Description:FIELD OF THE INVENTION

The present invention relates to the field of nanoparticles and aqueous compositions thereof. More particularly, the invention relates to bio adhesive calcium coated Poly Lactide-co-Glycolide (PLGA) nanoparticles encapsulating vitamin D and composition thereof. The invention also relates to the methods of preparations thereof.

BACKGROUND OF THE INVENTION

Vitamin D is a fat-soluble vitamin that plays an important role in calcium homeostasis and bone metabolism. It is produced in the skin upon exposure to sunlight. Though, sun exposure alone is thought to be adequate to achieve vitamin D sufficiency in the body, inadequate exposure to sunlight leads to its deficiency.

Vitamin D deficiency can lead to osteomalacia and rickets in children and osteomalacia in adults. The subclinical vitamin-D deficiency is associated with ill-defined generalized aches and pains, bone tenderness, osteoporosis, muscle weakness (especially proximal), unsteadiness, repeated falls, as well as neuropsychiatric symptoms including lethargy, fatigue, depression, immune dysfunction, type 1 and 2 diabetes mellitus, cardiovascular diseases, neurocognitive deficits, and various neoplasia such as prostate, breast, and colorectal cancers. The fortification of milk with vitamin D in the 1930s was effective in eradicating rickets in the world. However, subclinical vitamin D deficiency is still widely prevalent in both developed and developing countries with a worldwide prevalence of upto 1 billion.

Cholecalciferol (Vitamin D3), being a hydrophobic molecule, cannot be easily formulated in aqueous medium, beverages or food compositions. The absorption rate and time taken for absorption of Vitamin D3 is low, which leads to low bioavailability for a considerable period after administration.

Many approaches like food fortification, oral dosages were formulated to overcome Vitamin D deficiency, but inherent factors like very short shelf life of vitamin D fortified foods, over dosage or under dosage etc. pose severe risk for public health.

In view of the above-mentioned hurdles, the inventors have identified that water or aqueous beverages can serve as an important vehicle for delivery of vitamin D (US20210290640A1).

In order to adapt vitamin D for delivery through water or aqueous beverages, the inventors have contemplated an approach for development of bio adhesive / biocompatible calcium coated cationic vitamin D nanoparticles which are hydrophilic in nature and easily disperse in aqueous media.

These developed biocompatible cationic nanoparticles are cost-effective, have a considerable shelf life and a faster rate of absorption in the body which leads to a faster and sustained bioavailability. Biocompatible/Bio-adhesive delivery systems can further increase the residence time of active agent(s) at the site of absorption/action, provide sustained active agent (Vitamin D) release and minimize the degradation of Vitamin D in various body sites.

The present invention utilizes calcium ions to provide positive charge on the surface of the nanoparticle and provide mucoadhesive dosage forms.

The Vitamin D nanoparticles developed in the instant invention can be used for fortification of any aqueous medium or beverage, including bottled drinking water. Bottled drinking water fortified with vitamin D can work as a replacement for regular drinking water which can potentially lead to better patient compliance.

Orally administered particles rapidly undergo electrostatic interaction with negatively charged mucosal lining, eventually affecting the particle uptake, fate, absorption, distribution, and elimination in vivo, potentially. Attempts have been made to provide calcium ions on the surface of the PLGA nanoparticles encapsulating vitamin D to increase oral vitamin D bioavailability.
The calcium ions used for coating the negatively charged nanoparticles impart cationic nature to the nanoparticles which aids in mucoadhesive property of the formulation and also aids in sustaining the release of Vitamin D from the formulations in the intestine by withstanding the gut acids.

One of the prior arts known to the applicant is Ranga K. Dissanayake et al. (Enteric Coated Oral Delivery of Hydroxyapatite Nanoparticle for Modified Release Vitamin D3 Formulation: Journal of Nanomaterials Volume 2021, Article ID 9972475, 9 pages), wherein hydroxyapatite (HA) is utilized for oral delivery of Vitamin D (VD) due to its high affinity to bone tissue. The prior art discloses HA-VD composite nanoparticles as the codelivery system, providing both Vitamin D and Ca3(PO4)2. VD-loaded HA nanoparticles were further coated with a gastroresistant polymer, hypromellose phtalate-55 (HP-55), in order to protect the pH-sensitive HA from degradation at lower pHs.

This study suggests the use of HP-55 coated VD-loaded HA nanoparticles as a potent alternative for sustained and targeted oral delivery of VD with Ca2+ and PO43-. Further, the prior art teaches use of entero-coating consisting of specialized polymers for prevention of nanoparticle disintegration due to exposure to acidic gut (pH). Furthermore, the degradation of PLGA leaves behind acidic by-products which are undesirable for targeting Vitamin D in system. However, enteric coating is not required in the present invention, due to presence of positively charged calcium ions on the surface of the nanoparticle which are able to withstand the gut pH, preventing disintegration of the nanoparticle.

Further US20130295186A1, discloses the method for coating particles with calcium phosphate (CaP), wherein the particles are negatively charged. The method includes contacting the particles with a first solution containing calcium ions, removing the first solution to obtain a precipitate, and contacting the precipitate with a second solution containing phosphate ions to obtain CaP-coated particles, which are negatively charged on the surface. However, the nanoparticles containing CaP-coated particles are not suitable for intestinal muco-adhesion and absorption of active agent(s) (Vitamin D), due to lack of positive charge on the surface. Further the study does not provide any in-vivo studies to demonstrate the advantage of this coating.

Although the prior art discloses various techniques and formulations for the delivery of the active agent(s), the formulation has a setback due to its lack of adhesion at intestinal mucosa and extra entero-coating required to prevent disintegration of nanoparticle in stomach.

Hence there exists a need for development of bio adhesive nanoparticulate compositions which can alleviate the above problems as well as deliver Vitamin D at the targeted specific site of action.

