine chloride from the BNLT was more than BNNL in 24 hr. In BNNL, the drug was distributed within the film, reducing immediate surface contact with the nail. The delayed release of drug from the nanoparticles (BNNL) can also be ascribed to the encapsulation of drugs in the matrix of polycaprolactone which retards the fast release of drug from the nanoparticles. The resistance offered by the viscosity of nano nail lacquer formulation might also be attributing slow release from BNNL. Further, the calculated apparent permeability coefficient (Papp) for BNLT and BNNL formulation was 0.000023±0.07 and 0.000014cm/sec respectively.

Examples 3 (h) Morphological characterization of nanoparticles
Figure 2a showed the micrographs of the optimised batch of berberine chloride loaded polycaprolactone nanoparticles. The nanoparticles were spherical shaped, well dispersed and in the range of 100-150 nm. Figure 2b presented the photograph of polycaprolactone nanoparticles loaded in nail lacquer. The incorporation of nanoparticles in nail lacquer did not affect their shape. However, the micrograph showed the presence of coating of the polymer matrix of nail lacquer. The size of nanoparticles observed in the micrograph range from 130 nm to 430 nm.

Example 3 (i) Determination of Zone of inhibition
The in vitro antifungal activity of optimised nanoparticle formulation and BNNL was determined by zone of inhibition against the fungal strain of Candida albicans (MTCC 277). Figure 3 presents the ZOI of vehicle control, berberine chloride loaded nanoparticles (BNP), BNLT and BNNL respectively. The vehicle control showed no inhibitory effect on growth of Candida albicans. BNP and BNLT exhibited 28.19±0.23 mm, 41.26±0.23 mm respectively while BNNL showed the highest ZOI against Candida albicans (61.52±0.46 mm). The prolonged drug diffusion from BNNL and availability of greater surface area of nanoparticles to interact with fungal cells might be contributing to more effective inhibition of fungal growth. The observed results revealed that the antifungal activity of BNNL was not due to the presence of formulation components as ZOI of vehicle control was found to be negligible. Hence, it showed that berberine-loaded nano nail lacquer exhibited potent antifungal activity and could serve as a suitable formulation for the treatment of onychomycosis.

Examples 3 (j) Histopathological studies
Figure 4 showed the results of histological studies of the bovine hooves treated with blank, control and optimised formulations (BNLT and BNNL) respectively. Hoof treated with control showed no damage while the hooves treated with blank, BNLT and BNNL batch of nail lacquer showed presence of almost similar size of pores which might be attributed to the rupture of disulphide linkage of the keratin layers. Thioglycolic acid enhanced transungual flux by interacting with nail keratin and reducing disulfide bonds. This similarity in hoof histopathology of blank, BNLT and BNNL treatment may be due to presence of same permeation enhancer at same concentration (5% w/v) in formulations. As the permeation enhancer in the formulation is responsible for the formation of pore size. Therefore, pore size formed was also of almost same size.

Examples 3 (k) Stability Studies The stability study was carried out to analyse the quantitative changes in the formulation over a period of 90 days at room temperature (25±2ºC/60±5% RH). The results of stability studies showed that optimised formulations of nanoparticles had not shown any eloquent changes in particle size. BNNL formulation had not showed any significant change in drying time, non-volatile content, film adhesion and blush test. Table 1 presented the results of stability studies. The results revealed that optimised formulation of nanoparticles and berberine-loaded nano nail lacquer formulations were stable and exhibited promising properties over a period of 90 days.

Table 1 Stability study data of optimised nano loaded nail lacquer

Type of Formulation Parameters Period (days)
0 30 60 90
BNP Particle size
(nm±SD) 124±20.27 135±25.56 148±28.07 153±31.51
Entrapment efficiency
(%±SD) 87.31±6.59 87.02±5.67 85.98±7.25 85.34±9.62
BNNL Drying time (s±SD) 81.41±0.63 81.03±0.70 81.00±0.56 80.91±0.89
Non- volatile content (%±SD) 46.67±3.12 45.97±2.34 45.89±4.59 45.11±5.92
Adhesion studies
(%± SD) 4.93±0.18 4.90±0.73 4.76±0.40 4.12±0.89
Blush test Pass Pass Pass Pass

, Claims:We claim:

1. Nanoparticles comprising Berberine or its salt and polycaprolactone.

2. The nanoparticles as claimed in claim 1 wherein Berberine or its salt is present in an amount of 0.33%w/w and polycaprolactone is present in an amount of 0.33-0.60%w/w.

3. The nanoparticles as claimed in claim 1 wherein Berberine salt is selected from chloride, phosphate, diphosphate, acetate, sulfate or citrate salt.

4. The nanoparticles as claimed in claim 1 wherein the said nanoparticle has a particle size between of 100-150 nm.

5. A process for preparing nanoparticles as claimed in claim 1 said process comprising
(i) dissolving polycaprolactone in a suitable organic solvent to obtain a solution;
(ii) dissolving Berberine or its salt in a suitable organic solvent to obtain a solution;
(iii) mixing the solution of step (i) and (ii) to obtain an organic phase;
(iv) dissolving a stabilizer in water to obtain an aqueous phase;
(v) adding the organic phase dropwise to the aqueous phase under continuous stirring and sonication;
(vi) evaporating the organic phase to obtain a suspension; and
(vii) sonicating the suspension to obtain the nanoparticles.

6. The process as claimed in claim 5 wherein the suitable organic solvent is selected from methanol, ethanol, chloroform, acetone, benzene, hexane, ethyl acetate, dichloromethane, toluene, acetonitrile, carbon tetrachloride, isopropanol, hexane or cyclohexane.

7. The process as claimed in claim 5 wherein the stabilizer is selected from Tween 80, tween 20, Span, Ethyl cellulose and Eudragit.

8. A composition comprising the nanoparticles as claimed in claim 1.

9. The composition as claimed in claim 8 wherein the said composition is an oral, parenteral, topical, transdermal or Transungual composition.

10. The composition as claimed in claim 8 wherein the said composition is a pharmaceutical, neutraceutical, personal care or cosmetic composition.

11. The composition as claimed in claim 8 wherein the said composition is a transungual composition.

12. The composition as claimed in claim 8 wherein the said cosmetic composition is a nail lacquer.
Dated this the 5th day of November 2024.