In the present invention, the outer coating of positively charged calcium ions provides an anchoring site for attachment to the negatively charged mucosal lining in the intestine, for the delivery of active agent(s) (Vitamin D). The present invention overcomes the limitations of the prior arts by providing:
• Biocompatible and Bio adhesive nanoparticle-based delivery systems desirable to increase the residence time of active agent(s) (Vitamin D) at the site of absorption/action.
• Controlled sustained vitamin D release into the system because of better muco-adhesion of calcium coated PLGA particles.
• Minimizing the disaggregation of PLGA nanoparticles, calcium ions buffer the degradation environment at low pHs and deliver the active agent(s) (Vitamin D) at target / action site.
• Ease of processing the nanoparticles, due to absence of additional step to create the additional entero-coating.

OBJECTS OF THE INVENTION

The primary object of the present invention is to provide stable biocompatible and bio adhesive nanoparticle encapsulating Vitamin D.

Another object of the present invention is to provide biocompatible, and bio adhesive calcium coated PLGA nanoparticle encapsulating Vitamin D.

Another object of the present invention is to provide a cationic based delivery system comprising PLGA nanoparticles coated with calcium ions.

Another object of the present invention is to provide a composition comprising calcium coated nanoparticles with one or more pharmaceutically acceptable excipients or carriers.

Another object of the present invention is to provide a composition comprising calcium coated nanoparticles for sustained release and enhancement of bioavailability of lipid-soluble vitamin D.

Further object of the present invention is to provide a composition comprising calcium coated nanoparticles leading to higher level of serum bioavailability compared to conventional PLGA vitamin D nanoparticles.

Another object of the present invention is to provide a composition comprising calcium coated nanoparticles for providing protection w.r.t Vitamin D degradation from UVA and UVB radiations.

Yet another object of the invention is to provide a method for the preparation of encapsulated Vitamin D PLGA nanoparticles.

SUMMARY OF THE INVENTION

Recognizing the prior arts and need for delivery system to enhance the bioavailability of Vitamin D, in one aspect accordingly the present invention provides a calcium coated nanoparticle encapsulating Vitamin D, wherein the nanoparticle is Poly Lactide co Glycolide (PLGA) nanoparticle, and wherein calcium coating provides positive charge on the outer surface of nanoparticles.
The said calcium coated nanoparticles encapsulating Vitamin D has calcium ions present in an amount of 0.9-1.8 µmol/mg weight of nanoparticle.

In the said calcium coated nanoparticles encapsulating Vitamin D, the loading of vitamin D is about 75-95 wt% of nanoparticles.

In the said calcium coated nanoparticle encapsulating Vitamin D, the Vitamin D is cholecalciferol.

In the present calcium coated nanoparticles encapsulating Vitamin D, the positively charged nanoparticle is adapted to deliver Vitamin D to the negatively charged mucosal surface.

In another aspect, the present invention discloses the composition comprising calcium coated nanoparticle encapsulating Vitamin D and one or more pharmaceutically acceptable excipients or carriers.

The said composition comprising the calcium encapsulated PLGA nanoparticles contains the aqueous medium as the pharmaceutically acceptable excipients or carrier.

In the said composition, the calcium encapsulated PLGA nanoparticles exhibits higher level of serum bioavailability upto seven days after in vivo oral administration compared to conventional PLGA vitamin D nanoparticles.

The said composition, the calcium encapsulated PLGA nanoparticles exhibits the protection from UV A/ B radiations.

The composition comprising the calcium encapsulated PLGA nanoparticles is stable upto 250 days at both 27°C and 4°C.

The said composition comprising the calcium encapsulated PLGA nanoparticles, can be utilized for prevention or treatment of vitamin D deficiency.
Another aspect of the present invention discloses the method to prepare the calcium coated nanoparticles encapsulating Vitamin D which involves the steps of:
a) preparing a reaction mixture of PLGA nanoparticle with Vitamin D dissolved in organic Phase with stabilizer and calcium phosphate;
b) vortexing the mixture at 2000 rpm for 2-15 min, preferably 4-10 min to form coarse emulsion;
c) high pressure homogenization at 100-14000 bars, preferably 250-800 bars for 15 cycles of each 5–15 minutes to obtain a nano emulsion;
d) evaporating the solvent and forming nanoparticle with pressure of 100-900 psi preferably 200-750 psi;
e) ultracentrifugation thrice at 50000 rpm by suspending the pellet in deionized water; and
f) suspending the pellet in ultrapure water and lyophilization at 4°C.

In the above referenced method of the present invention, the concentration of PLGA nanoparticles is 1%. The present method utilizes Vitamin D at concentration of 1%.

The said method utilizes calcium phosphate at a concentration of 1.5% to provide positive charge on the outer surface.

Further the method for preparation of calcium coated nanoparticles encapsulating Vitamin D utilizes the stabilizer, wherein the stabilizer is Polyvinyl alcohol (PVA), at the concentration amount of 1.5%.

The obtained calcium coated nanoparticles encapsulating Vitamin D prepared using the above reference method, have an average particle (size) diameter between 150nm – 220nm.

The obtained calcium coated nanoparticles encapsulating Vitamin D prepared using the above reference method, have a positive zeta potential of 28.8mV.

Vitamin D utilized in the present method is Vitamin D3 (cholecalciferol).
BRIEF DESCRIPTION OF THE DRAWINGS:

Figure 1 depicts the mechanism of the cationic calcium coated PLGA nanoparticles in sustenance of the Vitamin D delivery.

Figure 2 depicts the diagram of cationic calcium coated PLGA nanoparticles.

Figure 3 depicts the graph showing the Zeta Potential distribution of the PLGA-cholecalciferol (Vitamin D3) nanoparticles and calcium coated PLGA-cholecalciferol (Vitamin D3) nanoparticles (Example 2).

Figure 4 depicts the quantification of bioavailability of vitamin D through PLGA-cholecalciferol (Vitamin D3) nanoparticles and calcium coated PLGA-cholecalciferol (Vitamin D3) nanoparticles after their oral administration to mice, using HPLC (Example 4).

Figure 5A depicts Free Vit D exposed to UV A.

Figure 5B depicts Free Vit D exposed to UV B.

Figure 5C depicts the protection of vitamin D encapsulated in calcium coated PLGA nanoparticles from UV A radiations (Example 5).

Figure 5D depicts the protection of vitamin D encapsulated in calcium coated PLGA nanoparticles from UV B radiations (Example 5).

DETAILED DESCRIPTION OF THE INVENTION:

The following detailed description refers to the accompanying drawings that show, by way of illustration, specific details and embodiments in which the invention may be practised. These embodiments are described in sufficient detail to enable those skilled in the art to practise the invention. Other embodiments may be utilized, and changes may be made without departing from the scope of the invention. The various embodiments are not necessarily mutually exclusive, as some embodiments can be combined with one or more other embodiments to form new embodiments.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods belong. Although any nanoparticles, compositions or methods similar or equivalent to those described herein can also be used in the practice or testing of the embodiments of the present invention.

Further the embodiments described herein can be understood more readily by reference to the following detailed description, examples, and drawings. Compositions and methods described herein are merely illustrative of the principles of the present invention and are not limited to the specific embodiments presented in the detailed description, examples, and drawings. Numerous modifications and adaptations will be readily apparent to those of skill in the art without departing from the spirit and scope of the invention.

Where a range of values is provided, it is understood that each intervening value between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within by the methods and compositions. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within by the methods and compositions, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods and compositions.

It is appreciated that certain features of the methods, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods and compositions, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination.

Conventional (non-mucoadhesive) formulations are unable to withstand the powerful involuntary muscle action as well as the thorough washing effect by some bodily fluids present, such as in the gastrointestinal lumen, ocular surface, urinary bladder, and other mucosal surfaces. This restriction causes a significant amount of the medications supplied to be lost at the application/absorption site. Not only may this raise the overall expense of the treatment programmes, but it could also result in therapy failure since the therapeutic medicine or active agent(s) (vitamin D) concentration cannot be attained.

The advantage of the bio/mucoadhesive delivery system is its ability to adhere to the mucous membranes. The residence time of medications and concentration gradient are both lengthened by the delivery system’s adherence to the intestinal mucosa. The combined effects may result in better patient compliance, cost-effective treatment, extended therapeutic benefits, increased bioavailability, and controlled active agent(s) (Vitamin D) release.

The conventional non-bio adhesive delivery systems often have poor residence on mucosal surfaces, which justifies the need for novel mucoadhesive materials. Present invention describes in detail the novel mucoadhesive target oriented nanoparticle-based system device to target Vitamin D at the action/ target site.

Vitamin D is a fat-soluble vitamin that is added to some foods, found naturally in a few, and sold as a dietary supplement. Additionally, it is created internally when ultraviolet (UV) rays from sunshine strike the skin and start the production of vitamin D. The biologically inactive vitamin D that is received through sunlight, meals, and supplements must go through two hydroxylations in order to become active in the body. Vitamin D undergoes its first hydroxylation in the liver, resulting in 25-hydroxyvitamin D [25(OH)D], sometimes referred to as “calcidiol”. The physiologically active 1,25-dihydroxyvitamin D [1,25(OH)2D], also known as “calcitriol”, is created by the second hydroxylation, which primarily takes place in the kidney.

Vitamin D promotes calcium absorption in the gut and maintains adequate serum calcium and phosphate concentrations to enable normal bone mineralization and to prevent hypercalcaemic tetany (involuntary contraction of muscles, leading to cramps and spasms). It is also needed for bone growth and bone remodelling by osteoblasts and osteoclasts. Vitamin D has other roles in the body, including reduction of inflammation as well as modulation of processes such as cell growth, neuromuscular and immune functions, and glucose metabolism. Many genes encoding proteins that regulate cell proliferation, differentiation, and apoptosis are modulated in part by vitamin D.

In foods and dietary supplements, vitamin D has two main forms, D2 (ergocalciferol) and D3 (cholecalciferol), that differ chemically only in their side-chain structures. Both are also naturally occurring forms that are produced in presence of the sun’s ultraviolet-B (UVB) rays. Vitamin D2 is produced in plants and fungi while Vitamin D3 is produced in animals, including humans. Both forms are well absorbed in the small intestine. Absorption occurs by simple passive diffusion and by a mechanism that involves intestinal membrane carrier proteins. The concurrent presence of fat in the gut enhances vitamin D absorption, but some vitamin D is absorbed even without dietary fat. Neither aging nor obesity alters vitamin D absorption from the gut.

The Recommended Dietary Allowance for adults 19 years and older is 600 IU (15 mcg) daily for men and women, and for adults >70 years it is 800 IU (20 mcg) daily. Many people may not be meeting the minimum requirement for the Vitamin D. Vitamin D deficiency may occur from lack in the diet, poor absorption, or having a metabolic need for higher amounts. If one is not eating enough vitamin D and does not receive enough ultraviolet sun exposure over an extended period, a deficiency may arise. People who cannot tolerate or do not eat milk, eggs, and fish, such as those with a lactose intolerance or who follow a vegan diet, are at a higher risk for deficiency. Vitamin D toxicity most often occurs from taking supplements.
The low amounts of the vitamin found in food are unlikely to reach a toxic level, and a high amount of sun exposure does not lead to toxicity because excess heat on the skin prevents D3 from forming. The target-oriented vitamin D delivery systems can provide maximum therapeutic benefit through controlled and predetermined release rate kinetics.

The system which further prevents Vitamin D degradation or inactivation during transit to target sites and prevents the body from adverse reactions can be utilized to meet the deficiency of Vitamin D in subjects. Though, both natural and synthetic materials can be utilized, synthetic nanoparticles are more desirable for therapeutic uses as they can be modified to meet the desired properties needed for controlled and targeted active agent(s) (Vitamin D) release.

These nanoparticles are minute solid particulates in the size range of 10-1000 nm and due to their colloidal nature can offer numerous advantages over conventional treatment strategies. Small sized particles possess large surface area and hence increase the dissolution properties of active agents with poor solubility.

A substance to be targeted can be either entrapped in the core, adsorbed on the surface or both. Nanoparticles can be prepared by varying the methodology for synthesis of these carriers. The potential of using nanoparticles for delivery of medicines and pharmaceutically active substance/ agent is massive. Because of their sub microscopic size, they have unique material characteristics, and the manufactured nanoparticles may find practical applications in a variety of areas, including medicine, engineering, catalysis, and environmental remediation.

In the present context, the particles to be coated may comprise a nanoparticle polymer or polymer mixture. Any polymeric material that is within the knowledge of the average skilled person can be used for this purpose. Such polymeric material can be of linear or branched polymers, homopolymers, block polymers, copolymers, or mixtures thereof.

The polymer may further be selected from but not limited to the group comprising of Poly (lactic-co-glycolic acid), poly (lactic acid), poly (ethylene glycol), poly(L-lactic), polycaprolactone, poly(N-isopropylacrylamide), pNIPAAm-poly(N,N'-methylenebisacrylamide) copolymer, poly(ethylene glycol), functionalized (pNIPAAm), polyvinyl alcohol (PVA), a hydroxylated poly(meth)acrylate, an ethylene-vinyl acetate copolymer, 2-hydroxyethyl methacrylate (HEMA), poly (maleic acid/octyl vinyl ether) (PMAOVE), a polyurethane, poly(acrylic acid), poly(stearyl acrylate) (PSA), poly(acrylamide) and copolymers thereof; or a polyolefin, and mixtures thereof.

Poly (lactic-co-glycolic acid) (PLGA) is one of the most effective biodegradable polymeric nanoparticles (NPs). The PLGA is a synthetic polymer nanoparticle that can be targeted to a specific site for the safe and effective delivery of payload. The term “payload” or “loading” as used herein means the percentage of active agent (vitamin D) present inside, or in the internal phase or core of the nanoparticle of the present invention.

In one of the embodiments of the invention, Vitamin D is encapsulated in Poly (lactic-co-glycolic acid) (PLGA). In another embodiment of the invention, the calcium coated PLGA nanoparticles encapsulate the Vitamin D, wherein the loading of vitamin D is about 75-95 wt % of nanoparticles (Example 1).

The calcium-based nanoparticles can be used to deliver various kind of active agents at the target site. In some embodiments the calcium coated nanoparticle is used to deliver the Vitamin D. In another embodiment of the invention, the calcium coated nanoparticle encapsulates Vitamin D which is Vitamin D3 (cholecalciferol). The term “encapsulated” as used herein means that the active agent (vitamin D) is located inside, or in the internal phase or core of the nanoparticle of the present invention and is completely surrounded by one or more polymeric component.

The PLGA Poly (lactic-co-glycolic acid) conventional (non-mucoadhesive) formulations lack the ability to withstand the gut pH and strong involuntary muscular movement at mucosal surfaces. The ability of bio/mucoadhesive delivery system to stick to the mucous layer of the mucous membranes makes it useful. Active agent(s) (Vitamin D) residence duration and concentration gradient are both lengthened by the delivery system’s adherence to intestinal mucosa. The combined effects may result in better patient compliance, cost-effective treatment, extended therapeutic benefits, increased bioavailability, and controlled active agent(s) (Vitamin D) release.

With the intension to develop a mucoadhesive delivery system that enhances the bioavailability of Vitamin D, the present invention provides a nanoparticle encapsulating Vitamin D coated with calcium particles. The calcium coating provides positive charge on the outer surface of nanoparticles (Figure 2).

Calcium (2+) is a divalent metal cation that is the metabolically active form of calcium which is not bound to proteins, and which circulates in the blood. Orally administered particles coated with calcium rapidly interact with mucosal walls, and this potentially affects particle uptake, fate, absorption, distribution, and elimination in vivo. The proper size, positive surface charge can help nanoparticle complete the process of transintestinal epithelial cell transport.

The positive charge on the surface of the NPs is advantageous to enter the intestinal epithelial cells. Positively charged calcium coating on the outer surface works as an anchor adapted to bind and deliver Vitamin D to the negatively charged mucosal lining of the target / action centre (Figure 1). Ionic interactions arise from electrostatic attraction between two groups of opposite charge. The muco-adhesion due to electrostatic interactions between the opposite charges increases the residence time of active agent(s) (Vitamin D) at the site of absorption/action site leading to controlled sustained active agent(s) (Vitamin D) release because of better muco-adhesion of calcium coated PLGA particles in system. Further, the positive charge on the surface of the nanoparticles was advantageous to enter the intestinal epithelial cells. Nanoparticles with a positive zeta potential tend to be taken up by endocytosis which indicates that endocytosis tends to favor NPs with a positive zeta potential.

The ionic charge due to calcium nanoparticles further minimizes the disaggregation of PLGA nanoparticles; calcium ions buffer the degradation environment at low pHs and deliver the payload at various sites of the body. The present invention provides ease of processing the nanoparticles, due to absence of additional step of creating additional entero-coating.

In one more embodiment of the invention positively charged calcium coated nanoparticle encapsulating Vitamin D is adapted to deliver the Vitamin D to the negatively charged mucosal surface. In one more of embodiments of the present invention the calcium coated nanoparticles encapsulating Vitamin D has calcium ions present in an amount of 0.9 – 1.8 µmol/mg wt of nanoparticle. The calcium ions present on the outer surface of the nanoparticles provide the positive zeta potential of 28.8mV.

Among the factors affecting absorption of particles, particle size appears to be the primary factor. The absorption efficiency of nanoparticles particles is clearly dependent on the size.

The particle size dependence on intestinal absorption is also observed for poly(lactide-co-glycolide) or PLGA particles by Desai et al. (Pharm. Res. 13:1838, 1996). In some embodiments of the invention, calcium coated nanoparticles encapsulating Vitamin D prepared using the above reference method, have an average particle (size) diameter between 150nm-220nm.

According to another aspect of the present invention, composition comprises of calcium coated polymeric nanoparticles of active agent (Vitamin D) and one or more pharmaceutically acceptable excipients or carriers.

The composition comprising nanoparticle comprising Vitamin D, can be used for fulfilling the daily needs of Vitamin D in subjects. As used herein, the term “subject” refers to an animal, preferably a mammal, including a human or non-human suffering from vitamin D deficiency.

The phrase “pharmaceutically acceptable excipients” or “carrier” is a compound compatible with the other ingredients of the composition and not disadvantageously deleterious to the intended recipient.

The pharmaceutically acceptable carriers or excipients can be sugars, such as lactose, glucose, sucrose; starches, such as corn starch or potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose or cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter or suppository waxes; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol or polyethylene glycol; esters, such as ethyl oleate or ethyl laurate; agar; buffering agents, such as magnesium hydroxide or aluminium hydroxide; alginic acid; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; purified water for injection; sterile saline solution; and other non-toxic compatible substances employed in pharmaceutical formulations.

Other suitable carriers or excipients that may be employed, in the pharmaceutical composition include, but are not limited to, dichloromethane, acetonitrile, ethyl acetate, acetone, propylene carbonate, water, glycerine, coconut fatty acid diethanolamide, medium and/or long chain fatty acids or glycerides, monoglycerides, diglycerides, triglycerides, structured triglycerides, soyabean oil, peanut oil, corn oil, corn oil mono glycerides, corn oil di glycerides, corn oil triglycerides, polyethylene glycol, caprylocaproylmacroglycerides, caproyl 90, propylene glycol, polyoxyethylenesorbitan fatty acid esters, polyoxyethylene castor oil derivatives, castor oil, cottonseed oil, olive oil, safflower oil, peppermint oil, coconut oil, palm seed oil, beeswax, oleic acid, methanol, ethanol, isopropyl alcohol, butanol, acetone, methyl isobutyl ketone, methyl ethyl ketone or combinations thereof.

The “composition” of present invention comprising nanoparticle can be administrated by systemic administration, or by parenteral administration.

Systemic administration can be performed through oral, intramuscular, intradermal, transdermal, or supradermal, subcutaneous, submucosal, intravenous etc. Parenteral administration can be performed through subcutaneous, submucosal injections, intravenous, intraperitoneal, intramuscular, intradermal, and infusion etc.

The dosage further may contain other substances such as suitable dispersing, solubilizing, wetting, emulsifying, stabilizing, suspending, thickening agent, preservatives, flavouring agents, anti-microbial or antibacterial or antifungals substances.

In certain embodiments, nanoparticles composition(s) may be administered in one or more dosage forms. The term “composition” includes oral dosage forms, syrup, oral suspension, oral solution, oral drop, oral emulsion, mixtures, linctus, emulsion, dispersions, sprays, elixir, spot on, micro formulations. Further the composition can be in other dosage forms such as but not limited to powder, tablets, capsules, pellets, beads, mini-tablets, pills, micro-pellets, small tablet units, disintegrating tablets, dispersible tablets, granules, sprinkles, microspheres and multiparticulates, sachets gels, bolus, depots, implants etc.

The term "composition" includes topical dosage forms, such as but not limited to, sprays, solutions, suspensions, ointments, drops, in-situ gel, aerosols, ointments, microspheres, creams, gels, patches, films and the like. Preferably, the pharmaceutical compositions of the present invention comprising polymeric nanoparticles of one or more active agent (Vitamin D) is provided in oral dosage forms.

In another embodiment of the invention, the composition comprising the calcium encapsulated PLGA nanoparticles contains the aqueous medium as the pharmaceutically acceptable excipients or carrier.

The term “aqueous medium” refers to a medium comprising water wherein water is preferably the dissolving medium. As used herein, the term includes bottled water, beverages, pharmaceutical compositions, food items having water as a substantial component etc.

Those skilled in the art will be able to determine, according to known methods, the appropriate amount, dose or dosage of the nanoparticle’s composition for administration to a subject considering factors such as age, weight, general health, the compositions administered, the route of administration, the nature and advancement of the vitamin D deficiency requiring treatment, and the presence of other medications. Determining such dosage adjustments is generally within the skill of those in the art of medication development.

In one of embodiments of the invention, the serum bioavailability of the composition comprising cationic calcium coated nanoparticles system was evaluated by orally administering the formulations to mice and periodically checking blood for serum level of Vitamin D. “Bioavailability” termed herein refers to the extent and rate at which the active moiety (Vitamin D) enters systemic circulation, thereby accessing the site of action.

The normal range of 25-hydroxy vitamin D {25(OH)D} is measured as nanograms per milliliter (ng/mL). A systematic review on vitamin D bioavailability showed that cationic nanoparticle vehicles produce greater increase in blood serum levels of 25-hydroxy vitamin D {25(OH)D} as compared to negatively charged PLGA nanoparticles.

This significant difference in serum levels can be attributed to the cationic nature of the nanoparticles wherein they form a weak ionic bond with the negatively charged mucosal linings of the gut and thereby their rapid clearance from the mucosa is avoided. This further leads to higher systemic bioavailability in comparison to the negatively charged formulations which get eliminated rapidly.

Mucoadhesive active agent(s) (Vitamin D) delivery systems are advantageous as they can adhere to the mucus layer of the mucous membrane. The adhesion of the delivery systems to mucosa (defined as mucoadhesion) increases the residence time of active agent(s) (Vitamin D), increases the concentration gradient and protects the vulnerable small molecular weight active agent(s) (Vitamin D) as well as peptide-based active agent(s) (Vitamin D). The overall effects could lead to controlled Vitamin D release, prolongation of therapeutic effects, enhancement in the bioavailability, cost-effective treatment and improved patient compliance.

In one or more embodiments of the invention, the composition comprising the calcium encapsulated PLGA nanoparticles, wherein calcium encapsulated PLGA nanoparticles exhibited higher level of serum bioavailability upto seven days after in vivo oral administration compared to conventional PLGA vitamin D nanoparticles. However, transmucosal active agent(s) (Vitamin D) delivery systems often have poor residence on mucosal surfaces, which justifies the need for novel mucoadhesive materials.

Further the present invention provides protection from UV A and UV B led degradation of nanoparticles. Spending longer in the sun will not increase your vitamin D levels. The body only needs a small amount of UV to make vitamin D. Any extra UV exposure will just add to skin damage, not vitamin D. Therefore, Vitamin D cannot be overdosed after threshold.

It is known in the art that Vitamin D degrades on exposure to UV rays. Hamdy, RC et al; studied the impact of direct sunlight on Vitamin D biodegradation in fortified milk. The study concluded that when the fortified milk was exposed to direct sunlight, there was a significant decrease up to about 80% of its original value over a period of 60 minutes. The vitamin D content of fortified milk kept unrefrigerated in shade also decreased over a period of 60 minutes, but the decrease was over by 10%, while the vitamin D content of samples kept refrigerated remained largely unchanged. (Direct Exposure to Sunlight Accelerates Vitamin D Biodegradation in Milk. J Clin Nutr Food Sci, 2(2): 063-067 (2019)).

The ability of this present nanoparticulate system in protecting vitamin D from UV A (3.0-3.2 eV), UV B (3.94-4.43 eV) radiations was evaluated. Vitamin D loaded nanoparticles were exposed to UV A and B radiations for 24 hours. The comparison between peak retention time of Vit D extracted from the UV exposed nanoparticles and the peak retention time of Vit D directly exposed to UV, implies that the nanoparticle system provided protection against UV-A and UV-B lead degradation of nanoparticles (Example 5).

In one more embodiment of the invention, the composition comprising the calcium encapsulated PLGA nanoparticles exhibited protection from exposure to UV A/ B radiations.

Further the composition was evaluated for its stability at 27°C and 4°C by monitoring the variations in the size and charge of the nanoparticles. The nanoparticles were found to be stable at 27? and 4?. In one or more embodiments of the invention the composition comprising the calcium encapsulated PLGA nanoparticles is stable up to 250 days at both 27? and 4?.

Another aspect of the invention discloses the method to prepare the calcium coated nanoparticles encapsulating Vitamin D which involves preparing:
a. preparing reaction mixture of PLGA nanoparticle with Vitamin D dissolved in organic phase of dichloromethane with stabilizer and calcium phosphate;
b. vortexing the mixture for 2-15 min, preferably 4-10 min to form coarse emulsion;
c. followed by the high pressure homogenization at 100-14000 bars; preferably 250-800 bars for several cycles to obtain a nano emulsion;
d. evaporating the solvent and forming nanoparticle with pressure of 100-900 psi preferably 200-750 psi;
e. ultracentrifugation thrice at 50000 rpm by suspending the pellet in deionized water; and
f. suspending the pellet in ultrapure water and lyophilization at 4°C.

In the above referenced aspect of the invention, the method of preparation of calcium coated nanoparticles encapsulating Vitamin D utilizes PLGA nanoparticles at the concentration of 1%. In some embodiment of the invention, the said method of preparation utilizes Vitamin D at a concentration of 1%. Further in some embodiment of the invention, the said method of preparation involves use of calcium phosphate at the concentration of 1.5% to provide positive charge on the outer surface. Further the said method of preparation comprises usage of a stabilizer, preferably Polyvinyl alcohol (PVA), and preferably at a concentration of 1.5%. In another embodiments of the present invention, Vitamin D utilized in the method for preparation of calcium coated nanoparticles encapsulating, is Vitamin D3 (cholecalciferol).

In another embodiment of the calcium coated nanoparticles encapsulating Vitamin D prepared using the above reference method, have an average particle (size) between 150nm-220nm.

The term average particle size as used herein refers to the average diameter of the particles.

The term “particles” as used herein refers to an individual particle of the polymeric nanoparticle comprising the active agent(s) (Vitamin D). In one or more embodiments of the present invention the calcium coated nanoparticles encapsulating Vitamin D prepared using the above reference method, have positive zeta potential of 28.8mV.

The present invention also provides a method of preventing or treating vitamin D deficiency by administering a pharmaceutical composition comprising polymeric nanoparticles of one or more active agent(s) (Vitamin D) to a patient in need thereof.

It may be well acknowledged to a person skilled in the art that the said composition, according to the present invention, may further comprise one or more active agents in particular other active agent(s) (Vitamin D), such as, but are not limited to, alkylating agents, antimetabolites, anti-microtubule agents, topoisomerase inhibitors or antitumor antibiotics, DNA linking agents, biological agents and bisphosphonates.

As will be apparent to those skilled in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited in any other order that is logically possible.

EXAMPLES:

Before the nanoparticles, compositions and methods of the present disclosure are described in greater detail, it is to be understood that the invention is not limited to particular embodiments and may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
Example 1:

Preparation of Vitamin D PLGA nanoparticles

The various components of nanoparticles including organic phase of dichloromethane with polymer, Vitamin D (1:1 W/W) and stabilizer as PVA (1.5% W/V), calcium phosphate (1.5%W/V) are combined and mixed together. This mixture is blended with vortex at 2000rpm for 2-15 min; preferably at 4-10 min to form a coarse emulsion with larger droplet size. The coarse emulsion was passed through high pressure homogenizer under high pressure of 100-14000 bars; preferably 250-800 bars for 15 cycles of each 5 minutes to 15 minutes to obtain a nano emulsion. The above obtained emulsion system was vacuum evaporated for removing the solvent and forming nanoparticles. A pressure of 100-900 psi was applied; more preferably 200-750 psi. The formed nanoparticles were ultra-centrifuged at 50000 rpm to remove excess stabilizer and the step was repeated thrice by resuspending the pellet in deionized water. The resulting nanoparticles pellet was resuspended in minimal volume of water, lyophilized and stored at 4ºC. The formed nanoparticle concentration was further determined by modified ion-pairing HPLC method using an evaporative light scattering detector, ELSD). The calcium content of nanoparticle was found to be 0.9-1.8 µmol/mg weight of nanoparticles. Concentration of Vitamin D was found to be about 75-95 wt % of nanoparticles.

The concentration of components is depicted in Table 1.

Table 1
S. No. Components Component Quantity (W/W or W/V) used in Process
1. PLGA 1%
2. Vitamin D 1%
3. PVA (polyvinyl Alcohol) 1.5%
4. Calcium phosphate 1.5%

Example 2:

Determination of Nanoparticle Size and Zeta Potential (surface charge)

The nanoparticles were analysed for particle size at 1:100 dilution factor with distilled water as diluent and zeta potential was measured using omega cuvettes of Litesizer 500. The average particle size of the final formulation was found to be ranging between 150-220nm with a positive zeta potential of 28.8mV. The calcium coating of positively charged calcium ions on the surface of PLGA nanoparticles encapsulating Vitamin D provides positive zeta potential. Figure 3 depicts the graph showing the zeta potential distribution of the PLGA-cholecalciferol (Vitamin D3 nanoparticles) and calcium coated PLGA-cholecalciferol (Vitamin D3 nanoparticles).

Example 3:

Stability Profile at Room Temperature (27°C) and at 4°C

Stability studies were conducted in order to assess the shelf-life of Vitamin D nanoparticulate composition. The Vitamin D formulation containing the amount of 40KIU/L was evaluated up to 250 days and the concentration of vitamin D in the solution was recorded at room temperature (27°C) and at temperature of 4ºC. Variations in the particle size and charge of the nanoparticles were monitored to study the stability. No significant variations in charge was found.

The results of particle size study are depicted in Table 2.
Table 2
Temp (°C)/ Size (nm) Day 1 Day 15 Day 35 Day 45 Day 60 Day 100 Day 150 Day 200 Day 250
27°C 192 ±
8.9 198.3 ±
0.24 201.1 ±
0.8 203.8±
0.15 204.8±
0.20 210.8±
0.15 212.8±
0.22 214.8 ±
0.25 218.8 ±
0.23
4°C 192 ±
8.9 198.4 ±
0.23 199.4 ±
0.20 199.5±
0.22 199.5±
0.25 200.5±
0.23 203.5±
0.20 2.05 ±
0.22 207.5 ±
0.23

At the end of 250 days, Vitamin D particles size variation was found to be around 218.8 ± 0.23 nm at 27°C, and 207.5 ± 0.23 nm at 4°C. Thus, the Vitamin D particles were found to be stable up to 250 days at both 27°C and 4°C. The system was found to be stable throughout the shelf life.

Example 4:

Quantification of bio availability of 25-hydroxyvitamin D [25(OH)D]/ Vitamin D

The bioavailability sustenance ability of these nanoparticulate systems was evaluated by orally administering the formulations to mice and periodically withdrawing blood on alternate days for seven days to monitor the serum Vitamin D (25 OH vitamin D) levels (nanograms per millilitre (ng/mL)) using HPLC quantification technique.

Table 3 depicts the comparative data for bioavailability of PLGA nanoparticles and Ca-PLGA nanoparticles:

Table 3
Day 1 Day 3 Day 5 Day 7
Mice group (n=5) PLGA Nanoparticle 26.15 ± 5.416 100.52 ± 3.44 3.47 ± 9.83 0.80 ± 2.422
Mice group 2
(n=5) Ca PLGA Nanoparticle 22.58 ± 6.68 99.00 ± 5.67 12.83 ± 6.47 2.314 ± 4.54
Control 0.512 ± 1.47 0.512 ± 1.47 0.512 ± 1.47 0.512 ± 1.47

From Table 3 it is clearly evident that the cationic nanoparticles (i.e Ca PLGA nanoparticles) were able to sustain the serum Vitamin D levels (ng/mL) longer in comparison to negatively charged PLGA nanoparticles. This significant difference in serum levels can be attributed to the cationic nature of the nanoparticles wherein they form a weak ionic bond with the negatively charged mucosal linings of the gut and thereby their rapid clearance from the mucosa is avoided. This further leads to higher systemic bioavailability in comparison to the negatively charged formulation-which gets eliminated rapidly.

Controls used for this experiment were the sera obtained from untreated mice (n=5) which were quantified for 25OH Vit D using HPLC and the obtained values were subtracted from the treated mice before plotting the graph.

Figure 4 depicts the quantification of bioavailability of 25-hydroxyvitamin D [25(OH)D]/ Vitamin D after oral administration to mice using HPLC.

Example 5:

UVA and UVB protection of nanoparticles

It is known in the art that Vitamin D degrades on exposure to UV rays. The ability of this present nanoparticulate system to protect vitamin D from UV A (3.0-3.2 eV), UV B (3.94-4.43 eV) radiations was evaluated.

The Vitamin D loaded nanoparticles (40K IU) were exposed to UV A and B radiations using simulator equipment DAAVALIN [integrating flex dosimetry control system-UV sensors R10000ABBX0606] for a particular period of time (24 hours) and then the nanoparticles were lysed using 1M NaOH solution, centrifuged at 8000rpm for 15min and the supernatant was collected. Vitamin D quantification for any degradation was carried out using HPLC method.

Figure 5A and 5B depict Free Vit D exposed to UV A and UV B, respectively.
Figure 5C and 5D depict the protection of vitamin D encapsulated in calcium coated PLGA nanoparticles from UVA and B radiations, respectively.

On comparing the peak retention time of Free Vit D exposed to UV A in figure 5A (2.240) and peak retention time of Vit D extracted from the UV exposed nanoparticles in figure 5C (7.753), it is clearly evident that the present nanoparticulate system provides protection to Vit D from UV A rays.

Similarly, on comparing the peak retention time of Free Vit D exposed to UV B in figure 5B (2.220 and 2.347) and peak retention time of Vit D extracted from the UV exposed nanoparticles in figure 5D (7.573), it is clearly evident that the present nanoparticulate system provides protection to Vit D from UV B rays.

From the results it is evident that the protection is offered by the nanoparticles to Vit D from UV rays.
, Claims:1. A calcium coated nanoparticle encapsulating Vitamin D, wherein the nanoparticle is Poly Lactide co Glycolide (PLGA) nanoparticle, and wherein calcium coating provides positive charge on the outer surface of nanoparticles.

2. The calcium coated nanoparticles encapsulating Vitamin D as claimed in Claim 1, wherein calcium ions are present in amount of 0.9-1.8 µmol/mg weight of nanoparticle.

3. The calcium coated nanoparticles encapsulating Vitamin D as claimed in Claim 1, wherein the loading of vitamin D is about 75-95 wt % of nanoparticles.

4. The calcium coated nanoparticle encapsulating Vitamin D as claimed in Claim 1, wherein Vitamin D is Vitamin D3 (cholecalciferol).

5. The calcium coated nanoparticle encapsulating Vitamin D as claimed in Claim 1, wherein positively charged nanoparticle is adapted to deliver the Vitamin D to the negatively charged mucosal surface.

6. A composition comprising the calcium nanoparticle as claimed in claim 1 to 5, and one or more pharmaceutically acceptable excipients or carriers.

7. The composition comprising the calcium encapsulated PLGA nanoparticles as claimed in Claim 6, wherein pharmaceutically acceptable excipients or carriers is an aqueous medium.

8. The composition comprising the calcium encapsulated PLGA nanoparticles as claimed in Claim 6 and Claim 7, wherein calcium encapsulated PLGA nanoparticles exhibits higher level of serum bioavailability upto seven days after in vivo oral administration compared to conventional PLGA vitamin D nanoparticles.
9. The composition comprising the calcium encapsulated PLGA nanoparticles as claimed in Claim 6 and Claim 7, wherein calcium encapsulated PLGA nanoparticles exhibits protection from exposure to UV A/ B radiations.

10. The composition comprising the calcium encapsulated PLGA nanoparticles as claimed in Claim 6 and Claim 7, wherein the composition is stable up to 250 days at both 27°C and 4°C.

11. The composition comprising the calcium encapsulated PLGA nanoparticles as claimed in Claim 6 and Claim 7, can be utilized for prevention or treatment of vitamin D deficiency.

12. A method for preparation of calcium coated nanoparticles encapsulating Vitamin D as claimed in Claim 1, by steps comprising:
a. preparing a reaction mixture of PLGA nanoparticle with Vitamin D dissolved in organic phase with stabilizer and calcium phosphate;
b. vortexing the mixture at 2000 rpm for 2-15 min, preferably 4-10 min to form coarse emulsion;
c. high pressure homogenization at 100-14000 bars, preferably 250-800 bars for 15 cycles of each 5 minutes to 15 minutes to obtain a nano emulsion;
d. evaporating the solvent and forming nanoparticle with pressure of 100-900 psi preferably 200-750 psi;
e. ultracentrifuging thrice at 50000 rpm by suspending the pellet in deionized water; and
f. suspending the pellet in ultrapure water and lyophilizing at 4°C.

13. The method for preparation of calcium coated nanoparticles encapsulating Vitamin D as claimed in Claim 12, wherein concentration of PLGA nanoparticles is 1%.

14. The method for preparation of calcium coated nanoparticles encapsulating Vitamin D as claimed in Claim 12, wherein concentration of Vitamin D is 1%.
15. The method for preparation of calcium coated nanoparticles encapsulating Vitamin D as claimed in Claim 12, wherein concentration of calcium phosphate is 1.5%.

16. The method for preparation of calcium coated nanoparticles encapsulating Vitamin D as claimed in Claim 12, wherein stabilizer is Polyvinyl alcohol (PVA).

17. The method for preparation of calcium coated nanoparticles encapsulating Vitamin D as claimed in Claim 12, wherein the concentration of Polyvinyl alcohol is 1.5%.

18. The method for preparation of calcium coated nanoparticles encapsulating Vitamin D as claimed in Claim 12, wherein the formed nanoparticles have an average particle (size) diameter between 150nm – 220nm.

19. The method for preparation of calcium coated nanoparticles encapsulating Vitamin D as claimed in claim 12, wherein the formed nanoparticles have a positive zeta potential of 28.8mV.

20. The method for preparation of calcium coated nanoparticles encapsulating Vitamin D as claimed in Claim 12, wherein Vitamin D is Vitamin D3 (cholecalciferol).